Bioanalytical Approaches for Early Assessment of Metabolite Exposures in Human and Animals to Address
MIST Fundamentals Strategy and Perspectives
Hongying Gao
Innovo Bioanalysis LLC
June 14 2018
Acknowledgements
R Scott Obach PDM Pfizer Inc
Shibing Deng Biostatistics Pfizer Inc
Safety Testing of Drug Metabolites Guidance
bull First FDA guidance was issued in Feb 2008 followed by ICH M3 (R2) in Jan 2010
bull Recent revision of FDA guidance was issued in Nov 2016
ndash Supersedes previous version and aligns with ICH M3(R2)
ndash 10 of total drug related exposure at steady state
ndash Approximately equal or greater exposure in animals
ndash Acknowledges the difficulties associated with metabolite synthesis
ndash Suggests early assessment of metabolite exposure in human and animals
Bioanalytical Challenges to Address MIST
bull Synthetic standards of metabolites
bull Simultaneous analysis of multiple analytes
bull Radiolabel ADME Studiesmdashonly single dose no steady state data
bull NMR-based standardsmdashbiosynthesis and isolation
Bioanalytical Approaches
Mixed Matrix Peak Area
Approaches (1)
Radiometric Calibrants (2)
NMR-Based Methods (3)
Conventional GLP Bioanalysis
Data Output
Animal vs Human Ratios
Absolute Concentrations
Absolute Concentrations
Absolute Concentrations
Reagent Needs
None Radio-labeled
Drug None
Metabolite Standards
Special Equipment Needs
None None NMR None
Resource Investment
Low Moderate High Very High
Uses MIST Coverage PK Data
MIST Coverage of total
PK Data MIST Coverage
PK Data MIST Coverage
MIST Tiered Approach
bull Use screening qualified or validated methods as function of stage of development and study type
Timmerman P et al 2010 Bioanalysis 2(7) 1185ndash1194
MIST Coverage is a Comparison Question
Animals have exposures of metabolites ge human
Not the exposure multiples
A simple LCMSMS method can provide the data to ascertain the exposure coverage without developing
and validating standard bioanalytical methods for every metabolite
Simple LCMSMS Peak Area Comparison
bull Principle Hamilton pool the time points Inject animal and human sample side-by-side on HPLC-MS compare peak area ratios of metabolites
bull Whatrsquos known bull HPLC retention time and fragmentation pattern for the
metabolite
bull Mixed matrix approach Routinely used in bioanalysis
Matrix effect is the same in samples provided the matrices are identical
bull Mix animal sample with control human matrix
bull Mix human sample with control animal matrix
bull Mix control animal and control human matrix to ensure no background interference
Time-Averaged Pooling (ldquoHamilton Poolsrdquo)
bull Plasma samples from different time points are mixed to provide a single sample in which the analyte concentration Cavg is AUC over the sampling interval
-0avg
AUCC
Hamilton Garnett Kline Clin Pharmacol Ther 1981
total
1-2-nn130201
V)t-(t)t-(t)t-(t)t-(t)t-(t
)t-(tV
010t
total
1-2-nn130201
1-n1nnt V
)t-(t)t-(t)t-(t)t-(t)t-(t
)t-(tV
total
1-2-nn130201
1-t V
)t-(t)t-(t)t-(t)t-(t)t-(t
)t-(tV
bull Can be used to reduce the number of samples to be analyzed and still yield exposure comparisons across species
Mixed Matrix Peak Area Comparison
Pooled Human Sample
Pooled Animal Sample
Mix with Opposite
Matrix Control
Control Mix
Add IS and
Solvent
Recover Supernatant Analyze by HPLC-MS
IS IS IS
Met
Met
Human Animal Matrix Control
(Human) AreaPeak IS
(Human) AreaPeak Metabolite
(Animal) AreaPeak IS
(Animal) AreaPeak Metabolite
Ratio Human to Animal
Control Mix
Mixed Human Sample
Mixed Animal Sample
Mixed Human Sample
Mixed Animal Sample
Gao et al DMD 2010 Gao and Obach DMD 2012
Protein Precipitation
bull Selectivity minus High separation resolution chromatography (a long gradient on
a large column) + MRM or high resolution MS (ge30K for parent ion) w product ion scan for major human metabolites (ge10 of total drug related material)
minus Low throughput is offset by the limited of samples
bull Sensitivity Typically not an issue minus Major human metabolites (ge10 of drug related total material)
and the animal is dosed at NOAEL minus MRM or high accurate MS for parent (plusmn 10ppm) is more
sensitive than full scan MSMS for metabolite ID
bull Matrix effect Normalized by mixed matrix approach
bull Linearity Serial dilutions of the samples in blank mixed plasma
How to Establish BA Parameters without Synthetic Standards
bull Stabilize the metabolites if procedures are known
bull Repetitive analysis of samples to cover the storage duration
bull Profile metabolitesstability reference (usually the parent drug) vs the storage time
bull Auto sampler stability re-injecting the supernatant 12-24 hr later
1day
dayX
sample) (Same AreaPeak reference Stable
sample) Same ( AreaPeak Metabolite
sample) (same AreaPeak reference Stable
species)(Any AreaPeak Metabolite
RatioStability
How to Establish Stability without Synthetic Standards
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
Variety of reaction types alterations in pKa multiple regioisomers
(AZT)
Blind Test 1 Stable Metabolites
M1 236gt134
M2 252gt209
Ziprasidonemetabolites MRM TIC in rat plasma 1
IS
Ziprasidone 413gt194
M9
M5
M10
Dilution Curve M1 236gt134
Dilution Curve M9 429gt280
2 B
80 B
Column Kinetex C18 150 X 46 mm 26 um
Parent Metabolite Potential Instability
Naproxen Naproxen Acyl Glucuronide
Acyl bond migration
Ester hydrolysis
Carbamazepine Carbamazepine-1011-Epoxide
Epoxide hydrolysis
Salicylic Acid 23-Dihydroxybenzoic Acid
Catechol oxidation
Sulfadiazine N-Acetylsulfadiazine
Amide hydrolysis
Methylphenidate Ritalinic Acid
Ester hydrolysis
Nicotine Nicotine-Δ1rsquo5rsquo-Iminium Ion and Cotinine
Imine Oxidation
N
O NH2
MeO
O
O
OH
OH
OH
O OH
N
N+
N
NO
N
N
NH
OMeO
NH
OHO
OH
O
OH
OH
NH
SNH
N
N
O
O
O
NH2
SNH
N
N
O
O
OH
O
OH
N
O NH2
O
MeO
OH
O
Blind Test 2 Unstable Metabolites
Long Term Storage Stability of Naproxen Acyl-Glucuronide Conjugate
0
05
1
15
2
0 50 100 150
(Nap
roxe
n-G
luN
apro
xen
) D
ay X
Day
1
Storage Time at -20ordmC (Day)
Naproxpen-GluNaproxen normalized byDay1 response
Stabilized Naproxen-GluNaproxenNormalized by Day 1
Linear (Naproxpen-GluNaproxen normalizedby Day1 response)
Linear (Stabilized Naproxen-GluNaproxenNormalized by Day 1)
n = 8
Untreated samples
A ratio between 08-12 is deemed stable
Stabilized samples
Gao H and Obach RS (2012) DMD 401290-1296
Correlation of Experimental MS Ratio vs Nominal Ratio
R 0994plusmn0005 Slope 099plusmn0082
MSNM Ratio
Metabolites
MS
NM
Ra
tio
10
15
20
1_O
H_T
acrine
2_O
H_T
acrine
3_M
eth
oxym
orp
hin
4_O
H_D
iclo
fenc
4_O
H_T
acrine
5_O
H_D
iclo
fenc
AZ
T
AZ
T_glu
c
Dextr
om
eth
orp
han
Dextr
orp
han
Dic
lofe
nac
Fexofe
nadin
e
M1
M10
M2
M5
M9
tacrine
Terf
enadin
e
Zip
rasid
one
Geo FC
Geo FC 95
Geo FC 99
Average 125
95 confidence 183
99 confidence 207
Precision Accuracy and Confidence Level bull Showed that a peak area ratio of 2 means that it is at least equal or
greater exposure
bull Increased replicates will increase confidence
95 Confidence Interval (~2SD)
Gao H Deng S and Obach RS (2010) DMD 382147-2156
Confidence Level Increases Dramatically with Measured Ratio
Ratio=4 plt2 ppm
Ratio=2 plt001
Measured animalhuman ratio by LCMSMS
Measured animalhuman ratio by LCMSMS
Probability of Nominal AnimalHuman ge1
What Drives High Confidence
bull Fundamental BA parameters are addressed Selectivity sensitivity linearity matrix effect stability precision and accuracy
bull SignalNoise Ratio Measured animalhuman 2
Measurement error (without cancellation) 025
SN (2-1)025=4
Limit of Detection A signal is real if SN=3
bull Minimal 3 independent animalhuman ratio measurements
bull Analysis reproducibility RSD of the ratio measurements (n=3) 33
bull Stability ratio for each metabolite plusmn20 (08-12) over the storage period
How to Evaluate Data Quality
MIST Coverage is a Comparison Question
Animals have exposures of metabolites ge human
Comparison 1 Giraffe vs Moose
Which one is taller
All that one needs to do is to have them stand next to each other The giraffe is much taller No need to know exact heights
Which one is taller
Too close to call just by looking at them -- The height measurements are needed
Comparison 2 Moose vs Whitetail Deer
If the animalhuman peak area ratio is ge 2 animal has exposure ge human with high confidence (plt001)
How often does it happen Most cases as animal is dosed at much higher
dose when corrected by body weight
A simple peak area ratio comparison is sufficient for most cases
Case Study
bull Drug A in project X at phase 1 14 days 35 mg BID in 6 human volunteers vs 5 days 300 mgkg in Sprague Dawley rats
bull Pooled plasma samples (Hamilton) across time points across subjects
bull 3 human metabolites Parent drug A 396gt203
m-hydroxy 412 ring opened 414 de-alkylated 304
bull HPLC method and major daughter ions were transferred from Biotransformation
bull Compare results with concentration measurements using the synthetic standard
MIST Coverage Evaluation Using RatHuman Ratio of Peak Area Ratios
Definitive Concentration Measurements Using Synthetic Standard
Species Parent De-alkylated Ring-opened m-Hydroxy
Human 0220 00406 000788 00210 Rat 770 0502 0292 000762
RatHuman ratio 1 35 124 371 0363
Human repeat 0184 00290 000506 00164
Rat repeat 734 0472 0266 000618 RatHuman
ratio 2 399 163 526 0378
Species m-Hydroxy (ngmL)
Human 743 plusmn 153
Rat 269 plusmn 258
Cavg RatHuman ratio 0362
Human Metabolite gt10 Drug-Related Material from a Human Radiolabel
ADME Study (or Metabolite Anticipated to Be gt10 from Other Human Studies)
Metabolite With Known Activity of
Interest
Use Standard Bioanalytical
Method
ICH Exempt Metabolite
Peak Area Ratio Method
Method Fails Criteria
Method Succeeds
Animal lt 2X Human
Animal ge 2X Human
No Further Work Needed
Coverage is Assured No Further Work
Needed
Prepare Authentic Standard of Metabolite
MIST Decision Tree
Gao H and Obach RS (2014) Bioanalysis 6(5) 641-650
Data Driven Strategy
bull Rigor of the analysis increases based on data Synthetic standard is needed only when the method fails or animal has less exposure than human
bull Conservative evaluation of MIST coverage
bull Assess MIST coverage at steady state without radio-label or synthetic standards enabling early assessment eg FIH
bull Time- and resource-saving strategy
Summary
bull LCMSMS peak area ratio comparison is scientifically sound and defensible method that can
ndash Ascertain the steady-state exposure of animals compared to humans (ie ldquoMIST coveragerdquo)
ndash Assess the MIST coverage without radio-label or synthetic standard
bull These methods are fit-for-purpose and can offer decision-making data useful for reliable estimation of metabolite exposure
bull Discussions with regulatory experts were positive and helpful
Gao et al (2013) AAPS Journal15(4) 970-973
Prerequisite for Current Methodology
bull Animal and human samples for side-by-side injection
minus In drug development first in human samples are typically ~1 year later than the toxicology study is done
bull Prior knowledge of human metabolites transferred from metabolite identification and scouting
minus Knowledge of major human metabolites may evolve only after in vivo human samples become available
minus Radio-labeled ADME is the most definitive study may take place late in the drug development process
Is it feasible to scan the samplesbank data and compare the data generated at different dates
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
MeO
O
O
OH
OH
OH
O OHNaproxen Acyl Glucuronide conjugate
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
(AZT)
Negative Control
Blind Test 3 Metabolite Cocktail
Gao et al Anal Chem 2015 87 11771-11776
Scanning and Banking DataSamples
bull Blind Test samples 22 metabolitesdrugs were spiked in human and rat plasmas (n=4 each)
bull No knowledge of the identities of the analytes and their concentrations in plasma were provided before sample analysis
bull Multiple analysis of the same samples (rat or human) using mixed matrix approach at Days 1 30 105 254 and 314
bull Treat the samples using the same IS solution in acetonitrile minus Freshly prepared to maintain the same concentration
bull Acquity UPLC generic gradient on C18 run time 25 min
bull Full scan and AIF scan on QExactive 70K at positive and negative polarity
bull Compare rat vs human samples analyzed at different dates
0 4 8 12 16 20 24 Time (min)
0
100 R
elat
ive
Ab
un
dan
ce
508
478 682
1132 1435 1450 763
773 243 1835 2120 2138 1164 1312 1638 288 534 1473 2338 802 2045 335 1062
42 43 44 45 46 47 48 49 50 51 52 53 54 55 Time (min)
0
100
Rel
ativ
e In
ten
sity
RT 508
RT 478
RT 495
RT 539
4 8 12 16 20 24 Time (min)
0
100 RT 624
RT 1313
RT 1113
RT 1626
RT 275 RT 879
RT 2008 RT 422
548
165 808
1 2
3
3
4
5
5
4
678
9 10 111213
14 16 15
1
2
3
4
56 7
Rel
ativ
e A
bu
nd
ance
Migrated naproxen acyl glucuronide
EIC 15 drugsmetabolites Resolution 70000 Extraction window 5ppm Polarity Positive
EIC 6 drugsmetabolites Resolution 70000 Extraction window 10 ppm Polarity Negative
2-OH Tacrine
4-OH Tacrine
1-OH Tacrine
Naproxen-Glu
Peak Area Ratio Measurement Variation
n=4 per species 5 independent analysis total n=40 per analyte
95 tile 037
Median 021
M1
0
DrugsMetabolites
Bank and Compare Data at Different Days
Gao et al Anal Chem 2015 87 11771-11776
Separation and Quantitative Analysis of Isomers
2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
100
Rela
tive I
nte
nsity
RT 1626
RT 275
RT 422 RT 1188 RT 632
Full Scan 4rsquo (or 5)-OH Diclofenac
XIC 3100032
Would high resolution ion mobility provide the orthogonal separation we need
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
Acknowledgements
R Scott Obach PDM Pfizer Inc
Shibing Deng Biostatistics Pfizer Inc
Safety Testing of Drug Metabolites Guidance
bull First FDA guidance was issued in Feb 2008 followed by ICH M3 (R2) in Jan 2010
bull Recent revision of FDA guidance was issued in Nov 2016
ndash Supersedes previous version and aligns with ICH M3(R2)
ndash 10 of total drug related exposure at steady state
ndash Approximately equal or greater exposure in animals
ndash Acknowledges the difficulties associated with metabolite synthesis
ndash Suggests early assessment of metabolite exposure in human and animals
Bioanalytical Challenges to Address MIST
bull Synthetic standards of metabolites
bull Simultaneous analysis of multiple analytes
bull Radiolabel ADME Studiesmdashonly single dose no steady state data
bull NMR-based standardsmdashbiosynthesis and isolation
Bioanalytical Approaches
Mixed Matrix Peak Area
Approaches (1)
Radiometric Calibrants (2)
NMR-Based Methods (3)
Conventional GLP Bioanalysis
Data Output
Animal vs Human Ratios
Absolute Concentrations
Absolute Concentrations
Absolute Concentrations
Reagent Needs
None Radio-labeled
Drug None
Metabolite Standards
Special Equipment Needs
None None NMR None
Resource Investment
Low Moderate High Very High
Uses MIST Coverage PK Data
MIST Coverage of total
PK Data MIST Coverage
PK Data MIST Coverage
MIST Tiered Approach
bull Use screening qualified or validated methods as function of stage of development and study type
Timmerman P et al 2010 Bioanalysis 2(7) 1185ndash1194
MIST Coverage is a Comparison Question
Animals have exposures of metabolites ge human
Not the exposure multiples
A simple LCMSMS method can provide the data to ascertain the exposure coverage without developing
and validating standard bioanalytical methods for every metabolite
Simple LCMSMS Peak Area Comparison
bull Principle Hamilton pool the time points Inject animal and human sample side-by-side on HPLC-MS compare peak area ratios of metabolites
bull Whatrsquos known bull HPLC retention time and fragmentation pattern for the
metabolite
bull Mixed matrix approach Routinely used in bioanalysis
Matrix effect is the same in samples provided the matrices are identical
bull Mix animal sample with control human matrix
bull Mix human sample with control animal matrix
bull Mix control animal and control human matrix to ensure no background interference
Time-Averaged Pooling (ldquoHamilton Poolsrdquo)
bull Plasma samples from different time points are mixed to provide a single sample in which the analyte concentration Cavg is AUC over the sampling interval
-0avg
AUCC
Hamilton Garnett Kline Clin Pharmacol Ther 1981
total
1-2-nn130201
V)t-(t)t-(t)t-(t)t-(t)t-(t
)t-(tV
010t
total
1-2-nn130201
1-n1nnt V
)t-(t)t-(t)t-(t)t-(t)t-(t
)t-(tV
total
1-2-nn130201
1-t V
)t-(t)t-(t)t-(t)t-(t)t-(t
)t-(tV
bull Can be used to reduce the number of samples to be analyzed and still yield exposure comparisons across species
Mixed Matrix Peak Area Comparison
Pooled Human Sample
Pooled Animal Sample
Mix with Opposite
Matrix Control
Control Mix
Add IS and
Solvent
Recover Supernatant Analyze by HPLC-MS
IS IS IS
Met
Met
Human Animal Matrix Control
(Human) AreaPeak IS
(Human) AreaPeak Metabolite
(Animal) AreaPeak IS
(Animal) AreaPeak Metabolite
Ratio Human to Animal
Control Mix
Mixed Human Sample
Mixed Animal Sample
Mixed Human Sample
Mixed Animal Sample
Gao et al DMD 2010 Gao and Obach DMD 2012
Protein Precipitation
bull Selectivity minus High separation resolution chromatography (a long gradient on
a large column) + MRM or high resolution MS (ge30K for parent ion) w product ion scan for major human metabolites (ge10 of total drug related material)
minus Low throughput is offset by the limited of samples
bull Sensitivity Typically not an issue minus Major human metabolites (ge10 of drug related total material)
and the animal is dosed at NOAEL minus MRM or high accurate MS for parent (plusmn 10ppm) is more
sensitive than full scan MSMS for metabolite ID
bull Matrix effect Normalized by mixed matrix approach
bull Linearity Serial dilutions of the samples in blank mixed plasma
How to Establish BA Parameters without Synthetic Standards
bull Stabilize the metabolites if procedures are known
bull Repetitive analysis of samples to cover the storage duration
bull Profile metabolitesstability reference (usually the parent drug) vs the storage time
bull Auto sampler stability re-injecting the supernatant 12-24 hr later
1day
dayX
sample) (Same AreaPeak reference Stable
sample) Same ( AreaPeak Metabolite
sample) (same AreaPeak reference Stable
species)(Any AreaPeak Metabolite
RatioStability
How to Establish Stability without Synthetic Standards
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
Variety of reaction types alterations in pKa multiple regioisomers
(AZT)
Blind Test 1 Stable Metabolites
M1 236gt134
M2 252gt209
Ziprasidonemetabolites MRM TIC in rat plasma 1
IS
Ziprasidone 413gt194
M9
M5
M10
Dilution Curve M1 236gt134
Dilution Curve M9 429gt280
2 B
80 B
Column Kinetex C18 150 X 46 mm 26 um
Parent Metabolite Potential Instability
Naproxen Naproxen Acyl Glucuronide
Acyl bond migration
Ester hydrolysis
Carbamazepine Carbamazepine-1011-Epoxide
Epoxide hydrolysis
Salicylic Acid 23-Dihydroxybenzoic Acid
Catechol oxidation
Sulfadiazine N-Acetylsulfadiazine
Amide hydrolysis
Methylphenidate Ritalinic Acid
Ester hydrolysis
Nicotine Nicotine-Δ1rsquo5rsquo-Iminium Ion and Cotinine
Imine Oxidation
N
O NH2
MeO
O
O
OH
OH
OH
O OH
N
N+
N
NO
N
N
NH
OMeO
NH
OHO
OH
O
OH
OH
NH
SNH
N
N
O
O
O
NH2
SNH
N
N
O
O
OH
O
OH
N
O NH2
O
MeO
OH
O
Blind Test 2 Unstable Metabolites
Long Term Storage Stability of Naproxen Acyl-Glucuronide Conjugate
0
05
1
15
2
0 50 100 150
(Nap
roxe
n-G
luN
apro
xen
) D
ay X
Day
1
Storage Time at -20ordmC (Day)
Naproxpen-GluNaproxen normalized byDay1 response
Stabilized Naproxen-GluNaproxenNormalized by Day 1
Linear (Naproxpen-GluNaproxen normalizedby Day1 response)
Linear (Stabilized Naproxen-GluNaproxenNormalized by Day 1)
n = 8
Untreated samples
A ratio between 08-12 is deemed stable
Stabilized samples
Gao H and Obach RS (2012) DMD 401290-1296
Correlation of Experimental MS Ratio vs Nominal Ratio
R 0994plusmn0005 Slope 099plusmn0082
MSNM Ratio
Metabolites
MS
NM
Ra
tio
10
15
20
1_O
H_T
acrine
2_O
H_T
acrine
3_M
eth
oxym
orp
hin
4_O
H_D
iclo
fenc
4_O
H_T
acrine
5_O
H_D
iclo
fenc
AZ
T
AZ
T_glu
c
Dextr
om
eth
orp
han
Dextr
orp
han
Dic
lofe
nac
Fexofe
nadin
e
M1
M10
M2
M5
M9
tacrine
Terf
enadin
e
Zip
rasid
one
Geo FC
Geo FC 95
Geo FC 99
Average 125
95 confidence 183
99 confidence 207
Precision Accuracy and Confidence Level bull Showed that a peak area ratio of 2 means that it is at least equal or
greater exposure
bull Increased replicates will increase confidence
95 Confidence Interval (~2SD)
Gao H Deng S and Obach RS (2010) DMD 382147-2156
Confidence Level Increases Dramatically with Measured Ratio
Ratio=4 plt2 ppm
Ratio=2 plt001
Measured animalhuman ratio by LCMSMS
Measured animalhuman ratio by LCMSMS
Probability of Nominal AnimalHuman ge1
What Drives High Confidence
bull Fundamental BA parameters are addressed Selectivity sensitivity linearity matrix effect stability precision and accuracy
bull SignalNoise Ratio Measured animalhuman 2
Measurement error (without cancellation) 025
SN (2-1)025=4
Limit of Detection A signal is real if SN=3
bull Minimal 3 independent animalhuman ratio measurements
bull Analysis reproducibility RSD of the ratio measurements (n=3) 33
bull Stability ratio for each metabolite plusmn20 (08-12) over the storage period
How to Evaluate Data Quality
MIST Coverage is a Comparison Question
Animals have exposures of metabolites ge human
Comparison 1 Giraffe vs Moose
Which one is taller
All that one needs to do is to have them stand next to each other The giraffe is much taller No need to know exact heights
Which one is taller
Too close to call just by looking at them -- The height measurements are needed
Comparison 2 Moose vs Whitetail Deer
If the animalhuman peak area ratio is ge 2 animal has exposure ge human with high confidence (plt001)
How often does it happen Most cases as animal is dosed at much higher
dose when corrected by body weight
A simple peak area ratio comparison is sufficient for most cases
Case Study
bull Drug A in project X at phase 1 14 days 35 mg BID in 6 human volunteers vs 5 days 300 mgkg in Sprague Dawley rats
bull Pooled plasma samples (Hamilton) across time points across subjects
bull 3 human metabolites Parent drug A 396gt203
m-hydroxy 412 ring opened 414 de-alkylated 304
bull HPLC method and major daughter ions were transferred from Biotransformation
bull Compare results with concentration measurements using the synthetic standard
MIST Coverage Evaluation Using RatHuman Ratio of Peak Area Ratios
Definitive Concentration Measurements Using Synthetic Standard
Species Parent De-alkylated Ring-opened m-Hydroxy
Human 0220 00406 000788 00210 Rat 770 0502 0292 000762
RatHuman ratio 1 35 124 371 0363
Human repeat 0184 00290 000506 00164
Rat repeat 734 0472 0266 000618 RatHuman
ratio 2 399 163 526 0378
Species m-Hydroxy (ngmL)
Human 743 plusmn 153
Rat 269 plusmn 258
Cavg RatHuman ratio 0362
Human Metabolite gt10 Drug-Related Material from a Human Radiolabel
ADME Study (or Metabolite Anticipated to Be gt10 from Other Human Studies)
Metabolite With Known Activity of
Interest
Use Standard Bioanalytical
Method
ICH Exempt Metabolite
Peak Area Ratio Method
Method Fails Criteria
Method Succeeds
Animal lt 2X Human
Animal ge 2X Human
No Further Work Needed
Coverage is Assured No Further Work
Needed
Prepare Authentic Standard of Metabolite
MIST Decision Tree
Gao H and Obach RS (2014) Bioanalysis 6(5) 641-650
Data Driven Strategy
bull Rigor of the analysis increases based on data Synthetic standard is needed only when the method fails or animal has less exposure than human
bull Conservative evaluation of MIST coverage
bull Assess MIST coverage at steady state without radio-label or synthetic standards enabling early assessment eg FIH
bull Time- and resource-saving strategy
Summary
bull LCMSMS peak area ratio comparison is scientifically sound and defensible method that can
ndash Ascertain the steady-state exposure of animals compared to humans (ie ldquoMIST coveragerdquo)
ndash Assess the MIST coverage without radio-label or synthetic standard
bull These methods are fit-for-purpose and can offer decision-making data useful for reliable estimation of metabolite exposure
bull Discussions with regulatory experts were positive and helpful
Gao et al (2013) AAPS Journal15(4) 970-973
Prerequisite for Current Methodology
bull Animal and human samples for side-by-side injection
minus In drug development first in human samples are typically ~1 year later than the toxicology study is done
bull Prior knowledge of human metabolites transferred from metabolite identification and scouting
minus Knowledge of major human metabolites may evolve only after in vivo human samples become available
minus Radio-labeled ADME is the most definitive study may take place late in the drug development process
Is it feasible to scan the samplesbank data and compare the data generated at different dates
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
MeO
O
O
OH
OH
OH
O OHNaproxen Acyl Glucuronide conjugate
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
(AZT)
Negative Control
Blind Test 3 Metabolite Cocktail
Gao et al Anal Chem 2015 87 11771-11776
Scanning and Banking DataSamples
bull Blind Test samples 22 metabolitesdrugs were spiked in human and rat plasmas (n=4 each)
bull No knowledge of the identities of the analytes and their concentrations in plasma were provided before sample analysis
bull Multiple analysis of the same samples (rat or human) using mixed matrix approach at Days 1 30 105 254 and 314
bull Treat the samples using the same IS solution in acetonitrile minus Freshly prepared to maintain the same concentration
bull Acquity UPLC generic gradient on C18 run time 25 min
bull Full scan and AIF scan on QExactive 70K at positive and negative polarity
bull Compare rat vs human samples analyzed at different dates
0 4 8 12 16 20 24 Time (min)
0
100 R
elat
ive
Ab
un
dan
ce
508
478 682
1132 1435 1450 763
773 243 1835 2120 2138 1164 1312 1638 288 534 1473 2338 802 2045 335 1062
42 43 44 45 46 47 48 49 50 51 52 53 54 55 Time (min)
0
100
Rel
ativ
e In
ten
sity
RT 508
RT 478
RT 495
RT 539
4 8 12 16 20 24 Time (min)
0
100 RT 624
RT 1313
RT 1113
RT 1626
RT 275 RT 879
RT 2008 RT 422
548
165 808
1 2
3
3
4
5
5
4
678
9 10 111213
14 16 15
1
2
3
4
56 7
Rel
ativ
e A
bu
nd
ance
Migrated naproxen acyl glucuronide
EIC 15 drugsmetabolites Resolution 70000 Extraction window 5ppm Polarity Positive
EIC 6 drugsmetabolites Resolution 70000 Extraction window 10 ppm Polarity Negative
2-OH Tacrine
4-OH Tacrine
1-OH Tacrine
Naproxen-Glu
Peak Area Ratio Measurement Variation
n=4 per species 5 independent analysis total n=40 per analyte
95 tile 037
Median 021
M1
0
DrugsMetabolites
Bank and Compare Data at Different Days
Gao et al Anal Chem 2015 87 11771-11776
Separation and Quantitative Analysis of Isomers
2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
100
Rela
tive I
nte
nsity
RT 1626
RT 275
RT 422 RT 1188 RT 632
Full Scan 4rsquo (or 5)-OH Diclofenac
XIC 3100032
Would high resolution ion mobility provide the orthogonal separation we need
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
Safety Testing of Drug Metabolites Guidance
bull First FDA guidance was issued in Feb 2008 followed by ICH M3 (R2) in Jan 2010
bull Recent revision of FDA guidance was issued in Nov 2016
ndash Supersedes previous version and aligns with ICH M3(R2)
ndash 10 of total drug related exposure at steady state
ndash Approximately equal or greater exposure in animals
ndash Acknowledges the difficulties associated with metabolite synthesis
ndash Suggests early assessment of metabolite exposure in human and animals
Bioanalytical Challenges to Address MIST
bull Synthetic standards of metabolites
bull Simultaneous analysis of multiple analytes
bull Radiolabel ADME Studiesmdashonly single dose no steady state data
bull NMR-based standardsmdashbiosynthesis and isolation
Bioanalytical Approaches
Mixed Matrix Peak Area
Approaches (1)
Radiometric Calibrants (2)
NMR-Based Methods (3)
Conventional GLP Bioanalysis
Data Output
Animal vs Human Ratios
Absolute Concentrations
Absolute Concentrations
Absolute Concentrations
Reagent Needs
None Radio-labeled
Drug None
Metabolite Standards
Special Equipment Needs
None None NMR None
Resource Investment
Low Moderate High Very High
Uses MIST Coverage PK Data
MIST Coverage of total
PK Data MIST Coverage
PK Data MIST Coverage
MIST Tiered Approach
bull Use screening qualified or validated methods as function of stage of development and study type
Timmerman P et al 2010 Bioanalysis 2(7) 1185ndash1194
MIST Coverage is a Comparison Question
Animals have exposures of metabolites ge human
Not the exposure multiples
A simple LCMSMS method can provide the data to ascertain the exposure coverage without developing
and validating standard bioanalytical methods for every metabolite
Simple LCMSMS Peak Area Comparison
bull Principle Hamilton pool the time points Inject animal and human sample side-by-side on HPLC-MS compare peak area ratios of metabolites
bull Whatrsquos known bull HPLC retention time and fragmentation pattern for the
metabolite
bull Mixed matrix approach Routinely used in bioanalysis
Matrix effect is the same in samples provided the matrices are identical
bull Mix animal sample with control human matrix
bull Mix human sample with control animal matrix
bull Mix control animal and control human matrix to ensure no background interference
Time-Averaged Pooling (ldquoHamilton Poolsrdquo)
bull Plasma samples from different time points are mixed to provide a single sample in which the analyte concentration Cavg is AUC over the sampling interval
-0avg
AUCC
Hamilton Garnett Kline Clin Pharmacol Ther 1981
total
1-2-nn130201
V)t-(t)t-(t)t-(t)t-(t)t-(t
)t-(tV
010t
total
1-2-nn130201
1-n1nnt V
)t-(t)t-(t)t-(t)t-(t)t-(t
)t-(tV
total
1-2-nn130201
1-t V
)t-(t)t-(t)t-(t)t-(t)t-(t
)t-(tV
bull Can be used to reduce the number of samples to be analyzed and still yield exposure comparisons across species
Mixed Matrix Peak Area Comparison
Pooled Human Sample
Pooled Animal Sample
Mix with Opposite
Matrix Control
Control Mix
Add IS and
Solvent
Recover Supernatant Analyze by HPLC-MS
IS IS IS
Met
Met
Human Animal Matrix Control
(Human) AreaPeak IS
(Human) AreaPeak Metabolite
(Animal) AreaPeak IS
(Animal) AreaPeak Metabolite
Ratio Human to Animal
Control Mix
Mixed Human Sample
Mixed Animal Sample
Mixed Human Sample
Mixed Animal Sample
Gao et al DMD 2010 Gao and Obach DMD 2012
Protein Precipitation
bull Selectivity minus High separation resolution chromatography (a long gradient on
a large column) + MRM or high resolution MS (ge30K for parent ion) w product ion scan for major human metabolites (ge10 of total drug related material)
minus Low throughput is offset by the limited of samples
bull Sensitivity Typically not an issue minus Major human metabolites (ge10 of drug related total material)
and the animal is dosed at NOAEL minus MRM or high accurate MS for parent (plusmn 10ppm) is more
sensitive than full scan MSMS for metabolite ID
bull Matrix effect Normalized by mixed matrix approach
bull Linearity Serial dilutions of the samples in blank mixed plasma
How to Establish BA Parameters without Synthetic Standards
bull Stabilize the metabolites if procedures are known
bull Repetitive analysis of samples to cover the storage duration
bull Profile metabolitesstability reference (usually the parent drug) vs the storage time
bull Auto sampler stability re-injecting the supernatant 12-24 hr later
1day
dayX
sample) (Same AreaPeak reference Stable
sample) Same ( AreaPeak Metabolite
sample) (same AreaPeak reference Stable
species)(Any AreaPeak Metabolite
RatioStability
How to Establish Stability without Synthetic Standards
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
Variety of reaction types alterations in pKa multiple regioisomers
(AZT)
Blind Test 1 Stable Metabolites
M1 236gt134
M2 252gt209
Ziprasidonemetabolites MRM TIC in rat plasma 1
IS
Ziprasidone 413gt194
M9
M5
M10
Dilution Curve M1 236gt134
Dilution Curve M9 429gt280
2 B
80 B
Column Kinetex C18 150 X 46 mm 26 um
Parent Metabolite Potential Instability
Naproxen Naproxen Acyl Glucuronide
Acyl bond migration
Ester hydrolysis
Carbamazepine Carbamazepine-1011-Epoxide
Epoxide hydrolysis
Salicylic Acid 23-Dihydroxybenzoic Acid
Catechol oxidation
Sulfadiazine N-Acetylsulfadiazine
Amide hydrolysis
Methylphenidate Ritalinic Acid
Ester hydrolysis
Nicotine Nicotine-Δ1rsquo5rsquo-Iminium Ion and Cotinine
Imine Oxidation
N
O NH2
MeO
O
O
OH
OH
OH
O OH
N
N+
N
NO
N
N
NH
OMeO
NH
OHO
OH
O
OH
OH
NH
SNH
N
N
O
O
O
NH2
SNH
N
N
O
O
OH
O
OH
N
O NH2
O
MeO
OH
O
Blind Test 2 Unstable Metabolites
Long Term Storage Stability of Naproxen Acyl-Glucuronide Conjugate
0
05
1
15
2
0 50 100 150
(Nap
roxe
n-G
luN
apro
xen
) D
ay X
Day
1
Storage Time at -20ordmC (Day)
Naproxpen-GluNaproxen normalized byDay1 response
Stabilized Naproxen-GluNaproxenNormalized by Day 1
Linear (Naproxpen-GluNaproxen normalizedby Day1 response)
Linear (Stabilized Naproxen-GluNaproxenNormalized by Day 1)
n = 8
Untreated samples
A ratio between 08-12 is deemed stable
Stabilized samples
Gao H and Obach RS (2012) DMD 401290-1296
Correlation of Experimental MS Ratio vs Nominal Ratio
R 0994plusmn0005 Slope 099plusmn0082
MSNM Ratio
Metabolites
MS
NM
Ra
tio
10
15
20
1_O
H_T
acrine
2_O
H_T
acrine
3_M
eth
oxym
orp
hin
4_O
H_D
iclo
fenc
4_O
H_T
acrine
5_O
H_D
iclo
fenc
AZ
T
AZ
T_glu
c
Dextr
om
