Evaluation of a LCEvaluation of a LC--MS/MS method MS/MS method for quantitative amino acid analysisfor quantitative amino acid analysis
Patrice K. Held, Ph.D.Patrice K. Held, Ph.D.
Assistant Professor of PathologyAssistant Professor of Pathology
University of Utah School of MedicineUniversity of Utah School of Medicine
Assistant Medical Director, Biochemical Genetics LaboratoryAssistant Medical Director, Biochemical Genetics Laboratory
Clinical Indications for Clinical Indications for Amino Acid AnalysisAmino Acid Analysis
Amino acid analysis should be considered in manyAmino acid analysis should be considered in manyclinical situations, includingclinical situations, including……
Lethargy, coma, seizures, or vomiting Lethargy, coma, seizures, or vomiting HyperammonemiaHyperammonemiaMetabolic acidosis or lactic Metabolic acidosis or lactic acidemiaacidemiaMetabolic Metabolic decompensationdecompensationUnexplained developmental delayUnexplained developmental delayAbnormal amino acid results by newborn screeningAbnormal amino acid results by newborn screeningDiet monitoring in patients with known metabolic Diet monitoring in patients with known metabolic disordersdisorders
Ion Exchange Chromatography
InjectionPort
Inlet
Separation Detection
IE column UV detectorDerivatization
SamplePrep
Sample PrepSample Prep Ion Exchange ChromatographyIon Exchange Chromatography
PlasmaPlasmaSamples are Samples are deproteinizeddeproteinized with with sulphosalycilicsulphosalycilic acid prior acid prior to injectionto injection
CSFCSFSamples are Samples are deproteinizeddeproteinized with with sulphosalycilicsulphosalycilic acid prior acid prior to injectionto injection
UrineUrineCreatinineCreatinine is measured and samples are lyophilized and is measured and samples are lyophilized and resuspendedresuspended to a certain concentration per gram of to a certain concentration per gram of creatininecreatinineSamples are Samples are deproteinizeddeproteinized with with sulphosalycilicsulphosalycilic acid prior acid prior to injectionto injection
Separation is affected by:1) pKa
values of the side chains R
2) interaction of the side chains with the matrix3) temperature4) ionic strength
+ Li++ +H3
N-CHRCOOHMatrix-SO3-Li+ Matrix-SO3
-+H3
N-CHRCOOH
SeparationSeparation Ion Exchange ChromatographyIon Exchange Chromatography
NinhydrinNinhydrin detectiondetectionEluting amino acids are mixed with Eluting amino acids are mixed with ninhydrinninhydrin and and flow through a reaction coil. The high temperature flow through a reaction coil. The high temperature in the coil facilitates the development of a purple in the coil facilitates the development of a purple color.color.
The The derivatizedderivatized amino acids go through a UV amino acids go through a UV detector. The concentration of the amino acids is detector. The concentration of the amino acids is proportional to the intensity of the purple color.proportional to the intensity of the purple color.
