comprehensive investigation of the utilization of sfc/esi positive mode ms for chiral and achiral...

©2015 Waters Corporation 1

Comprehensive Investigation of the Utilization

of SFC/ ESI Positive Mode MS

for Chiral and Achiral Bioanalytical Studies

Paul D. Rainville Ph.D.


Challenges in DMPK

Sensitivity

Sample type

Selectivity Regulatory

Robustness


Selectivity - RPLC vs. CC

Time0.50 1.00 1.50 2.00 2.50 3.00 3.50

%

0

3.19

1.31

0.85

1.83

1.48

2.17

2.00

Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80

%

4

15Aug2011_fresh solution_replicate 4 MRM of 6 Channels ES+ TIC

2.07e50.86

0.57

0.96

1.51

1.36

1.30

Ranitidine

Lidocaine

OmeprazoleClopidogrel

test mix 12.5pg/50pg

Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80

%

3

09Sep2011_008 MRM of 7 Channels ES+ TIC

1.68e6

1.44

0.73

0.48

1.23

0.851.29

1.61

WarfarinTolbutamide

Alprazolam


Clopidogrel - UPLC

Parents of 184m/z - UPLC

Reversed-phase separation Analyte co-elution with background phospholipids

Even the most sensitive MS can suffer from matrix interferences, especially in a region that contains endogenous interferences

Simeone J, Rainville P, Waters Tech Brief


Clopidogrel – UPC2

Parents of 184m/z - UPC2

Selectivity Analyte separation from background phospholipids

UPC2 provides orthogonal selectivity to RP-LC, moving the analyte of interest away from endogenous matrix interferences

Clopidogrel - UPLC

Parents of 184m/z - UPLC


General Met ID use case Buspirone UPLC

Parent Drug

+O

+2O

Researchers typically have to estimate based on parent drug retention that they enough room for unknown polar metabolites to be retained and separated


General Met ID use case Buspirone UPC2

Inversion of retention, all metabolites elute after the parent drug, most metabolites are MORE retentive than parent

Parent Drug

+O

+2O


Time-0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00

%

0

-0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00

%

0

100

-0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00

%

0

100R S

R S

Incubated sample t = 0

Incubated sample t = 60

propranolol

4-hydroxypropranolol

S propranolol

S 4-hydroxypropranolol

Rat Separation of chiral metabolites of propranolol

Pure standards

“It should be appreciated that toxicity or unusual pharmacologic properties might reside not in the parent isomer, but in an isomer-specific metabolite” Development of New Stereoisomeric Drugs 5/1/1992 http://www.fda.gov/drugs/guidancecomplianceregulatoryinformation/guidances/ucm122883.htm


Time0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90

%

19

0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90

%

0

100

0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90%

0

1000.60

0.860.91

0.60

0.56

0.82 0.85

0.520.15

0.12

0.030.05

0.430.310.29

0.280.19

0.27

0.360.41

0.46

0.57 0.60

0.64

0.650.75 0.860.82

0.75

0.77

0.960.94

UPLC - PPT 1µL injection

UPLC - PPT 3µL injection

Minimal fronting – peak shape is adequate

Caffeine

Direct Injection of Highly Organic Extracts

UPLC

Caffeine 1µL

Max Injection Volume

Direct injection of protein PPT samples (3:1 ACN crash) can be difficult with RP-LC, as highly organic extracts affect peak shape as injection

volume increases

3 more molecules Ranitidine 1 µL, poor peak shape

Fluconazole 3 µL

Acetaminophen 1 µL, poor peak shape Larger injection

volumes cause peak distortion (splitting)


With UPC2 no solvent effect is observed even for 7 µL injection

Higher retention of polar molecule

Direct Injection of Highly Organic Extracts

UPLC UPC2

Caffeine 1µL 7µL

Ranitidine

1µL, poor peak shape

10µL

Fluconazole 3µL

5µL

Acetaminophen 1 µL, poor peak shape

7µL

Max Injection Volume

Caffeine

UPC2 - PPT 7 µL injection

UPC2 - PPT 1 µL injection

Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00

%

3

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00

%

1

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00

%

0

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00

%

0

1.02

1.01

1.02

1.06

Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00

%

3

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00

%

1

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00

%

0

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00

%

0

1.02

1.01

1.02

1.06


Typical Sample Preparation Procedures for Lipid Analysis

Liquid-liquid extraction using chloroform/MeOH (2/1)

–Folch method / Blight and Dyer method

RPLC: phase transfer required to be able to injected onto RP system

UPC2: phase transfer process can be eliminated by injecting directly

onto a UPC2 system


Comparison of GC/MS and UPC2 methods for the determination of fatty acids in whole blood samples

Fatty acid profiling of biological samples has gained tremendous

importance in order to understand patient dietary lipid profiles

in relation to disease states.

GC/MS, or GC/FID methods have become important tools

Decreased need for sample preparation has been implemented

in this study

In this study a simplified sample preparation method was

compared using UPC2-MS with a classical derivatization method

GC/MS


Last FAME eluted

after 8.5 minutes

Time between

injections 18 minutes

GC-MS FAME method C16 – C22


Analysis of FFA in blood

All FFA

eluted in

<1.5 min.

Time

between

injections

4.5 minutes

Further

posID of all

FFA needed,

but not

pursued in

this study

due to lack

of standards


Prostaglandins: Background

PGE2 and 8-iso PGE2 are diastereomers

Both contain 20 carbon atoms with a 5-carbon ring.