eth
orp
han
Dextr
orp
han
Dic
lofe
nac
Fexofe
nadin
e
M1
M10
M2
M5
M9
tacrine
Terf
enadin
e
Zip
rasid
one
Geo FC
Geo FC 95
Geo FC 99
Average 125
95 confidence 183
99 confidence 207
Precision Accuracy and Confidence Level bull Showed that a peak area ratio of 2 means that it is at least equal or
greater exposure
bull Increased replicates will increase confidence
95 Confidence Interval (~2SD)
Gao H Deng S and Obach RS (2010) DMD 382147-2156
Confidence Level Increases Dramatically with Measured Ratio
Ratio=4 plt2 ppm
Ratio=2 plt001
Measured animalhuman ratio by LCMSMS
Measured animalhuman ratio by LCMSMS
Probability of Nominal AnimalHuman ge1
What Drives High Confidence
bull Fundamental BA parameters are addressed Selectivity sensitivity linearity matrix effect stability precision and accuracy
bull SignalNoise Ratio Measured animalhuman 2
Measurement error (without cancellation) 025
SN (2-1)025=4
Limit of Detection A signal is real if SN=3
bull Minimal 3 independent animalhuman ratio measurements
bull Analysis reproducibility RSD of the ratio measurements (n=3) 33
bull Stability ratio for each metabolite plusmn20 (08-12) over the storage period
How to Evaluate Data Quality
MIST Coverage is a Comparison Question
Animals have exposures of metabolites ge human
Comparison 1 Giraffe vs Moose
Which one is taller
All that one needs to do is to have them stand next to each other The giraffe is much taller No need to know exact heights
Which one is taller
Too close to call just by looking at them -- The height measurements are needed
Comparison 2 Moose vs Whitetail Deer
If the animalhuman peak area ratio is ge 2 animal has exposure ge human with high confidence (plt001)
How often does it happen Most cases as animal is dosed at much higher
dose when corrected by body weight
A simple peak area ratio comparison is sufficient for most cases
Case Study
bull Drug A in project X at phase 1 14 days 35 mg BID in 6 human volunteers vs 5 days 300 mgkg in Sprague Dawley rats
bull Pooled plasma samples (Hamilton) across time points across subjects
bull 3 human metabolites Parent drug A 396gt203
m-hydroxy 412 ring opened 414 de-alkylated 304
bull HPLC method and major daughter ions were transferred from Biotransformation
bull Compare results with concentration measurements using the synthetic standard
MIST Coverage Evaluation Using RatHuman Ratio of Peak Area Ratios
Definitive Concentration Measurements Using Synthetic Standard
Species Parent De-alkylated Ring-opened m-Hydroxy
Human 0220 00406 000788 00210 Rat 770 0502 0292 000762
RatHuman ratio 1 35 124 371 0363
Human repeat 0184 00290 000506 00164
Rat repeat 734 0472 0266 000618 RatHuman
ratio 2 399 163 526 0378
Species m-Hydroxy (ngmL)
Human 743 plusmn 153
Rat 269 plusmn 258
Cavg RatHuman ratio 0362
Human Metabolite gt10 Drug-Related Material from a Human Radiolabel
ADME Study (or Metabolite Anticipated to Be gt10 from Other Human Studies)
Metabolite With Known Activity of
Interest
Use Standard Bioanalytical
Method
ICH Exempt Metabolite
Peak Area Ratio Method
Method Fails Criteria
Method Succeeds
Animal lt 2X Human
Animal ge 2X Human
No Further Work Needed
Coverage is Assured No Further Work
Needed
Prepare Authentic Standard of Metabolite
MIST Decision Tree
Gao H and Obach RS (2014) Bioanalysis 6(5) 641-650
Data Driven Strategy
bull Rigor of the analysis increases based on data Synthetic standard is needed only when the method fails or animal has less exposure than human
bull Conservative evaluation of MIST coverage
bull Assess MIST coverage at steady state without radio-label or synthetic standards enabling early assessment eg FIH
bull Time- and resource-saving strategy
Summary
bull LCMSMS peak area ratio comparison is scientifically sound and defensible method that can
ndash Ascertain the steady-state exposure of animals compared to humans (ie ldquoMIST coveragerdquo)
ndash Assess the MIST coverage without radio-label or synthetic standard
bull These methods are fit-for-purpose and can offer decision-making data useful for reliable estimation of metabolite exposure
bull Discussions with regulatory experts were positive and helpful
Gao et al (2013) AAPS Journal15(4) 970-973
Prerequisite for Current Methodology
bull Animal and human samples for side-by-side injection
minus In drug development first in human samples are typically ~1 year later than the toxicology study is done
bull Prior knowledge of human metabolites transferred from metabolite identification and scouting
minus Knowledge of major human metabolites may evolve only after in vivo human samples become available
minus Radio-labeled ADME is the most definitive study may take place late in the drug development process
Is it feasible to scan the samplesbank data and compare the data generated at different dates
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
MeO
O
O
OH
OH
OH
O OHNaproxen Acyl Glucuronide conjugate
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
(AZT)
Negative Control
Blind Test 3 Metabolite Cocktail
Gao et al Anal Chem 2015 87 11771-11776
Scanning and Banking DataSamples
bull Blind Test samples 22 metabolitesdrugs were spiked in human and rat plasmas (n=4 each)
bull No knowledge of the identities of the analytes and their concentrations in plasma were provided before sample analysis
bull Multiple analysis of the same samples (rat or human) using mixed matrix approach at Days 1 30 105 254 and 314
bull Treat the samples using the same IS solution in acetonitrile minus Freshly prepared to maintain the same concentration
bull Acquity UPLC generic gradient on C18 run time 25 min
bull Full scan and AIF scan on QExactive 70K at positive and negative polarity
bull Compare rat vs human samples analyzed at different dates
0 4 8 12 16 20 24 Time (min)
0
100 R
elat
ive
Ab
un
dan
ce
508
478 682
1132 1435 1450 763
773 243 1835 2120 2138 1164 1312 1638 288 534 1473 2338 802 2045 335 1062
42 43 44 45 46 47 48 49 50 51 52 53 54 55 Time (min)
0
100
Rel
ativ
e In
ten
sity
RT 508
RT 478
RT 495
RT 539
4 8 12 16 20 24 Time (min)
0
100 RT 624
RT 1313
RT 1113
RT 1626
RT 275 RT 879
RT 2008 RT 422
548
165 808
1 2
3
3
4
5
5
4
678
9 10 111213
14 16 15
1
2
3
4
56 7
Rel
ativ
e A
bu
nd
ance
Migrated naproxen acyl glucuronide
EIC 15 drugsmetabolites Resolution 70000 Extraction window 5ppm Polarity Positive
EIC 6 drugsmetabolites Resolution 70000 Extraction window 10 ppm Polarity Negative
2-OH Tacrine
4-OH Tacrine
1-OH Tacrine
Naproxen-Glu
Peak Area Ratio Measurement Variation
n=4 per species 5 independent analysis total n=40 per analyte
95 tile 037
Median 021
M1
0
DrugsMetabolites
Bank and Compare Data at Different Days
Gao et al Anal Chem 2015 87 11771-11776
Separation and Quantitative Analysis of Isomers
2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
100
Rela
tive I
nte
nsity
RT 1626
RT 275
RT 422 RT 1188 RT 632
Full Scan 4rsquo (or 5)-OH Diclofenac
XIC 3100032
Would high resolution ion mobility provide the orthogonal separation we need
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
Bioanalytical Challenges to Address MIST
bull Synthetic standards of metabolites
bull Simultaneous analysis of multiple analytes
bull Radiolabel ADME Studiesmdashonly single dose no steady state data
bull NMR-based standardsmdashbiosynthesis and isolation
Bioanalytical Approaches
Mixed Matrix Peak Area
Approaches (1)
Radiometric Calibrants (2)
NMR-Based Methods (3)
Conventional GLP Bioanalysis
Data Output
Animal vs Human Ratios
Absolute Concentrations
Absolute Concentrations
Absolute Concentrations
Reagent Needs
None Radio-labeled
Drug None
Metabolite Standards
Special Equipment Needs
None None NMR None
Resource Investment
Low Moderate High Very High
Uses MIST Coverage PK Data
MIST Coverage of total
PK Data MIST Coverage
PK Data MIST Coverage
MIST Tiered Approach
bull Use screening qualified or validated methods as function of stage of development and study type
Timmerman P et al 2010 Bioanalysis 2(7) 1185ndash1194
MIST Coverage is a Comparison Question
Animals have exposures of metabolites ge human
Not the exposure multiples
A simple LCMSMS method can provide the data to ascertain the exposure coverage without developing
and validating standard bioanalytical methods for every metabolite
Simple LCMSMS Peak Area Comparison
bull Principle Hamilton pool the time points Inject animal and human sample side-by-side on HPLC-MS compare peak area ratios of metabolites
bull Whatrsquos known bull HPLC retention time and fragmentation pattern for the
metabolite
bull Mixed matrix approach Routinely used in bioanalysis
Matrix effect is the same in samples provided the matrices are identical
bull Mix animal sample with control human matrix
bull Mix human sample with control animal matrix
bull Mix control animal and control human matrix to ensure no background interference
Time-Averaged Pooling (ldquoHamilton Poolsrdquo)
bull Plasma samples from different time points are mixed to provide a single sample in which the analyte concentration Cavg is AUC over the sampling interval
-0avg
AUCC
Hamilton Garnett Kline Clin Pharmacol Ther 1981
total
1-2-nn130201
V)t-(t)t-(t)t-(t)t-(t)t-(t
)t-(tV
010t
total
1-2-nn130201
1-n1nnt V
)t-(t)t-(t)t-(t)t-(t)t-(t
)t-(tV
total
1-2-nn130201
1-t V
)t-(t)t-(t)t-(t)t-(t)t-(t
)t-(tV
bull Can be used to reduce the number of samples to be analyzed and still yield exposure comparisons across species
Mixed Matrix Peak Area Comparison
Pooled Human Sample
Pooled Animal Sample
Mix with Opposite
Matrix Control
Control Mix
Add IS and
Solvent
Recover Supernatant Analyze by HPLC-MS
IS IS IS
Met
Met
Human Animal Matrix Control
(Human) AreaPeak IS
(Human) AreaPeak Metabolite
(Animal) AreaPeak IS
(Animal) AreaPeak Metabolite
Ratio Human to Animal
Control Mix
Mixed Human Sample
Mixed Animal Sample
Mixed Human Sample
Mixed Animal Sample
Gao et al DMD 2010 Gao and Obach DMD 2012
Protein Precipitation
bull Selectivity minus High separation resolution chromatography (a long gradient on
a large column) + MRM or high resolution MS (ge30K for parent ion) w product ion scan for major human metabolites (ge10 of total drug related material)
minus Low throughput is offset by the limited of samples
bull Sensitivity Typically not an issue minus Major human metabolites (ge10 of drug related total material)
and the animal is dosed at NOAEL minus MRM or high accurate MS for parent (plusmn 10ppm) is more
sensitive than full scan MSMS for metabolite ID
bull Matrix effect Normalized by mixed matrix approach
bull Linearity Serial dilutions of the samples in blank mixed plasma
How to Establish BA Parameters without Synthetic Standards
bull Stabilize the metabolites if procedures are known
bull Repetitive analysis of samples to cover the storage duration
bull Profile metabolitesstability reference (usually the parent drug) vs the storage time
bull Auto sampler stability re-injecting the supernatant 12-24 hr later
1day
dayX
sample) (Same AreaPeak reference Stable
sample) Same ( AreaPeak Metabolite
sample) (same AreaPeak reference Stable
species)(Any AreaPeak Metabolite
RatioStability
How to Establish Stability without Synthetic Standards
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
Variety of reaction types alterations in pKa multiple regioisomers
(AZT)
Blind Test 1 Stable Metabolites
M1 236gt134
M2 252gt209
Ziprasidonemetabolites MRM TIC in rat plasma 1
IS
Ziprasidone 413gt194
M9
M5
M10
Dilution Curve M1 236gt134
Dilution Curve M9 429gt280
2 B
80 B
Column Kinetex C18 150 X 46 mm 26 um
Parent Metabolite Potential Instability
Naproxen Naproxen Acyl Glucuronide
Acyl bond migration
Ester hydrolysis
Carbamazepine Carbamazepine-1011-Epoxide
Epoxide hydrolysis
Salicylic Acid 23-Dihydroxybenzoic Acid
Catechol oxidation
Sulfadiazine N-Acetylsulfadiazine
Amide hydrolysis
Methylphenidate Ritalinic Acid
Ester hydrolysis
Nicotine Nicotine-Δ1rsquo5rsquo-Iminium Ion and Cotinine
Imine Oxidation
N
O NH2
MeO
O
O
OH
OH
OH
O OH
N
N+
N
NO
N
N
NH
OMeO
NH
OHO
OH
O
OH
OH
NH
SNH
N
N
O
O
O
NH2
SNH
N
N
O
O
OH
O
OH
N
O NH2
O
MeO
OH
O
Blind Test 2 Unstable Metabolites
Long Term Storage Stability of Naproxen Acyl-Glucuronide Conjugate
0
05
1
15
2
0 50 100 150
(Nap
roxe
n-G
luN
apro
xen
) D
ay X
Day
1
Storage Time at -20ordmC (Day)
Naproxpen-GluNaproxen normalized byDay1 response
Stabilized Naproxen-GluNaproxenNormalized by Day 1
Linear (Naproxpen-GluNaproxen normalizedby Day1 response)
Linear (Stabilized Naproxen-GluNaproxenNormalized by Day 1)
n = 8
Untreated samples
A ratio between 08-12 is deemed stable
Stabilized samples
Gao H and Obach RS (2012) DMD 401290-1296
Correlation of Experimental MS Ratio vs Nominal Ratio
R 0994plusmn0005 Slope 099plusmn0082
MSNM Ratio
Metabolites
MS
NM
Ra
tio
10
15
20
1_O
H_T
acrine
2_O
H_T
acrine
3_M
eth
oxym
orp
hin
4_O
H_D
iclo
fenc
4_O
H_T
acrine
5_O
H_D
iclo
fenc
AZ
T
AZ
T_glu
c
Dextr
om
eth
orp
han
Dextr
orp
han
Dic
lofe
nac
Fexofe
nadin
e
M1
M10
M2
M5
M9
tacrine
Terf
enadin
e
Zip
rasid
one
Geo FC
Geo FC 95
Geo FC 99
Average 125
95 confidence 183
99 confidence 207
Precision Accuracy and Confidence Level bull Showed that a peak area ratio of 2 means that it is at least equal or
greater exposure
bull Increased replicates will increase confidence
95 Confidence Interval (~2SD)
Gao H Deng S and Obach RS (2010) DMD 382147-2156
Confidence Level Increases Dramatically with Measured Ratio
Ratio=4 plt2 ppm
Ratio=2 plt001
Measured animalhuman ratio by LCMSMS
Measured animalhuman ratio by LCMSMS
Probability of Nominal AnimalHuman ge1
What Drives High Confidence
bull Fundamental BA parameters are addressed Selectivity sensitivity linearity matrix effect stability precision and accuracy
bull SignalNoise Ratio Measured animalhuman 2
Measurement error (without cancellation) 025
SN (2-1)025=4
Limit of Detection A signal is real if SN=3
bull Minimal 3 independent animalhuman ratio measurements
bull Analysis reproducibility RSD of the ratio measurements (n=3) 33
bull Stability ratio for each metabolite plusmn20 (08-12) over the storage period
How to Evaluate Data Quality
MIST Coverage is a Comparison Question
Animals have exposures of metabolites ge human
Comparison 1 Giraffe vs Moose
Which one is taller
All that one needs to do is to have them stand next to each other The giraffe is much taller No need to know exact heights
Which one is taller
Too close to call just by looking at them -- The height measurements are needed
Comparison 2 Moose vs Whitetail Deer
If the animalhuman peak area ratio is ge 2 animal has exposure ge human with high confidence (plt001)
How often does it happen Most cases as animal is dosed at much higher
dose when corrected by body weight
A simple peak area ratio comparison is sufficient for most cases
Case Study
bull Drug A in project X at phase 1 14 days 35 mg BID in 6 human volunteers vs 5 days 300 mgkg in Sprague Dawley rats
bull Pooled plasma samples (Hamilton) across time points across subjects
bull 3 human metabolites Parent drug A 396gt203
m-hydroxy 412 ring opened 414 de-alkylated 304
bull HPLC method and major daughter ions were transferred from Biotransformation
bull Compare results with concentration measurements using the synthetic standard
MIST Coverage Evaluation Using RatHuman Ratio of Peak Area Ratios
Definitive Concentration Measurements Using Synthetic Standard
Species Parent De-alkylated Ring-opened m-Hydroxy
Human 0220 00406 000788 00210 Rat 770 0502 0292 000762
RatHuman ratio 1 35 124 371 0363
Human repeat 0184 00290 000506 00164
Rat repeat 734 0472 0266 000618 RatHuman
ratio 2 399 163 526 0378
Species m-Hydroxy (ngmL)
Human 743 plusmn 153
Rat 269 plusmn 258
Cavg RatHuman ratio 0362
Human Metabolite gt10 Drug-Related Material from a Human Radiolabel
ADME Study (or Metabolite Anticipated to Be gt10 from Other Human Studies)
Metabolite With Known Activity of
Interest
Use Standard Bioanalytical
Method
ICH Exempt Metabolite
Peak Area Ratio Method
Method Fails Criteria
Method Succeeds
Animal lt 2X Human
Animal ge 2X Human
No Further Work Needed
Coverage is Assured No Further Work
Needed
Prepare Authentic Standard of Metabolite
MIST Decision Tree
Gao H and Obach RS (2014) Bioanalysis 6(5) 641-650
Data Driven Strategy
bull Rigor of the analysis increases based on data Synthetic standard is needed only when the method fails or animal has less exposure than human
bull Conservative evaluation of MIST coverage
bull Assess MIST coverage at steady state without radio-label or synthetic standards enabling early assessment eg FIH
bull Time- and resource-saving strategy
Summary
bull LCMSMS peak area ratio comparison is scientifically sound and defensible method that can
ndash Ascertain the steady-state exposure of animals compared to humans (ie ldquoMIST coveragerdquo)
ndash Assess the MIST coverage without radio-label or synthetic standard
bull These methods are fit-for-purpose and can offer decision-making data useful for reliable estimation of metabolite exposure
bull Discussions with regulatory experts were positive and helpful
Gao et al (2013) AAPS Journal15(4) 970-973
Prerequisite for Current Methodology
bull Animal and human samples for side-by-side injection
minus In drug development first in human samples are typically ~1 year later than the toxicology study is done
bull Prior knowledge of human metabolites transferred from metabolite identification and scouting
minus Knowledge of major human metabolites may evolve only after in vivo human samples become available
minus Radio-labeled ADME is the most definitive study may take place late in the drug development process
Is it feasible to scan the samplesbank data and compare the data generated at different dates
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
MeO
O
O
OH
OH
OH
O OHNaproxen Acyl Glucuronide conjugate
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
(AZT)
Negative Control
Blind Test 3 Metabolite Cocktail
Gao et al Anal Chem 2015 87 11771-11776
Scanning and Banking DataSamples
bull Blind Test samples 22 metabolitesdrugs were spiked in human and rat plasmas (n=4 each)
bull No knowledge of the identities of the analytes and their concentrations in plasma were provided before sample analysis
bull Multiple analysis of the same samples (rat or human) using mixed matrix approach at Days 1 30 105 254 and 314
bull Treat the samples using the same IS solution in acetonitrile minus Freshly prepared to maintain the same concentration
bull Acquity UPLC generic gradient on C18 run time 25 min
bull Full scan and AIF scan on QExactive 70K at positive and negative polarity
bull Compare rat vs human samples analyzed at different dates
0 4 8 12 16 20 24 Time (min)
0
100 R
elat
ive
Ab
un
dan
ce
508
478 682
1132 1435 1450 763
773 243 1835 2120 2138 1164 1312 1638 288 534 1473 2338 802 2045 335 1062
42 43 44 45 46 47 48 49 50 51 52 53 54 55 Time (min)
0
100
Rel
ativ
e In
ten
sity
RT 508
RT 478
RT 495
RT 539
4 8 12 16 20 24 Time (min)
0
100 RT 624
RT 1313
RT 1113
RT 1626
RT 275 RT 879
RT 2008 RT 422
548
165 808
1 2
3
3
4
5
5
4
678
9 10 111213
14 16 15
1
2
3
4
56 7
Rel
ativ
e A
bu
nd
ance
Migrated naproxen acyl glucuronide
EIC 15 drugsmetabolites Resolution 70000 Extraction window 5ppm Polarity Positive
EIC 6 drugsmetabolites Resolution 70000 Extraction window 10 ppm Polarity Negative
2-OH Tacrine
4-OH Tacrine
1-OH Tacrine
Naproxen-Glu
Peak Area Ratio Measurement Variation
n=4 per species 5 independent analysis total n=40 per analyte
95 tile 037
Median 021
M1
0
DrugsMetabolites
Bank and Compare Data at Different Days
Gao et al Anal Chem 2015 87 11771-11776
Separation and Quantitative Analysis of Isomers
2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
100
Rela
tive I
nte
nsity
RT 1626
RT 275
RT 422 RT 1188 RT 632
Full Scan 4rsquo (or 5)-OH Diclofenac
XIC 3100032
Would high resolution ion mobility provide the orthogonal separation we need
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
Bioanalytical Approaches
Mixed Matrix Peak Area
Approaches (1)
Radiometric Calibrants (2)
NMR-Based Methods (3)
Conventional GLP Bioanalysis
Data Output
Animal vs Human Ratios
Absolute Concentrations
Absolute Concentrations
Absolute Concentrations
Reagent Needs
None Radio-labeled
Drug None
Metabolite Standards
Special Equipment Needs
None None NMR None
Resource Investment
Low Moderate High Very High
Uses MIST Coverage PK Data
MIST Coverage of total
PK Data MIST Coverage
PK Data MIST Coverage
MIST Tiered Approach
bull Use screening qualified or validated methods as function of stage of development and study type
Timmerman P et al 2010 Bioanalysis 2(7) 1185ndash1194
MIST Coverage is a Comparison Question
Animals have exposures of metabolites ge human
Not the exposure multiples
A simple LCMSMS method can provide the data to ascertain the exposure coverage without developing
and validating standard bioanalytical methods for every metabolite
Simple LCMSMS Peak Area Comparison
bull Principle Hamilton pool the time points Inject animal and human sample side-by-side on HPLC-MS compare peak area ratios of metabolites
bull Whatrsquos known bull HPLC retention time and fragmentation pattern for the
metabolite
bull Mixed matrix approach Routinely used in bioanalysis
Matrix effect is the same in samples provided the matrices are identical
bull Mix animal sample with control human matrix
bull Mix human sample with control animal matrix
bull Mix control animal and control human matrix to ensure no background interference
Time-Averaged Pooling (ldquoHamilton Poolsrdquo)
bull Plasma samples from different time points are mixed to provide a single sample in which the analyte concentration Cavg is AUC over the sampling interval
-0avg
AUCC
Hamilton Garnett Kline Clin Pharmacol Ther 1981
total
1-2-nn130201
V)t-(t)t-(t)t-(t)t-(t)t-(t
)t-(tV
010t
total
1-2-nn130201
1-n1nnt V
)t-(t)t-(t)t-(t)t-(t)t-(t
)t-(tV
total
1-2-nn130201
1-t V
)t-(t)t-(t)t-(t)t-(t)t-(t
)t-(tV
bull Can be used to reduce the number of samples to be analyzed and still yield exposure comparisons across species
Mixed Matrix Peak Area Comparison
Pooled Human Sample
Pooled Animal Sample
Mix with Opposite
Matrix Control
Control Mix
Add IS and
Solvent
Recover Supernatant Analyze by HPLC-MS
IS IS IS
Met
Met
Human Animal Matrix Control
(Human) AreaPeak IS
(Human) AreaPeak Metabolite
(Animal) AreaPeak IS
(Animal) AreaPeak Metabolite
Ratio Human to Animal
Control Mix
Mixed Human Sample
Mixed Animal Sample
Mixed Human Sample
Mixed Animal Sample
Gao et al DMD 2010 Gao and Obach DMD 2012
Protein Precipitation
bull Selectivity minus High separation resolution chromatography (a long gradient on
a large column) + MRM or high resolution MS (ge30K for parent ion) w product ion scan for major human metabolites (ge10 of total drug related material)
minus Low throughput is offset by the limited of samples
bull Sensitivity Typically not an issue minus Major human metabolites (ge10 of drug related total material)
and the animal is dosed at NOAEL minus MRM or high accurate MS for parent (plusmn 10ppm) is more
sensitive than full scan MSMS for metabolite ID
bull Matrix effect Normalized by mixed matrix approach
bull Linearity Serial dilutions of the samples in blank mixed plasma
How to Establish BA Parameters without Synthetic Standards
bull Stabilize the metabolites if procedures are known
bull Repetitive analysis of samples to cover the storage duration
bull Profile metabolitesstability reference (usually the parent drug) vs the storage time
bull Auto sampler stability re-injecting the supernatant 12-24 hr later
1day
dayX
sample) (Same AreaPeak reference Stable
sample) Same ( AreaPeak Metabolite
sample) (same AreaPeak reference Stable
species)(Any AreaPeak Metabolite
RatioStability
How to Establish Stability without Synthetic Standards
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
Variety of reaction types alterations in pKa multiple regioisomers
(AZT)
Blind Test 1 Stable Metabolites
M1 236gt134
M2 252gt209
Ziprasidonemetabolites MRM TIC in rat plasma 1
IS
Ziprasidone 413gt194
M9
M5
M10
Dilution Curve M1 236gt134
Dilution Curve M9 429gt280
2 B
80 B
Column Kinetex C18 150 X 46 mm 26 um
Parent Metabolite Potential Instability
Naproxen Naproxen Acyl Glucuronide
Acyl bond migration
Ester hydrolysis
Carbamazepine Carbamazepine-1011-Epoxide
Epoxide hydrolysis
Salicylic Acid 23-Dihydroxybenzoic Acid
Catechol oxidation
Sulfadiazine N-Acetylsulfadiazine
Amide hydrolysis
Methylphenidate Ritalinic Acid
Ester hydrolysis
Nicotine Nicotine-Δ1rsquo5rsquo-Iminium Ion and Cotinine
Imine Oxidation
N
O NH2
MeO
O
O
OH
OH
OH
O OH
N
N+
N
NO
N
N
NH
OMeO
NH
OHO
OH
O
OH
OH
NH
SNH
N
N
O
O
O
NH2
SNH
N
N
O
O
OH
O
OH
N
O NH2
O
MeO
OH
O
Blind Test 2 Unstable Metabolites
Long Term Storage Stability of Naproxen Acyl-Glucuronide Conjugate
0
05
1
15
2
0 50 100 150
(Nap
roxe
n-G
luN
apro
xen
) D
ay X
Day
1
Storage Time at -20ordmC (Day)
Naproxpen-GluNaproxen normalized byDay1 response
Stabilized Naproxen-GluNaproxenNormalized by Day 1
Linear (Naproxpen-GluNaproxen normalizedby Day1 response)
Linear (Stabilized Naproxen-GluNaproxenNormalized by Day 1)
n = 8
Untreated samples
A ratio between 08-12 is deemed stable
Stabilized samples
Gao H and Obach RS (2012) DMD 401290-1296
Correlation of Experimental MS Ratio vs Nominal Ratio
R 0994plusmn0005 Slope 099plusmn0082
MSNM Ratio
Metabolites
MS
NM
Ra
tio
10
15
20
1_O
H_T
acrine
2_O
H_T
acrine
3_M
eth
oxym
orp
hin
4_O
H_D
iclo
fenc
4_O
H_T
acrine
5_O
H_D
iclo
fenc
AZ
T
AZ
T_glu
c
Dextr
om
eth
orp
han
Dextr
orp
han
Dic
lofe
nac
Fexofe
nadin
e
M1
M10
M2
M5
M9
tacrine
Terf
enadin
e
Zip
rasid
one
Geo FC
Geo FC 95
Geo FC 99
Average 125
95 confidence 183
99 confidence 207
Precision Accuracy and Confidence Level bull Showed that a peak area ratio of 2 means that it is at least equal or
greater exposure
bull Increased replicates will increase confidence
95 Confidence Interval (~2SD)
Gao H Deng S and Obach RS (2010) DMD 382147-2156
Confidence Level Increases Dramatically with Measured Ratio
Ratio=4 plt2 ppm
Ratio=2 plt001
Measured animalhuman ratio by LCMSMS
Measured animalhuman ratio by LCMSMS
Probability of Nominal AnimalHuman ge1
What Drives High Confidence
bull Fundamental BA parameters are addressed Selectivity sensitivity linearity matrix effect stability precision and accuracy
bull SignalNoise Ratio Measured animalhuman 2
Measurement error (without cancellation) 025
SN (2-1)025=4
Limit of Detection A signal is real if SN=3
bull Minimal 3 independent animalhuman ratio measurements
bull Analysis reproducibility RSD of the ratio measurements (n=3) 33
bull Stability ratio for each metabolite plusmn20 (08-12) over the storage period
How to Evaluate Data Quality
MIST Coverage is a Comparison Question
Animals have exposures of metabolites ge human
Comparison 1 Giraffe vs Moose
Which one is taller
All that one needs to do is to have them stand next to each other The giraffe is much taller No need to know exact heights
Which one is taller
Too close to call just by looking at them -- The height measurements are needed
Comparison 2 Moose vs Whitetail Deer
If the animalhuman peak area ratio is ge 2 animal has exposure ge human with high confidence (plt001)
How often does it happen Most cases as animal is dosed at much higher
dose when corrected by body weight
A simple peak area ratio comparison is sufficient for most cases
Case Study
bull Drug A in project X at phase 1 14 days 35 mg BID in 6 human volunteers vs 5 days 300 mgkg in Sprague Dawley rats
bull Pooled plasma samples (Hamilton) across time points across subjects
bull 3 human metabolites Parent drug A 396gt203
m-hydroxy 412 ring opened 414 de-alkylated 304
bull HPLC method and major daughter ions were transferred from Biotransformation
bull Compare results with concentration measurements using the synthetic standard
MIST Coverage Evaluation Using RatHuman Ratio of Peak Area Ratios
Definitive Concentration Measurements Using Synthetic Standard
Species Parent De-alkylated Ring-opened m-Hydroxy
Human 0220 00406 000788 00210 Rat 770 0502 0292 000762
RatHuman ratio 1 35 124 371 0363
Human repeat 0184 00290 000506 00164
Rat repeat 734 0472 0266 000618 RatHuman
ratio 2 399 163 526 0378
Species m-Hydroxy (ngmL)
Human 743 plusmn 153
Rat 269 plusmn 258
Cavg RatHuman ratio 0362
Human Metabolite gt10 Drug-Related Material from a Human Radiolabel
ADME Study (or Metabolite Anticipated to Be gt10 from Other Human Studies)
Metabolite With Known Activity of
Interest
Use Standard Bioanalytical
Method
ICH Exempt Metabolite
Peak Area Ratio Method
Method Fails Criteria
Method Succeeds
Animal lt 2X Human
Animal ge 2X Human
No Further Work Needed
Coverage is Assured No Further Work
Needed
Prepare Authentic Standard of Metabolite
MIST Decision Tree
Gao H and Obach RS (2014) Bioanalysis 6(5) 641-650
Data Driven Strategy
bull Rigor of the analysis increases based on data Synthetic standard is needed only when the method fails or animal has less exposure than human
bull Conservative evaluation of MIST coverage
bull Assess MIST coverage at steady state without radio-label or synthetic standards enabling early assessment eg FIH
bull Time- and resource-saving strategy
Summary
bull LCMSMS peak area ratio comparison is scientifically sound and defensible method that can
ndash Ascertain the steady-state exposure of animals compared to humans (ie ldquoMIST coveragerdquo)
ndash Assess the MIST coverage without radio-label or synthetic standard
bull These methods are fit-for-purpose and can offer decision-making data useful for reliable estimation of metabolite exposure
bull Discussions with regulatory experts were positive and helpful
Gao et al (2013) AAPS Journal15(4) 970-973
Prerequisite for Current Methodology
bull Animal and human samples for side-by-side injection
minus In drug development first in human samples are typically ~1 year later than the toxicology study is done
bull Prior knowledge of human metabolites transferred from metabolite identification and scouting
minus Knowledge of major human metabolites may evolve only after in vivo human samples become available
minus Radio-labeled ADME is the most definitive study may take place late in the drug development process
Is it feasible to scan the samplesbank data and compare the data generated at different dates
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
MeO
O
O
OH
OH
OH
O OHNaproxen Acyl Glucuronide conjugate
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
(AZT)
Negative Control
Blind Test 3 Metabolite Cocktail
Gao et al Anal Chem 2015 87 11771-11776
Scanning and Banking DataSamples
bull Blind Test samples 22 metabolitesdrugs were spiked in human and rat plasmas (n=4 each)
bull No knowledge of the identities of the analytes and their concentrations in plasma were provided before sample analysis
bull Multiple analysis of the same samples (rat or human) using mixed matrix approach at Days 1 30 105 254 and 314
bull Treat the samples using the same IS solution in acetonitrile minus Freshly prepared to maintain the same concentration
bull Acquity UPLC generic gradient on C18 run time 25 min
bull Full scan and AIF scan on QExactive 70K at positive and negative polarity
bull Compare rat vs human samples analyzed at different dates
0 4 8 12 16 20 24 Time (min)
0
100 R
elat
ive
Ab
un
dan
ce
508
478 682
1132 1435 1450 763
773 243 1835 2120 2138 1164 1312 1638 288 534 1473 2338 802 2045 335 1062
42 43 44 45 46 47 48 49 50 51 52 53 54 55 Time (min)
0
100
Rel
ativ
e In
ten
sity
RT 508
RT 478
RT 495
RT 539
4 8 12 16 20 24 Time (min)
0
100 RT 624
RT 1313
RT 1113
RT 1626
RT 275 RT 879
RT 2008 RT 422
548
165 808
1 2
3
3
4
5
5
4
678
9 10 111213
14 16 15
1
2
3
4
56 7
Rel
ativ
e A
bu
nd
ance
Migrated naproxen acyl glucuronide
EIC 15 drugsmetabolites Resolution 70000 Extraction window 5ppm Polarity Positive
EIC 6 drugsmetabolites Resolution 70000 Extraction window 10 ppm Polarity Negative
2-OH Tacrine
4-OH Tacrine
1-OH Tacrine
Naproxen-Glu
Peak Area Ratio Measurement Variation
n=4 per species 5 independent analysis total n=40 per analyte
95 tile 037
Median 021
M1
0
DrugsMetabolites
Bank and Compare Data at Different Days
Gao et al Anal Chem 2015 87 11771-11776
Separation and Quantitative Analysis of Isomers
2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
100
Rela
tive I
nte
nsity
RT 1626
RT 275
RT 422 RT 1188 RT 632
Full Scan 4rsquo (or 5)-OH Diclofenac
XIC 3100032
Would high resolution ion mobility provide the orthogonal separation we need
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
MIST Tiered Approach
bull Use screening qualified or validated methods as function of stage of development and study type
Timmerman P et al 2010 Bioanalysis 2(7) 1185ndash1194
MIST Coverage is a Comparison Question
Animals have exposures of metabolites ge human
Not the exposure multiples
A simple LCMSMS method can provide the data to ascertain the exposure coverage without developing
and validating standard bioanalytical methods for every metabolite
Simple LCMSMS Peak Area Comparison
bull Principle Hamilton pool the time points Inject animal and human sample side-by-side on HPLC-MS compare peak area ratios of metabolites
bull Whatrsquos known bull HPLC retention time and fragmentation pattern for the
metabolite
bull Mixed matrix approach Routinely used in bioanalysis
Matrix effect is the same in samples provided the matrices are identical
bull Mix animal sample with control human matrix