2 wavelengths are used: 570 nm (amino acids) and 2 wavelengths are used: 570 nm (amino acids) and 440 nm (440 nm (iminoimino acids, such as acids, such as prolineproline and and hydroxyprolinehydroxyproline))
DetectionDetection Ion Exchange ChromatographyIon Exchange Chromatography
IECIEC--Plasma Amino Acid ProfilePlasma Amino Acid Profile
Minutes
0 10 20 30 40 50 60 70 80 90 100 110 120 130
mVo
lts
125
150
175
200
225
250
275
300
325
350
375
400
425
450
mVo
lts
125
150
175
200
225
250
275
300
325
350
375
400
425
450
Phs
er Taur
Pea
Ure
a
Asp
Thr
Ser
Asn
Glu
Gln
(Sar
c)
AA
AA
G
lyA
la
Cit
aaba
Val
Cys Met
(Cys
th 1
)(C
ysth
2)
Ile
Leu
Ty
r(b
-ala
)
Phe
(aai
ba)
(Hcy
s)
(Gab
a)
(Eth
an)
Am
m
(Hyl
ys)
ISO
rn
Lys
1-M
his His
3-M
his
(Ans
)
(Car
) Arg
Hyp
ro
Pro
IS
570nmPL150 09.029.126663 DALTON L
Name
440nmPL150 09.029.126663 DALTON L
Name
Ion Exchange ChromatographyIon Exchange ChromatographyAdvantagesAdvantages
ReproducibilityReproducibilityHigh resolutionHigh resolutionMinimal sample preparationMinimal sample preparationLarge linear dynamic range Large linear dynamic range
1 1 umolumol/L to 4000 /L to 4000 umolumol/L/Lapproximately 3approximately 3--4 orders of magnitude4 orders of magnitude
Identification of atypical amino acidsIdentification of atypical amino acids
DisadvantagesDisadvantagesRun time (2.5 hours per sample)Run time (2.5 hours per sample)Interferences within urine samples Interferences within urine samples Identification of atypical amino acidsIdentification of atypical amino acids
Amino Acid Analysis byAmino Acid Analysis by Tandem Mass SpectrometryTandem Mass Spectrometry
Automated tandem mass spectrometry for mass newborn screening foAutomated tandem mass spectrometry for mass newborn screening for disorders in fatty acid, r disorders in fatty acid, organic acid, and amino acid metabolism.organic acid, and amino acid metabolism.Naylor EW, Naylor EW, ChaceChace
DH.DH.J Child NeurolJ Child Neurol. 1999 Nov;14 . 1999 Nov;14 SupplSuppl
1:S41:S4--8.8.
Development of a method for rapid Development of a method for rapid quantitationquantitation
of amino acids by liquid chromatographyof amino acids by liquid chromatography--tandem tandem mass spectrometry (LCmass spectrometry (LC--MSMS) in plasma.MSMS) in plasma.Casetta B, Casetta B, TagliacozziTagliacozzi
D, D, ShushanShushan
B, B, FedericiFederici
G.G.ClinClin
ChemChem
Lab MedLab Med. 2000 May;38(5):391. 2000 May;38(5):391--401.401.
Rapid comprehensive amino acid analysis by liquid chromatographyRapid comprehensive amino acid analysis by liquid chromatography/tandem mass spectrometry: /tandem mass spectrometry: comparison to comparison to cationcation
exchange with postexchange with post--column column ninhydrinninhydrin
detection.detection.DietzenDietzen
DJ, DJ, WeindelWeindel
AL, AL, CarayannopoulosCarayannopoulos
MO, MO, LandtLandt
M, M, NormansellNormansell
ET, ET, ReimschiselReimschisel
TE, Smith CH.TE, Smith CH.Rapid Rapid CommunCommun
Mass Mass SpectromSpectrom. 2008 Nov;22(22):3481. 2008 Nov;22(22):3481--8.8.
iTRAQ Reagent ReactioniTRAQ Reagent Reaction
LabeledAminoAcid
AminoAcid
NH2 O H
O
R
OHN
O
O
+
iTRAQReagent
N
N
ON
O
O
O
N
N
O
NH
O H
O
R
Reporter group(mass 114 or 115)
Balance group(mass 30 or 31)
Amino acid Reactive group
Courtesy of Applied Biosystems
+
iTRAQiTRAQ