Prostaglandin E2 (PGE2)

8-iso Prostaglandin E2 (8-iso PGE2)


Challenges for PG Separation

Prostaglandins (8-iso PGE2 and PGE2) can be separated with

non-chiral columns

Very long chromatographic time (>40min)

Separation of 8-iso PGE2 and PGE2 on Luna C18 column ( 150x2mm, Phenomenex) coupled to QQQ

Stephen A. Brose, Brock T. Thuen, and Mikhail Y. Golovko J Lipid Res. 2011 April; 52(4): 850–859.


Fast Separation of Prostaglandin Diastereomers Using UPC2

UPC2 separation of prostaglandins on a non-chiral BEH column

2 min time scale

PGE2

8-iso PGE2

8-iso PGE2 + PGE2

Prostaglandin E2 8-iso Prostaglandin E2


Eicosanoids: Background

12R and 12S-HETE are enantiomers (chiral)

Biologically important in inflammation (ω-6 eicosanoids pro-

and ω-3 are anti-inflammatory).

Separation of such enantiomers is difficult by RP-LC even with a

50 min gradient

Karen A. Massey, Anna Nicolaou, Free Radic Biol Med. 2013 Jun;59:45-55.


Fast Separation of Eicosanoid Enantiomers Using UPC2

Separation of 12(R)-HETE and 12-(S)-HETE on Chiralpak IA-3 and Chiralpak ID-3 columns.

12(R)-HETE 12(S)-HETE 12(R)-HETE 12(S)-HETE

column ID-3 column IA-3


Simplifying BioA Workflows

Convergence chromatography simplifies the DMPK workflow by:

– Reducing sample preparation and analysis times

o Direct injection of organic solvent extracts (PPT, LLE, SPE, etc.)

Add Extraction Solvent

Transfer to new vessel

Evaporate to dryness Risk for thermally unstable analytes/metabolites to degrade

Reconstitute in aqueous to match RP starting conditions

Solubility issues may cause incomplete dissolution

Directly inject extract onto UPC2 system

• Removes two steps which may lead to losses in sensitivity and cause reproducibility issues

• Reduction in extraction time

For a PPT extraction, direct injection removes the need to dilute sample (and impact sensitivity) with aqueous prior to injection

A typical LLE workflow

Vortex then Centrifuge

http://www.google.com/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=NNE2k5tJiWMRaM&tbnid=6NUvubOU6y2e1M:&ved=0CAUQjRw&url=http://www.3tailer.com/venous-blood-collection-tube-bd-vacutainer-plasma-tube-3-ml-13-x-75-mm-light-green-top-bx-100&ei=4ALfUY2ANdi14AOZ9oHQCw&bvm=bv.48705608,d.dmg&psig=AFQjCNGx7NXZTezDLEKIMmfrmszi2_oB5Q&ust=1373656125262816


%CV averaged over three days, N = 18

meets guidelines for method validation Sensitivity better than that achieved with UPLC-Xevo TQ-S

Linear with r2 in the range of 25 – 5000 pg/mL

Avg. QC LLOQ

Avg. QC LOW Avg. QC MID Avg. QC HIGH

5.13 6.70 4.90 5.90

Regulated Bioanalysis with UPC2 3 day validation studies of Clopidogrel with LLE

QC LLOQ

QC LOW QC MID

QC HIGH

25.0 pg/mL

75.0 pg/mL

350 pg/mL

3500 pg/mL

23.6 71.6 355 3384

23.4 76.5 358 3422

25.0 67.2 363 3299

22.0 63.6 349 3237

25.9 72.8 333 3558

23.3 74.0 346 3629

Mean 23.9 71.0 351 3422

St Dev 1.38 4.73 10.5 150

% CV 5.8 6.7 3.0 4.4

% Bias -4.5 -5.4 -6.5 -8.8

QC LLOQ

QC LOW QC MID

QC HIGH

25.0 pg/mL

75.0 pg/mL

350 pg/mL

3500 pg/mL

21.4 74.6 404 3464

22.3 80.4 379 3577

21.8 66.8 390 3395

20.5 68.9 370 3387

21.7 74.2 343 3647

21.3 69.9 361 3459

Mean 21.5 72.5 374 3488

St Dev 0.60 4.94 21.5 103

% CV 2.8 6.8 5.7 3.0

% Bias -14.0 -3.4 -0.1 -7.0

QC LLOQ

QC LOW QC MID

QC HIGH

25.0 pg/mL

75.0 pg/mL

350 pg/mL

3500 pg/mL

28.0 69.8 358 3083

27.1 63.2 337 3684

25.1 74.3 340 3257

27.7 68.3 346 3940

30.3 75.7 395 3967

29.9 67.7 347 3857

Mean 28.0 69.8 354 3631

St Dev 1.91 4.59 21.3 375

% CV 6.8 6.6 6.0 10.3

% Bias 12.1 -6.9 -5.7 -3.2


Convergence Chromatography/MS in DMPK

Provides an orthogonal separation technique to reversed-phase chromatography – Reduce potential matrix interferences

– May provide better retention for polar metabolites

Simplifies sample analysis workflows: – Combines multiple techniques (GC/NP/RP) into ONE analytical

platform

– Reduces sample prep and analysis times to streamline the analytical workflow

o Direct injection of organic solvents/extracts

o Reduces solvent usage

o No derivatization required for free fatty acid analysis

Separates compounds with structural similarity – Optical isomers, positional isomers, structural analogs, conjugates


Acknowledgements

Giorgis Isaacs

Jennifer Simeone

Michael Jones

Steven Lai

Hernando Olivos

Adam Ladak

comprehensive investigation of the utilization of sfc/esi positive mode ms for chiral and achiral...

Science

waters corporation

injection uplc ppt

mz uplc

parent parent drug o

parent drug retention

parent isomer

waters tech brief

clopidogrel upc2 parents