bull Mix human sample with control animal matrix
bull Mix control animal and control human matrix to ensure no background interference
Time-Averaged Pooling (ldquoHamilton Poolsrdquo)
bull Plasma samples from different time points are mixed to provide a single sample in which the analyte concentration Cavg is AUC over the sampling interval
-0avg
AUCC
Hamilton Garnett Kline Clin Pharmacol Ther 1981
total
1-2-nn130201
V)t-(t)t-(t)t-(t)t-(t)t-(t
)t-(tV
010t
total
1-2-nn130201
1-n1nnt V
)t-(t)t-(t)t-(t)t-(t)t-(t
)t-(tV
total
1-2-nn130201
1-t V
)t-(t)t-(t)t-(t)t-(t)t-(t
)t-(tV
bull Can be used to reduce the number of samples to be analyzed and still yield exposure comparisons across species
Mixed Matrix Peak Area Comparison
Pooled Human Sample
Pooled Animal Sample
Mix with Opposite
Matrix Control
Control Mix
Add IS and
Solvent
Recover Supernatant Analyze by HPLC-MS
IS IS IS
Met
Met
Human Animal Matrix Control
(Human) AreaPeak IS
(Human) AreaPeak Metabolite
(Animal) AreaPeak IS
(Animal) AreaPeak Metabolite
Ratio Human to Animal
Control Mix
Mixed Human Sample
Mixed Animal Sample
Mixed Human Sample
Mixed Animal Sample
Gao et al DMD 2010 Gao and Obach DMD 2012
Protein Precipitation
bull Selectivity minus High separation resolution chromatography (a long gradient on
a large column) + MRM or high resolution MS (ge30K for parent ion) w product ion scan for major human metabolites (ge10 of total drug related material)
minus Low throughput is offset by the limited of samples
bull Sensitivity Typically not an issue minus Major human metabolites (ge10 of drug related total material)
and the animal is dosed at NOAEL minus MRM or high accurate MS for parent (plusmn 10ppm) is more
sensitive than full scan MSMS for metabolite ID
bull Matrix effect Normalized by mixed matrix approach
bull Linearity Serial dilutions of the samples in blank mixed plasma
How to Establish BA Parameters without Synthetic Standards
bull Stabilize the metabolites if procedures are known
bull Repetitive analysis of samples to cover the storage duration
bull Profile metabolitesstability reference (usually the parent drug) vs the storage time
bull Auto sampler stability re-injecting the supernatant 12-24 hr later
1day
dayX
sample) (Same AreaPeak reference Stable
sample) Same ( AreaPeak Metabolite
sample) (same AreaPeak reference Stable
species)(Any AreaPeak Metabolite
RatioStability
How to Establish Stability without Synthetic Standards
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
Variety of reaction types alterations in pKa multiple regioisomers
(AZT)
Blind Test 1 Stable Metabolites
M1 236gt134
M2 252gt209
Ziprasidonemetabolites MRM TIC in rat plasma 1
IS
Ziprasidone 413gt194
M9
M5
M10
Dilution Curve M1 236gt134
Dilution Curve M9 429gt280
2 B
80 B
Column Kinetex C18 150 X 46 mm 26 um
Parent Metabolite Potential Instability
Naproxen Naproxen Acyl Glucuronide
Acyl bond migration
Ester hydrolysis
Carbamazepine Carbamazepine-1011-Epoxide
Epoxide hydrolysis
Salicylic Acid 23-Dihydroxybenzoic Acid
Catechol oxidation
Sulfadiazine N-Acetylsulfadiazine
Amide hydrolysis
Methylphenidate Ritalinic Acid
Ester hydrolysis
Nicotine Nicotine-Δ1rsquo5rsquo-Iminium Ion and Cotinine
Imine Oxidation
N
O NH2
MeO
O
O
OH
OH
OH
O OH
N
N+
N
NO
N
N
NH
OMeO
NH
OHO
OH
O
OH
OH
NH
SNH
N
N
O
O
O
NH2
SNH
N
N
O
O
OH
O
OH
N
O NH2
O
MeO
OH
O
Blind Test 2 Unstable Metabolites
Long Term Storage Stability of Naproxen Acyl-Glucuronide Conjugate
0
05
1
15
2
0 50 100 150
(Nap
roxe
n-G
luN
apro
xen
) D
ay X
Day
1
Storage Time at -20ordmC (Day)
Naproxpen-GluNaproxen normalized byDay1 response
Stabilized Naproxen-GluNaproxenNormalized by Day 1
Linear (Naproxpen-GluNaproxen normalizedby Day1 response)
Linear (Stabilized Naproxen-GluNaproxenNormalized by Day 1)
n = 8
Untreated samples
A ratio between 08-12 is deemed stable
Stabilized samples
Gao H and Obach RS (2012) DMD 401290-1296
Correlation of Experimental MS Ratio vs Nominal Ratio
R 0994plusmn0005 Slope 099plusmn0082
MSNM Ratio
Metabolites
MS
NM
Ra
tio
10
15
20
1_O
H_T
acrine
2_O
H_T
acrine
3_M
eth
oxym
orp
hin
4_O
H_D
iclo
fenc
4_O
H_T
acrine
5_O
H_D
iclo
fenc
AZ
T
AZ
T_glu
c
Dextr
om
eth
orp
han
Dextr
orp
han
Dic
lofe
nac
Fexofe
nadin
e
M1
M10
M2
M5
M9
tacrine
Terf
enadin
e
Zip
rasid
one
Geo FC
Geo FC 95
Geo FC 99
Average 125
95 confidence 183
99 confidence 207
Precision Accuracy and Confidence Level bull Showed that a peak area ratio of 2 means that it is at least equal or
greater exposure
bull Increased replicates will increase confidence
95 Confidence Interval (~2SD)
Gao H Deng S and Obach RS (2010) DMD 382147-2156
Confidence Level Increases Dramatically with Measured Ratio
Ratio=4 plt2 ppm
Ratio=2 plt001
Measured animalhuman ratio by LCMSMS
Measured animalhuman ratio by LCMSMS
Probability of Nominal AnimalHuman ge1
What Drives High Confidence
bull Fundamental BA parameters are addressed Selectivity sensitivity linearity matrix effect stability precision and accuracy
bull SignalNoise Ratio Measured animalhuman 2
Measurement error (without cancellation) 025
SN (2-1)025=4
Limit of Detection A signal is real if SN=3
bull Minimal 3 independent animalhuman ratio measurements
bull Analysis reproducibility RSD of the ratio measurements (n=3) 33
bull Stability ratio for each metabolite plusmn20 (08-12) over the storage period
How to Evaluate Data Quality
MIST Coverage is a Comparison Question
Animals have exposures of metabolites ge human
Comparison 1 Giraffe vs Moose
Which one is taller
All that one needs to do is to have them stand next to each other The giraffe is much taller No need to know exact heights
Which one is taller
Too close to call just by looking at them -- The height measurements are needed
Comparison 2 Moose vs Whitetail Deer
If the animalhuman peak area ratio is ge 2 animal has exposure ge human with high confidence (plt001)
How often does it happen Most cases as animal is dosed at much higher
dose when corrected by body weight
A simple peak area ratio comparison is sufficient for most cases
Case Study
bull Drug A in project X at phase 1 14 days 35 mg BID in 6 human volunteers vs 5 days 300 mgkg in Sprague Dawley rats
bull Pooled plasma samples (Hamilton) across time points across subjects
bull 3 human metabolites Parent drug A 396gt203
m-hydroxy 412 ring opened 414 de-alkylated 304
bull HPLC method and major daughter ions were transferred from Biotransformation
bull Compare results with concentration measurements using the synthetic standard
MIST Coverage Evaluation Using RatHuman Ratio of Peak Area Ratios
Definitive Concentration Measurements Using Synthetic Standard
Species Parent De-alkylated Ring-opened m-Hydroxy
Human 0220 00406 000788 00210 Rat 770 0502 0292 000762
RatHuman ratio 1 35 124 371 0363
Human repeat 0184 00290 000506 00164
Rat repeat 734 0472 0266 000618 RatHuman
ratio 2 399 163 526 0378
Species m-Hydroxy (ngmL)
Human 743 plusmn 153
Rat 269 plusmn 258
Cavg RatHuman ratio 0362
Human Metabolite gt10 Drug-Related Material from a Human Radiolabel
ADME Study (or Metabolite Anticipated to Be gt10 from Other Human Studies)
Metabolite With Known Activity of
Interest
Use Standard Bioanalytical
Method
ICH Exempt Metabolite
Peak Area Ratio Method
Method Fails Criteria
Method Succeeds
Animal lt 2X Human
Animal ge 2X Human
No Further Work Needed
Coverage is Assured No Further Work
Needed
Prepare Authentic Standard of Metabolite
MIST Decision Tree
Gao H and Obach RS (2014) Bioanalysis 6(5) 641-650
Data Driven Strategy
bull Rigor of the analysis increases based on data Synthetic standard is needed only when the method fails or animal has less exposure than human
bull Conservative evaluation of MIST coverage
bull Assess MIST coverage at steady state without radio-label or synthetic standards enabling early assessment eg FIH
bull Time- and resource-saving strategy
Summary
bull LCMSMS peak area ratio comparison is scientifically sound and defensible method that can
ndash Ascertain the steady-state exposure of animals compared to humans (ie ldquoMIST coveragerdquo)
ndash Assess the MIST coverage without radio-label or synthetic standard
bull These methods are fit-for-purpose and can offer decision-making data useful for reliable estimation of metabolite exposure
bull Discussions with regulatory experts were positive and helpful
Gao et al (2013) AAPS Journal15(4) 970-973
Prerequisite for Current Methodology
bull Animal and human samples for side-by-side injection
minus In drug development first in human samples are typically ~1 year later than the toxicology study is done
bull Prior knowledge of human metabolites transferred from metabolite identification and scouting
minus Knowledge of major human metabolites may evolve only after in vivo human samples become available
minus Radio-labeled ADME is the most definitive study may take place late in the drug development process
Is it feasible to scan the samplesbank data and compare the data generated at different dates
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
MeO
O
O
OH
OH
OH
O OHNaproxen Acyl Glucuronide conjugate
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
(AZT)
Negative Control
Blind Test 3 Metabolite Cocktail
Gao et al Anal Chem 2015 87 11771-11776
Scanning and Banking DataSamples
bull Blind Test samples 22 metabolitesdrugs were spiked in human and rat plasmas (n=4 each)
bull No knowledge of the identities of the analytes and their concentrations in plasma were provided before sample analysis
bull Multiple analysis of the same samples (rat or human) using mixed matrix approach at Days 1 30 105 254 and 314
bull Treat the samples using the same IS solution in acetonitrile minus Freshly prepared to maintain the same concentration
bull Acquity UPLC generic gradient on C18 run time 25 min
bull Full scan and AIF scan on QExactive 70K at positive and negative polarity
bull Compare rat vs human samples analyzed at different dates
0 4 8 12 16 20 24 Time (min)
0
100 R
elat
ive
Ab
un
dan
ce
508
478 682
1132 1435 1450 763
773 243 1835 2120 2138 1164 1312 1638 288 534 1473 2338 802 2045 335 1062
42 43 44 45 46 47 48 49 50 51 52 53 54 55 Time (min)
0
100
Rel
ativ
e In
ten
sity
RT 508
RT 478
RT 495
RT 539
4 8 12 16 20 24 Time (min)
0
100 RT 624
RT 1313
RT 1113
RT 1626
RT 275 RT 879
RT 2008 RT 422
548
165 808
1 2
3
3
4
5
5
4
678
9 10 111213
14 16 15
1
2
3
4
56 7
Rel
ativ
e A
bu
nd
ance
Migrated naproxen acyl glucuronide
EIC 15 drugsmetabolites Resolution 70000 Extraction window 5ppm Polarity Positive
EIC 6 drugsmetabolites Resolution 70000 Extraction window 10 ppm Polarity Negative
2-OH Tacrine
4-OH Tacrine
1-OH Tacrine
Naproxen-Glu
Peak Area Ratio Measurement Variation
n=4 per species 5 independent analysis total n=40 per analyte
95 tile 037
Median 021
M1
0
DrugsMetabolites
Bank and Compare Data at Different Days
Gao et al Anal Chem 2015 87 11771-11776
Separation and Quantitative Analysis of Isomers
2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
100
Rela
tive I
nte
nsity
RT 1626
RT 275
RT 422 RT 1188 RT 632
Full Scan 4rsquo (or 5)-OH Diclofenac
XIC 3100032
Would high resolution ion mobility provide the orthogonal separation we need
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
MIST Coverage is a Comparison Question
Animals have exposures of metabolites ge human
Not the exposure multiples
A simple LCMSMS method can provide the data to ascertain the exposure coverage without developing
and validating standard bioanalytical methods for every metabolite
Simple LCMSMS Peak Area Comparison
bull Principle Hamilton pool the time points Inject animal and human sample side-by-side on HPLC-MS compare peak area ratios of metabolites
bull Whatrsquos known bull HPLC retention time and fragmentation pattern for the
metabolite
bull Mixed matrix approach Routinely used in bioanalysis
Matrix effect is the same in samples provided the matrices are identical
bull Mix animal sample with control human matrix
bull Mix human sample with control animal matrix
bull Mix control animal and control human matrix to ensure no background interference
Time-Averaged Pooling (ldquoHamilton Poolsrdquo)
bull Plasma samples from different time points are mixed to provide a single sample in which the analyte concentration Cavg is AUC over the sampling interval
-0avg
AUCC
Hamilton Garnett Kline Clin Pharmacol Ther 1981
total
1-2-nn130201
V)t-(t)t-(t)t-(t)t-(t)t-(t
)t-(tV
010t
total
1-2-nn130201
1-n1nnt V
)t-(t)t-(t)t-(t)t-(t)t-(t
)t-(tV
total
1-2-nn130201
1-t V
)t-(t)t-(t)t-(t)t-(t)t-(t
)t-(tV
bull Can be used to reduce the number of samples to be analyzed and still yield exposure comparisons across species
Mixed Matrix Peak Area Comparison
Pooled Human Sample
Pooled Animal Sample
Mix with Opposite
Matrix Control
Control Mix
Add IS and
Solvent
Recover Supernatant Analyze by HPLC-MS
IS IS IS
Met
Met
Human Animal Matrix Control
(Human) AreaPeak IS
(Human) AreaPeak Metabolite
(Animal) AreaPeak IS
(Animal) AreaPeak Metabolite
Ratio Human to Animal
Control Mix
Mixed Human Sample
Mixed Animal Sample
Mixed Human Sample
Mixed Animal Sample
Gao et al DMD 2010 Gao and Obach DMD 2012
Protein Precipitation
bull Selectivity minus High separation resolution chromatography (a long gradient on
a large column) + MRM or high resolution MS (ge30K for parent ion) w product ion scan for major human metabolites (ge10 of total drug related material)
minus Low throughput is offset by the limited of samples
bull Sensitivity Typically not an issue minus Major human metabolites (ge10 of drug related total material)
and the animal is dosed at NOAEL minus MRM or high accurate MS for parent (plusmn 10ppm) is more
sensitive than full scan MSMS for metabolite ID
bull Matrix effect Normalized by mixed matrix approach
bull Linearity Serial dilutions of the samples in blank mixed plasma
How to Establish BA Parameters without Synthetic Standards
bull Stabilize the metabolites if procedures are known
bull Repetitive analysis of samples to cover the storage duration
bull Profile metabolitesstability reference (usually the parent drug) vs the storage time
bull Auto sampler stability re-injecting the supernatant 12-24 hr later
1day
dayX
sample) (Same AreaPeak reference Stable
sample) Same ( AreaPeak Metabolite
sample) (same AreaPeak reference Stable
species)(Any AreaPeak Metabolite
RatioStability
How to Establish Stability without Synthetic Standards
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
Variety of reaction types alterations in pKa multiple regioisomers
(AZT)
Blind Test 1 Stable Metabolites
M1 236gt134
M2 252gt209
Ziprasidonemetabolites MRM TIC in rat plasma 1
IS
Ziprasidone 413gt194
M9
M5
M10
Dilution Curve M1 236gt134
Dilution Curve M9 429gt280
2 B
80 B
Column Kinetex C18 150 X 46 mm 26 um
Parent Metabolite Potential Instability
Naproxen Naproxen Acyl Glucuronide
Acyl bond migration
Ester hydrolysis
Carbamazepine Carbamazepine-1011-Epoxide
Epoxide hydrolysis
Salicylic Acid 23-Dihydroxybenzoic Acid
Catechol oxidation
Sulfadiazine N-Acetylsulfadiazine
Amide hydrolysis
Methylphenidate Ritalinic Acid
Ester hydrolysis
Nicotine Nicotine-Δ1rsquo5rsquo-Iminium Ion and Cotinine
Imine Oxidation
N
O NH2
MeO
O
O
OH
OH
OH
O OH
N
N+
N
NO
N
N
NH
OMeO
NH
OHO
OH
O
OH
OH
NH
SNH
N
N
O
O
O
NH2
SNH
N
N
O
O
OH
O
OH
N
O NH2
O
MeO
OH
O
Blind Test 2 Unstable Metabolites
Long Term Storage Stability of Naproxen Acyl-Glucuronide Conjugate
0
05
1
15
2
0 50 100 150
(Nap
roxe
n-G
luN
apro
xen
) D
ay X
Day
1
Storage Time at -20ordmC (Day)
Naproxpen-GluNaproxen normalized byDay1 response
Stabilized Naproxen-GluNaproxenNormalized by Day 1
Linear (Naproxpen-GluNaproxen normalizedby Day1 response)
Linear (Stabilized Naproxen-GluNaproxenNormalized by Day 1)
n = 8
Untreated samples
A ratio between 08-12 is deemed stable
Stabilized samples
Gao H and Obach RS (2012) DMD 401290-1296
Correlation of Experimental MS Ratio vs Nominal Ratio
R 0994plusmn0005 Slope 099plusmn0082
MSNM Ratio
Metabolites
MS
NM
Ra
tio
10
15
20
1_O
H_T
acrine
2_O
H_T
acrine
3_M
eth
oxym
orp
hin
4_O
H_D
iclo
fenc
4_O
H_T
acrine
5_O
H_D
iclo
fenc
AZ
T
AZ
T_glu
c
Dextr
om
eth
orp
han
Dextr
orp
han
Dic
lofe
nac
Fexofe
nadin
e
M1
M10
M2
M5
M9
tacrine
Terf
enadin
e
Zip
rasid
one
Geo FC
Geo FC 95
Geo FC 99
Average 125
95 confidence 183
99 confidence 207
Precision Accuracy and Confidence Level bull Showed that a peak area ratio of 2 means that it is at least equal or
greater exposure
bull Increased replicates will increase confidence
95 Confidence Interval (~2SD)
Gao H Deng S and Obach RS (2010) DMD 382147-2156
Confidence Level Increases Dramatically with Measured Ratio
Ratio=4 plt2 ppm
Ratio=2 plt001
Measured animalhuman ratio by LCMSMS
Measured animalhuman ratio by LCMSMS
Probability of Nominal AnimalHuman ge1
What Drives High Confidence
bull Fundamental BA parameters are addressed Selectivity sensitivity linearity matrix effect stability precision and accuracy
bull SignalNoise Ratio Measured animalhuman 2
Measurement error (without cancellation) 025
SN (2-1)025=4
Limit of Detection A signal is real if SN=3
bull Minimal 3 independent animalhuman ratio measurements
bull Analysis reproducibility RSD of the ratio measurements (n=3) 33
bull Stability ratio for each metabolite plusmn20 (08-12) over the storage period
How to Evaluate Data Quality
MIST Coverage is a Comparison Question
Animals have exposures of metabolites ge human
Comparison 1 Giraffe vs Moose
Which one is taller
All that one needs to do is to have them stand next to each other The giraffe is much taller No need to know exact heights
Which one is taller
Too close to call just by looking at them -- The height measurements are needed
Comparison 2 Moose vs Whitetail Deer
If the animalhuman peak area ratio is ge 2 animal has exposure ge human with high confidence (plt001)
How often does it happen Most cases as animal is dosed at much higher
dose when corrected by body weight
A simple peak area ratio comparison is sufficient for most cases
Case Study
bull Drug A in project X at phase 1 14 days 35 mg BID in 6 human volunteers vs 5 days 300 mgkg in Sprague Dawley rats
bull Pooled plasma samples (Hamilton) across time points across subjects
bull 3 human metabolites Parent drug A 396gt203
m-hydroxy 412 ring opened 414 de-alkylated 304
bull HPLC method and major daughter ions were transferred from Biotransformation
bull Compare results with concentration measurements using the synthetic standard
MIST Coverage Evaluation Using RatHuman Ratio of Peak Area Ratios
Definitive Concentration Measurements Using Synthetic Standard
Species Parent De-alkylated Ring-opened m-Hydroxy
Human 0220 00406 000788 00210 Rat 770 0502 0292 000762
RatHuman ratio 1 35 124 371 0363
Human repeat 0184 00290 000506 00164
Rat repeat 734 0472 0266 000618 RatHuman
ratio 2 399 163 526 0378
Species m-Hydroxy (ngmL)
Human 743 plusmn 153
Rat 269 plusmn 258
Cavg RatHuman ratio 0362
Human Metabolite gt10 Drug-Related Material from a Human Radiolabel
ADME Study (or Metabolite Anticipated to Be gt10 from Other Human Studies)
Metabolite With Known Activity of
Interest
Use Standard Bioanalytical
Method
ICH Exempt Metabolite
Peak Area Ratio Method
Method Fails Criteria
Method Succeeds
Animal lt 2X Human
Animal ge 2X Human
No Further Work Needed
Coverage is Assured No Further Work
Needed
Prepare Authentic Standard of Metabolite
MIST Decision Tree
Gao H and Obach RS (2014) Bioanalysis 6(5) 641-650
Data Driven Strategy
bull Rigor of the analysis increases based on data Synthetic standard is needed only when the method fails or animal has less exposure than human
bull Conservative evaluation of MIST coverage
bull Assess MIST coverage at steady state without radio-label or synthetic standards enabling early assessment eg FIH
bull Time- and resource-saving strategy
Summary
bull LCMSMS peak area ratio comparison is scientifically sound and defensible method that can
ndash Ascertain the steady-state exposure of animals compared to humans (ie ldquoMIST coveragerdquo)
ndash Assess the MIST coverage without radio-label or synthetic standard
bull These methods are fit-for-purpose and can offer decision-making data useful for reliable estimation of metabolite exposure
bull Discussions with regulatory experts were positive and helpful
Gao et al (2013) AAPS Journal15(4) 970-973
Prerequisite for Current Methodology
bull Animal and human samples for side-by-side injection
minus In drug development first in human samples are typically ~1 year later than the toxicology study is done
bull Prior knowledge of human metabolites transferred from metabolite identification and scouting
minus Knowledge of major human metabolites may evolve only after in vivo human samples become available
minus Radio-labeled ADME is the most definitive study may take place late in the drug development process
Is it feasible to scan the samplesbank data and compare the data generated at different dates
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
MeO
O
O
OH
OH
OH
O OHNaproxen Acyl Glucuronide conjugate
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
(AZT)
Negative Control
Blind Test 3 Metabolite Cocktail
Gao et al Anal Chem 2015 87 11771-11776
Scanning and Banking DataSamples
bull Blind Test samples 22 metabolitesdrugs were spiked in human and rat plasmas (n=4 each)
bull No knowledge of the identities of the analytes and their concentrations in plasma were provided before sample analysis
bull Multiple analysis of the same samples (rat or human) using mixed matrix approach at Days 1 30 105 254 and 314
bull Treat the samples using the same IS solution in acetonitrile minus Freshly prepared to maintain the same concentration
bull Acquity UPLC generic gradient on C18 run time 25 min
bull Full scan and AIF scan on QExactive 70K at positive and negative polarity
bull Compare rat vs human samples analyzed at different dates
0 4 8 12 16 20 24 Time (min)
0
100 R
elat
ive
Ab
un
dan
ce
508
478 682
1132 1435 1450 763
773 243 1835 2120 2138 1164 1312 1638 288 534 1473 2338 802 2045 335 1062
42 43 44 45 46 47 48 49 50 51 52 53 54 55 Time (min)
0
100
Rel
ativ
e In
ten
sity
RT 508
RT 478
RT 495
RT 539
4 8 12 16 20 24 Time (min)
0
100 RT 624
RT 1313
RT 1113
RT 1626
RT 275 RT 879
RT 2008 RT 422
548
165 808
1 2
3
3
4
5
5
4
678
9 10 111213
14 16 15
1
2
3
4
56 7
Rel
ativ
e A
bu
nd
ance
Migrated naproxen acyl glucuronide
EIC 15 drugsmetabolites Resolution 70000 Extraction window 5ppm Polarity Positive
EIC 6 drugsmetabolites Resolution 70000 Extraction window 10 ppm Polarity Negative
2-OH Tacrine
4-OH Tacrine
1-OH Tacrine
Naproxen-Glu
Peak Area Ratio Measurement Variation
n=4 per species 5 independent analysis total n=40 per analyte
95 tile 037
Median 021
M1
0
DrugsMetabolites
Bank and Compare Data at Different Days
Gao et al Anal Chem 2015 87 11771-11776
Separation and Quantitative Analysis of Isomers
2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
100
Rela
tive I
nte
nsity
RT 1626
RT 275
RT 422 RT 1188 RT 632
Full Scan 4rsquo (or 5)-OH Diclofenac
XIC 3100032
Would high resolution ion mobility provide the orthogonal separation we need
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
Simple LCMSMS Peak Area Comparison
bull Principle Hamilton pool the time points Inject animal and human sample side-by-side on HPLC-MS compare peak area ratios of metabolites
bull Whatrsquos known bull HPLC retention time and fragmentation pattern for the
metabolite
bull Mixed matrix approach Routinely used in bioanalysis
Matrix effect is the same in samples provided the matrices are identical
bull Mix animal sample with control human matrix
bull Mix human sample with control animal matrix
bull Mix control animal and control human matrix to ensure no background interference
Time-Averaged Pooling (ldquoHamilton Poolsrdquo)
bull Plasma samples from different time points are mixed to provide a single sample in which the analyte concentration Cavg is AUC over the sampling interval
-0avg
AUCC
Hamilton Garnett Kline Clin Pharmacol Ther 1981
total
1-2-nn130201
V)t-(t)t-(t)t-(t)t-(t)t-(t
)t-(tV
010t
total
1-2-nn130201
1-n1nnt V
)t-(t)t-(t)t-(t)t-(t)t-(t
)t-(tV
total
1-2-nn130201
1-t V
)t-(t)t-(t)t-(t)t-(t)t-(t
)t-(tV
bull Can be used to reduce the number of samples to be analyzed and still yield exposure comparisons across species
Mixed Matrix Peak Area Comparison
Pooled Human Sample
Pooled Animal Sample
Mix with Opposite
Matrix Control
Control Mix
Add IS and
Solvent
Recover Supernatant Analyze by HPLC-MS
IS IS IS
Met
Met
Human Animal Matrix Control
(Human) AreaPeak IS
(Human) AreaPeak Metabolite
(Animal) AreaPeak IS
(Animal) AreaPeak Metabolite
Ratio Human to Animal
Control Mix
Mixed Human Sample
Mixed Animal Sample
Mixed Human Sample
Mixed Animal Sample
Gao et al DMD 2010 Gao and Obach DMD 2012
Protein Precipitation
bull Selectivity minus High separation resolution chromatography (a long gradient on
a large column) + MRM or high resolution MS (ge30K for parent ion) w product ion scan for major human metabolites (ge10 of total drug related material)
minus Low throughput is offset by the limited of samples
bull Sensitivity Typically not an issue minus Major human metabolites (ge10 of drug related total material)
and the animal is dosed at NOAEL minus MRM or high accurate MS for parent (plusmn 10ppm) is more
sensitive than full scan MSMS for metabolite ID
bull Matrix effect Normalized by mixed matrix approach
bull Linearity Serial dilutions of the samples in blank mixed plasma
How to Establish BA Parameters without Synthetic Standards
bull Stabilize the metabolites if procedures are known
bull Repetitive analysis of samples to cover the storage duration
bull Profile metabolitesstability reference (usually the parent drug) vs the storage time
bull Auto sampler stability re-injecting the supernatant 12-24 hr later
1day
dayX
sample) (Same AreaPeak reference Stable
sample) Same ( AreaPeak Metabolite
sample) (same AreaPeak reference Stable
species)(Any AreaPeak Metabolite
RatioStability
How to Establish Stability without Synthetic Standards
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
Variety of reaction types alterations in pKa multiple regioisomers
(AZT)
Blind Test 1 Stable Metabolites
M1 236gt134
M2 252gt209
Ziprasidonemetabolites MRM TIC in rat plasma 1
IS
Ziprasidone 413gt194
M9
M5
M10
Dilution Curve M1 236gt134
Dilution Curve M9 429gt280
2 B
80 B
Column Kinetex C18 150 X 46 mm 26 um
Parent Metabolite Potential Instability
Naproxen Naproxen Acyl Glucuronide
Acyl bond migration
Ester hydrolysis
Carbamazepine Carbamazepine-1011-Epoxide
Epoxide hydrolysis
Salicylic Acid 23-Dihydroxybenzoic Acid
Catechol oxidation
Sulfadiazine N-Acetylsulfadiazine
Amide hydrolysis
Methylphenidate Ritalinic Acid
Ester hydrolysis
Nicotine Nicotine-Δ1rsquo5rsquo-Iminium Ion and Cotinine
Imine Oxidation
N
O NH2
MeO
O
O
OH
OH
OH
O OH
N
N+
N
NO
N
N
NH
OMeO
NH
OHO
OH
O
OH
OH
NH
SNH
N
N
O
O
O
NH2
SNH
N
N
O
O
OH
O
OH
N
O NH2
O
MeO
OH
O
Blind Test 2 Unstable Metabolites
Long Term Storage Stability of Naproxen Acyl-Glucuronide Conjugate
0
05
1
15
2
0 50 100 150
(Nap
roxe
n-G
luN
apro
xen
) D
ay X
Day
1
Storage Time at -20ordmC (Day)
Naproxpen-GluNaproxen normalized byDay1 response
Stabilized Naproxen-GluNaproxenNormalized by Day 1
Linear (Naproxpen-GluNaproxen normalizedby Day1 response)
Linear (Stabilized Naproxen-GluNaproxenNormalized by Day 1)
n = 8
Untreated samples
A ratio between 08-12 is deemed stable
Stabilized samples
Gao H and Obach RS (2012) DMD 401290-1296
Correlation of Experimental MS Ratio vs Nominal Ratio
R 0994plusmn0005 Slope 099plusmn0082
MSNM Ratio
Metabolites
MS
NM
Ra
tio
10
15
20
1_O
H_T
acrine
2_O
H_T
acrine
3_M
eth
oxym
orp
hin
4_O
H_D
iclo
fenc
4_O
H_T
acrine
5_O
H_D
iclo
fenc
AZ
T
AZ
T_glu
c
Dextr
om
eth
orp
han
Dextr
orp
han
Dic
lofe
nac
Fexofe
nadin
e
M1
M10
M2
M5
M9
tacrine
Terf
enadin
e
Zip
rasid
one
Geo FC
Geo FC 95
Geo FC 99
Average 125
95 confidence 183
99 confidence 207
Precision Accuracy and Confidence Level bull Showed that a peak area ratio of 2 means that it is at least equal or
greater exposure
bull Increased replicates will increase confidence
95 Confidence Interval (~2SD)
Gao H Deng S and Obach RS (2010) DMD 382147-2156
Confidence Level Increases Dramatically with Measured Ratio
Ratio=4 plt2 ppm
Ratio=2 plt001
Measured animalhuman ratio by LCMSMS
Measured animalhuman ratio by LCMSMS
Probability of Nominal AnimalHuman ge1
What Drives High Confidence
bull Fundamental BA parameters are addressed Selectivity sensitivity linearity matrix effect stability precision and accuracy
bull SignalNoise Ratio Measured animalhuman 2
Measurement error (without cancellation) 025
SN (2-1)025=4
Limit of Detection A signal is real if SN=3
bull Minimal 3 independent animalhuman ratio measurements
bull Analysis reproducibility RSD of the ratio measurements (n=3) 33
bull Stability ratio for each metabolite plusmn20 (08-12) over the storage period
How to Evaluate Data Quality
MIST Coverage is a Comparison Question
Animals have exposures of metabolites ge human
Comparison 1 Giraffe vs Moose
Which one is taller
All that one needs to do is to have them stand next to each other The giraffe is much taller No need to know exact heights
Which one is taller
Too close to call just by looking at them -- The height measurements are needed
Comparison 2 Moose vs Whitetail Deer
If the animalhuman peak area ratio is ge 2 animal has exposure ge human with high confidence (plt001)
How often does it happen Most cases as animal is dosed at much higher
dose when corrected by body weight
A simple peak area ratio comparison is sufficient for most cases
Case Study
bull Drug A in project X at phase 1 14 days 35 mg BID in 6 human volunteers vs 5 days 300 mgkg in Sprague Dawley rats
bull Pooled plasma samples (Hamilton) across time points across subjects
bull 3 human metabolites Parent drug A 396gt203
m-hydroxy 412 ring opened 414 de-alkylated 304
bull HPLC method and major daughter ions were transferred from Biotransformation
bull Compare results with concentration measurements using the synthetic standard
MIST Coverage Evaluation Using RatHuman Ratio of Peak Area Ratios
Definitive Concentration Measurements Using Synthetic Standard
Species Parent De-alkylated Ring-opened m-Hydroxy
Human 0220 00406 000788 00210 Rat 770 0502 0292 000762
RatHuman ratio 1 35 124 371 0363
Human repeat 0184 00290 000506 00164
Rat repeat 734 0472 0266 000618 RatHuman
ratio 2 399 163 526 0378
Species m-Hydroxy (ngmL)
Human 743 plusmn 153
Rat 269 plusmn 258
Cavg RatHuman ratio 0362
Human Metabolite gt10 Drug-Related Material from a Human Radiolabel
ADME Study (or Metabolite Anticipated to Be gt10 from Other Human Studies)
Metabolite With Known Activity of
Interest
Use Standard Bioanalytical
Method
ICH Exempt Metabolite
Peak Area Ratio Method
Method Fails Criteria
Method Succeeds
Animal lt 2X Human
Animal ge 2X Human
No Further Work Needed
Coverage is Assured No Further Work
Needed
Prepare Authentic Standard of Metabolite
MIST Decision Tree
Gao H and Obach RS (2014) Bioanalysis 6(5) 641-650
Data Driven Strategy
bull Rigor of the analysis increases based on data Synthetic standard is needed only when the method fails or animal has less exposure than human
bull Conservative evaluation of MIST coverage
bull Assess MIST coverage at steady state without radio-label or synthetic standards enabling early assessment eg FIH
bull Time- and resource-saving strategy
Summary
bull LCMSMS peak area ratio comparison is scientifically sound and defensible method that can
ndash Ascertain the steady-state exposure of animals compared to humans (ie ldquoMIST coveragerdquo)
ndash Assess the MIST coverage without radio-label or synthetic standard
bull These methods are fit-for-purpose and can offer decision-making data useful for reliable estimation of metabolite exposure
bull Discussions with regulatory experts were positive and helpful
Gao et al (2013) AAPS Journal15(4) 970-973
Prerequisite for Current Methodology
bull Animal and human samples for side-by-side injection
minus In drug development first in human samples are typically ~1 year later than the toxicology study is done
bull Prior knowledge of human metabolites transferred from metabolite identification and scouting
minus Knowledge of major human metabolites may evolve only after in vivo human samples become available
minus Radio-labeled ADME is the most definitive study may take place late in the drug development process
Is it feasible to scan the samplesbank data and compare the data generated at different dates
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
MeO
O
O
OH
OH
OH
O OHNaproxen Acyl Glucuronide conjugate
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
(AZT)
Negative Control
Blind Test 3 Metabolite Cocktail
Gao et al Anal Chem 2015 87 11771-11776
Scanning and Banking DataSamples
bull Blind Test samples 22 metabolitesdrugs were spiked in human and rat plasmas (n=4 each)
bull No knowledge of the identities of the analytes and their concentrations in plasma were provided before sample analysis
bull Multiple analysis of the same samples (rat or human) using mixed matrix approach at Days 1 30 105 254 and 314
bull Treat the samples using the same IS solution in acetonitrile minus Freshly prepared to maintain the same concentration
bull Acquity UPLC generic gradient on C18 run time 25 min
bull Full scan and AIF scan on QExactive 70K at positive and negative polarity
bull Compare rat vs human samples analyzed at different dates
0 4 8 12 16 20 24 Time (min)
0
100 R
elat
ive
Ab
un
dan
ce
508
478 682
1132 1435 1450 763
773 243 1835 2120 2138 1164 1312 1638 288 534 1473 2338 802 2045 335 1062
42 43 44 45 46 47 48 49 50 51 52 53 54 55 Time (min)
0
100
Rel
ativ
e In
ten
sity
RT 508
RT 478
RT 495
RT 539
4 8 12 16 20 24 Time (min)
0
100 RT 624
RT 1313
RT 1113
RT 1626
RT 275 RT 879
RT 2008 RT 422
548
165 808
1 2
3
3
4
5
5
4
678
9 10 111213
14 16 15
1
2
3
4
56 7
Rel
ativ
e A
bu
nd
ance
Migrated naproxen acyl glucuronide
EIC 15 drugsmetabolites Resolution 70000 Extraction window 5ppm Polarity Positive
EIC 6 drugsmetabolites Resolution 70000 Extraction window 10 ppm Polarity Negative
2-OH Tacrine
4-OH Tacrine
1-OH Tacrine
Naproxen-Glu
Peak Area Ratio Measurement Variation
n=4 per species 5 independent analysis total n=40 per analyte
95 tile 037
Median 021
M1
0
DrugsMetabolites
Bank and Compare Data at Different Days
Gao et al Anal Chem 2015 87 11771-11776
Separation and Quantitative Analysis of Isomers
2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
100
Rela
tive I
nte
nsity
RT 1626
RT 275
RT 422 RT 1188 RT 632
Full Scan 4rsquo (or 5)-OH Diclofenac
XIC 3100032
Would high resolution ion mobility provide the orthogonal separation we need
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
Time-Averaged Pooling (ldquoHamilton Poolsrdquo)
bull Plasma samples from different time points are mixed to provide a single sample in which the analyte concentration Cavg is AUC over the sampling interval
-0avg
AUCC
Hamilton Garnett Kline Clin Pharmacol Ther 1981
total
1-2-nn130201
V)t-(t)t-(t)t-(t)t-(t)t-(t
)t-(tV
010t
total
1-2-nn130201
1-n1nnt V
)t-(t)t-(t)t-(t)t-(t)t-(t
)t-(tV
total
1-2-nn130201
1-t V
)t-(t)t-(t)t-(t)t-(t)t-(t
)t-(tV
bull Can be used to reduce the number of samples to be analyzed and still yield exposure comparisons across species
Mixed Matrix Peak Area Comparison
Pooled Human Sample
Pooled Animal Sample
Mix with Opposite
Matrix Control
Control Mix
Add IS and
Solvent
Recover Supernatant Analyze by HPLC-MS
IS IS IS
Met
Met
Human Animal Matrix Control
(Human) AreaPeak IS
(Human) AreaPeak Metabolite
(Animal) AreaPeak IS
(Animal) AreaPeak Metabolite
Ratio Human to Animal
Control Mix
Mixed Human Sample
Mixed Animal Sample
Mixed Human Sample
Mixed Animal Sample
Gao et al DMD 2010 Gao and Obach DMD 2012
Protein Precipitation
bull Selectivity minus High separation resolution chromatography (a long gradient on
a large column) + MRM or high resolution MS (ge30K for parent ion) w product ion scan for major human metabolites (ge10 of total drug related material)
minus Low throughput is offset by the limited of samples
bull Sensitivity Typically not an issue minus Major human metabolites (ge10 of drug related total material)
and the animal is dosed at NOAEL minus MRM or high accurate MS for parent (plusmn 10ppm) is more
sensitive than full scan MSMS for metabolite ID
bull Matrix effect Normalized by mixed matrix approach
bull Linearity Serial dilutions of the samples in blank mixed plasma
How to Establish BA Parameters without Synthetic Standards
bull Stabilize the metabolites if procedures are known
bull Repetitive analysis of samples to cover the storage duration
bull Profile metabolitesstability reference (usually the parent drug) vs the storage time
bull Auto sampler stability re-injecting the supernatant 12-24 hr later
1day
dayX
sample) (Same AreaPeak reference Stable
sample) Same ( AreaPeak Metabolite
sample) (same AreaPeak reference Stable
species)(Any AreaPeak Metabolite
RatioStability
How to Establish Stability without Synthetic Standards
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
Variety of reaction types alterations in pKa multiple regioisomers
(AZT)
Blind Test 1 Stable Metabolites
M1 236gt134
M2 252gt209
Ziprasidonemetabolites MRM TIC in rat plasma 1
IS
Ziprasidone 413gt194
M9
M5
M10
Dilution Curve M1 236gt134
Dilution Curve M9 429gt280
2 B
80 B
Column Kinetex C18 150 X 46 mm 26 um
Parent Metabolite Potential Instability
Naproxen Naproxen Acyl Glucuronide
Acyl bond migration
Ester hydrolysis
Carbamazepine Carbamazepine-1011-Epoxide
Epoxide hydrolysis
Salicylic Acid 23-Dihydroxybenzoic Acid
Catechol oxidation
Sulfadiazine N-Acetylsulfadiazine
Amide hydrolysis
Methylphenidate Ritalinic Acid
Ester hydrolysis
Nicotine Nicotine-Δ1rsquo5rsquo-Iminium Ion and Cotinine
Imine Oxidation
N
O NH2
MeO
O
O
OH
OH
OH
O OH
N
N+
N
NO
N
N
NH
OMeO
NH
OHO
OH
O
OH
OH
NH
SNH
N
N
O
O
O
NH2
SNH
N
N
O
O
OH
O
OH
N
O NH2
O
MeO
OH
O
Blind Test 2 Unstable Metabolites
Long Term Storage Stability of Naproxen Acyl-Glucuronide Conjugate
0
05
1
15
2
0 50 100 150
(Nap
roxe
n-G
luN
apro
xen
) D
ay X
Day
1
Storage Time at -20ordmC (Day)
Naproxpen-GluNaproxen normalized byDay1 response
Stabilized Naproxen-GluNaproxenNormalized by Day 1
Linear (Naproxpen-GluNaproxen normalizedby Day1 response)
Linear (Stabilized Naproxen-GluNaproxenNormalized by Day 1)
n = 8
Untreated samples
A ratio between 08-12 is deemed stable
Stabilized samples
Gao H and Obach RS (2012) DMD 401290-1296
Correlation of Experimental MS Ratio vs Nominal Ratio
R 0994plusmn0005 Slope 099plusmn0082
MSNM Ratio
Metabolites
MS
NM
Ra
tio
10
15
20
1_O
H_T
acrine
2_O
H_T
acrine
3_M
eth
oxym
orp
hin
4_O
H_D
iclo
fenc
4_O
H_T
acrine
5_O
H_D
iclo
fenc
AZ
T
AZ
T_glu
c
Dextr
om
eth
orp
han
Dextr
orp
han
Dic
lofe
nac
Fexofe
nadin
e
M1
M10
M2
M5
M9
tacrine
Terf
enadin
e
Zip
rasid
one
Geo FC
Geo FC 95
Geo FC 99
Average 125
95 confidence 183
99 confidence 207
Precision Accuracy and Confidence Level bull Showed that a peak area ratio of 2 means that it is at least equal or
greater exposure
bull Increased replicates will increase confidence
95 Confidence Interval (~2SD)
Gao H Deng S and Obach RS (2010) DMD 382147-2156
Confidence Level Increases Dramatically with Measured Ratio
Ratio=4 plt2 ppm
Ratio=2 plt001
Measured animalhuman ratio by LCMSMS
Measured animalhuman ratio by LCMSMS
Probability of Nominal AnimalHuman ge1
What Drives High Confidence
bull Fundamental BA parameters are addressed Selectivity sensitivity linearity matrix effect stability precision and accuracy
bull SignalNoise Ratio Measured animalhuman 2
Measurement error (without cancellation) 025
SN (2-1)025=4
Limit of Detection A signal is real if SN=3
bull Minimal 3 independent animalhuman ratio measurements
bull Analysis reproducibility RSD of the ratio measurements (n=3) 33
bull Stability ratio for each metabolite plusmn20 (08-12) over the storage period
How to Evaluate Data Quality
MIST Coverage is a Comparison Question
Animals have exposures of metabolites ge human
Comparison 1 Giraffe vs Moose
Which one is taller
All that one needs to do is to have them stand next to each other The giraffe is much taller No need to know exact heights
Which one is taller
Too close to call just by looking at them -- The height measurements are needed
Comparison 2 Moose vs Whitetail Deer
If the animalhuman peak area ratio is ge 2 animal has exposure ge human with high confidence (plt001)
How often does it happen Most cases as animal is dosed at much higher
dose when corrected by body weight
A simple peak area ratio comparison is sufficient for most cases
Case Study
bull Drug A in project X at phase 1 14 days 35 mg BID in 6 human volunteers vs 5 days 300 mgkg in Sprague Dawley rats
bull Pooled plasma samples (Hamilton) across time points across subjects
bull 3 human metabolites Parent drug A 396gt203
m-hydroxy 412 ring opened 414 de-alkylated 304
bull HPLC method and major daughter ions were transferred from Biotransformation
bull Compare results with concentration measurements using the synthetic standard
MIST Coverage Evaluation Using RatHuman Ratio of Peak Area Ratios
Definitive Concentration Measurements Using Synthetic Standard
Species Parent De-alkylated Ring-opened m-Hydroxy
Human 0220 00406 000788 00210 Rat 770 0502 0292 000762
RatHuman ratio 1 35 124 371 0363
Human repeat 0184 00290 000506 00164
Rat repeat 734 0472 0266 000618 RatHuman
ratio 2 399 163 526 0378
Species m-Hydroxy (ngmL)
Human 743 plusmn 153
Rat 269 plusmn 258
Cavg RatHuman ratio 0362
Human Metabolite gt10 Drug-Related Material from a Human Radiolabel
ADME Study (or Metabolite Anticipated to Be gt10 from Other Human Studies)
Metabolite With Known Activity of
Interest
Use Standard Bioanalytical
Method
ICH Exempt Metabolite
Peak Area Ratio Method
Method Fails Criteria
Method Succeeds
Animal lt 2X Human
Animal ge 2X Human
No Further Work Needed
Coverage is Assured No Further Work
Needed
Prepare Authentic Standard of Metabolite
MIST Decision Tree
Gao H and Obach RS (2014) Bioanalysis 6(5) 641-650
Data Driven Strategy
bull Rigor of the analysis increases based on data Synthetic standard is needed only when the method fails or animal has less exposure than human
bull Conservative evaluation of MIST coverage
bull Assess MIST coverage at steady state without radio-label or synthetic standards enabling early assessment eg FIH
bull Time- and resource-saving strategy
Summary
bull LCMSMS peak area ratio comparison is scientifically sound and defensible method that can
ndash Ascertain the steady-state exposure of animals compared to humans (ie ldquoMIST coveragerdquo)
ndash Assess the MIST coverage without radio-label or synthetic standard
bull These methods are fit-for-purpose and can offer decision-making data useful for reliable estimation of metabolite exposure
bull Discussions with regulatory experts were positive and helpful
Gao et al (2013) AAPS Journal15(4) 970-973
Prerequisite for Current Methodology
bull Animal and human samples for side-by-side injection
minus In drug development first in human samples are typically ~1 year later than the toxicology study is done
bull Prior knowledge of human metabolites transferred from metabolite identification and scouting
minus Knowledge of major human metabolites may evolve only after in vivo human samples become available
minus Radio-labeled ADME is the most definitive study may take place late in the drug development process
Is it feasible to scan the samplesbank data and compare the data generated at different dates
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
MeO
O
O
OH
OH
OH
O OHNaproxen Acyl Glucuronide conjugate
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
(AZT)
Negative Control
Blind Test 3 Metabolite Cocktail
Gao et al Anal Chem 2015 87 11771-11776
Scanning and Banking DataSamples
bull Blind Test samples 22 metabolitesdrugs were spiked in human and rat plasmas (n=4 each)
bull No knowledge of the identities of the analytes and their concentrations in plasma were provided before sample analysis
bull Multiple analysis of the same samples (rat or human) using mixed matrix approach at Days 1 30 105 254 and 314
bull Treat the samples using the same IS solution in acetonitrile minus Freshly prepared to maintain the same concentration
bull Acquity UPLC generic gradient on C18 run time 25 min
bull Full scan and AIF scan on QExactive 70K at positive and negative polarity
bull Compare rat vs human samples analyzed at different dates
0 4 8 12 16 20 24 Time (min)
0
100 R
elat
ive
Ab
un
dan
ce
508
478 682
1132 1435 1450 763
773 243 1835 2120 2138 1164 1312 1638 288 534 1473 2338 802 2045 335 1062
42 43 44 45 46 47 48 49 50 51 52 53 54 55 Time (min)
0
100
Rel
ativ
e In
ten
sity
RT 508
RT 478
RT 495
RT 539
4 8 12 16 20 24 Time (min)
0
100 RT 624
RT 1313
RT 1113
RT 1626
RT 275 RT 879
RT 2008 RT 422
548
165 808
1 2
3
3
4
5
5
4
678
9 10 111213
14 16 15
1
2
3
4
56 7
Rel
ativ
e A
bu
nd
ance
Migrated naproxen acyl glucuronide
EIC 15 drugsmetabolites Resolution 70000 Extraction window 5ppm Polarity Positive
EIC 6 drugsmetabolites Resolution 70000 Extraction window 10 ppm Polarity Negative
2-OH Tacrine
4-OH Tacrine
1-OH Tacrine
Naproxen-Glu
Peak Area Ratio Measurement Variation
n=4 per species 5 independent analysis total n=40 per analyte
95 tile 037
Median 021
M1
0
DrugsMetabolites
Bank and Compare Data at Different Days
Gao et al Anal Chem 2015 87 11771-11776
Separation and Quantitative Analysis of Isomers
2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
100
Rela
tive I
nte
nsity
RT 1626
RT 275
RT 422 RT 1188 RT 632
Full Scan 4rsquo (or 5)-OH Diclofenac
XIC 3100032
Would high resolution ion mobility provide the orthogonal separation we need
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
Mixed Matrix Peak Area Comparison
Pooled Human Sample
Pooled Animal Sample
Mix with Opposite
Matrix Control
Control Mix
Add IS and
Solvent
Recover Supernatant Analyze by HPLC-MS
IS IS IS
Met
Met
Human Animal Matrix Control
(Human) AreaPeak IS
(Human) AreaPeak Metabolite
(Animal) AreaPeak IS
(Animal) AreaPeak Metabolite
Ratio Human to Animal
Control Mix
Mixed Human Sample
Mixed Animal Sample
Mixed Human Sample
Mixed Animal Sample
Gao et al DMD 2010 Gao and Obach DMD 2012
Protein Precipitation
bull Selectivity minus High separation resolution chromatography (a long gradient on
a large column) + MRM or high resolution MS (ge30K for parent ion) w product ion scan for major human metabolites (ge10 of total drug related material)
minus Low throughput is offset by the limited of samples
bull Sensitivity Typically not an issue minus Major human metabolites (ge10 of drug related total material)
and the animal is dosed at NOAEL minus MRM or high accurate MS for parent (plusmn 10ppm) is more
sensitive than full scan MSMS for metabolite ID
bull Matrix effect Normalized by mixed matrix approach
bull Linearity Serial dilutions of the samples in blank mixed plasma
How to Establish BA Parameters without Synthetic Standards
bull Stabilize the metabolites if procedures are known
bull Repetitive analysis of samples to cover the storage duration
bull Profile metabolitesstability reference (usually the parent drug) vs the storage time
bull Auto sampler stability re-injecting the supernatant 12-24 hr later
1day
dayX
sample) (Same AreaPeak reference Stable
sample) Same ( AreaPeak Metabolite
sample) (same AreaPeak reference Stable
species)(Any AreaPeak Metabolite
RatioStability
How to Establish Stability without Synthetic Standards
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
Variety of reaction types alterations in pKa multiple regioisomers
(AZT)
Blind Test 1 Stable Metabolites
M1 236gt134
M2 252gt209
Ziprasidonemetabolites MRM TIC in rat plasma 1
IS
Ziprasidone 413gt194
M9
M5
M10
Dilution Curve M1 236gt134
Dilution Curve M9 429gt280
2 B
80 B
Column Kinetex C18 150 X 46 mm 26 um
Parent Metabolite Potential Instability
Naproxen Naproxen Acyl Glucuronide
Acyl bond migration
Ester hydrolysis
Carbamazepine Carbamazepine-1011-Epoxide
Epoxide hydrolysis
Salicylic Acid 23-Dihydroxybenzoic Acid
Catechol oxidation
Sulfadiazine N-Acetylsulfadiazine
Amide hydrolysis
Methylphenidate Ritalinic Acid
Ester hydrolysis
Nicotine Nicotine-Δ1rsquo5rsquo-Iminium Ion and Cotinine
Imine Oxidation
N
O NH2
MeO
O
O
OH
OH
OH
O OH
N
N+
N
NO
N
N
NH
OMeO
NH
OHO
OH
O
OH
OH
NH
SNH
N
N
O
O
O
NH2
SNH
N
N
O
O
OH
O
OH
N
O NH2
O
MeO
OH
O
Blind Test 2 Unstable Metabolites
Long Term Storage Stability of Naproxen Acyl-Glucuronide Conjugate
0
05
1
15
2
0 50 100 150
(Nap
roxe
n-G
luN
apro
xen
) D
ay X
Day
1
Storage Time at -20ordmC (Day)
Naproxpen-GluNaproxen normalized byDay1 response
Stabilized Naproxen-GluNaproxenNormalized by Day 1
Linear (Naproxpen-GluNaproxen normalizedby Day1 response)
Linear (Stabilized Naproxen-GluNaproxenNormalized by Day 1)
n = 8
Untreated samples
A ratio between 08-12 is deemed stable
Stabilized samples
Gao H and Obach RS (2012) DMD 401290-1296
Correlation of Experimental MS Ratio vs Nominal Ratio
R 0994plusmn0005 Slope 099plusmn0082
MSNM Ratio
Metabolites
MS
NM
Ra
tio
10
15
20
1_O
H_T
acrine
2_O
H_T
acrine
3_M
eth
oxym
orp
hin
4_O
H_D
iclo
fenc
4_O
H_T
acrine
5_O
H_D
iclo
fenc
AZ
T
AZ
T_glu
c
Dextr
om
eth
orp
han
Dextr
orp
han
Dic
lofe
nac
Fexofe
nadin
e
M1
M10
M2
M5
M9
tacrine
Terf
enadin
e
Zip
rasid
one
Geo FC
Geo FC 95
Geo FC 99
Average 125
95 confidence 183
99 confidence 207
Precision Accuracy and Confidence Level bull Showed that a peak area ratio of 2 means that it is at least equal or
greater exposure
bull Increased replicates will increase confidence
95 Confidence Interval (~2SD)
Gao H Deng S and Obach RS (2010) DMD 382147-2156
Confidence Level Increases Dramatically with Measured Ratio
Ratio=4 plt2 ppm
Ratio=2 plt001
Measured animalhuman ratio by LCMSMS
Measured animalhuman ratio by LCMSMS
Probability of Nominal AnimalHuman ge1
What Drives High Confidence
bull Fundamental BA parameters are addressed Selectivity sensitivity linearity matrix effect stability precision and accuracy
bull SignalNoise Ratio Measured animalhuman 2
Measurement error (without cancellation) 025
SN (2-1)025=4
Limit of Detection A signal is real if SN=3
bull Minimal 3 independent animalhuman ratio measurements
bull Analysis reproducibility RSD of the ratio measurements (n=3) 33
bull Stability ratio for each metabolite plusmn20 (08-12) over the storage period
How to Evaluate Data Quality
MIST Coverage is a Comparison Question
Animals have exposures of metabolites ge human
Comparison 1 Giraffe vs Moose
Which one is taller
All that one needs to do is to have them stand next to each other The giraffe is much taller No need to know exact heights
Which one is taller
Too close to call just by looking at them -- The height measurements are needed
Comparison 2 Moose vs Whitetail Deer
If the animalhuman peak area ratio is ge 2 animal has exposure ge human with high confidence (plt001)
How often does it happen Most cases as animal is dosed at much higher
dose when corrected by body weight
A simple peak area ratio comparison is sufficient for most cases
Case Study
bull Drug A in project X at phase 1 14 days 35 mg BID in 6 human volunteers vs 5 days 300 mgkg in Sprague Dawley rats
bull Pooled plasma samples (Hamilton) across time points across subjects
bull 3 human metabolites Parent drug A 396gt203
m-hydroxy 412 ring opened 414 de-alkylated 304
bull HPLC method and major daughter ions were transferred from Biotransformation
bull Compare results with concentration measurements using the synthetic standard
MIST Coverage Evaluation Using RatHuman Ratio of Peak Area Ratios
Definitive Concentration Measurements Using Synthetic Standard
Species Parent De-alkylated Ring-opened m-Hydroxy
Human 0220 00406 000788 00210 Rat 770 0502 0292 000762
RatHuman ratio 1 35 124 371 0363
Human repeat 0184 00290 000506 00164
Rat repeat 734 0472 0266 000618 RatHuman
ratio 2 399 163 526 0378
Species m-Hydroxy (ngmL)
Human 743 plusmn 153
Rat 269 plusmn 258
Cavg RatHuman ratio 0362
Human Metabolite gt10 Drug-Related Material from a Human Radiolabel
ADME Study (or Metabolite Anticipated to Be gt10 from Other Human Studies)
Metabolite With Known Activity of
Interest
Use Standard Bioanalytical
Method
ICH Exempt Metabolite
Peak Area Ratio Method
Method Fails Criteria
Method Succeeds
Animal lt 2X Human
Animal ge 2X Human
No Further Work Needed
Coverage is Assured No Further Work
Needed
Prepare Authentic Standard of Metabolite
MIST Decision Tree
Gao H and Obach RS (2014) Bioanalysis 6(5) 641-650
Data Driven Strategy
bull Rigor of the analysis increases based on data Synthetic standard is needed only when the method fails or animal has less exposure than human
bull Conservative evaluation of MIST coverage
bull Assess MIST coverage at steady state without radio-label or synthetic standards enabling early assessment eg FIH
bull Time- and resource-saving strategy
Summary
bull LCMSMS peak area ratio comparison is scientifically sound and defensible method that can
ndash Ascertain the steady-state exposure of animals compared to humans (ie ldquoMIST coveragerdquo)
ndash Assess the MIST coverage without radio-label or synthetic standard
bull These methods are fit-for-purpose and can offer decision-making data useful for reliable estimation of metabolite exposure
bull Discussions with regulatory experts were positive and helpful
Gao et al (2013) AAPS Journal15(4) 970-973
Prerequisite for Current Methodology
bull Animal and human samples for side-by-side injection
minus In drug development first in human samples are typically ~1 year later than the toxicology study is done
bull Prior knowledge of human metabolites transferred from metabolite identification and scouting
minus Knowledge of major human metabolites may evolve only after in vivo human samples become available
minus Radio-labeled ADME is the most definitive study may take place late in the drug development process
Is it feasible to scan the samplesbank data and compare the data generated at different dates
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
MeO
O
O
OH
OH
OH
O OHNaproxen Acyl Glucuronide conjugate
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
(AZT)
Negative Control
Blind Test 3 Metabolite Cocktail
Gao et al Anal Chem 2015 87 11771-11776
Scanning and Banking DataSamples
bull Blind Test samples 22 metabolitesdrugs were spiked in human and rat plasmas (n=4 each)
bull No knowledge of the identities of the analytes and their concentrations in plasma were provided before sample analysis
bull Multiple analysis of the same samples (rat or human) using mixed matrix approach at Days 1 30 105 254 and 314
bull Treat the samples using the same IS solution in acetonitrile minus Freshly prepared to maintain the same concentration
bull Acquity UPLC generic gradient on C18 run time 25 min
bull Full scan and AIF scan on QExactive 70K at positive and negative polarity
bull Compare rat vs human samples analyzed at different dates
0 4 8 12 16 20 24 Time (min)
0
100 R
elat
ive
Ab
un
dan
ce
508
478 682
1132 1435 1450 763
773 243 1835 2120 2138 1164 1312 1638 288 534 1473 2338 802 2045 335 1062
42 43 44 45 46 47 48 49 50 51 52 53 54 55 Time (min)
0
100
Rel
ativ
e In
ten
sity
RT 508
RT 478
RT 495
RT 539
4 8 12 16 20 24 Time (min)
0
100 RT 624
RT 1313
RT 1113
RT 1626
RT 275 RT 879
RT 2008 RT 422
548
165 808
1 2
3
3
4
5
5
4
678
9 10 111213
14 16 15
1
2
3
4
56 7
Rel
ativ
e A
bu
nd
ance
Migrated naproxen acyl glucuronide
EIC 15 drugsmetabolites Resolution 70000 Extraction window 5ppm Polarity Positive
EIC 6 drugsmetabolites Resolution 70000 Extraction window 10 ppm Polarity Negative
2-OH Tacrine
4-OH Tacrine
1-OH Tacrine
Naproxen-Glu
Peak Area Ratio Measurement Variation
n=4 per species 5 independent analysis total n=40 per analyte
95 tile 037
Median 021
M1
0
DrugsMetabolites
Bank and Compare Data at Different Days
Gao et al Anal Chem 2015 87 11771-11776
Separation and Quantitative Analysis of Isomers
2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
100
Rela
tive I
nte
nsity
RT 1626
RT 275
RT 422 RT 1188 RT 632
Full Scan 4rsquo (or 5)-OH Diclofenac
XIC 3100032
Would high resolution ion mobility provide the orthogonal separation we need
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
bull Selectivity minus High separation resolution chromatography (a long gradient on
a large column) + MRM or high resolution MS (ge30K for parent ion) w product ion scan for major human metabolites (ge10 of total drug related material)
minus Low throughput is offset by the limited of samples
bull Sensitivity Typically not an issue minus Major human metabolites (ge10 of drug related total material)
and the animal is dosed at NOAEL minus MRM or high accurate MS for parent (plusmn 10ppm) is more
sensitive than full scan MSMS for metabolite ID
bull Matrix effect Normalized by mixed matrix approach
bull Linearity Serial dilutions of the samples in blank mixed plasma
How to Establish BA Parameters without Synthetic Standards
bull Stabilize the metabolites if procedures are known
bull Repetitive analysis of samples to cover the storage duration
bull Profile metabolitesstability reference (usually the parent drug) vs the storage time
bull Auto sampler stability re-injecting the supernatant 12-24 hr later
1day
dayX
sample) (Same AreaPeak reference Stable
sample) Same ( AreaPeak Metabolite
sample) (same AreaPeak reference Stable
species)(Any AreaPeak Metabolite
RatioStability
How to Establish Stability without Synthetic Standards
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
Variety of reaction types alterations in pKa multiple regioisomers
(AZT)
Blind Test 1 Stable Metabolites
M1 236gt134
M2 252gt209
Ziprasidonemetabolites MRM TIC in rat plasma 1
IS
Ziprasidone 413gt194
M9
M5
M10
Dilution Curve M1 236gt134
Dilution Curve M9 429gt280
2 B
80 B
Column Kinetex C18 150 X 46 mm 26 um
Parent Metabolite Potential Instability
Naproxen Naproxen Acyl Glucuronide
Acyl bond migration
Ester hydrolysis
Carbamazepine Carbamazepine-1011-Epoxide
Epoxide hydrolysis
Salicylic Acid 23-Dihydroxybenzoic Acid
Catechol oxidation
Sulfadiazine N-Acetylsulfadiazine
Amide hydrolysis
Methylphenidate Ritalinic Acid
Ester hydrolysis
Nicotine Nicotine-Δ1rsquo5rsquo-Iminium Ion and Cotinine
Imine Oxidation
N
O NH2
MeO
O
O
OH
OH
OH
O OH
N
N+
N
NO
N
N
NH
OMeO
NH
OHO
OH
O
OH
OH
NH
SNH
N
N
O
O
O
NH2
SNH
N
N
O
O
OH
O
OH
N
O NH2
O
MeO
OH
O
Blind Test 2 Unstable Metabolites
Long Term Storage Stability of Naproxen Acyl-Glucuronide Conjugate
0
05
1
15
2
0 50 100 150
(Nap
roxe
n-G
luN
apro
xen
) D
ay X
Day
1
Storage Time at -20ordmC (Day)
Naproxpen-GluNaproxen normalized byDay1 response
Stabilized Naproxen-GluNaproxenNormalized by Day 1
Linear (Naproxpen-GluNaproxen normalizedby Day1 response)
Linear (Stabilized Naproxen-GluNaproxenNormalized by Day 1)
n = 8
Untreated samples
A ratio between 08-12 is deemed stable
Stabilized samples
Gao H and Obach RS (2012) DMD 401290-1296
Correlation of Experimental MS Ratio vs Nominal Ratio
R 0994plusmn0005 Slope 099plusmn0082
MSNM Ratio
Metabolites
MS
NM
Ra
tio
10
15
20
1_O
H_T
acrine
2_O
H_T
acrine
3_M
eth
oxym
orp
hin
4_O
H_D
iclo
fenc
4_O
H_T
acrine
5_O
H_D
iclo
fenc
AZ
T
AZ
T_glu
c
Dextr
om
eth
orp
han
Dextr
orp
han
Dic
lofe
nac
Fexofe
nadin
e
M1
M10
M2
M5
M9
tacrine
Terf
enadin
e
Zip
rasid
one
Geo FC
Geo FC 95
Geo FC 99
Average 125
95 confidence 183
99 confidence 207
Precision Accuracy and Confidence Level bull Showed that a peak area ratio of 2 means that it is at least equal or
greater exposure
bull Increased replicates will increase confidence
95 Confidence Interval (~2SD)
Gao H Deng S and Obach RS (2010) DMD 382147-2156
Confidence Level Increases Dramatically with Measured Ratio
Ratio=4 plt2 ppm
Ratio=2 plt001
Measured animalhuman ratio by LCMSMS
Measured animalhuman ratio by LCMSMS
Probability of Nominal AnimalHuman ge1
What Drives High Confidence
bull Fundamental BA parameters are addressed Selectivity sensitivity linearity matrix effect stability precision and accuracy
bull SignalNoise Ratio Measured animalhuman 2
Measurement error (without cancellation) 025
SN (2-1)025=4
Limit of Detection A signal is real if SN=3
bull Minimal 3 independent animalhuman ratio measurements
bull Analysis reproducibility RSD of the ratio measurements (n=3) 33
bull Stability ratio for each metabolite plusmn20 (08-12) over the storage period
How to Evaluate Data Quality
MIST Coverage is a Comparison Question
Animals have exposures of metabolites ge human
Comparison 1 Giraffe vs Moose
Which one is taller
All that one needs to do is to have them stand next to each other The giraffe is much taller No need to know exact heights
Which one is taller
Too close to call just by looking at them -- The height measurements are needed
Comparison 2 Moose vs Whitetail Deer
If the animalhuman peak area ratio is ge 2 animal has exposure ge human with high confidence (plt001)
How often does it happen Most cases as animal is dosed at much higher
dose when corrected by body weight
A simple peak area ratio comparison is sufficient for most cases
Case Study
bull Drug A in project X at phase 1 14 days 35 mg BID in 6 human volunteers vs 5 days 300 mgkg in Sprague Dawley rats
bull Pooled plasma samples (Hamilton) across time points across subjects
bull 3 human metabolites Parent drug A 396gt203
m-hydroxy 412 ring opened 414 de-alkylated 304
bull HPLC method and major daughter ions were transferred from Biotransformation
bull Compare results with concentration measurements using the synthetic standard
MIST Coverage Evaluation Using RatHuman Ratio of Peak Area Ratios
Definitive Concentration Measurements Using Synthetic Standard
Species Parent De-alkylated Ring-opened m-Hydroxy
Human 0220 00406 000788 00210 Rat 770 0502 0292 000762
RatHuman ratio 1 35 124 371 0363
Human repeat 0184 00290 000506 00164
Rat repeat 734 0472 0266 000618 RatHuman
ratio 2 399 163 526 0378
Species m-Hydroxy (ngmL)
Human 743 plusmn 153
Rat 269 plusmn 258
Cavg RatHuman ratio 0362
Human Metabolite gt10 Drug-Related Material from a Human Radiolabel
ADME Study (or Metabolite Anticipated to Be gt10 from Other Human Studies)
Metabolite With Known Activity of
Interest
Use Standard Bioanalytical
Method
ICH Exempt Metabolite
Peak Area Ratio Method
Method Fails Criteria
Method Succeeds
Animal lt 2X Human
Animal ge 2X Human
No Further Work Needed
Coverage is Assured No Further Work
Needed
Prepare Authentic Standard of Metabolite
MIST Decision Tree
Gao H and Obach RS (2014) Bioanalysis 6(5) 641-650
Data Driven Strategy
bull Rigor of the analysis increases based on data Synthetic standard is needed only when the method fails or animal has less exposure than human
bull Conservative evaluation of MIST coverage
bull Assess MIST coverage at steady state without radio-label or synthetic standards enabling early assessment eg FIH
bull Time- and resource-saving strategy
Summary
bull LCMSMS peak area ratio comparison is scientifically sound and defensible method that can
ndash Ascertain the steady-state exposure of animals compared to humans (ie ldquoMIST coveragerdquo)
ndash Assess the MIST coverage without radio-label or synthetic standard
bull These methods are fit-for-purpose and can offer decision-making data useful for reliable estimation of metabolite exposure
bull Discussions with regulatory experts were positive and helpful
Gao et al (2013) AAPS Journal15(4) 970-973
Prerequisite for Current Methodology
bull Animal and human samples for side-by-side injection
minus In drug development first in human samples are typically ~1 year later than the toxicology study is done
bull Prior knowledge of human metabolites transferred from metabolite identification and scouting
minus Knowledge of major human metabolites may evolve only after in vivo human samples become available
minus Radio-labeled ADME is the most definitive study may take place late in the drug development process
Is it feasible to scan the samplesbank data and compare the data generated at different dates
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
MeO
O
O
OH
OH
OH
O OHNaproxen Acyl Glucuronide conjugate
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
(AZT)
Negative Control
Blind Test 3 Metabolite Cocktail
Gao et al Anal Chem 2015 87 11771-11776
Scanning and Banking DataSamples
bull Blind Test samples 22 metabolitesdrugs were spiked in human and rat plasmas (n=4 each)
bull No knowledge of the identities of the analytes and their concentrations in plasma were provided before sample analysis
bull Multiple analysis of the same samples (rat or human) using mixed matrix approach at Days 1 30 105 254 and 314
bull Treat the samples using the same IS solution in acetonitrile minus Freshly prepared to maintain the same concentration
bull Acquity UPLC generic gradient on C18 run time 25 min
bull Full scan and AIF scan on QExactive 70K at positive and negative polarity
bull Compare rat vs human samples analyzed at different dates
0 4 8 12 16 20 24 Time (min)
0
100 R
elat
ive
Ab
un
dan
ce
508
478 682
1132 1435 1450 763
773 243 1835 2120 2138 1164 1312 1638 288 534 1473 2338 802 2045 335 1062
42 43 44 45 46 47 48 49 50 51 52 53 54 55 Time (min)
0
100
Rel
ativ
e In
ten
sity
RT 508
RT 478
RT 495
RT 539
4 8 12 16 20 24 Time (min)
0
100 RT 624
RT 1313
RT 1113
RT 1626
RT 275 RT 879
RT 2008 RT 422
548
165 808
1 2
3
3
4
5
5
4
678
9 10 111213
14 16 15
1
2
3
4
56 7
Rel
ativ
e A
bu
nd
ance
Migrated naproxen acyl glucuronide
EIC 15 drugsmetabolites Resolution 70000 Extraction window 5ppm Polarity Positive
EIC 6 drugsmetabolites Resolution 70000 Extraction window 10 ppm Polarity Negative
2-OH Tacrine
4-OH Tacrine
1-OH Tacrine
Naproxen-Glu
Peak Area Ratio Measurement Variation
n=4 per species 5 independent analysis total n=40 per analyte
95 tile 037
Median 021
M1
0
DrugsMetabolites
Bank and Compare Data at Different Days
Gao et al Anal Chem 2015 87 11771-11776
Separation and Quantitative Analysis of Isomers
2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
100
Rela
tive I
nte
nsity
RT 1626
RT 275
RT 422 RT 1188 RT 632
Full Scan 4rsquo (or 5)-OH Diclofenac
XIC 3100032
Would high resolution ion mobility provide the orthogonal separation we need
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
bull Stabilize the metabolites if procedures are known
bull Repetitive analysis of samples to cover the storage duration
bull Profile metabolitesstability reference (usually the parent drug) vs the storage time
bull Auto sampler stability re-injecting the supernatant 12-24 hr later
1day
dayX
sample) (Same AreaPeak reference Stable
sample) Same ( AreaPeak Metabolite
sample) (same AreaPeak reference Stable
species)(Any AreaPeak Metabolite
RatioStability
How to Establish Stability without Synthetic Standards
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
Variety of reaction types alterations in pKa multiple regioisomers
(AZT)
Blind Test 1 Stable Metabolites
M1 236gt134
M2 252gt209
Ziprasidonemetabolites MRM TIC in rat plasma 1
IS
Ziprasidone 413gt194
M9
M5
M10
Dilution Curve M1 236gt134
Dilution Curve M9 429gt280
2 B
80 B
Column Kinetex C18 150 X 46 mm 26 um
Parent Metabolite Potential Instability
Naproxen Naproxen Acyl Glucuronide
Acyl bond migration
Ester hydrolysis
Carbamazepine Carbamazepine-1011-Epoxide
Epoxide hydrolysis
Salicylic Acid 23-Dihydroxybenzoic Acid
Catechol oxidation
Sulfadiazine N-Acetylsulfadiazine
Amide hydrolysis
Methylphenidate Ritalinic Acid
Ester hydrolysis
Nicotine Nicotine-Δ1rsquo5rsquo-Iminium Ion and Cotinine
Imine Oxidation
N
O NH2
MeO
O
O
OH
OH
OH
O OH
N
N+
N
NO
N
N
NH
OMeO
NH
OHO
OH
O
OH
OH
NH
SNH
N
N
O
O
O
NH2
SNH
N
N
O
O
OH
O
OH
N
O NH2
O
MeO
OH
O
Blind Test 2 Unstable Metabolites
Long Term Storage Stability of Naproxen Acyl-Glucuronide Conjugate
0
05
1
15
2
0 50 100 150
(Nap
roxe
n-G
luN
apro
xen
) D
ay X
Day
1
Storage Time at -20ordmC (Day)
Naproxpen-GluNaproxen normalized byDay1 response
Stabilized Naproxen-GluNaproxenNormalized by Day 1
Linear (Naproxpen-GluNaproxen normalizedby Day1 response)
Linear (Stabilized Naproxen-GluNaproxenNormalized by Day 1)
n = 8
Untreated samples
A ratio between 08-12 is deemed stable
Stabilized samples
Gao H and Obach RS (2012) DMD 401290-1296
Correlation of Experimental MS Ratio vs Nominal Ratio
R 0994plusmn0005 Slope 099plusmn0082
MSNM Ratio
Metabolites
MS
NM
Ra
tio
10
15
20
1_O
H_T
acrine
2_O
H_T
acrine
3_M
eth
oxym
orp
hin
4_O
H_D
iclo
fenc
4_O
H_T
acrine
5_O
H_D
iclo
fenc
AZ
T
AZ
T_glu
c
Dextr
om
eth
orp
han
Dextr
orp
han
Dic
lofe
nac
Fexofe
nadin
e
M1
M10
M2
M5
M9
tacrine
Terf
enadin
e
Zip
rasid
one
Geo FC
Geo FC 95
Geo FC 99
Average 125
95 confidence 183
99 confidence 207
Precision Accuracy and Confidence Level bull Showed that a peak area ratio of 2 means that it is at least equal or
greater exposure
bull Increased replicates will increase confidence
95 Confidence Interval (~2SD)
Gao H Deng S and Obach RS (2010) DMD 382147-2156
Confidence Level Increases Dramatically with Measured Ratio
Ratio=4 plt2 ppm
Ratio=2 plt001
Measured animalhuman ratio by LCMSMS
Measured animalhuman ratio by LCMSMS
Probability of Nominal AnimalHuman ge1
What Drives High Confidence
bull Fundamental BA parameters are addressed Selectivity sensitivity linearity matrix effect stability precision and accuracy
bull SignalNoise Ratio Measured animalhuman 2
Measurement error (without cancellation) 025
SN (2-1)025=4
Limit of Detection A signal is real if SN=3
bull Minimal 3 independent animalhuman ratio measurements
bull Analysis reproducibility RSD of the ratio measurements (n=3) 33
bull Stability ratio for each metabolite plusmn20 (08-12) over the storage period
How to Evaluate Data Quality
MIST Coverage is a Comparison Question
Animals have exposures of metabolites ge human
Comparison 1 Giraffe vs Moose
Which one is taller
All that one needs to do is to have them stand next to each other The giraffe is much taller No need to know exact heights
Which one is taller
Too close to call just by looking at them -- The height measurements are needed
Comparison 2 Moose vs Whitetail Deer
If the animalhuman peak area ratio is ge 2 animal has exposure ge human with high confidence (plt001)
How often does it happen Most cases as animal is dosed at much higher
dose when corrected by body weight
A simple peak area ratio comparison is sufficient for most cases
Case Study
bull Drug A in project X at phase 1 14 days 35 mg BID in 6 human volunteers vs 5 days 300 mgkg in Sprague Dawley rats
bull Pooled plasma samples (Hamilton) across time points across subjects
bull 3 human metabolites Parent drug A 396gt203
m-hydroxy 412 ring opened 414 de-alkylated 304
bull HPLC method and major daughter ions were transferred from Biotransformation
bull Compare results with concentration measurements using the synthetic standard
MIST Coverage Evaluation Using RatHuman Ratio of Peak Area Ratios
Definitive Concentration Measurements Using Synthetic Standard
Species Parent De-alkylated Ring-opened m-Hydroxy
Human 0220 00406 000788 00210 Rat 770 0502 0292 000762
RatHuman ratio 1 35 124 371 0363
Human repeat 0184 00290 000506 00164
Rat repeat 734 0472 0266 000618 RatHuman
ratio 2 399 163 526 0378
Species m-Hydroxy (ngmL)
Human 743 plusmn 153
Rat 269 plusmn 258
Cavg RatHuman ratio 0362
Human Metabolite gt10 Drug-Related Material from a Human Radiolabel
ADME Study (or Metabolite Anticipated to Be gt10 from Other Human Studies)
Metabolite With Known Activity of
Interest
Use Standard Bioanalytical
Method
ICH Exempt Metabolite
Peak Area Ratio Method
Method Fails Criteria
Method Succeeds
Animal lt 2X Human
Animal ge 2X Human
No Further Work Needed
Coverage is Assured No Further Work
Needed
Prepare Authentic Standard of Metabolite
MIST Decision Tree
Gao H and Obach RS (2014) Bioanalysis 6(5) 641-650
Data Driven Strategy
bull Rigor of the analysis increases based on data Synthetic standard is needed only when the method fails or animal has less exposure than human
bull Conservative evaluation of MIST coverage
bull Assess MIST coverage at steady state without radio-label or synthetic standards enabling early assessment eg FIH
bull Time- and resource-saving strategy
Summary
bull LCMSMS peak area ratio comparison is scientifically sound and defensible method that can
ndash Ascertain the steady-state exposure of animals compared to humans (ie ldquoMIST coveragerdquo)
ndash Assess the MIST coverage without radio-label or synthetic standard
bull These methods are fit-for-purpose and can offer decision-making data useful for reliable estimation of metabolite exposure
bull Discussions with regulatory experts were positive and helpful
Gao et al (2013) AAPS Journal15(4) 970-973
Prerequisite for Current Methodology
bull Animal and human samples for side-by-side injection
minus In drug development first in human samples are typically ~1 year later than the toxicology study is done
bull Prior knowledge of human metabolites transferred from metabolite identification and scouting
minus Knowledge of major human metabolites may evolve only after in vivo human samples become available
minus Radio-labeled ADME is the most definitive study may take place late in the drug development process
Is it feasible to scan the samplesbank data and compare the data generated at different dates
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
MeO
O
O
OH
OH
OH
O OHNaproxen Acyl Glucuronide conjugate
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
(AZT)
Negative Control
Blind Test 3 Metabolite Cocktail
Gao et al Anal Chem 2015 87 11771-11776
Scanning and Banking DataSamples
bull Blind Test samples 22 metabolitesdrugs were spiked in human and rat plasmas (n=4 each)
bull No knowledge of the identities of the analytes and their concentrations in plasma were provided before sample analysis
bull Multiple analysis of the same samples (rat or human) using mixed matrix approach at Days 1 30 105 254 and 314
bull Treat the samples using the same IS solution in acetonitrile minus Freshly prepared to maintain the same concentration
bull Acquity UPLC generic gradient on C18 run time 25 min
bull Full scan and AIF scan on QExactive 70K at positive and negative polarity
bull Compare rat vs human samples analyzed at different dates
0 4 8 12 16 20 24 Time (min)
0
100 R
elat
ive
Ab
un
dan
ce
508
478 682
1132 1435 1450 763
773 243 1835 2120 2138 1164 1312 1638 288 534 1473 2338 802 2045 335 1062
42 43 44 45 46 47 48 49 50 51 52 53 54 55 Time (min)
0
100
Rel
ativ
e In
ten
sity
RT 508
RT 478
RT 495
RT 539
4 8 12 16 20 24 Time (min)
0
100 RT 624
RT 1313
RT 1113
RT 1626
RT 275 RT 879
RT 2008 RT 422
548
165 808
1 2
3
3
4
5
5
4
678
9 10 111213
14 16 15
1
2
3
4
56 7
Rel
ativ
e A
bu
nd
ance
Migrated naproxen acyl glucuronide
EIC 15 drugsmetabolites Resolution 70000 Extraction window 5ppm Polarity Positive
EIC 6 drugsmetabolites Resolution 70000 Extraction window 10 ppm Polarity Negative
2-OH Tacrine
4-OH Tacrine
1-OH Tacrine
Naproxen-Glu
Peak Area Ratio Measurement Variation
n=4 per species 5 independent analysis total n=40 per analyte
95 tile 037
Median 021
M1
0
DrugsMetabolites
Bank and Compare Data at Different Days
Gao et al Anal Chem 2015 87 11771-11776
Separation and Quantitative Analysis of Isomers
2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
100
Rela
tive I
nte
nsity
RT 1626
RT 275
RT 422 RT 1188 RT 632
Full Scan 4rsquo (or 5)-OH Diclofenac
XIC 3100032
Would high resolution ion mobility provide the orthogonal separation we need
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
Variety of reaction types alterations in pKa multiple regioisomers
(AZT)
Blind Test 1 Stable Metabolites
M1 236gt134
M2 252gt209
Ziprasidonemetabolites MRM TIC in rat plasma 1
IS
Ziprasidone 413gt194
M9
M5
M10
Dilution Curve M1 236gt134
Dilution Curve M9 429gt280
2 B
80 B
Column Kinetex C18 150 X 46 mm 26 um
Parent Metabolite Potential Instability
Naproxen Naproxen Acyl Glucuronide
Acyl bond migration
Ester hydrolysis
Carbamazepine Carbamazepine-1011-Epoxide
Epoxide hydrolysis
Salicylic Acid 23-Dihydroxybenzoic Acid
Catechol oxidation
Sulfadiazine N-Acetylsulfadiazine
Amide hydrolysis
Methylphenidate Ritalinic Acid
Ester hydrolysis
Nicotine Nicotine-Δ1rsquo5rsquo-Iminium Ion and Cotinine
Imine Oxidation
N
O NH2
MeO
O
O
OH
OH
OH
O OH
N
N+
N
NO
N
N
NH
OMeO
NH
OHO
OH
O
OH
OH
NH
SNH
N
N
O
O
O
NH2
SNH
N
N
O
O
OH
O
OH
N
O NH2
O
MeO
OH
O
Blind Test 2 Unstable Metabolites
Long Term Storage Stability of Naproxen Acyl-Glucuronide Conjugate
0
05
1
15
2
0 50 100 150
(Nap
roxe
n-G
luN
apro
xen
) D
ay X
Day
1
Storage Time at -20ordmC (Day)
Naproxpen-GluNaproxen normalized byDay1 response
Stabilized Naproxen-GluNaproxenNormalized by Day 1
Linear (Naproxpen-GluNaproxen normalizedby Day1 response)
Linear (Stabilized Naproxen-GluNaproxenNormalized by Day 1)
n = 8
Untreated samples
A ratio between 08-12 is deemed stable
Stabilized samples
Gao H and Obach RS (2012) DMD 401290-1296
Correlation of Experimental MS Ratio vs Nominal Ratio
R 0994plusmn0005 Slope 099plusmn0082
MSNM Ratio
Metabolites
MS
NM
Ra
tio
10
15
20
1_O
H_T
acrine
2_O
H_T
acrine
3_M
eth
oxym
orp
hin
4_O
H_D
iclo
fenc
4_O
H_T
acrine
5_O
H_D
iclo
fenc
AZ
T
AZ
T_glu
c
Dextr
om
eth
orp
han
Dextr
orp
han
Dic
lofe
nac
Fexofe
nadin
e
M1
M10
M2
M5
M9
tacrine
Terf
enadin
e
Zip
rasid
one
Geo FC
Geo FC 95
Geo FC 99
Average 125
95 confidence 183
99 confidence 207
Precision Accuracy and Confidence Level bull Showed that a peak area ratio of 2 means that it is at least equal or
greater exposure
bull Increased replicates will increase confidence
95 Confidence Interval (~2SD)
Gao H Deng S and Obach RS (2010) DMD 382147-2156
Confidence Level Increases Dramatically with Measured Ratio
Ratio=4 plt2 ppm
Ratio=2 plt001
Measured animalhuman ratio by LCMSMS
Measured animalhuman ratio by LCMSMS
Probability of Nominal AnimalHuman ge1
What Drives High Confidence
bull Fundamental BA parameters are addressed Selectivity sensitivity linearity matrix effect stability precision and accuracy
bull SignalNoise Ratio Measured animalhuman 2
Measurement error (without cancellation) 025
SN (2-1)025=4
Limit of Detection A signal is real if SN=3
bull Minimal 3 independent animalhuman ratio measurements
bull Analysis reproducibility RSD of the ratio measurements (n=3) 33
bull Stability ratio for each metabolite plusmn20 (08-12) over the storage period
How to Evaluate Data Quality
MIST Coverage is a Comparison Question
Animals have exposures of metabolites ge human
Comparison 1 Giraffe vs Moose
Which one is taller
All that one needs to do is to have them stand next to each other The giraffe is much taller No need to know exact heights
Which one is taller
Too close to call just by looking at them -- The height measurements are needed
Comparison 2 Moose vs Whitetail Deer
If the animalhuman peak area ratio is ge 2 animal has exposure ge human with high confidence (plt001)
How often does it happen Most cases as animal is dosed at much higher
dose when corrected by body weight
A simple peak area ratio comparison is sufficient for most cases
Case Study
bull Drug A in project X at phase 1 14 days 35 mg BID in 6 human volunteers vs 5 days 300 mgkg in Sprague Dawley rats
bull Pooled plasma samples (Hamilton) across time points across subjects
bull 3 human metabolites Parent drug A 396gt203
m-hydroxy 412 ring opened 414 de-alkylated 304
bull HPLC method and major daughter ions were transferred from Biotransformation
bull Compare results with concentration measurements using the synthetic standard
MIST Coverage Evaluation Using RatHuman Ratio of Peak Area Ratios
Definitive Concentration Measurements Using Synthetic Standard
Species Parent De-alkylated Ring-opened m-Hydroxy
Human 0220 00406 000788 00210 Rat 770 0502 0292 000762
RatHuman ratio 1 35 124 371 0363
Human repeat 0184 00290 000506 00164
Rat repeat 734 0472 0266 000618 RatHuman
ratio 2 399 163 526 0378
Species m-Hydroxy (ngmL)
Human 743 plusmn 153
Rat 269 plusmn 258
Cavg RatHuman ratio 0362
Human Metabolite gt10 Drug-Related Material from a Human Radiolabel
ADME Study (or Metabolite Anticipated to Be gt10 from Other Human Studies)
Metabolite With Known Activity of
Interest
Use Standard Bioanalytical
Method
ICH Exempt Metabolite
Peak Area Ratio Method
Method Fails Criteria
Method Succeeds
Animal lt 2X Human
Animal ge 2X Human
No Further Work Needed
Coverage is Assured No Further Work
Needed
Prepare Authentic Standard of Metabolite
MIST Decision Tree
Gao H and Obach RS (2014) Bioanalysis 6(5) 641-650
Data Driven Strategy
bull Rigor of the analysis increases based on data Synthetic standard is needed only when the method fails or animal has less exposure than human
bull Conservative evaluation of MIST coverage
bull Assess MIST coverage at steady state without radio-label or synthetic standards enabling early assessment eg FIH
bull Time- and resource-saving strategy
Summary
bull LCMSMS peak area ratio comparison is scientifically sound and defensible method that can
ndash Ascertain the steady-state exposure of animals compared to humans (ie ldquoMIST coveragerdquo)
ndash Assess the MIST coverage without radio-label or synthetic standard
bull These methods are fit-for-purpose and can offer decision-making data useful for reliable estimation of metabolite exposure
bull Discussions with regulatory experts were positive and helpful
Gao et al (2013) AAPS Journal15(4) 970-973
Prerequisite for Current Methodology
bull Animal and human samples for side-by-side injection
minus In drug development first in human samples are typically ~1 year later than the toxicology study is done
bull Prior knowledge of human metabolites transferred from metabolite identification and scouting
minus Knowledge of major human metabolites may evolve only after in vivo human samples become available
minus Radio-labeled ADME is the most definitive study may take place late in the drug development process
Is it feasible to scan the samplesbank data and compare the data generated at different dates
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
MeO
O
O
OH
OH
OH
O OHNaproxen Acyl Glucuronide conjugate
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
(AZT)
Negative Control
Blind Test 3 Metabolite Cocktail
Gao et al Anal Chem 2015 87 11771-11776
Scanning and Banking DataSamples
bull Blind Test samples 22 metabolitesdrugs were spiked in human and rat plasmas (n=4 each)
bull No knowledge of the identities of the analytes and their concentrations in plasma were provided before sample analysis
bull Multiple analysis of the same samples (rat or human) using mixed matrix approach at Days 1 30 105 254 and 314
bull Treat the samples using the same IS solution in acetonitrile minus Freshly prepared to maintain the same concentration
bull Acquity UPLC generic gradient on C18 run time 25 min
bull Full scan and AIF scan on QExactive 70K at positive and negative polarity
bull Compare rat vs human samples analyzed at different dates
0 4 8 12 16 20 24 Time (min)
0
100 R
elat
ive
Ab
un
dan
ce
508
478 682
1132 1435 1450 763
773 243 1835 2120 2138 1164 1312 1638 288 534 1473 2338 802 2045 335 1062
42 43 44 45 46 47 48 49 50 51 52 53 54 55 Time (min)
0
100
Rel
ativ
e In
ten
sity
RT 508
RT 478
RT 495
RT 539
4 8 12 16 20 24 Time (min)
0
100 RT 624
RT 1313
RT 1113
RT 1626
RT 275 RT 879
RT 2008 RT 422
548
165 808
1 2
3
3
4
5
5
4
678
9 10 111213
14 16 15
1
2
3
4
56 7
Rel
ativ
e A
bu
nd
ance
Migrated naproxen acyl glucuronide
EIC 15 drugsmetabolites Resolution 70000 Extraction window 5ppm Polarity Positive
EIC 6 drugsmetabolites Resolution 70000 Extraction window 10 ppm Polarity Negative
2-OH Tacrine
4-OH Tacrine
1-OH Tacrine
Naproxen-Glu
Peak Area Ratio Measurement Variation
n=4 per species 5 independent analysis total n=40 per analyte
95 tile 037
Median 021
M1
0
DrugsMetabolites
Bank and Compare Data at Different Days
Gao et al Anal Chem 2015 87 11771-11776
Separation and Quantitative Analysis of Isomers
2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
100
Rela
tive I
nte
nsity
RT 1626
RT 275
RT 422 RT 1188 RT 632
Full Scan 4rsquo (or 5)-OH Diclofenac
XIC 3100032
Would high resolution ion mobility provide the orthogonal separation we need
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
M1 236gt134
M2 252gt209
Ziprasidonemetabolites MRM TIC in rat plasma 1
IS
Ziprasidone 413gt194
M9
M5
M10
Dilution Curve M1 236gt134
Dilution Curve M9 429gt280
2 B
80 B
Column Kinetex C18 150 X 46 mm 26 um
Parent Metabolite Potential Instability
Naproxen Naproxen Acyl Glucuronide
Acyl bond migration
Ester hydrolysis
Carbamazepine Carbamazepine-1011-Epoxide
Epoxide hydrolysis
Salicylic Acid 23-Dihydroxybenzoic Acid
Catechol oxidation
Sulfadiazine N-Acetylsulfadiazine
Amide hydrolysis
Methylphenidate Ritalinic Acid
Ester hydrolysis
Nicotine Nicotine-Δ1rsquo5rsquo-Iminium Ion and Cotinine
Imine Oxidation
N
O NH2
MeO
O
O
OH
OH
OH
O OH
N
N+
N
NO
N
N
NH
OMeO
NH
OHO
OH
O
OH
OH
NH
SNH
N
N
O
O
O
NH2
SNH
N
N
O
O
OH
O
OH
N
O NH2
O
MeO
OH
O
Blind Test 2 Unstable Metabolites
Long Term Storage Stability of Naproxen Acyl-Glucuronide Conjugate
0
05
1
15
2
0 50 100 150
(Nap
roxe
n-G
luN
apro
xen
) D
ay X
Day
1
Storage Time at -20ordmC (Day)
Naproxpen-GluNaproxen normalized byDay1 response
Stabilized Naproxen-GluNaproxenNormalized by Day 1
Linear (Naproxpen-GluNaproxen normalizedby Day1 response)
Linear (Stabilized Naproxen-GluNaproxenNormalized by Day 1)
n = 8
Untreated samples
A ratio between 08-12 is deemed stable
Stabilized samples
Gao H and Obach RS (2012) DMD 401290-1296
Correlation of Experimental MS Ratio vs Nominal Ratio
R 0994plusmn0005 Slope 099plusmn0082
MSNM Ratio
Metabolites
MS
NM
Ra
tio
10
15
20
1_O
H_T
acrine
2_O
H_T
acrine
3_M
eth
oxym
orp
hin
4_O
H_D
iclo
fenc
4_O
H_T
acrine
5_O
H_D
iclo
fenc
AZ
T
AZ
T_glu
c
Dextr
om
eth
orp
han
Dextr
orp
han
Dic
lofe
nac
Fexofe
nadin
e
M1
M10
M2
M5
M9
tacrine
Terf
enadin
e
Zip
rasid
one
Geo FC
Geo FC 95
Geo FC 99
Average 125
95 confidence 183
99 confidence 207
Precision Accuracy and Confidence Level bull Showed that a peak area ratio of 2 means that it is at least equal or
greater exposure
bull Increased replicates will increase confidence
95 Confidence Interval (~2SD)
Gao H Deng S and Obach RS (2010) DMD 382147-2156
Confidence Level Increases Dramatically with Measured Ratio
Ratio=4 plt2 ppm
Ratio=2 plt001
Measured animalhuman ratio by LCMSMS
Measured animalhuman ratio by LCMSMS
Probability of Nominal AnimalHuman ge1
What Drives High Confidence
bull Fundamental BA parameters are addressed Selectivity sensitivity linearity matrix effect stability precision and accuracy
bull SignalNoise Ratio Measured animalhuman 2
Measurement error (without cancellation) 025
SN (2-1)025=4
Limit of Detection A signal is real if SN=3
bull Minimal 3 independent animalhuman ratio measurements
bull Analysis reproducibility RSD of the ratio measurements (n=3) 33
bull Stability ratio for each metabolite plusmn20 (08-12) over the storage period
How to Evaluate Data Quality
MIST Coverage is a Comparison Question
Animals have exposures of metabolites ge human
Comparison 1 Giraffe vs Moose
Which one is taller
All that one needs to do is to have them stand next to each other The giraffe is much taller No need to know exact heights
Which one is taller
Too close to call just by looking at them -- The height measurements are needed
Comparison 2 Moose vs Whitetail Deer
If the animalhuman peak area ratio is ge 2 animal has exposure ge human with high confidence (plt001)
How often does it happen Most cases as animal is dosed at much higher
dose when corrected by body weight
A simple peak area ratio comparison is sufficient for most cases
Case Study
bull Drug A in project X at phase 1 14 days 35 mg BID in 6 human volunteers vs 5 days 300 mgkg in Sprague Dawley rats
bull Pooled plasma samples (Hamilton) across time points across subjects
bull 3 human metabolites Parent drug A 396gt203
m-hydroxy 412 ring opened 414 de-alkylated 304
bull HPLC method and major daughter ions were transferred from Biotransformation
bull Compare results with concentration measurements using the synthetic standard
MIST Coverage Evaluation Using RatHuman Ratio of Peak Area Ratios
Definitive Concentration Measurements Using Synthetic Standard
Species Parent De-alkylated Ring-opened m-Hydroxy
Human 0220 00406 000788 00210 Rat 770 0502 0292 000762
RatHuman ratio 1 35 124 371 0363
Human repeat 0184 00290 000506 00164
Rat repeat 734 0472 0266 000618 RatHuman
ratio 2 399 163 526 0378
Species m-Hydroxy (ngmL)
Human 743 plusmn 153
Rat 269 plusmn 258
Cavg RatHuman ratio 0362
Human Metabolite gt10 Drug-Related Material from a Human Radiolabel
ADME Study (or Metabolite Anticipated to Be gt10 from Other Human Studies)
Metabolite With Known Activity of
Interest
Use Standard Bioanalytical
Method
ICH Exempt Metabolite
Peak Area Ratio Method
Method Fails Criteria
Method Succeeds
Animal lt 2X Human
Animal ge 2X Human
No Further Work Needed
Coverage is Assured No Further Work
Needed
Prepare Authentic Standard of Metabolite
MIST Decision Tree
Gao H and Obach RS (2014) Bioanalysis 6(5) 641-650
Data Driven Strategy
bull Rigor of the analysis increases based on data Synthetic standard is needed only when the method fails or animal has less exposure than human
bull Conservative evaluation of MIST coverage
bull Assess MIST coverage at steady state without radio-label or synthetic standards enabling early assessment eg FIH
bull Time- and resource-saving strategy
Summary
bull LCMSMS peak area ratio comparison is scientifically sound and defensible method that can
ndash Ascertain the steady-state exposure of animals compared to humans (ie ldquoMIST coveragerdquo)
ndash Assess the MIST coverage without radio-label or synthetic standard
bull These methods are fit-for-purpose and can offer decision-making data useful for reliable estimation of metabolite exposure
bull Discussions with regulatory experts were positive and helpful
Gao et al (2013) AAPS Journal15(4) 970-973
Prerequisite for Current Methodology
bull Animal and human samples for side-by-side injection
minus In drug development first in human samples are typically ~1 year later than the toxicology study is done
bull Prior knowledge of human metabolites transferred from metabolite identification and scouting
minus Knowledge of major human metabolites may evolve only after in vivo human samples become available
minus Radio-labeled ADME is the most definitive study may take place late in the drug development process
Is it feasible to scan the samplesbank data and compare the data generated at different dates
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
MeO
O
O
OH
OH
OH
O OHNaproxen Acyl Glucuronide conjugate
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
(AZT)
Negative Control
Blind Test 3 Metabolite Cocktail
Gao et al Anal Chem 2015 87 11771-11776
Scanning and Banking DataSamples
bull Blind Test samples 22 metabolitesdrugs were spiked in human and rat plasmas (n=4 each)
bull No knowledge of the identities of the analytes and their concentrations in plasma were provided before sample analysis
bull Multiple analysis of the same samples (rat or human) using mixed matrix approach at Days 1 30 105 254 and 314
bull Treat the samples using the same IS solution in acetonitrile minus Freshly prepared to maintain the same concentration
bull Acquity UPLC generic gradient on C18 run time 25 min
bull Full scan and AIF scan on QExactive 70K at positive and negative polarity
bull Compare rat vs human samples analyzed at different dates
0 4 8 12 16 20 24 Time (min)
0
100 R
elat
ive
Ab
un
dan
ce
508
478 682
1132 1435 1450 763
773 243 1835 2120 2138 1164 1312 1638 288 534 1473 2338 802 2045 335 1062
42 43 44 45 46 47 48 49 50 51 52 53 54 55 Time (min)
0
100
Rel
ativ
e In
ten
sity
RT 508
RT 478
RT 495
RT 539
4 8 12 16 20 24 Time (min)
0
100 RT 624
RT 1313
RT 1113
RT 1626
RT 275 RT 879
RT 2008 RT 422
548
165 808
1 2
3
3
4
5
5
4
678
9 10 111213
14 16 15
1
2
3
4
56 7
Rel
ativ
e A
bu
nd
ance
Migrated naproxen acyl glucuronide
EIC 15 drugsmetabolites Resolution 70000 Extraction window 5ppm Polarity Positive
EIC 6 drugsmetabolites Resolution 70000 Extraction window 10 ppm Polarity Negative
2-OH Tacrine
4-OH Tacrine
1-OH Tacrine
Naproxen-Glu
Peak Area Ratio Measurement Variation
n=4 per species 5 independent analysis total n=40 per analyte
95 tile 037
Median 021
M1
0
DrugsMetabolites
Bank and Compare Data at Different Days
Gao et al Anal Chem 2015 87 11771-11776
Separation and Quantitative Analysis of Isomers
2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
100
Rela
tive I
nte
nsity
RT 1626
RT 275
RT 422 RT 1188 RT 632
Full Scan 4rsquo (or 5)-OH Diclofenac
XIC 3100032
Would high resolution ion mobility provide the orthogonal separation we need
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
Parent Metabolite Potential Instability
Naproxen Naproxen Acyl Glucuronide
Acyl bond migration
Ester hydrolysis
Carbamazepine Carbamazepine-1011-Epoxide
Epoxide hydrolysis
Salicylic Acid 23-Dihydroxybenzoic Acid
Catechol oxidation
Sulfadiazine N-Acetylsulfadiazine
Amide hydrolysis
Methylphenidate Ritalinic Acid
Ester hydrolysis
Nicotine Nicotine-Δ1rsquo5rsquo-Iminium Ion and Cotinine
Imine Oxidation
N
O NH2
MeO
O
O
OH
OH
OH
O OH
N
N+
N
NO
N
N
NH
OMeO
NH
OHO
OH
O
OH
OH
NH
SNH
N
N
O
O
O
NH2
SNH
N
N
O
O
OH
O
OH
N
O NH2
O
MeO
OH
O
Blind Test 2 Unstable Metabolites
Long Term Storage Stability of Naproxen Acyl-Glucuronide Conjugate
0
05
1
15
2
0 50 100 150
(Nap
roxe
n-G
luN
apro
xen
) D
ay X
Day
1
Storage Time at -20ordmC (Day)
Naproxpen-GluNaproxen normalized byDay1 response
Stabilized Naproxen-GluNaproxenNormalized by Day 1
Linear (Naproxpen-GluNaproxen normalizedby Day1 response)
Linear (Stabilized Naproxen-GluNaproxenNormalized by Day 1)
n = 8
Untreated samples
A ratio between 08-12 is deemed stable
Stabilized samples
Gao H and Obach RS (2012) DMD 401290-1296
Correlation of Experimental MS Ratio vs Nominal Ratio
R 0994plusmn0005 Slope 099plusmn0082
MSNM Ratio
Metabolites
MS
NM
Ra
tio
10
15
20
1_O
H_T
acrine
2_O
H_T
acrine
3_M
eth
oxym
orp
hin
4_O
H_D
iclo
fenc
4_O
H_T
acrine
5_O
H_D
iclo
fenc
AZ
T
AZ
T_glu
c
Dextr
om
eth
orp
han
Dextr
orp
han
Dic
lofe
nac
Fexofe
nadin
e
M1
M10
M2
M5
M9
tacrine
Terf
enadin
e
Zip
rasid
one
Geo FC
Geo FC 95
Geo FC 99
Average 125
95 confidence 183
99 confidence 207
Precision Accuracy and Confidence Level bull Showed that a peak area ratio of 2 means that it is at least equal or
greater exposure
bull Increased replicates will increase confidence
95 Confidence Interval (~2SD)
Gao H Deng S and Obach RS (2010) DMD 382147-2156
Confidence Level Increases Dramatically with Measured Ratio
Ratio=4 plt2 ppm
Ratio=2 plt001
Measured animalhuman ratio by LCMSMS
Measured animalhuman ratio by LCMSMS
Probability of Nominal AnimalHuman ge1
What Drives High Confidence
bull Fundamental BA parameters are addressed Selectivity sensitivity linearity matrix effect stability precision and accuracy
bull SignalNoise Ratio Measured animalhuman 2
Measurement error (without cancellation) 025
SN (2-1)025=4
Limit of Detection A signal is real if SN=3
bull Minimal 3 independent animalhuman ratio measurements
bull Analysis reproducibility RSD of the ratio measurements (n=3) 33
bull Stability ratio for each metabolite plusmn20 (08-12) over the storage period
How to Evaluate Data Quality
MIST Coverage is a Comparison Question
Animals have exposures of metabolites ge human
Comparison 1 Giraffe vs Moose
Which one is taller
All that one needs to do is to have them stand next to each other The giraffe is much taller No need to know exact heights
Which one is taller
Too close to call just by looking at them -- The height measurements are needed
Comparison 2 Moose vs Whitetail Deer
If the animalhuman peak area ratio is ge 2 animal has exposure ge human with high confidence (plt001)
How often does it happen Most cases as animal is dosed at much higher
dose when corrected by body weight
A simple peak area ratio comparison is sufficient for most cases
Case Study
bull Drug A in project X at phase 1 14 days 35 mg BID in 6 human volunteers vs 5 days 300 mgkg in Sprague Dawley rats
bull Pooled plasma samples (Hamilton) across time points across subjects
bull 3 human metabolites Parent drug A 396gt203
m-hydroxy 412 ring opened 414 de-alkylated 304
bull HPLC method and major daughter ions were transferred from Biotransformation
bull Compare results with concentration measurements using the synthetic standard
MIST Coverage Evaluation Using RatHuman Ratio of Peak Area Ratios
Definitive Concentration Measurements Using Synthetic Standard
Species Parent De-alkylated Ring-opened m-Hydroxy
Human 0220 00406 000788 00210 Rat 770 0502 0292 000762
RatHuman ratio 1 35 124 371 0363
Human repeat 0184 00290 000506 00164
Rat repeat 734 0472 0266 000618 RatHuman
ratio 2 399 163 526 0378
Species m-Hydroxy (ngmL)
Human 743 plusmn 153
Rat 269 plusmn 258
Cavg RatHuman ratio 0362
Human Metabolite gt10 Drug-Related Material from a Human Radiolabel
ADME Study (or Metabolite Anticipated to Be gt10 from Other Human Studies)
Metabolite With Known Activity of
Interest
Use Standard Bioanalytical
Method
ICH Exempt Metabolite
Peak Area Ratio Method
Method Fails Criteria
Method Succeeds
Animal lt 2X Human
Animal ge 2X Human
No Further Work Needed
Coverage is Assured No Further Work
Needed
Prepare Authentic Standard of Metabolite
MIST Decision Tree
Gao H and Obach RS (2014) Bioanalysis 6(5) 641-650
Data Driven Strategy
bull Rigor of the analysis increases based on data Synthetic standard is needed only when the method fails or animal has less exposure than human
bull Conservative evaluation of MIST coverage
bull Assess MIST coverage at steady state without radio-label or synthetic standards enabling early assessment eg FIH
bull Time- and resource-saving strategy
Summary
bull LCMSMS peak area ratio comparison is scientifically sound and defensible method that can
ndash Ascertain the steady-state exposure of animals compared to humans (ie ldquoMIST coveragerdquo)
ndash Assess the MIST coverage without radio-label or synthetic standard
bull These methods are fit-for-purpose and can offer decision-making data useful for reliable estimation of metabolite exposure
bull Discussions with regulatory experts were positive and helpful
Gao et al (2013) AAPS Journal15(4) 970-973
Prerequisite for Current Methodology
bull Animal and human samples for side-by-side injection
minus In drug development first in human samples are typically ~1 year later than the toxicology study is done
bull Prior knowledge of human metabolites transferred from metabolite identification and scouting
minus Knowledge of major human metabolites may evolve only after in vivo human samples become available
minus Radio-labeled ADME is the most definitive study may take place late in the drug development process
Is it feasible to scan the samplesbank data and compare the data generated at different dates
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
MeO
O
O
OH
OH
OH
O OHNaproxen Acyl Glucuronide conjugate
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
(AZT)
Negative Control
Blind Test 3 Metabolite Cocktail
Gao et al Anal Chem 2015 87 11771-11776
Scanning and Banking DataSamples
bull Blind Test samples 22 metabolitesdrugs were spiked in human and rat plasmas (n=4 each)
bull No knowledge of the identities of the analytes and their concentrations in plasma were provided before sample analysis
bull Multiple analysis of the same samples (rat or human) using mixed matrix approach at Days 1 30 105 254 and 314
bull Treat the samples using the same IS solution in acetonitrile minus Freshly prepared to maintain the same concentration
bull Acquity UPLC generic gradient on C18 run time 25 min
bull Full scan and AIF scan on QExactive 70K at positive and negative polarity
bull Compare rat vs human samples analyzed at different dates
0 4 8 12 16 20 24 Time (min)
0
100 R
elat
ive
Ab
un
dan
ce
508
478 682
1132 1435 1450 763
773 243 1835 2120 2138 1164 1312 1638 288 534 1473 2338 802 2045 335 1062
42 43 44 45 46 47 48 49 50 51 52 53 54 55 Time (min)
0
100
Rel
ativ
e In
ten
sity
RT 508
RT 478
RT 495
RT 539
4 8 12 16 20 24 Time (min)
0
100 RT 624
RT 1313
RT 1113
RT 1626
RT 275 RT 879
RT 2008 RT 422
548
165 808
1 2
3
3
4
5
5
4
678
9 10 111213
14 16 15
1
2
3
4
56 7
Rel
ativ
e A
bu
nd
ance
Migrated naproxen acyl glucuronide
EIC 15 drugsmetabolites Resolution 70000 Extraction window 5ppm Polarity Positive
EIC 6 drugsmetabolites Resolution 70000 Extraction window 10 ppm Polarity Negative
2-OH Tacrine
4-OH Tacrine
1-OH Tacrine
Naproxen-Glu
Peak Area Ratio Measurement Variation
n=4 per species 5 independent analysis total n=40 per analyte
95 tile 037
Median 021
M1
0
DrugsMetabolites
Bank and Compare Data at Different Days
Gao et al Anal Chem 2015 87 11771-11776
Separation and Quantitative Analysis of Isomers
2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
100
Rela
tive I
nte
nsity
RT 1626
RT 275
RT 422 RT 1188 RT 632
Full Scan 4rsquo (or 5)-OH Diclofenac
XIC 3100032
Would high resolution ion mobility provide the orthogonal separation we need
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
Long Term Storage Stability of Naproxen Acyl-Glucuronide Conjugate
0
05
1
15
2
0 50 100 150
(Nap
roxe
n-G
luN
apro
xen
) D
ay X
Day
1
Storage Time at -20ordmC (Day)
Naproxpen-GluNaproxen normalized byDay1 response
Stabilized Naproxen-GluNaproxenNormalized by Day 1
Linear (Naproxpen-GluNaproxen normalizedby Day1 response)
Linear (Stabilized Naproxen-GluNaproxenNormalized by Day 1)
n = 8
Untreated samples
A ratio between 08-12 is deemed stable
Stabilized samples
Gao H and Obach RS (2012) DMD 401290-1296
Correlation of Experimental MS Ratio vs Nominal Ratio
R 0994plusmn0005 Slope 099plusmn0082
MSNM Ratio
Metabolites
MS
NM
Ra
tio
10
15
20
1_O
H_T
acrine
2_O
H_T
acrine
3_M
eth
oxym
orp
hin
4_O
H_D
iclo
fenc
4_O
H_T
acrine
5_O
H_D
iclo
fenc
AZ
T
AZ
T_glu
c
Dextr
om
eth
orp
han
Dextr
orp
han
Dic
lofe
nac
Fexofe
nadin
e
M1
M10
M2
M5
M9
tacrine
Terf
enadin
e
Zip
rasid
one
Geo FC
Geo FC 95
Geo FC 99
Average 125
95 confidence 183
99 confidence 207
Precision Accuracy and Confidence Level bull Showed that a peak area ratio of 2 means that it is at least equal or
greater exposure
bull Increased replicates will increase confidence
95 Confidence Interval (~2SD)
Gao H Deng S and Obach RS (2010) DMD 382147-2156
Confidence Level Increases Dramatically with Measured Ratio
Ratio=4 plt2 ppm
Ratio=2 plt001
Measured animalhuman ratio by LCMSMS
Measured animalhuman ratio by LCMSMS
Probability of Nominal AnimalHuman ge1
What Drives High Confidence
bull Fundamental BA parameters are addressed Selectivity sensitivity linearity matrix effect stability precision and accuracy
bull SignalNoise Ratio Measured animalhuman 2
Measurement error (without cancellation) 025
SN (2-1)025=4
Limit of Detection A signal is real if SN=3
bull Minimal 3 independent animalhuman ratio measurements
bull Analysis reproducibility RSD of the ratio measurements (n=3) 33
bull Stability ratio for each metabolite plusmn20 (08-12) over the storage period
How to Evaluate Data Quality
MIST Coverage is a Comparison Question
Animals have exposures of metabolites ge human
Comparison 1 Giraffe vs Moose
Which one is taller
All that one needs to do is to have them stand next to each other The giraffe is much taller No need to know exact heights
Which one is taller
Too close to call just by looking at them -- The height measurements are needed
Comparison 2 Moose vs Whitetail Deer
If the animalhuman peak area ratio is ge 2 animal has exposure ge human with high confidence (plt001)
How often does it happen Most cases as animal is dosed at much higher
dose when corrected by body weight
A simple peak area ratio comparison is sufficient for most cases
Case Study
bull Drug A in project X at phase 1 14 days 35 mg BID in 6 human volunteers vs 5 days 300 mgkg in Sprague Dawley rats
bull Pooled plasma samples (Hamilton) across time points across subjects
bull 3 human metabolites Parent drug A 396gt203
m-hydroxy 412 ring opened 414 de-alkylated 304
bull HPLC method and major daughter ions were transferred from Biotransformation
bull Compare results with concentration measurements using the synthetic standard
MIST Coverage Evaluation Using RatHuman Ratio of Peak Area Ratios
Definitive Concentration Measurements Using Synthetic Standard
Species Parent De-alkylated Ring-opened m-Hydroxy
Human 0220 00406 000788 00210 Rat 770 0502 0292 000762
RatHuman ratio 1 35 124 371 0363
Human repeat 0184 00290 000506 00164
Rat repeat 734 0472 0266 000618 RatHuman
ratio 2 399 163 526 0378
Species m-Hydroxy (ngmL)
Human 743 plusmn 153
Rat 269 plusmn 258
Cavg RatHuman ratio 0362
Human Metabolite gt10 Drug-Related Material from a Human Radiolabel
ADME Study (or Metabolite Anticipated to Be gt10 from Other Human Studies)
Metabolite With Known Activity of
Interest
Use Standard Bioanalytical
Method
ICH Exempt Metabolite
Peak Area Ratio Method
Method Fails Criteria
Method Succeeds
Animal lt 2X Human
Animal ge 2X Human
No Further Work Needed
Coverage is Assured No Further Work
Needed
Prepare Authentic Standard of Metabolite
MIST Decision Tree
Gao H and Obach RS (2014) Bioanalysis 6(5) 641-650
Data Driven Strategy
bull Rigor of the analysis increases based on data Synthetic standard is needed only when the method fails or animal has less exposure than human
bull Conservative evaluation of MIST coverage
bull Assess MIST coverage at steady state without radio-label or synthetic standards enabling early assessment eg FIH
bull Time- and resource-saving strategy
Summary
bull LCMSMS peak area ratio comparison is scientifically sound and defensible method that can
ndash Ascertain the steady-state exposure of animals compared to humans (ie ldquoMIST coveragerdquo)
ndash Assess the MIST coverage without radio-label or synthetic standard
bull These methods are fit-for-purpose and can offer decision-making data useful for reliable estimation of metabolite exposure
bull Discussions with regulatory experts were positive and helpful
Gao et al (2013) AAPS Journal15(4) 970-973
Prerequisite for Current Methodology
bull Animal and human samples for side-by-side injection
minus In drug development first in human samples are typically ~1 year later than the toxicology study is done
bull Prior knowledge of human metabolites transferred from metabolite identification and scouting
minus Knowledge of major human metabolites may evolve only after in vivo human samples become available
minus Radio-labeled ADME is the most definitive study may take place late in the drug development process
Is it feasible to scan the samplesbank data and compare the data generated at different dates
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
MeO
O
O
OH
OH
OH
O OHNaproxen Acyl Glucuronide conjugate
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
(AZT)
Negative Control
Blind Test 3 Metabolite Cocktail
Gao et al Anal Chem 2015 87 11771-11776
Scanning and Banking DataSamples
bull Blind Test samples 22 metabolitesdrugs were spiked in human and rat plasmas (n=4 each)
bull No knowledge of the identities of the analytes and their concentrations in plasma were provided before sample analysis
bull Multiple analysis of the same samples (rat or human) using mixed matrix approach at Days 1 30 105 254 and 314
bull Treat the samples using the same IS solution in acetonitrile minus Freshly prepared to maintain the same concentration
bull Acquity UPLC generic gradient on C18 run time 25 min
bull Full scan and AIF scan on QExactive 70K at positive and negative polarity
bull Compare rat vs human samples analyzed at different dates
0 4 8 12 16 20 24 Time (min)
0
100 R
elat
ive
Ab
un
dan
ce
508
478 682
1132 1435 1450 763
773 243 1835 2120 2138 1164 1312 1638 288 534 1473 2338 802 2045 335 1062
42 43 44 45 46 47 48 49 50 51 52 53 54 55 Time (min)
0
100
Rel
ativ
e In
ten
sity
RT 508
RT 478
RT 495
RT 539
4 8 12 16 20 24 Time (min)
0
100 RT 624
RT 1313
RT 1113
RT 1626
RT 275 RT 879
RT 2008 RT 422
548
165 808
1 2
3
3
4
5
5
4
678
9 10 111213
14 16 15
1
2
3
4
56 7
Rel
ativ
e A
bu
nd
ance
Migrated naproxen acyl glucuronide
EIC 15 drugsmetabolites Resolution 70000 Extraction window 5ppm Polarity Positive
EIC 6 drugsmetabolites Resolution 70000 Extraction window 10 ppm Polarity Negative
2-OH Tacrine
4-OH Tacrine
1-OH Tacrine
Naproxen-Glu
Peak Area Ratio Measurement Variation
n=4 per species 5 independent analysis total n=40 per analyte
95 tile 037
Median 021
M1
0
DrugsMetabolites
Bank and Compare Data at Different Days
Gao et al Anal Chem 2015 87 11771-11776
Separation and Quantitative Analysis of Isomers
2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
100
Rela
tive I
nte
nsity
RT 1626
RT 275
RT 422 RT 1188 RT 632
Full Scan 4rsquo (or 5)-OH Diclofenac
XIC 3100032
Would high resolution ion mobility provide the orthogonal separation we need
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
Correlation of Experimental MS Ratio vs Nominal Ratio
R 0994plusmn0005 Slope 099plusmn0082
MSNM Ratio
Metabolites
MS
NM
Ra
tio
10
15
20
1_O
H_T
acrine
2_O
H_T
acrine
3_M
eth
oxym
orp
hin
4_O
H_D
iclo
fenc
4_O
H_T
acrine
5_O
H_D
iclo
fenc
AZ
T
AZ
T_glu
c
Dextr
om
eth
orp
han
Dextr
orp
han
Dic
lofe
nac
Fexofe
nadin
e
M1
M10
M2
M5
M9
tacrine
Terf
enadin
e
Zip
rasid
one
Geo FC
Geo FC 95
Geo FC 99
Average 125
95 confidence 183
99 confidence 207
Precision Accuracy and Confidence Level bull Showed that a peak area ratio of 2 means that it is at least equal or
greater exposure
bull Increased replicates will increase confidence
95 Confidence Interval (~2SD)
Gao H Deng S and Obach RS (2010) DMD 382147-2156
Confidence Level Increases Dramatically with Measured Ratio
Ratio=4 plt2 ppm
Ratio=2 plt001
Measured animalhuman ratio by LCMSMS
Measured animalhuman ratio by LCMSMS
Probability of Nominal AnimalHuman ge1
What Drives High Confidence
bull Fundamental BA parameters are addressed Selectivity sensitivity linearity matrix effect stability precision and accuracy
bull SignalNoise Ratio Measured animalhuman 2
Measurement error (without cancellation) 025
SN (2-1)025=4
Limit of Detection A signal is real if SN=3
bull Minimal 3 independent animalhuman ratio measurements
bull Analysis reproducibility RSD of the ratio measurements (n=3) 33
bull Stability ratio for each metabolite plusmn20 (08-12) over the storage period
How to Evaluate Data Quality
MIST Coverage is a Comparison Question
Animals have exposures of metabolites ge human
Comparison 1 Giraffe vs Moose
Which one is taller
All that one needs to do is to have them stand next to each other The giraffe is much taller No need to know exact heights
Which one is taller
Too close to call just by looking at them -- The height measurements are needed
Comparison 2 Moose vs Whitetail Deer
If the animalhuman peak area ratio is ge 2 animal has exposure ge human with high confidence (plt001)
How often does it happen Most cases as animal is dosed at much higher
dose when corrected by body weight
A simple peak area ratio comparison is sufficient for most cases
Case Study
bull Drug A in project X at phase 1 14 days 35 mg BID in 6 human volunteers vs 5 days 300 mgkg in Sprague Dawley rats
bull Pooled plasma samples (Hamilton) across time points across subjects
bull 3 human metabolites Parent drug A 396gt203
m-hydroxy 412 ring opened 414 de-alkylated 304
bull HPLC method and major daughter ions were transferred from Biotransformation
bull Compare results with concentration measurements using the synthetic standard
MIST Coverage Evaluation Using RatHuman Ratio of Peak Area Ratios
Definitive Concentration Measurements Using Synthetic Standard
Species Parent De-alkylated Ring-opened m-Hydroxy
Human 0220 00406 000788 00210 Rat 770 0502 0292 000762
RatHuman ratio 1 35 124 371 0363
Human repeat 0184 00290 000506 00164
Rat repeat 734 0472 0266 000618 RatHuman
ratio 2 399 163 526 0378
Species m-Hydroxy (ngmL)
Human 743 plusmn 153
Rat 269 plusmn 258
Cavg RatHuman ratio 0362
Human Metabolite gt10 Drug-Related Material from a Human Radiolabel
ADME Study (or Metabolite Anticipated to Be gt10 from Other Human Studies)
Metabolite With Known Activity of
Interest
Use Standard Bioanalytical
Method
ICH Exempt Metabolite
Peak Area Ratio Method
Method Fails Criteria
Method Succeeds
Animal lt 2X Human
Animal ge 2X Human
No Further Work Needed
Coverage is Assured No Further Work
Needed
Prepare Authentic Standard of Metabolite
MIST Decision Tree
Gao H and Obach RS (2014) Bioanalysis 6(5) 641-650
Data Driven Strategy
bull Rigor of the analysis increases based on data Synthetic standard is needed only when the method fails or animal has less exposure than human
bull Conservative evaluation of MIST coverage
bull Assess MIST coverage at steady state without radio-label or synthetic standards enabling early assessment eg FIH
bull Time- and resource-saving strategy
Summary
bull LCMSMS peak area ratio comparison is scientifically sound and defensible method that can
ndash Ascertain the steady-state exposure of animals compared to humans (ie ldquoMIST coveragerdquo)
ndash Assess the MIST coverage without radio-label or synthetic standard
bull These methods are fit-for-purpose and can offer decision-making data useful for reliable estimation of metabolite exposure
bull Discussions with regulatory experts were positive and helpful
Gao et al (2013) AAPS Journal15(4) 970-973
Prerequisite for Current Methodology
bull Animal and human samples for side-by-side injection
minus In drug development first in human samples are typically ~1 year later than the toxicology study is done
bull Prior knowledge of human metabolites transferred from metabolite identification and scouting
minus Knowledge of major human metabolites may evolve only after in vivo human samples become available
minus Radio-labeled ADME is the most definitive study may take place late in the drug development process
Is it feasible to scan the samplesbank data and compare the data generated at different dates
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
MeO
O
O
OH
OH
OH
O OHNaproxen Acyl Glucuronide conjugate
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
(AZT)
Negative Control
Blind Test 3 Metabolite Cocktail
Gao et al Anal Chem 2015 87 11771-11776
Scanning and Banking DataSamples
bull Blind Test samples 22 metabolitesdrugs were spiked in human and rat plasmas (n=4 each)
bull No knowledge of the identities of the analytes and their concentrations in plasma were provided before sample analysis
bull Multiple analysis of the same samples (rat or human) using mixed matrix approach at Days 1 30 105 254 and 314
bull Treat the samples using the same IS solution in acetonitrile minus Freshly prepared to maintain the same concentration
bull Acquity UPLC generic gradient on C18 run time 25 min
bull Full scan and AIF scan on QExactive 70K at positive and negative polarity
bull Compare rat vs human samples analyzed at different dates
0 4 8 12 16 20 24 Time (min)
0
100 R
elat
ive
Ab
un
dan
ce
508
478 682
1132 1435 1450 763
773 243 1835 2120 2138 1164 1312 1638 288 534 1473 2338 802 2045 335 1062
42 43 44 45 46 47 48 49 50 51 52 53 54 55 Time (min)
0
100
Rel
ativ
e In
ten
sity
RT 508
RT 478
RT 495
RT 539
4 8 12 16 20 24 Time (min)
0
100 RT 624
RT 1313
RT 1113
RT 1626
RT 275 RT 879
RT 2008 RT 422
548
165 808
1 2
3
3
4
5
5
4
678
9 10 111213
14 16 15
1
2
3
4
56 7
Rel
ativ
e A
bu
nd
ance
Migrated naproxen acyl glucuronide
EIC 15 drugsmetabolites Resolution 70000 Extraction window 5ppm Polarity Positive
EIC 6 drugsmetabolites Resolution 70000 Extraction window 10 ppm Polarity Negative
2-OH Tacrine
4-OH Tacrine
1-OH Tacrine
Naproxen-Glu
Peak Area Ratio Measurement Variation
n=4 per species 5 independent analysis total n=40 per analyte
95 tile 037
Median 021
M1
0
DrugsMetabolites
Bank and Compare Data at Different Days
Gao et al Anal Chem 2015 87 11771-11776
Separation and Quantitative Analysis of Isomers
2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
100
Rela
tive I
nte
nsity
RT 1626
RT 275
RT 422 RT 1188 RT 632
Full Scan 4rsquo (or 5)-OH Diclofenac
XIC 3100032
Would high resolution ion mobility provide the orthogonal separation we need
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
MSNM Ratio
Metabolites
MS
NM
Ra
tio
10
15
20
1_O
H_T
acrine
2_O
H_T
acrine
3_M
eth
oxym
orp
hin
4_O
H_D
iclo
fenc
4_O
H_T
acrine
5_O
H_D
iclo
fenc
AZ
T
AZ
T_glu
c
Dextr
om
eth
orp
han
Dextr
orp
han
Dic
lofe
nac
Fexofe
nadin
e
M1
M10
M2
M5
M9
tacrine
Terf
enadin
e
Zip
rasid
one
Geo FC
Geo FC 95
Geo FC 99
Average 125
95 confidence 183
99 confidence 207
Precision Accuracy and Confidence Level bull Showed that a peak area ratio of 2 means that it is at least equal or
greater exposure
bull Increased replicates will increase confidence
95 Confidence Interval (~2SD)
Gao H Deng S and Obach RS (2010) DMD 382147-2156
Confidence Level Increases Dramatically with Measured Ratio
Ratio=4 plt2 ppm
Ratio=2 plt001
Measured animalhuman ratio by LCMSMS
Measured animalhuman ratio by LCMSMS
Probability of Nominal AnimalHuman ge1
What Drives High Confidence
bull Fundamental BA parameters are addressed Selectivity sensitivity linearity matrix effect stability precision and accuracy
bull SignalNoise Ratio Measured animalhuman 2
Measurement error (without cancellation) 025
SN (2-1)025=4
Limit of Detection A signal is real if SN=3
bull Minimal 3 independent animalhuman ratio measurements
bull Analysis reproducibility RSD of the ratio measurements (n=3) 33
bull Stability ratio for each metabolite plusmn20 (08-12) over the storage period
How to Evaluate Data Quality
MIST Coverage is a Comparison Question
Animals have exposures of metabolites ge human
Comparison 1 Giraffe vs Moose
Which one is taller
All that one needs to do is to have them stand next to each other The giraffe is much taller No need to know exact heights
Which one is taller
Too close to call just by looking at them -- The height measurements are needed
Comparison 2 Moose vs Whitetail Deer
If the animalhuman peak area ratio is ge 2 animal has exposure ge human with high confidence (plt001)
How often does it happen Most cases as animal is dosed at much higher
dose when corrected by body weight
A simple peak area ratio comparison is sufficient for most cases
Case Study
bull Drug A in project X at phase 1 14 days 35 mg BID in 6 human volunteers vs 5 days 300 mgkg in Sprague Dawley rats
bull Pooled plasma samples (Hamilton) across time points across subjects
bull 3 human metabolites Parent drug A 396gt203
m-hydroxy 412 ring opened 414 de-alkylated 304
bull HPLC method and major daughter ions were transferred from Biotransformation
bull Compare results with concentration measurements using the synthetic standard
MIST Coverage Evaluation Using RatHuman Ratio of Peak Area Ratios
Definitive Concentration Measurements Using Synthetic Standard
Species Parent De-alkylated Ring-opened m-Hydroxy
Human 0220 00406 000788 00210 Rat 770 0502 0292 000762
RatHuman ratio 1 35 124 371 0363
Human repeat 0184 00290 000506 00164
Rat repeat 734 0472 0266 000618 RatHuman
ratio 2 399 163 526 0378
Species m-Hydroxy (ngmL)
Human 743 plusmn 153
Rat 269 plusmn 258
Cavg RatHuman ratio 0362
Human Metabolite gt10 Drug-Related Material from a Human Radiolabel
ADME Study (or Metabolite Anticipated to Be gt10 from Other Human Studies)
Metabolite With Known Activity of
Interest
Use Standard Bioanalytical
Method
ICH Exempt Metabolite
Peak Area Ratio Method
Method Fails Criteria
Method Succeeds
Animal lt 2X Human
Animal ge 2X Human
No Further Work Needed
Coverage is Assured No Further Work
Needed
Prepare Authentic Standard of Metabolite
MIST Decision Tree
Gao H and Obach RS (2014) Bioanalysis 6(5) 641-650
Data Driven Strategy
bull Rigor of the analysis increases based on data Synthetic standard is needed only when the method fails or animal has less exposure than human
bull Conservative evaluation of MIST coverage
bull Assess MIST coverage at steady state without radio-label or synthetic standards enabling early assessment eg FIH
bull Time- and resource-saving strategy
Summary
bull LCMSMS peak area ratio comparison is scientifically sound and defensible method that can
ndash Ascertain the steady-state exposure of animals compared to humans (ie ldquoMIST coveragerdquo)
ndash Assess the MIST coverage without radio-label or synthetic standard
bull These methods are fit-for-purpose and can offer decision-making data useful for reliable estimation of metabolite exposure
bull Discussions with regulatory experts were positive and helpful
Gao et al (2013) AAPS Journal15(4) 970-973
Prerequisite for Current Methodology
bull Animal and human samples for side-by-side injection
minus In drug development first in human samples are typically ~1 year later than the toxicology study is done
bull Prior knowledge of human metabolites transferred from metabolite identification and scouting
minus Knowledge of major human metabolites may evolve only after in vivo human samples become available
minus Radio-labeled ADME is the most definitive study may take place late in the drug development process
Is it feasible to scan the samplesbank data and compare the data generated at different dates
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
MeO
O
O
OH
OH
OH
O OHNaproxen Acyl Glucuronide conjugate
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
(AZT)
Negative Control
Blind Test 3 Metabolite Cocktail
Gao et al Anal Chem 2015 87 11771-11776
Scanning and Banking DataSamples
bull Blind Test samples 22 metabolitesdrugs were spiked in human and rat plasmas (n=4 each)
bull No knowledge of the identities of the analytes and their concentrations in plasma were provided before sample analysis
bull Multiple analysis of the same samples (rat or human) using mixed matrix approach at Days 1 30 105 254 and 314
bull Treat the samples using the same IS solution in acetonitrile minus Freshly prepared to maintain the same concentration
bull Acquity UPLC generic gradient on C18 run time 25 min
bull Full scan and AIF scan on QExactive 70K at positive and negative polarity
bull Compare rat vs human samples analyzed at different dates
0 4 8 12 16 20 24 Time (min)
0
100 R
elat
ive
Ab
un
dan
ce
508
478 682
1132 1435 1450 763
773 243 1835 2120 2138 1164 1312 1638 288 534 1473 2338 802 2045 335 1062
42 43 44 45 46 47 48 49 50 51 52 53 54 55 Time (min)
0
100
Rel
ativ
e In
ten
sity
RT 508
RT 478
RT 495
RT 539
4 8 12 16 20 24 Time (min)
0
100 RT 624
RT 1313
RT 1113
RT 1626
RT 275 RT 879
RT 2008 RT 422
548
165 808
1 2
3
3
4
5
5
4
678
9 10 111213
14 16 15
1
2
3
4
56 7
Rel
ativ
e A
bu
nd
ance
Migrated naproxen acyl glucuronide
EIC 15 drugsmetabolites Resolution 70000 Extraction window 5ppm Polarity Positive
EIC 6 drugsmetabolites Resolution 70000 Extraction window 10 ppm Polarity Negative
2-OH Tacrine
4-OH Tacrine
1-OH Tacrine
Naproxen-Glu
Peak Area Ratio Measurement Variation
n=4 per species 5 independent analysis total n=40 per analyte
95 tile 037
Median 021
M1
0
DrugsMetabolites
Bank and Compare Data at Different Days
Gao et al Anal Chem 2015 87 11771-11776
Separation and Quantitative Analysis of Isomers
2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
100
Rela
tive I
nte
nsity
RT 1626
RT 275
RT 422 RT 1188 RT 632
Full Scan 4rsquo (or 5)-OH Diclofenac
XIC 3100032
Would high resolution ion mobility provide the orthogonal separation we need
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
Confidence Level Increases Dramatically with Measured Ratio
Ratio=4 plt2 ppm
Ratio=2 plt001
Measured animalhuman ratio by LCMSMS
Measured animalhuman ratio by LCMSMS
Probability of Nominal AnimalHuman ge1
What Drives High Confidence
bull Fundamental BA parameters are addressed Selectivity sensitivity linearity matrix effect stability precision and accuracy
bull SignalNoise Ratio Measured animalhuman 2
Measurement error (without cancellation) 025
SN (2-1)025=4
Limit of Detection A signal is real if SN=3
bull Minimal 3 independent animalhuman ratio measurements
bull Analysis reproducibility RSD of the ratio measurements (n=3) 33
bull Stability ratio for each metabolite plusmn20 (08-12) over the storage period
How to Evaluate Data Quality
MIST Coverage is a Comparison Question
Animals have exposures of metabolites ge human
Comparison 1 Giraffe vs Moose
Which one is taller
All that one needs to do is to have them stand next to each other The giraffe is much taller No need to know exact heights
Which one is taller
Too close to call just by looking at them -- The height measurements are needed
Comparison 2 Moose vs Whitetail Deer
If the animalhuman peak area ratio is ge 2 animal has exposure ge human with high confidence (plt001)
How often does it happen Most cases as animal is dosed at much higher
dose when corrected by body weight
A simple peak area ratio comparison is sufficient for most cases
Case Study
bull Drug A in project X at phase 1 14 days 35 mg BID in 6 human volunteers vs 5 days 300 mgkg in Sprague Dawley rats
bull Pooled plasma samples (Hamilton) across time points across subjects
bull 3 human metabolites Parent drug A 396gt203
m-hydroxy 412 ring opened 414 de-alkylated 304
bull HPLC method and major daughter ions were transferred from Biotransformation
bull Compare results with concentration measurements using the synthetic standard
MIST Coverage Evaluation Using RatHuman Ratio of Peak Area Ratios
Definitive Concentration Measurements Using Synthetic Standard
Species Parent De-alkylated Ring-opened m-Hydroxy
Human 0220 00406 000788 00210 Rat 770 0502 0292 000762
RatHuman ratio 1 35 124 371 0363
Human repeat 0184 00290 000506 00164
Rat repeat 734 0472 0266 000618 RatHuman
ratio 2 399 163 526 0378
Species m-Hydroxy (ngmL)
Human 743 plusmn 153
Rat 269 plusmn 258
Cavg RatHuman ratio 0362
Human Metabolite gt10 Drug-Related Material from a Human Radiolabel
ADME Study (or Metabolite Anticipated to Be gt10 from Other Human Studies)
Metabolite With Known Activity of
Interest
Use Standard Bioanalytical
Method
ICH Exempt Metabolite
Peak Area Ratio Method
Method Fails Criteria
Method Succeeds
Animal lt 2X Human
Animal ge 2X Human
No Further Work Needed
Coverage is Assured No Further Work
Needed
Prepare Authentic Standard of Metabolite
MIST Decision Tree
Gao H and Obach RS (2014) Bioanalysis 6(5) 641-650
Data Driven Strategy
bull Rigor of the analysis increases based on data Synthetic standard is needed only when the method fails or animal has less exposure than human
bull Conservative evaluation of MIST coverage
bull Assess MIST coverage at steady state without radio-label or synthetic standards enabling early assessment eg FIH
bull Time- and resource-saving strategy
Summary
bull LCMSMS peak area ratio comparison is scientifically sound and defensible method that can
ndash Ascertain the steady-state exposure of animals compared to humans (ie ldquoMIST coveragerdquo)
ndash Assess the MIST coverage without radio-label or synthetic standard
bull These methods are fit-for-purpose and can offer decision-making data useful for reliable estimation of metabolite exposure
bull Discussions with regulatory experts were positive and helpful
Gao et al (2013) AAPS Journal15(4) 970-973
Prerequisite for Current Methodology
bull Animal and human samples for side-by-side injection
minus In drug development first in human samples are typically ~1 year later than the toxicology study is done
bull Prior knowledge of human metabolites transferred from metabolite identification and scouting
minus Knowledge of major human metabolites may evolve only after in vivo human samples become available
minus Radio-labeled ADME is the most definitive study may take place late in the drug development process
Is it feasible to scan the samplesbank data and compare the data generated at different dates
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
MeO
O
O
OH
OH
OH
O OHNaproxen Acyl Glucuronide conjugate
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
(AZT)
Negative Control
Blind Test 3 Metabolite Cocktail
Gao et al Anal Chem 2015 87 11771-11776
Scanning and Banking DataSamples
bull Blind Test samples 22 metabolitesdrugs were spiked in human and rat plasmas (n=4 each)
bull No knowledge of the identities of the analytes and their concentrations in plasma were provided before sample analysis
bull Multiple analysis of the same samples (rat or human) using mixed matrix approach at Days 1 30 105 254 and 314
bull Treat the samples using the same IS solution in acetonitrile minus Freshly prepared to maintain the same concentration
bull Acquity UPLC generic gradient on C18 run time 25 min
bull Full scan and AIF scan on QExactive 70K at positive and negative polarity
bull Compare rat vs human samples analyzed at different dates
0 4 8 12 16 20 24 Time (min)
0
100 R
elat
ive
Ab
un
dan
ce
508
478 682
1132 1435 1450 763
773 243 1835 2120 2138 1164 1312 1638 288 534 1473 2338 802 2045 335 1062
42 43 44 45 46 47 48 49 50 51 52 53 54 55 Time (min)
0
100
Rel
ativ
e In
ten
sity
RT 508
RT 478
RT 495
RT 539
4 8 12 16 20 24 Time (min)
0
100 RT 624
RT 1313
RT 1113
RT 1626
RT 275 RT 879
RT 2008 RT 422
548
165 808
1 2
3
3
4
5
5
4
678
9 10 111213
14 16 15
1
2
3
4
56 7
Rel
ativ
e A
bu
nd
ance
Migrated naproxen acyl glucuronide
EIC 15 drugsmetabolites Resolution 70000 Extraction window 5ppm Polarity Positive
EIC 6 drugsmetabolites Resolution 70000 Extraction window 10 ppm Polarity Negative
2-OH Tacrine
4-OH Tacrine
1-OH Tacrine
Naproxen-Glu
Peak Area Ratio Measurement Variation
n=4 per species 5 independent analysis total n=40 per analyte
95 tile 037
Median 021
M1
0
DrugsMetabolites
Bank and Compare Data at Different Days
Gao et al Anal Chem 2015 87 11771-11776
Separation and Quantitative Analysis of Isomers
2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
100
Rela
tive I
nte
nsity
RT 1626
RT 275
RT 422 RT 1188 RT 632
Full Scan 4rsquo (or 5)-OH Diclofenac
XIC 3100032
Would high resolution ion mobility provide the orthogonal separation we need
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
What Drives High Confidence
bull Fundamental BA parameters are addressed Selectivity sensitivity linearity matrix effect stability precision and accuracy
bull SignalNoise Ratio Measured animalhuman 2
Measurement error (without cancellation) 025
SN (2-1)025=4
Limit of Detection A signal is real if SN=3
bull Minimal 3 independent animalhuman ratio measurements
bull Analysis reproducibility RSD of the ratio measurements (n=3) 33
bull Stability ratio for each metabolite plusmn20 (08-12) over the storage period
How to Evaluate Data Quality
MIST Coverage is a Comparison Question
Animals have exposures of metabolites ge human
Comparison 1 Giraffe vs Moose
Which one is taller
All that one needs to do is to have them stand next to each other The giraffe is much taller No need to know exact heights
Which one is taller
Too close to call just by looking at them -- The height measurements are needed
Comparison 2 Moose vs Whitetail Deer
If the animalhuman peak area ratio is ge 2 animal has exposure ge human with high confidence (plt001)
How often does it happen Most cases as animal is dosed at much higher
dose when corrected by body weight
A simple peak area ratio comparison is sufficient for most cases
Case Study
bull Drug A in project X at phase 1 14 days 35 mg BID in 6 human volunteers vs 5 days 300 mgkg in Sprague Dawley rats
bull Pooled plasma samples (Hamilton) across time points across subjects
bull 3 human metabolites Parent drug A 396gt203
m-hydroxy 412 ring opened 414 de-alkylated 304
bull HPLC method and major daughter ions were transferred from Biotransformation
bull Compare results with concentration measurements using the synthetic standard
MIST Coverage Evaluation Using RatHuman Ratio of Peak Area Ratios
Definitive Concentration Measurements Using Synthetic Standard
Species Parent De-alkylated Ring-opened m-Hydroxy
Human 0220 00406 000788 00210 Rat 770 0502 0292 000762
RatHuman ratio 1 35 124 371 0363
Human repeat 0184 00290 000506 00164
Rat repeat 734 0472 0266 000618 RatHuman
ratio 2 399 163 526 0378
Species m-Hydroxy (ngmL)
Human 743 plusmn 153
Rat 269 plusmn 258
Cavg RatHuman ratio 0362
Human Metabolite gt10 Drug-Related Material from a Human Radiolabel
ADME Study (or Metabolite Anticipated to Be gt10 from Other Human Studies)
Metabolite With Known Activity of
Interest
Use Standard Bioanalytical
Method
ICH Exempt Metabolite
Peak Area Ratio Method
Method Fails Criteria
Method Succeeds
Animal lt 2X Human
Animal ge 2X Human
No Further Work Needed
Coverage is Assured No Further Work
Needed
Prepare Authentic Standard of Metabolite
MIST Decision Tree
Gao H and Obach RS (2014) Bioanalysis 6(5) 641-650
Data Driven Strategy
bull Rigor of the analysis increases based on data Synthetic standard is needed only when the method fails or animal has less exposure than human
bull Conservative evaluation of MIST coverage
bull Assess MIST coverage at steady state without radio-label or synthetic standards enabling early assessment eg FIH
bull Time- and resource-saving strategy
Summary
bull LCMSMS peak area ratio comparison is scientifically sound and defensible method that can
ndash Ascertain the steady-state exposure of animals compared to humans (ie ldquoMIST coveragerdquo)
ndash Assess the MIST coverage without radio-label or synthetic standard
bull These methods are fit-for-purpose and can offer decision-making data useful for reliable estimation of metabolite exposure
bull Discussions with regulatory experts were positive and helpful
Gao et al (2013) AAPS Journal15(4) 970-973
Prerequisite for Current Methodology
bull Animal and human samples for side-by-side injection
minus In drug development first in human samples are typically ~1 year later than the toxicology study is done
bull Prior knowledge of human metabolites transferred from metabolite identification and scouting
minus Knowledge of major human metabolites may evolve only after in vivo human samples become available
minus Radio-labeled ADME is the most definitive study may take place late in the drug development process
Is it feasible to scan the samplesbank data and compare the data generated at different dates
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
MeO
O
O
OH
OH
OH
O OHNaproxen Acyl Glucuronide conjugate
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
(AZT)
Negative Control
Blind Test 3 Metabolite Cocktail
Gao et al Anal Chem 2015 87 11771-11776
Scanning and Banking DataSamples
bull Blind Test samples 22 metabolitesdrugs were spiked in human and rat plasmas (n=4 each)
bull No knowledge of the identities of the analytes and their concentrations in plasma were provided before sample analysis
bull Multiple analysis of the same samples (rat or human) using mixed matrix approach at Days 1 30 105 254 and 314
bull Treat the samples using the same IS solution in acetonitrile minus Freshly prepared to maintain the same concentration
bull Acquity UPLC generic gradient on C18 run time 25 min
bull Full scan and AIF scan on QExactive 70K at positive and negative polarity
bull Compare rat vs human samples analyzed at different dates
0 4 8 12 16 20 24 Time (min)
0
100 R
elat
ive
Ab
un
dan
ce
508
478 682
1132 1435 1450 763
773 243 1835 2120 2138 1164 1312 1638 288 534 1473 2338 802 2045 335 1062
42 43 44 45 46 47 48 49 50 51 52 53 54 55 Time (min)
0
100
Rel
ativ
e In
ten
sity
RT 508
RT 478
RT 495
RT 539
4 8 12 16 20 24 Time (min)
0
100 RT 624
RT 1313
RT 1113
RT 1626
RT 275 RT 879
RT 2008 RT 422
548
165 808
1 2
3
3
4
5
5
4
678
9 10 111213
14 16 15
1
2
3
4
56 7
Rel
ativ
e A
bu
nd
ance
Migrated naproxen acyl glucuronide
EIC 15 drugsmetabolites Resolution 70000 Extraction window 5ppm Polarity Positive
EIC 6 drugsmetabolites Resolution 70000 Extraction window 10 ppm Polarity Negative
2-OH Tacrine
4-OH Tacrine
1-OH Tacrine
Naproxen-Glu
Peak Area Ratio Measurement Variation
n=4 per species 5 independent analysis total n=40 per analyte
95 tile 037
Median 021
M1
0
DrugsMetabolites
Bank and Compare Data at Different Days
Gao et al Anal Chem 2015 87 11771-11776
Separation and Quantitative Analysis of Isomers
2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
100
Rela
tive I
nte
nsity
RT 1626
RT 275
RT 422 RT 1188 RT 632
Full Scan 4rsquo (or 5)-OH Diclofenac
XIC 3100032
Would high resolution ion mobility provide the orthogonal separation we need
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
bull Minimal 3 independent animalhuman ratio measurements
bull Analysis reproducibility RSD of the ratio measurements (n=3) 33
bull Stability ratio for each metabolite plusmn20 (08-12) over the storage period
How to Evaluate Data Quality
MIST Coverage is a Comparison Question
Animals have exposures of metabolites ge human
Comparison 1 Giraffe vs Moose
Which one is taller
All that one needs to do is to have them stand next to each other The giraffe is much taller No need to know exact heights
Which one is taller
Too close to call just by looking at them -- The height measurements are needed
Comparison 2 Moose vs Whitetail Deer
If the animalhuman peak area ratio is ge 2 animal has exposure ge human with high confidence (plt001)
How often does it happen Most cases as animal is dosed at much higher
dose when corrected by body weight
A simple peak area ratio comparison is sufficient for most cases
Case Study
bull Drug A in project X at phase 1 14 days 35 mg BID in 6 human volunteers vs 5 days 300 mgkg in Sprague Dawley rats
bull Pooled plasma samples (Hamilton) across time points across subjects
bull 3 human metabolites Parent drug A 396gt203
m-hydroxy 412 ring opened 414 de-alkylated 304
bull HPLC method and major daughter ions were transferred from Biotransformation
bull Compare results with concentration measurements using the synthetic standard
MIST Coverage Evaluation Using RatHuman Ratio of Peak Area Ratios
Definitive Concentration Measurements Using Synthetic Standard
Species Parent De-alkylated Ring-opened m-Hydroxy
Human 0220 00406 000788 00210 Rat 770 0502 0292 000762
RatHuman ratio 1 35 124 371 0363
Human repeat 0184 00290 000506 00164
Rat repeat 734 0472 0266 000618 RatHuman
ratio 2 399 163 526 0378
Species m-Hydroxy (ngmL)
Human 743 plusmn 153
Rat 269 plusmn 258
Cavg RatHuman ratio 0362
Human Metabolite gt10 Drug-Related Material from a Human Radiolabel
ADME Study (or Metabolite Anticipated to Be gt10 from Other Human Studies)
Metabolite With Known Activity of
Interest
Use Standard Bioanalytical
Method
ICH Exempt Metabolite
Peak Area Ratio Method
Method Fails Criteria
Method Succeeds
Animal lt 2X Human
Animal ge 2X Human
No Further Work Needed
Coverage is Assured No Further Work
Needed
Prepare Authentic Standard of Metabolite
MIST Decision Tree
Gao H and Obach RS (2014) Bioanalysis 6(5) 641-650
Data Driven Strategy
bull Rigor of the analysis increases based on data Synthetic standard is needed only when the method fails or animal has less exposure than human
bull Conservative evaluation of MIST coverage
bull Assess MIST coverage at steady state without radio-label or synthetic standards enabling early assessment eg FIH
bull Time- and resource-saving strategy
Summary
bull LCMSMS peak area ratio comparison is scientifically sound and defensible method that can
ndash Ascertain the steady-state exposure of animals compared to humans (ie ldquoMIST coveragerdquo)
ndash Assess the MIST coverage without radio-label or synthetic standard
bull These methods are fit-for-purpose and can offer decision-making data useful for reliable estimation of metabolite exposure
bull Discussions with regulatory experts were positive and helpful
Gao et al (2013) AAPS Journal15(4) 970-973
Prerequisite for Current Methodology
bull Animal and human samples for side-by-side injection
minus In drug development first in human samples are typically ~1 year later than the toxicology study is done
bull Prior knowledge of human metabolites transferred from metabolite identification and scouting
minus Knowledge of major human metabolites may evolve only after in vivo human samples become available
minus Radio-labeled ADME is the most definitive study may take place late in the drug development process
Is it feasible to scan the samplesbank data and compare the data generated at different dates
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
MeO
O
O
OH
OH
OH
O OHNaproxen Acyl Glucuronide conjugate
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
(AZT)
Negative Control
Blind Test 3 Metabolite Cocktail
Gao et al Anal Chem 2015 87 11771-11776
Scanning and Banking DataSamples
bull Blind Test samples 22 metabolitesdrugs were spiked in human and rat plasmas (n=4 each)
bull No knowledge of the identities of the analytes and their concentrations in plasma were provided before sample analysis
bull Multiple analysis of the same samples (rat or human) using mixed matrix approach at Days 1 30 105 254 and 314
bull Treat the samples using the same IS solution in acetonitrile minus Freshly prepared to maintain the same concentration
bull Acquity UPLC generic gradient on C18 run time 25 min
bull Full scan and AIF scan on QExactive 70K at positive and negative polarity
bull Compare rat vs human samples analyzed at different dates
0 4 8 12 16 20 24 Time (min)
0
100 R
elat
ive
Ab
un
dan
ce
508
478 682
1132 1435 1450 763
773 243 1835 2120 2138 1164 1312 1638 288 534 1473 2338 802 2045 335 1062
42 43 44 45 46 47 48 49 50 51 52 53 54 55 Time (min)
0
100
Rel
ativ
e In
ten
sity
RT 508
RT 478
RT 495
RT 539
4 8 12 16 20 24 Time (min)
0
100 RT 624
RT 1313
RT 1113
RT 1626
RT 275 RT 879
RT 2008 RT 422
548
165 808
1 2
3
3
4
5
5
4
678
9 10 111213
14 16 15
1
2
3
4
56 7
Rel
ativ
e A
bu
nd
ance
Migrated naproxen acyl glucuronide
EIC 15 drugsmetabolites Resolution 70000 Extraction window 5ppm Polarity Positive
EIC 6 drugsmetabolites Resolution 70000 Extraction window 10 ppm Polarity Negative
2-OH Tacrine
4-OH Tacrine
1-OH Tacrine
Naproxen-Glu
Peak Area Ratio Measurement Variation
n=4 per species 5 independent analysis total n=40 per analyte
95 tile 037
Median 021
M1
0
DrugsMetabolites
Bank and Compare Data at Different Days
Gao et al Anal Chem 2015 87 11771-11776
Separation and Quantitative Analysis of Isomers
2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
100
Rela
tive I
nte
nsity
RT 1626
RT 275
RT 422 RT 1188 RT 632
Full Scan 4rsquo (or 5)-OH Diclofenac
XIC 3100032
Would high resolution ion mobility provide the orthogonal separation we need
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
MIST Coverage is a Comparison Question
Animals have exposures of metabolites ge human
Comparison 1 Giraffe vs Moose
Which one is taller
All that one needs to do is to have them stand next to each other The giraffe is much taller No need to know exact heights
Which one is taller
Too close to call just by looking at them -- The height measurements are needed
Comparison 2 Moose vs Whitetail Deer
If the animalhuman peak area ratio is ge 2 animal has exposure ge human with high confidence (plt001)
How often does it happen Most cases as animal is dosed at much higher
dose when corrected by body weight
A simple peak area ratio comparison is sufficient for most cases
Case Study
bull Drug A in project X at phase 1 14 days 35 mg BID in 6 human volunteers vs 5 days 300 mgkg in Sprague Dawley rats
bull Pooled plasma samples (Hamilton) across time points across subjects
bull 3 human metabolites Parent drug A 396gt203
m-hydroxy 412 ring opened 414 de-alkylated 304
bull HPLC method and major daughter ions were transferred from Biotransformation
bull Compare results with concentration measurements using the synthetic standard
MIST Coverage Evaluation Using RatHuman Ratio of Peak Area Ratios
Definitive Concentration Measurements Using Synthetic Standard
Species Parent De-alkylated Ring-opened m-Hydroxy
Human 0220 00406 000788 00210 Rat 770 0502 0292 000762
RatHuman ratio 1 35 124 371 0363
Human repeat 0184 00290 000506 00164
Rat repeat 734 0472 0266 000618 RatHuman
ratio 2 399 163 526 0378
Species m-Hydroxy (ngmL)
Human 743 plusmn 153
Rat 269 plusmn 258
Cavg RatHuman ratio 0362
Human Metabolite gt10 Drug-Related Material from a Human Radiolabel
ADME Study (or Metabolite Anticipated to Be gt10 from Other Human Studies)
Metabolite With Known Activity of
Interest
Use Standard Bioanalytical
Method
ICH Exempt Metabolite
Peak Area Ratio Method
Method Fails Criteria
Method Succeeds
Animal lt 2X Human
Animal ge 2X Human
No Further Work Needed
Coverage is Assured No Further Work
Needed
Prepare Authentic Standard of Metabolite
MIST Decision Tree
Gao H and Obach RS (2014) Bioanalysis 6(5) 641-650
Data Driven Strategy
bull Rigor of the analysis increases based on data Synthetic standard is needed only when the method fails or animal has less exposure than human
bull Conservative evaluation of MIST coverage
bull Assess MIST coverage at steady state without radio-label or synthetic standards enabling early assessment eg FIH
bull Time- and resource-saving strategy
Summary
bull LCMSMS peak area ratio comparison is scientifically sound and defensible method that can
ndash Ascertain the steady-state exposure of animals compared to humans (ie ldquoMIST coveragerdquo)
ndash Assess the MIST coverage without radio-label or synthetic standard
bull These methods are fit-for-purpose and can offer decision-making data useful for reliable estimation of metabolite exposure
bull Discussions with regulatory experts were positive and helpful
Gao et al (2013) AAPS Journal15(4) 970-973
Prerequisite for Current Methodology
bull Animal and human samples for side-by-side injection
minus In drug development first in human samples are typically ~1 year later than the toxicology study is done
bull Prior knowledge of human metabolites transferred from metabolite identification and scouting
minus Knowledge of major human metabolites may evolve only after in vivo human samples become available
minus Radio-labeled ADME is the most definitive study may take place late in the drug development process
Is it feasible to scan the samplesbank data and compare the data generated at different dates
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
MeO
O
O
OH
OH
OH
O OHNaproxen Acyl Glucuronide conjugate
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
(AZT)
Negative Control
Blind Test 3 Metabolite Cocktail
Gao et al Anal Chem 2015 87 11771-11776
Scanning and Banking DataSamples
bull Blind Test samples 22 metabolitesdrugs were spiked in human and rat plasmas (n=4 each)
bull No knowledge of the identities of the analytes and their concentrations in plasma were provided before sample analysis
bull Multiple analysis of the same samples (rat or human) using mixed matrix approach at Days 1 30 105 254 and 314
bull Treat the samples using the same IS solution in acetonitrile minus Freshly prepared to maintain the same concentration
bull Acquity UPLC generic gradient on C18 run time 25 min
bull Full scan and AIF scan on QExactive 70K at positive and negative polarity
bull Compare rat vs human samples analyzed at different dates
0 4 8 12 16 20 24 Time (min)
0
100 R
elat
ive
Ab
un
dan
ce
508
478 682
1132 1435 1450 763
773 243 1835 2120 2138 1164 1312 1638 288 534 1473 2338 802 2045 335 1062
42 43 44 45 46 47 48 49 50 51 52 53 54 55 Time (min)
0
100
Rel
ativ
e In
ten
sity
RT 508
RT 478
RT 495
RT 539
4 8 12 16 20 24 Time (min)
0
100 RT 624
RT 1313
RT 1113
RT 1626
RT 275 RT 879
RT 2008 RT 422
548
165 808
1 2
3
3
4
5
5
4
678
9 10 111213
14 16 15
1
2
3
4
56 7
Rel
ativ
e A
bu
nd
ance
Migrated naproxen acyl glucuronide
EIC 15 drugsmetabolites Resolution 70000 Extraction window 5ppm Polarity Positive
EIC 6 drugsmetabolites Resolution 70000 Extraction window 10 ppm Polarity Negative
2-OH Tacrine
4-OH Tacrine
1-OH Tacrine
Naproxen-Glu
Peak Area Ratio Measurement Variation
n=4 per species 5 independent analysis total n=40 per analyte
95 tile 037
Median 021
M1
0
DrugsMetabolites
Bank and Compare Data at Different Days
Gao et al Anal Chem 2015 87 11771-11776
Separation and Quantitative Analysis of Isomers
2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
100
Rela
tive I
nte
nsity
RT 1626
RT 275
RT 422 RT 1188 RT 632
Full Scan 4rsquo (or 5)-OH Diclofenac
XIC 3100032
Would high resolution ion mobility provide the orthogonal separation we need
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
Comparison 1 Giraffe vs Moose
Which one is taller
All that one needs to do is to have them stand next to each other The giraffe is much taller No need to know exact heights
Which one is taller
Too close to call just by looking at them -- The height measurements are needed
Comparison 2 Moose vs Whitetail Deer
If the animalhuman peak area ratio is ge 2 animal has exposure ge human with high confidence (plt001)
How often does it happen Most cases as animal is dosed at much higher
dose when corrected by body weight
A simple peak area ratio comparison is sufficient for most cases
Case Study
bull Drug A in project X at phase 1 14 days 35 mg BID in 6 human volunteers vs 5 days 300 mgkg in Sprague Dawley rats
bull Pooled plasma samples (Hamilton) across time points across subjects
bull 3 human metabolites Parent drug A 396gt203
m-hydroxy 412 ring opened 414 de-alkylated 304
bull HPLC method and major daughter ions were transferred from Biotransformation
bull Compare results with concentration measurements using the synthetic standard
MIST Coverage Evaluation Using RatHuman Ratio of Peak Area Ratios
Definitive Concentration Measurements Using Synthetic Standard
Species Parent De-alkylated Ring-opened m-Hydroxy
Human 0220 00406 000788 00210 Rat 770 0502 0292 000762
RatHuman ratio 1 35 124 371 0363
Human repeat 0184 00290 000506 00164
Rat repeat 734 0472 0266 000618 RatHuman
ratio 2 399 163 526 0378
Species m-Hydroxy (ngmL)
Human 743 plusmn 153
Rat 269 plusmn 258
Cavg RatHuman ratio 0362
Human Metabolite gt10 Drug-Related Material from a Human Radiolabel
ADME Study (or Metabolite Anticipated to Be gt10 from Other Human Studies)
Metabolite With Known Activity of
Interest
Use Standard Bioanalytical
Method
ICH Exempt Metabolite
Peak Area Ratio Method
Method Fails Criteria
Method Succeeds
Animal lt 2X Human
Animal ge 2X Human
No Further Work Needed
Coverage is Assured No Further Work
Needed
Prepare Authentic Standard of Metabolite
MIST Decision Tree
Gao H and Obach RS (2014) Bioanalysis 6(5) 641-650
Data Driven Strategy
bull Rigor of the analysis increases based on data Synthetic standard is needed only when the method fails or animal has less exposure than human
bull Conservative evaluation of MIST coverage
bull Assess MIST coverage at steady state without radio-label or synthetic standards enabling early assessment eg FIH
bull Time- and resource-saving strategy
Summary
bull LCMSMS peak area ratio comparison is scientifically sound and defensible method that can
ndash Ascertain the steady-state exposure of animals compared to humans (ie ldquoMIST coveragerdquo)
ndash Assess the MIST coverage without radio-label or synthetic standard
bull These methods are fit-for-purpose and can offer decision-making data useful for reliable estimation of metabolite exposure
bull Discussions with regulatory experts were positive and helpful
Gao et al (2013) AAPS Journal15(4) 970-973
Prerequisite for Current Methodology
bull Animal and human samples for side-by-side injection
minus In drug development first in human samples are typically ~1 year later than the toxicology study is done
bull Prior knowledge of human metabolites transferred from metabolite identification and scouting
minus Knowledge of major human metabolites may evolve only after in vivo human samples become available
minus Radio-labeled ADME is the most definitive study may take place late in the drug development process
Is it feasible to scan the samplesbank data and compare the data generated at different dates
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
MeO
O
O
OH
OH
OH
O OHNaproxen Acyl Glucuronide conjugate
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
(AZT)
Negative Control
Blind Test 3 Metabolite Cocktail
Gao et al Anal Chem 2015 87 11771-11776
Scanning and Banking DataSamples
bull Blind Test samples 22 metabolitesdrugs were spiked in human and rat plasmas (n=4 each)
bull No knowledge of the identities of the analytes and their concentrations in plasma were provided before sample analysis
bull Multiple analysis of the same samples (rat or human) using mixed matrix approach at Days 1 30 105 254 and 314
bull Treat the samples using the same IS solution in acetonitrile minus Freshly prepared to maintain the same concentration
bull Acquity UPLC generic gradient on C18 run time 25 min
bull Full scan and AIF scan on QExactive 70K at positive and negative polarity
bull Compare rat vs human samples analyzed at different dates
0 4 8 12 16 20 24 Time (min)
0
100 R
elat
ive
Ab
un
dan
ce
508
478 682
1132 1435 1450 763
773 243 1835 2120 2138 1164 1312 1638 288 534 1473 2338 802 2045 335 1062
42 43 44 45 46 47 48 49 50 51 52 53 54 55 Time (min)
0
100
Rel
ativ
e In
ten
sity
RT 508
RT 478
RT 495
RT 539
4 8 12 16 20 24 Time (min)
0
100 RT 624
RT 1313
RT 1113
RT 1626
RT 275 RT 879
RT 2008 RT 422
548
165 808
1 2
3
3
4
5
5
4
678
9 10 111213
14 16 15
1
2
3
4
56 7
Rel
ativ
e A
bu
nd
ance
Migrated naproxen acyl glucuronide
EIC 15 drugsmetabolites Resolution 70000 Extraction window 5ppm Polarity Positive
EIC 6 drugsmetabolites Resolution 70000 Extraction window 10 ppm Polarity Negative
2-OH Tacrine
4-OH Tacrine
1-OH Tacrine
Naproxen-Glu
Peak Area Ratio Measurement Variation
n=4 per species 5 independent analysis total n=40 per analyte
95 tile 037
Median 021
M1
0
DrugsMetabolites
Bank and Compare Data at Different Days
Gao et al Anal Chem 2015 87 11771-11776
Separation and Quantitative Analysis of Isomers
2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
100
Rela
tive I
nte
nsity
RT 1626
RT 275
RT 422 RT 1188 RT 632
Full Scan 4rsquo (or 5)-OH Diclofenac
XIC 3100032
Would high resolution ion mobility provide the orthogonal separation we need
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
Which one is taller
Too close to call just by looking at them -- The height measurements are needed
Comparison 2 Moose vs Whitetail Deer
If the animalhuman peak area ratio is ge 2 animal has exposure ge human with high confidence (plt001)
How often does it happen Most cases as animal is dosed at much higher
dose when corrected by body weight
A simple peak area ratio comparison is sufficient for most cases
Case Study
bull Drug A in project X at phase 1 14 days 35 mg BID in 6 human volunteers vs 5 days 300 mgkg in Sprague Dawley rats
bull Pooled plasma samples (Hamilton) across time points across subjects
bull 3 human metabolites Parent drug A 396gt203
m-hydroxy 412 ring opened 414 de-alkylated 304
bull HPLC method and major daughter ions were transferred from Biotransformation
bull Compare results with concentration measurements using the synthetic standard
MIST Coverage Evaluation Using RatHuman Ratio of Peak Area Ratios
Definitive Concentration Measurements Using Synthetic Standard
Species Parent De-alkylated Ring-opened m-Hydroxy
Human 0220 00406 000788 00210 Rat 770 0502 0292 000762
RatHuman ratio 1 35 124 371 0363
Human repeat 0184 00290 000506 00164
Rat repeat 734 0472 0266 000618 RatHuman
ratio 2 399 163 526 0378
Species m-Hydroxy (ngmL)
Human 743 plusmn 153
Rat 269 plusmn 258
Cavg RatHuman ratio 0362
Human Metabolite gt10 Drug-Related Material from a Human Radiolabel
ADME Study (or Metabolite Anticipated to Be gt10 from Other Human Studies)
Metabolite With Known Activity of
Interest
Use Standard Bioanalytical
Method
ICH Exempt Metabolite
Peak Area Ratio Method
Method Fails Criteria
Method Succeeds
Animal lt 2X Human
Animal ge 2X Human
No Further Work Needed
Coverage is Assured No Further Work
Needed
Prepare Authentic Standard of Metabolite
MIST Decision Tree
Gao H and Obach RS (2014) Bioanalysis 6(5) 641-650
Data Driven Strategy
bull Rigor of the analysis increases based on data Synthetic standard is needed only when the method fails or animal has less exposure than human
bull Conservative evaluation of MIST coverage
bull Assess MIST coverage at steady state without radio-label or synthetic standards enabling early assessment eg FIH
bull Time- and resource-saving strategy
Summary
bull LCMSMS peak area ratio comparison is scientifically sound and defensible method that can
ndash Ascertain the steady-state exposure of animals compared to humans (ie ldquoMIST coveragerdquo)
ndash Assess the MIST coverage without radio-label or synthetic standard
bull These methods are fit-for-purpose and can offer decision-making data useful for reliable estimation of metabolite exposure
bull Discussions with regulatory experts were positive and helpful
Gao et al (2013) AAPS Journal15(4) 970-973
Prerequisite for Current Methodology
bull Animal and human samples for side-by-side injection
minus In drug development first in human samples are typically ~1 year later than the toxicology study is done
bull Prior knowledge of human metabolites transferred from metabolite identification and scouting
minus Knowledge of major human metabolites may evolve only after in vivo human samples become available
minus Radio-labeled ADME is the most definitive study may take place late in the drug development process
Is it feasible to scan the samplesbank data and compare the data generated at different dates
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
MeO
O
O
OH
OH
OH
O OHNaproxen Acyl Glucuronide conjugate
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
(AZT)
Negative Control
Blind Test 3 Metabolite Cocktail
Gao et al Anal Chem 2015 87 11771-11776
Scanning and Banking DataSamples
bull Blind Test samples 22 metabolitesdrugs were spiked in human and rat plasmas (n=4 each)
bull No knowledge of the identities of the analytes and their concentrations in plasma were provided before sample analysis
bull Multiple analysis of the same samples (rat or human) using mixed matrix approach at Days 1 30 105 254 and 314
bull Treat the samples using the same IS solution in acetonitrile minus Freshly prepared to maintain the same concentration
bull Acquity UPLC generic gradient on C18 run time 25 min
bull Full scan and AIF scan on QExactive 70K at positive and negative polarity
bull Compare rat vs human samples analyzed at different dates
0 4 8 12 16 20 24 Time (min)
0
100 R
elat
ive
Ab
un
dan
ce
508
478 682
1132 1435 1450 763
773 243 1835 2120 2138 1164 1312 1638 288 534 1473 2338 802 2045 335 1062
42 43 44 45 46 47 48 49 50 51 52 53 54 55 Time (min)
0
100
Rel
ativ
e In
ten
sity
RT 508
RT 478
RT 495
RT 539
4 8 12 16 20 24 Time (min)
0
100 RT 624
RT 1313
RT 1113
RT 1626
RT 275 RT 879
RT 2008 RT 422
548
165 808
1 2
3
3
4
5
5
4
678
9 10 111213
14 16 15
1
2
3
4
56 7
Rel
ativ
e A
bu
nd
ance
Migrated naproxen acyl glucuronide
EIC 15 drugsmetabolites Resolution 70000 Extraction window 5ppm Polarity Positive
EIC 6 drugsmetabolites Resolution 70000 Extraction window 10 ppm Polarity Negative
2-OH Tacrine
4-OH Tacrine
1-OH Tacrine
Naproxen-Glu
Peak Area Ratio Measurement Variation
n=4 per species 5 independent analysis total n=40 per analyte
95 tile 037
Median 021
M1
0
DrugsMetabolites
Bank and Compare Data at Different Days
Gao et al Anal Chem 2015 87 11771-11776
Separation and Quantitative Analysis of Isomers
2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
100
Rela
tive I
nte
nsity
RT 1626
RT 275
RT 422 RT 1188 RT 632
Full Scan 4rsquo (or 5)-OH Diclofenac
XIC 3100032
Would high resolution ion mobility provide the orthogonal separation we need
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
If the animalhuman peak area ratio is ge 2 animal has exposure ge human with high confidence (plt001)
How often does it happen Most cases as animal is dosed at much higher
dose when corrected by body weight
A simple peak area ratio comparison is sufficient for most cases
Case Study
bull Drug A in project X at phase 1 14 days 35 mg BID in 6 human volunteers vs 5 days 300 mgkg in Sprague Dawley rats
bull Pooled plasma samples (Hamilton) across time points across subjects
bull 3 human metabolites Parent drug A 396gt203
m-hydroxy 412 ring opened 414 de-alkylated 304
bull HPLC method and major daughter ions were transferred from Biotransformation
bull Compare results with concentration measurements using the synthetic standard
MIST Coverage Evaluation Using RatHuman Ratio of Peak Area Ratios
Definitive Concentration Measurements Using Synthetic Standard
Species Parent De-alkylated Ring-opened m-Hydroxy
Human 0220 00406 000788 00210 Rat 770 0502 0292 000762
RatHuman ratio 1 35 124 371 0363
Human repeat 0184 00290 000506 00164
Rat repeat 734 0472 0266 000618 RatHuman
ratio 2 399 163 526 0378
Species m-Hydroxy (ngmL)
Human 743 plusmn 153
Rat 269 plusmn 258
Cavg RatHuman ratio 0362
Human Metabolite gt10 Drug-Related Material from a Human Radiolabel
ADME Study (or Metabolite Anticipated to Be gt10 from Other Human Studies)
Metabolite With Known Activity of
Interest
Use Standard Bioanalytical
Method
ICH Exempt Metabolite
Peak Area Ratio Method
Method Fails Criteria
Method Succeeds
Animal lt 2X Human
Animal ge 2X Human
No Further Work Needed
Coverage is Assured No Further Work
Needed
Prepare Authentic Standard of Metabolite
MIST Decision Tree
Gao H and Obach RS (2014) Bioanalysis 6(5) 641-650
Data Driven Strategy
bull Rigor of the analysis increases based on data Synthetic standard is needed only when the method fails or animal has less exposure than human
bull Conservative evaluation of MIST coverage
bull Assess MIST coverage at steady state without radio-label or synthetic standards enabling early assessment eg FIH
bull Time- and resource-saving strategy
Summary
bull LCMSMS peak area ratio comparison is scientifically sound and defensible method that can
ndash Ascertain the steady-state exposure of animals compared to humans (ie ldquoMIST coveragerdquo)
ndash Assess the MIST coverage without radio-label or synthetic standard
bull These methods are fit-for-purpose and can offer decision-making data useful for reliable estimation of metabolite exposure
bull Discussions with regulatory experts were positive and helpful
Gao et al (2013) AAPS Journal15(4) 970-973
Prerequisite for Current Methodology
bull Animal and human samples for side-by-side injection
minus In drug development first in human samples are typically ~1 year later than the toxicology study is done
bull Prior knowledge of human metabolites transferred from metabolite identification and scouting
minus Knowledge of major human metabolites may evolve only after in vivo human samples become available
minus Radio-labeled ADME is the most definitive study may take place late in the drug development process
Is it feasible to scan the samplesbank data and compare the data generated at different dates
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
MeO
O
O
OH
OH
OH
O OHNaproxen Acyl Glucuronide conjugate
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
(AZT)
Negative Control
Blind Test 3 Metabolite Cocktail
Gao et al Anal Chem 2015 87 11771-11776
Scanning and Banking DataSamples
bull Blind Test samples 22 metabolitesdrugs were spiked in human and rat plasmas (n=4 each)
bull No knowledge of the identities of the analytes and their concentrations in plasma were provided before sample analysis
bull Multiple analysis of the same samples (rat or human) using mixed matrix approach at Days 1 30 105 254 and 314
bull Treat the samples using the same IS solution in acetonitrile minus Freshly prepared to maintain the same concentration
bull Acquity UPLC generic gradient on C18 run time 25 min
bull Full scan and AIF scan on QExactive 70K at positive and negative polarity
bull Compare rat vs human samples analyzed at different dates
0 4 8 12 16 20 24 Time (min)
0
100 R
elat
ive
Ab
un
dan
ce
508
478 682
1132 1435 1450 763
773 243 1835 2120 2138 1164 1312 1638 288 534 1473 2338 802 2045 335 1062
42 43 44 45 46 47 48 49 50 51 52 53 54 55 Time (min)
0
100
Rel
ativ
e In
ten
sity
RT 508
RT 478
RT 495
RT 539
4 8 12 16 20 24 Time (min)
0
100 RT 624
RT 1313
RT 1113
RT 1626
RT 275 RT 879
RT 2008 RT 422
548
165 808
1 2
3
3
4
5
5
4
678
9 10 111213
14 16 15
1
2
3
4
56 7
Rel
ativ
e A
bu
nd
ance
Migrated naproxen acyl glucuronide
EIC 15 drugsmetabolites Resolution 70000 Extraction window 5ppm Polarity Positive
EIC 6 drugsmetabolites Resolution 70000 Extraction window 10 ppm Polarity Negative
2-OH Tacrine
4-OH Tacrine
1-OH Tacrine
Naproxen-Glu
Peak Area Ratio Measurement Variation
n=4 per species 5 independent analysis total n=40 per analyte
95 tile 037
Median 021
M1
0
DrugsMetabolites
Bank and Compare Data at Different Days
Gao et al Anal Chem 2015 87 11771-11776
Separation and Quantitative Analysis of Isomers
2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
100
Rela
tive I
nte
nsity
RT 1626
RT 275
RT 422 RT 1188 RT 632
Full Scan 4rsquo (or 5)-OH Diclofenac
XIC 3100032
Would high resolution ion mobility provide the orthogonal separation we need
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
Case Study
bull Drug A in project X at phase 1 14 days 35 mg BID in 6 human volunteers vs 5 days 300 mgkg in Sprague Dawley rats
bull Pooled plasma samples (Hamilton) across time points across subjects
bull 3 human metabolites Parent drug A 396gt203
m-hydroxy 412 ring opened 414 de-alkylated 304
bull HPLC method and major daughter ions were transferred from Biotransformation
bull Compare results with concentration measurements using the synthetic standard
MIST Coverage Evaluation Using RatHuman Ratio of Peak Area Ratios
Definitive Concentration Measurements Using Synthetic Standard
Species Parent De-alkylated Ring-opened m-Hydroxy
Human 0220 00406 000788 00210 Rat 770 0502 0292 000762
RatHuman ratio 1 35 124 371 0363
Human repeat 0184 00290 000506 00164
Rat repeat 734 0472 0266 000618 RatHuman
ratio 2 399 163 526 0378
Species m-Hydroxy (ngmL)
Human 743 plusmn 153
Rat 269 plusmn 258
Cavg RatHuman ratio 0362
Human Metabolite gt10 Drug-Related Material from a Human Radiolabel
ADME Study (or Metabolite Anticipated to Be gt10 from Other Human Studies)
Metabolite With Known Activity of
Interest
Use Standard Bioanalytical
Method
ICH Exempt Metabolite
Peak Area Ratio Method
Method Fails Criteria
Method Succeeds
Animal lt 2X Human
Animal ge 2X Human
No Further Work Needed
Coverage is Assured No Further Work
Needed
Prepare Authentic Standard of Metabolite
MIST Decision Tree
Gao H and Obach RS (2014) Bioanalysis 6(5) 641-650
Data Driven Strategy
bull Rigor of the analysis increases based on data Synthetic standard is needed only when the method fails or animal has less exposure than human
bull Conservative evaluation of MIST coverage
bull Assess MIST coverage at steady state without radio-label or synthetic standards enabling early assessment eg FIH
bull Time- and resource-saving strategy
Summary
bull LCMSMS peak area ratio comparison is scientifically sound and defensible method that can
ndash Ascertain the steady-state exposure of animals compared to humans (ie ldquoMIST coveragerdquo)
ndash Assess the MIST coverage without radio-label or synthetic standard
bull These methods are fit-for-purpose and can offer decision-making data useful for reliable estimation of metabolite exposure
bull Discussions with regulatory experts were positive and helpful
Gao et al (2013) AAPS Journal15(4) 970-973
Prerequisite for Current Methodology
bull Animal and human samples for side-by-side injection
minus In drug development first in human samples are typically ~1 year later than the toxicology study is done
bull Prior knowledge of human metabolites transferred from metabolite identification and scouting
minus Knowledge of major human metabolites may evolve only after in vivo human samples become available
minus Radio-labeled ADME is the most definitive study may take place late in the drug development process
Is it feasible to scan the samplesbank data and compare the data generated at different dates
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
MeO
O
O
OH
OH
OH
O OHNaproxen Acyl Glucuronide conjugate
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
(AZT)
Negative Control
Blind Test 3 Metabolite Cocktail
Gao et al Anal Chem 2015 87 11771-11776
Scanning and Banking DataSamples
bull Blind Test samples 22 metabolitesdrugs were spiked in human and rat plasmas (n=4 each)
bull No knowledge of the identities of the analytes and their concentrations in plasma were provided before sample analysis
bull Multiple analysis of the same samples (rat or human) using mixed matrix approach at Days 1 30 105 254 and 314
bull Treat the samples using the same IS solution in acetonitrile minus Freshly prepared to maintain the same concentration
bull Acquity UPLC generic gradient on C18 run time 25 min
bull Full scan and AIF scan on QExactive 70K at positive and negative polarity
bull Compare rat vs human samples analyzed at different dates
0 4 8 12 16 20 24 Time (min)
0
100 R
elat
ive
Ab
un
dan
ce
508
478 682
1132 1435 1450 763
773 243 1835 2120 2138 1164 1312 1638 288 534 1473 2338 802 2045 335 1062
42 43 44 45 46 47 48 49 50 51 52 53 54 55 Time (min)
0
100
Rel
ativ
e In
ten
sity
RT 508
RT 478
RT 495
RT 539
4 8 12 16 20 24 Time (min)
0
100 RT 624
RT 1313
RT 1113
RT 1626
RT 275 RT 879
RT 2008 RT 422
548
165 808
1 2
3
3
4
5
5
4
678
9 10 111213
14 16 15
1
2
3
4
56 7
Rel
ativ
e A
bu
nd
ance
Migrated naproxen acyl glucuronide
EIC 15 drugsmetabolites Resolution 70000 Extraction window 5ppm Polarity Positive
EIC 6 drugsmetabolites Resolution 70000 Extraction window 10 ppm Polarity Negative
2-OH Tacrine
4-OH Tacrine
1-OH Tacrine
Naproxen-Glu
Peak Area Ratio Measurement Variation
n=4 per species 5 independent analysis total n=40 per analyte
95 tile 037
Median 021
M1
0
DrugsMetabolites
Bank and Compare Data at Different Days
Gao et al Anal Chem 2015 87 11771-11776
Separation and Quantitative Analysis of Isomers
2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
100
Rela
tive I
nte
nsity
RT 1626
RT 275
RT 422 RT 1188 RT 632
Full Scan 4rsquo (or 5)-OH Diclofenac
XIC 3100032
Would high resolution ion mobility provide the orthogonal separation we need
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
MIST Coverage Evaluation Using RatHuman Ratio of Peak Area Ratios
Definitive Concentration Measurements Using Synthetic Standard
Species Parent De-alkylated Ring-opened m-Hydroxy
Human 0220 00406 000788 00210 Rat 770 0502 0292 000762
RatHuman ratio 1 35 124 371 0363
Human repeat 0184 00290 000506 00164
Rat repeat 734 0472 0266 000618 RatHuman
ratio 2 399 163 526 0378
Species m-Hydroxy (ngmL)
Human 743 plusmn 153
Rat 269 plusmn 258
Cavg RatHuman ratio 0362
Human Metabolite gt10 Drug-Related Material from a Human Radiolabel
ADME Study (or Metabolite Anticipated to Be gt10 from Other Human Studies)
Metabolite With Known Activity of
Interest
Use Standard Bioanalytical
Method
ICH Exempt Metabolite
Peak Area Ratio Method
Method Fails Criteria
Method Succeeds
Animal lt 2X Human
Animal ge 2X Human
No Further Work Needed
Coverage is Assured No Further Work
Needed
Prepare Authentic Standard of Metabolite
MIST Decision Tree
Gao H and Obach RS (2014) Bioanalysis 6(5) 641-650
Data Driven Strategy
bull Rigor of the analysis increases based on data Synthetic standard is needed only when the method fails or animal has less exposure than human
bull Conservative evaluation of MIST coverage
bull Assess MIST coverage at steady state without radio-label or synthetic standards enabling early assessment eg FIH
bull Time- and resource-saving strategy
Summary
bull LCMSMS peak area ratio comparison is scientifically sound and defensible method that can
ndash Ascertain the steady-state exposure of animals compared to humans (ie ldquoMIST coveragerdquo)
ndash Assess the MIST coverage without radio-label or synthetic standard
bull These methods are fit-for-purpose and can offer decision-making data useful for reliable estimation of metabolite exposure
bull Discussions with regulatory experts were positive and helpful
Gao et al (2013) AAPS Journal15(4) 970-973
Prerequisite for Current Methodology
bull Animal and human samples for side-by-side injection
minus In drug development first in human samples are typically ~1 year later than the toxicology study is done
bull Prior knowledge of human metabolites transferred from metabolite identification and scouting
minus Knowledge of major human metabolites may evolve only after in vivo human samples become available
minus Radio-labeled ADME is the most definitive study may take place late in the drug development process
Is it feasible to scan the samplesbank data and compare the data generated at different dates
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
MeO
O
O
OH
OH
OH
O OHNaproxen Acyl Glucuronide conjugate
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
(AZT)
Negative Control
Blind Test 3 Metabolite Cocktail
Gao et al Anal Chem 2015 87 11771-11776
Scanning and Banking DataSamples
bull Blind Test samples 22 metabolitesdrugs were spiked in human and rat plasmas (n=4 each)
bull No knowledge of the identities of the analytes and their concentrations in plasma were provided before sample analysis
bull Multiple analysis of the same samples (rat or human) using mixed matrix approach at Days 1 30 105 254 and 314
bull Treat the samples using the same IS solution in acetonitrile minus Freshly prepared to maintain the same concentration
bull Acquity UPLC generic gradient on C18 run time 25 min
bull Full scan and AIF scan on QExactive 70K at positive and negative polarity
bull Compare rat vs human samples analyzed at different dates
0 4 8 12 16 20 24 Time (min)
0
100 R
elat
ive
Ab
un
dan
ce
508
478 682
1132 1435 1450 763
773 243 1835 2120 2138 1164 1312 1638 288 534 1473 2338 802 2045 335 1062
42 43 44 45 46 47 48 49 50 51 52 53 54 55 Time (min)
0
100
Rel
ativ
e In
ten
sity
RT 508
RT 478
RT 495
RT 539
4 8 12 16 20 24 Time (min)
0
100 RT 624
RT 1313
RT 1113
RT 1626
RT 275 RT 879
RT 2008 RT 422
548
165 808
1 2
3
3
4
5
5
4
678
9 10 111213
14 16 15
1
2
3
4
56 7
Rel
ativ
e A
bu
nd
ance
Migrated naproxen acyl glucuronide
EIC 15 drugsmetabolites Resolution 70000 Extraction window 5ppm Polarity Positive
EIC 6 drugsmetabolites Resolution 70000 Extraction window 10 ppm Polarity Negative
2-OH Tacrine
4-OH Tacrine
1-OH Tacrine
Naproxen-Glu
Peak Area Ratio Measurement Variation
n=4 per species 5 independent analysis total n=40 per analyte
95 tile 037
Median 021
M1
0
DrugsMetabolites
Bank and Compare Data at Different Days
Gao et al Anal Chem 2015 87 11771-11776
Separation and Quantitative Analysis of Isomers
2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
100
Rela
tive I
nte
nsity
RT 1626
RT 275
RT 422 RT 1188 RT 632
Full Scan 4rsquo (or 5)-OH Diclofenac
XIC 3100032
Would high resolution ion mobility provide the orthogonal separation we need
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
Human Metabolite gt10 Drug-Related Material from a Human Radiolabel
ADME Study (or Metabolite Anticipated to Be gt10 from Other Human Studies)
Metabolite With Known Activity of
Interest
Use Standard Bioanalytical
Method
ICH Exempt Metabolite
Peak Area Ratio Method
Method Fails Criteria
Method Succeeds
Animal lt 2X Human
Animal ge 2X Human
No Further Work Needed
Coverage is Assured No Further Work
Needed
Prepare Authentic Standard of Metabolite
MIST Decision Tree
Gao H and Obach RS (2014) Bioanalysis 6(5) 641-650
Data Driven Strategy
bull Rigor of the analysis increases based on data Synthetic standard is needed only when the method fails or animal has less exposure than human
bull Conservative evaluation of MIST coverage
bull Assess MIST coverage at steady state without radio-label or synthetic standards enabling early assessment eg FIH
bull Time- and resource-saving strategy
Summary
bull LCMSMS peak area ratio comparison is scientifically sound and defensible method that can
ndash Ascertain the steady-state exposure of animals compared to humans (ie ldquoMIST coveragerdquo)
ndash Assess the MIST coverage without radio-label or synthetic standard
bull These methods are fit-for-purpose and can offer decision-making data useful for reliable estimation of metabolite exposure
bull Discussions with regulatory experts were positive and helpful
Gao et al (2013) AAPS Journal15(4) 970-973
Prerequisite for Current Methodology
bull Animal and human samples for side-by-side injection
minus In drug development first in human samples are typically ~1 year later than the toxicology study is done
bull Prior knowledge of human metabolites transferred from metabolite identification and scouting
minus Knowledge of major human metabolites may evolve only after in vivo human samples become available
minus Radio-labeled ADME is the most definitive study may take place late in the drug development process
Is it feasible to scan the samplesbank data and compare the data generated at different dates
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
MeO
O
O
OH
OH
OH
O OHNaproxen Acyl Glucuronide conjugate
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
(AZT)
Negative Control
Blind Test 3 Metabolite Cocktail
Gao et al Anal Chem 2015 87 11771-11776
Scanning and Banking DataSamples
bull Blind Test samples 22 metabolitesdrugs were spiked in human and rat plasmas (n=4 each)
bull No knowledge of the identities of the analytes and their concentrations in plasma were provided before sample analysis
bull Multiple analysis of the same samples (rat or human) using mixed matrix approach at Days 1 30 105 254 and 314
bull Treat the samples using the same IS solution in acetonitrile minus Freshly prepared to maintain the same concentration
bull Acquity UPLC generic gradient on C18 run time 25 min
bull Full scan and AIF scan on QExactive 70K at positive and negative polarity
bull Compare rat vs human samples analyzed at different dates
0 4 8 12 16 20 24 Time (min)
0
100 R
elat
ive
Ab
un
dan
ce
508
478 682
1132 1435 1450 763
773 243 1835 2120 2138 1164 1312 1638 288 534 1473 2338 802 2045 335 1062
42 43 44 45 46 47 48 49 50 51 52 53 54 55 Time (min)
0
100
Rel
ativ
e In
ten
sity
RT 508
RT 478
RT 495
RT 539
4 8 12 16 20 24 Time (min)
0
100 RT 624
RT 1313
RT 1113
RT 1626
RT 275 RT 879
RT 2008 RT 422
548
165 808
1 2
3
3
4
5
5
4
678
9 10 111213
14 16 15
1
2
3
4
56 7
Rel
ativ
e A
bu
nd
ance
Migrated naproxen acyl glucuronide
EIC 15 drugsmetabolites Resolution 70000 Extraction window 5ppm Polarity Positive
EIC 6 drugsmetabolites Resolution 70000 Extraction window 10 ppm Polarity Negative
2-OH Tacrine
4-OH Tacrine
1-OH Tacrine
Naproxen-Glu
Peak Area Ratio Measurement Variation
n=4 per species 5 independent analysis total n=40 per analyte
95 tile 037
Median 021
M1
0
DrugsMetabolites
Bank and Compare Data at Different Days
Gao et al Anal Chem 2015 87 11771-11776
Separation and Quantitative Analysis of Isomers
2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
100
Rela
tive I
nte
nsity
RT 1626
RT 275
RT 422 RT 1188 RT 632
Full Scan 4rsquo (or 5)-OH Diclofenac
XIC 3100032
Would high resolution ion mobility provide the orthogonal separation we need
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
Data Driven Strategy
bull Rigor of the analysis increases based on data Synthetic standard is needed only when the method fails or animal has less exposure than human
bull Conservative evaluation of MIST coverage
bull Assess MIST coverage at steady state without radio-label or synthetic standards enabling early assessment eg FIH
bull Time- and resource-saving strategy
Summary
bull LCMSMS peak area ratio comparison is scientifically sound and defensible method that can
ndash Ascertain the steady-state exposure of animals compared to humans (ie ldquoMIST coveragerdquo)
ndash Assess the MIST coverage without radio-label or synthetic standard
bull These methods are fit-for-purpose and can offer decision-making data useful for reliable estimation of metabolite exposure
bull Discussions with regulatory experts were positive and helpful
Gao et al (2013) AAPS Journal15(4) 970-973
Prerequisite for Current Methodology
bull Animal and human samples for side-by-side injection
minus In drug development first in human samples are typically ~1 year later than the toxicology study is done
bull Prior knowledge of human metabolites transferred from metabolite identification and scouting
minus Knowledge of major human metabolites may evolve only after in vivo human samples become available
minus Radio-labeled ADME is the most definitive study may take place late in the drug development process
Is it feasible to scan the samplesbank data and compare the data generated at different dates
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
MeO
O
O
OH
OH
OH
O OHNaproxen Acyl Glucuronide conjugate
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
(AZT)
Negative Control
Blind Test 3 Metabolite Cocktail
Gao et al Anal Chem 2015 87 11771-11776
Scanning and Banking DataSamples
bull Blind Test samples 22 metabolitesdrugs were spiked in human and rat plasmas (n=4 each)
bull No knowledge of the identities of the analytes and their concentrations in plasma were provided before sample analysis
bull Multiple analysis of the same samples (rat or human) using mixed matrix approach at Days 1 30 105 254 and 314
bull Treat the samples using the same IS solution in acetonitrile minus Freshly prepared to maintain the same concentration
bull Acquity UPLC generic gradient on C18 run time 25 min
bull Full scan and AIF scan on QExactive 70K at positive and negative polarity
bull Compare rat vs human samples analyzed at different dates
0 4 8 12 16 20 24 Time (min)
0
100 R
elat
ive
Ab
un
dan
ce
508
478 682
1132 1435 1450 763
773 243 1835 2120 2138 1164 1312 1638 288 534 1473 2338 802 2045 335 1062
42 43 44 45 46 47 48 49 50 51 52 53 54 55 Time (min)
0
100
Rel
ativ
e In
ten
sity
RT 508
RT 478
RT 495
RT 539
4 8 12 16 20 24 Time (min)
0
100 RT 624
RT 1313
RT 1113
RT 1626
RT 275 RT 879
RT 2008 RT 422
548
165 808
1 2
3
3
4
5
5
4
678
9 10 111213
14 16 15
1
2
3
4
56 7
Rel
ativ
e A
bu
nd
ance
Migrated naproxen acyl glucuronide
EIC 15 drugsmetabolites Resolution 70000 Extraction window 5ppm Polarity Positive
EIC 6 drugsmetabolites Resolution 70000 Extraction window 10 ppm Polarity Negative
2-OH Tacrine
4-OH Tacrine
1-OH Tacrine
Naproxen-Glu
Peak Area Ratio Measurement Variation
n=4 per species 5 independent analysis total n=40 per analyte
95 tile 037
Median 021
M1
0
DrugsMetabolites
Bank and Compare Data at Different Days
Gao et al Anal Chem 2015 87 11771-11776
Separation and Quantitative Analysis of Isomers
2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
100
Rela
tive I
nte
nsity
RT 1626
RT 275
RT 422 RT 1188 RT 632
Full Scan 4rsquo (or 5)-OH Diclofenac
XIC 3100032
Would high resolution ion mobility provide the orthogonal separation we need
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
Summary
bull LCMSMS peak area ratio comparison is scientifically sound and defensible method that can
ndash Ascertain the steady-state exposure of animals compared to humans (ie ldquoMIST coveragerdquo)
ndash Assess the MIST coverage without radio-label or synthetic standard
bull These methods are fit-for-purpose and can offer decision-making data useful for reliable estimation of metabolite exposure
bull Discussions with regulatory experts were positive and helpful
Gao et al (2013) AAPS Journal15(4) 970-973
Prerequisite for Current Methodology
bull Animal and human samples for side-by-side injection
minus In drug development first in human samples are typically ~1 year later than the toxicology study is done
bull Prior knowledge of human metabolites transferred from metabolite identification and scouting
minus Knowledge of major human metabolites may evolve only after in vivo human samples become available
minus Radio-labeled ADME is the most definitive study may take place late in the drug development process
Is it feasible to scan the samplesbank data and compare the data generated at different dates
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
MeO
O
O
OH
OH
OH
O OHNaproxen Acyl Glucuronide conjugate
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
(AZT)
Negative Control
Blind Test 3 Metabolite Cocktail
Gao et al Anal Chem 2015 87 11771-11776
Scanning and Banking DataSamples
bull Blind Test samples 22 metabolitesdrugs were spiked in human and rat plasmas (n=4 each)
bull No knowledge of the identities of the analytes and their concentrations in plasma were provided before sample analysis
bull Multiple analysis of the same samples (rat or human) using mixed matrix approach at Days 1 30 105 254 and 314
bull Treat the samples using the same IS solution in acetonitrile minus Freshly prepared to maintain the same concentration
bull Acquity UPLC generic gradient on C18 run time 25 min
bull Full scan and AIF scan on QExactive 70K at positive and negative polarity
bull Compare rat vs human samples analyzed at different dates
0 4 8 12 16 20 24 Time (min)
0
100 R
elat
ive
Ab
un
dan
ce
508
478 682
1132 1435 1450 763
773 243 1835 2120 2138 1164 1312 1638 288 534 1473 2338 802 2045 335 1062
42 43 44 45 46 47 48 49 50 51 52 53 54 55 Time (min)
0
100
Rel
ativ
e In
ten
sity
RT 508
RT 478
RT 495
RT 539
4 8 12 16 20 24 Time (min)
0
100 RT 624
RT 1313
RT 1113
RT 1626
RT 275 RT 879
RT 2008 RT 422
548
165 808
1 2
3
3
4
5
5
4
678
9 10 111213
14 16 15
1
2
3
4
56 7
Rel
ativ
e A
bu
nd
ance
Migrated naproxen acyl glucuronide
EIC 15 drugsmetabolites Resolution 70000 Extraction window 5ppm Polarity Positive
EIC 6 drugsmetabolites Resolution 70000 Extraction window 10 ppm Polarity Negative
2-OH Tacrine
4-OH Tacrine
1-OH Tacrine
Naproxen-Glu
Peak Area Ratio Measurement Variation
n=4 per species 5 independent analysis total n=40 per analyte
95 tile 037
Median 021
M1
0
DrugsMetabolites
Bank and Compare Data at Different Days
Gao et al Anal Chem 2015 87 11771-11776
Separation and Quantitative Analysis of Isomers
2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
100
Rela
tive I
nte
nsity
RT 1626
RT 275
RT 422 RT 1188 RT 632
Full Scan 4rsquo (or 5)-OH Diclofenac
XIC 3100032
Would high resolution ion mobility provide the orthogonal separation we need
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
Prerequisite for Current Methodology
bull Animal and human samples for side-by-side injection
minus In drug development first in human samples are typically ~1 year later than the toxicology study is done
bull Prior knowledge of human metabolites transferred from metabolite identification and scouting
minus Knowledge of major human metabolites may evolve only after in vivo human samples become available
minus Radio-labeled ADME is the most definitive study may take place late in the drug development process
Is it feasible to scan the samplesbank data and compare the data generated at different dates
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
MeO
O
O
OH
OH
OH
O OHNaproxen Acyl Glucuronide conjugate
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
(AZT)
Negative Control
Blind Test 3 Metabolite Cocktail
Gao et al Anal Chem 2015 87 11771-11776
Scanning and Banking DataSamples
bull Blind Test samples 22 metabolitesdrugs were spiked in human and rat plasmas (n=4 each)
bull No knowledge of the identities of the analytes and their concentrations in plasma were provided before sample analysis
bull Multiple analysis of the same samples (rat or human) using mixed matrix approach at Days 1 30 105 254 and 314
bull Treat the samples using the same IS solution in acetonitrile minus Freshly prepared to maintain the same concentration
bull Acquity UPLC generic gradient on C18 run time 25 min
bull Full scan and AIF scan on QExactive 70K at positive and negative polarity
bull Compare rat vs human samples analyzed at different dates
0 4 8 12 16 20 24 Time (min)
0
100 R
elat
ive
Ab
un
dan
ce
508
478 682
1132 1435 1450 763
773 243 1835 2120 2138 1164 1312 1638 288 534 1473 2338 802 2045 335 1062
42 43 44 45 46 47 48 49 50 51 52 53 54 55 Time (min)
0
100
Rel
ativ
e In
ten
sity
RT 508
RT 478
RT 495
RT 539
4 8 12 16 20 24 Time (min)
0
100 RT 624
RT 1313
RT 1113
RT 1626
RT 275 RT 879
RT 2008 RT 422
548
165 808
1 2
3
3
4
5
5
4
678
9 10 111213
14 16 15
1
2
3
4
56 7
Rel
ativ
e A
bu
nd
ance
Migrated naproxen acyl glucuronide
EIC 15 drugsmetabolites Resolution 70000 Extraction window 5ppm Polarity Positive
EIC 6 drugsmetabolites Resolution 70000 Extraction window 10 ppm Polarity Negative
2-OH Tacrine
4-OH Tacrine
1-OH Tacrine
Naproxen-Glu
Peak Area Ratio Measurement Variation
n=4 per species 5 independent analysis total n=40 per analyte
95 tile 037
Median 021
M1
0
DrugsMetabolites
Bank and Compare Data at Different Days
Gao et al Anal Chem 2015 87 11771-11776
Separation and Quantitative Analysis of Isomers
2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
100
Rela
tive I
nte
nsity
RT 1626
RT 275
RT 422 RT 1188 RT 632
Full Scan 4rsquo (or 5)-OH Diclofenac
XIC 3100032
Would high resolution ion mobility provide the orthogonal separation we need
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
Is it feasible to scan the samplesbank data and compare the data generated at different dates
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
MeO
O
O
OH
OH
OH
O OHNaproxen Acyl Glucuronide conjugate
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
(AZT)
Negative Control
Blind Test 3 Metabolite Cocktail
Gao et al Anal Chem 2015 87 11771-11776
Scanning and Banking DataSamples
bull Blind Test samples 22 metabolitesdrugs were spiked in human and rat plasmas (n=4 each)
bull No knowledge of the identities of the analytes and their concentrations in plasma were provided before sample analysis
bull Multiple analysis of the same samples (rat or human) using mixed matrix approach at Days 1 30 105 254 and 314
bull Treat the samples using the same IS solution in acetonitrile minus Freshly prepared to maintain the same concentration
bull Acquity UPLC generic gradient on C18 run time 25 min
bull Full scan and AIF scan on QExactive 70K at positive and negative polarity
bull Compare rat vs human samples analyzed at different dates
0 4 8 12 16 20 24 Time (min)
0
100 R
elat
ive
Ab
un
dan
ce
508
478 682
1132 1435 1450 763
773 243 1835 2120 2138 1164 1312 1638 288 534 1473 2338 802 2045 335 1062
42 43 44 45 46 47 48 49 50 51 52 53 54 55 Time (min)
0
100
Rel
ativ
e In
ten
sity
RT 508
RT 478
RT 495
RT 539
4 8 12 16 20 24 Time (min)
0
100 RT 624
RT 1313
RT 1113
RT 1626
RT 275 RT 879
RT 2008 RT 422
548
165 808
1 2
3
3
4
5
5
4
678
9 10 111213
14 16 15
1
2
3
4
56 7
Rel
ativ
e A
bu
nd
ance
Migrated naproxen acyl glucuronide
EIC 15 drugsmetabolites Resolution 70000 Extraction window 5ppm Polarity Positive
EIC 6 drugsmetabolites Resolution 70000 Extraction window 10 ppm Polarity Negative
2-OH Tacrine
4-OH Tacrine
1-OH Tacrine
Naproxen-Glu
Peak Area Ratio Measurement Variation
n=4 per species 5 independent analysis total n=40 per analyte
95 tile 037
Median 021
M1
0
DrugsMetabolites
Bank and Compare Data at Different Days
Gao et al Anal Chem 2015 87 11771-11776
Separation and Quantitative Analysis of Isomers
2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
100
Rela
tive I
nte
nsity
RT 1626
RT 275
RT 422 RT 1188 RT 632
Full Scan 4rsquo (or 5)-OH Diclofenac
XIC 3100032
Would high resolution ion mobility provide the orthogonal separation we need
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
SN
N N
N
O
Cl
NH
Cl
Cl
O
OH
N
OCH3
CH3
NOH
OH
N
NH2
NH N O OH
N3
O
O
ziprasidone
diclofenac tacrine
terfenadine
dextromethorphan
zidovudine
MeO
O
O
OH
OH
OH
O OHNaproxen Acyl Glucuronide conjugate
S-Oxidation Reduction+Methylation N-Dealkylation
Hydroxylation Hydroxylation
Oxidation to Carboxylic Acid
Demethylation Glucuronidation
(AZT)
Negative Control
Blind Test 3 Metabolite Cocktail
Gao et al Anal Chem 2015 87 11771-11776
Scanning and Banking DataSamples
bull Blind Test samples 22 metabolitesdrugs were spiked in human and rat plasmas (n=4 each)
bull No knowledge of the identities of the analytes and their concentrations in plasma were provided before sample analysis
bull Multiple analysis of the same samples (rat or human) using mixed matrix approach at Days 1 30 105 254 and 314
bull Treat the samples using the same IS solution in acetonitrile minus Freshly prepared to maintain the same concentration
bull Acquity UPLC generic gradient on C18 run time 25 min
bull Full scan and AIF scan on QExactive 70K at positive and negative polarity
bull Compare rat vs human samples analyzed at different dates
0 4 8 12 16 20 24 Time (min)
0
100 R
elat
ive
Ab
un
dan
ce
508
478 682
1132 1435 1450 763
773 243 1835 2120 2138 1164 1312 1638 288 534 1473 2338 802 2045 335 1062
42 43 44 45 46 47 48 49 50 51 52 53 54 55 Time (min)
0
100
Rel
ativ
e In
ten
sity
RT 508
RT 478
RT 495
RT 539
4 8 12 16 20 24 Time (min)
0
100 RT 624
RT 1313
RT 1113
RT 1626
RT 275 RT 879
RT 2008 RT 422
548
165 808
1 2
3
3
4
5
5
4
678
9 10 111213
14 16 15
1
2
3
4
56 7
Rel
ativ
e A
bu
nd
ance
Migrated naproxen acyl glucuronide
EIC 15 drugsmetabolites Resolution 70000 Extraction window 5ppm Polarity Positive
EIC 6 drugsmetabolites Resolution 70000 Extraction window 10 ppm Polarity Negative
2-OH Tacrine
4-OH Tacrine
1-OH Tacrine
Naproxen-Glu
Peak Area Ratio Measurement Variation
n=4 per species 5 independent analysis total n=40 per analyte
95 tile 037
Median 021
M1
0
DrugsMetabolites
Bank and Compare Data at Different Days
Gao et al Anal Chem 2015 87 11771-11776
Separation and Quantitative Analysis of Isomers
2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
100
Rela
tive I
nte
nsity
RT 1626
RT 275
RT 422 RT 1188 RT 632
Full Scan 4rsquo (or 5)-OH Diclofenac
XIC 3100032
Would high resolution ion mobility provide the orthogonal separation we need
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
Scanning and Banking DataSamples
bull Blind Test samples 22 metabolitesdrugs were spiked in human and rat plasmas (n=4 each)
bull No knowledge of the identities of the analytes and their concentrations in plasma were provided before sample analysis
bull Multiple analysis of the same samples (rat or human) using mixed matrix approach at Days 1 30 105 254 and 314
bull Treat the samples using the same IS solution in acetonitrile minus Freshly prepared to maintain the same concentration
bull Acquity UPLC generic gradient on C18 run time 25 min
bull Full scan and AIF scan on QExactive 70K at positive and negative polarity
bull Compare rat vs human samples analyzed at different dates
0 4 8 12 16 20 24 Time (min)
0
100 R
elat
ive
Ab
un
dan
ce
508
478 682
1132 1435 1450 763
773 243 1835 2120 2138 1164 1312 1638 288 534 1473 2338 802 2045 335 1062
42 43 44 45 46 47 48 49 50 51 52 53 54 55 Time (min)
0
100
Rel
ativ
e In
ten
sity
RT 508
RT 478
RT 495
RT 539
4 8 12 16 20 24 Time (min)
0
100 RT 624
RT 1313
RT 1113
RT 1626
RT 275 RT 879
RT 2008 RT 422
548
165 808
1 2
3
3
4
5
5
4
678
9 10 111213
14 16 15
1
2
3
4
56 7
Rel
ativ
e A
bu
nd
ance
Migrated naproxen acyl glucuronide
EIC 15 drugsmetabolites Resolution 70000 Extraction window 5ppm Polarity Positive
EIC 6 drugsmetabolites Resolution 70000 Extraction window 10 ppm Polarity Negative
2-OH Tacrine
4-OH Tacrine
1-OH Tacrine
Naproxen-Glu
Peak Area Ratio Measurement Variation
n=4 per species 5 independent analysis total n=40 per analyte
95 tile 037
Median 021
M1
0
DrugsMetabolites
Bank and Compare Data at Different Days
Gao et al Anal Chem 2015 87 11771-11776
Separation and Quantitative Analysis of Isomers
2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
100
Rela
tive I
nte
nsity
RT 1626
RT 275
RT 422 RT 1188 RT 632
Full Scan 4rsquo (or 5)-OH Diclofenac
XIC 3100032
Would high resolution ion mobility provide the orthogonal separation we need
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
0 4 8 12 16 20 24 Time (min)
0
100 R
elat
ive
Ab
un
dan
ce
508
478 682
1132 1435 1450 763
773 243 1835 2120 2138 1164 1312 1638 288 534 1473 2338 802 2045 335 1062
42 43 44 45 46 47 48 49 50 51 52 53 54 55 Time (min)
0
100
Rel
ativ
e In
ten
sity
RT 508
RT 478
RT 495
RT 539
4 8 12 16 20 24 Time (min)
0
100 RT 624
RT 1313
RT 1113
RT 1626
RT 275 RT 879
RT 2008 RT 422
548
165 808
1 2
3
3
4
5
5
4
678
9 10 111213
14 16 15
1
2
3
4
56 7
Rel
ativ
e A
bu
nd
ance
Migrated naproxen acyl glucuronide
EIC 15 drugsmetabolites Resolution 70000 Extraction window 5ppm Polarity Positive
EIC 6 drugsmetabolites Resolution 70000 Extraction window 10 ppm Polarity Negative
2-OH Tacrine
4-OH Tacrine
1-OH Tacrine
Naproxen-Glu
Peak Area Ratio Measurement Variation
n=4 per species 5 independent analysis total n=40 per analyte
95 tile 037
Median 021
M1
0
DrugsMetabolites
Bank and Compare Data at Different Days
Gao et al Anal Chem 2015 87 11771-11776
Separation and Quantitative Analysis of Isomers
2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
100
Rela
tive I
nte
nsity
RT 1626
RT 275
RT 422 RT 1188 RT 632
Full Scan 4rsquo (or 5)-OH Diclofenac
XIC 3100032
Would high resolution ion mobility provide the orthogonal separation we need
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
Peak Area Ratio Measurement Variation
n=4 per species 5 independent analysis total n=40 per analyte
95 tile 037
Median 021
M1
0
DrugsMetabolites
Bank and Compare Data at Different Days
Gao et al Anal Chem 2015 87 11771-11776
Separation and Quantitative Analysis of Isomers
2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
100
Rela
tive I
nte
nsity
RT 1626
RT 275
RT 422 RT 1188 RT 632
Full Scan 4rsquo (or 5)-OH Diclofenac
XIC 3100032
Would high resolution ion mobility provide the orthogonal separation we need
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
Bank and Compare Data at Different Days
Gao et al Anal Chem 2015 87 11771-11776
Separation and Quantitative Analysis of Isomers
2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
100
Rela
tive I
nte
nsity
RT 1626
RT 275
RT 422 RT 1188 RT 632
Full Scan 4rsquo (or 5)-OH Diclofenac
XIC 3100032
Would high resolution ion mobility provide the orthogonal separation we need
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
Separation and Quantitative Analysis of Isomers
2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0
100
Rela
tive I
nte
nsity
RT 1626
RT 275
RT 422 RT 1188 RT 632
Full Scan 4rsquo (or 5)-OH Diclofenac
XIC 3100032
Would high resolution ion mobility provide the orthogonal separation we need
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
Ideal Paradigm to Address MIST
Un-biased scan the sample using super LCMS
Process post-acquisition data when the knowledge of analytes is available
Bank Data
Address biological questions using peak
area ratio measurements at different dates
Calibrate the ion response using a synthetic standard
when concentration measurement is needed
Samples may degrade data will not
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
Necessity is the mother of invention
Laziness is the father of invention
Efficiency
Challenges in real life applications drive innovation and technology advances
Next generation LCMS will change how we do analysis in pharmabiotech industry research labs
Gao et al (2017) JASMS 281248-1249
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
Looking for innovative BA approaches to address DMPK issues
Contact hongyinggaoinnovobioanalysiscom
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
Backups
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
Rat Day1
Human Day 1 Rat same day (n=5)
Human
Rat Day105
Human Day 1
Rat Day 314
Human Day 1
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites
1
2
3
4
5
6
Tre
ate
d n
apro
xe
n
Tre
ate
d n
apro
xe
n a
cyl
glu
cu
ron
ide
Untr
eate
d n
ap
roxen
Untr
eate
d n
ap
roxen
acyl
glu
cu
ron
ide
Tre
ate
d s
alic
ylic
acid
Tre
ate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Un
treate
d s
alic
ylic
acid
Untr
eate
d 2
3-
dih
yd
roxyb
en
zo
ic a
cid
Carb
am
aze
pin
e
Carb
am
aze
pin
e-1
01
1-
epo
xid
e
Sulfa
dia
zin
e
N-A
ce
tyl sulfa
dia
zin
e
Tre
ate
d m
eth
ylp
he
nid
ate
Tre
ate
d r
italin
ic a
cid
Untr
eate
dm
eth
ylp
hen
ida
te
Untr
eate
d r
ita
linic
acid
Nic
otin
e I
min
ium
io
n
Cotinin
e
MS
NM
Ra
tio
Measurement Error for Unstable Metabolites