Reagent MethodologyReagent Methodology
115 30 AARG Amino Acid(patient)
Label 115 30 Amino Acid(patient)
114 31 AARG Amino Acid(standard)
Label 114 31 Amino Acid(standard)
0.0E+00
1.0E+04
2.0E+04
3.0E+04
4.0E+04
5.0E+04
3.0 3.2 3.4 3.6 3.8 4.0
Time (min)
Standard
Sample
0.0E+00
1.0E+04
2.0E+04
3.0E+04
4.0E+04
5.0E+04
3.0 3.2 3.4 3.6 3.8 4.0
Time (min)
Standard
Sample
0.0E+00
1.0E+04
2.0E+04
3.0E+04
4.0E+04
5.0E+04
3.0 3.2 3.4 3.6 3.8 4.0
Time (min)
Standard (transition 345→114)
Patient (transition 345→115)×
114standardAmountArea
AreaAmount ×patient
×= 115
Courtesy of Applied Biosystems
Details of the Analysis MethodDetails of the Analysis MethodAPI 4000API 4000 with Shimadzu HPLCwith Shimadzu HPLCMobile Phase AMobile Phase A-- 0.1% formic acid, 0.01% 0.1% formic acid, 0.01% heptafluorobutyricheptafluorobutyric acid in wateracid in waterMobile Phase BMobile Phase B-- 0.1% formic acid, 0.01% 0.1% formic acid, 0.01% heptafluorobutyricheptafluorobutyric acid in acid in methanolmethanol
C18 Column heated to 50 C18 Column heated to 50 °°CCFlow rate 0.8 mL/minFlow rate 0.8 mL/min
XIC of +MRM (26 pairs): 286.1/114.1 amu from Sample 2 (SST002) of 081117.wiff (Turbo Spray) Max. 1440.0 cps.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 2578 155 232 309 385 462 539 616 693 770 847 924 1000 1077 1154 1231 1308 1385 1462 1539 1615 1692 1769 1846 1923
Time, min
0.0
5.0e4
1.0e5
1.5e5
2.0e5
2.5e5
3.0e5
3.5e5
4.0e5
4.5e5
5.0e5
5.5e5
6.0e5
6.5e5
7.0e5
7.5e5
8.0e5
4.392.58 14.4213.5612.066.44 6.675.79 8.673.93 11.030.25 16.15 23.30
Sample Name: "250" Sample ID: "" File: "081118.wiff"Peak Name: "Phe" Mass(es): "310.2/115.1 amu"Comment: "" Annotation: ""
Sample Index: 3 Sample Type: Standard Concentration: 0.0000 umol/L Calculated Conc: 0.00 umol/L Acq. Date: 11/18/2008 Acq. Time: 1:52:07 PM Modified: No Proc. Algorithm: IntelliQuan - MQII Noise Percentage: 50 Base. Sub. Window: 1.00 minPeak-Split. Factor: 2 Report Largest Peak: No Min. Peak Height: 500.00 cpsMin. Peak Width: 0.00 secSmoothing Width: 3 pointsRT Window: 240. secExpected RT: 10.4 minUse Relative RT: No Int. Type: Valley Retention Time: 10.1 minArea: 8.4721e+006 countsHeight: 1.52e+006 cpsStart Time: 9.90 minEnd Time: 10.6 min
8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.016 55 94 133 171 210 249 288 327
Time, min
0.0
5.0e4
1.0e5
1.5e5
2.0e5
2.5e5
3.0e5
3.5e5
4.0e5
4.5e5
5.0e5
5.5e5
6.0e5
6.5e5
7.0e5
7.5e5
8.0e5
8.5e5
9.0e5
9.5e5
1.0e6
1.1e6
1.1e6
1.2e6
1.2e6
1.3e6
1.3e6
1.4e6
1.4e6
1.5e6
1.5e6
Intensity, cps
10.13
Sample Name: "250" Sample ID: "" File: "081118.wiff"Peak Name: "ISTD (Phe)(IS)" Mass(es): "310.2/114.1 amu"Comment: "" Annotation: ""
Sample Index: 3 Sample Type: Standard Concentration: 1.0000 umol/L Calculated Conc: N/A Acq. Date: 11/18/2008 Acq. Time: 1:52:07 PM Modified: No Proc. Algorithm: IntelliQuan - MQII Noise Percentage: 50 Base. Sub. Window: 1.00 minPeak-Split. Factor: 2 Report Largest Peak: No Min. Peak Height: 500.00 cpsMin. Peak Width: 0.00 secSmoothing Width: 3 pointsRT Window: 240. secExpected RT: 10.4 minUse Relative RT: No Int. Type: Valley Retention Time: 10.1 minArea: 3.1623e+006 countsHeight: 5.75e+005 cpsStart Time: 9.90 minEnd Time: 10.5 min
8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.016 55 94 133 171 210 249 288 327
Time, min
0.0
2.0e4
4.0e4
6.0e4
8.0e4
1.0e5
1.2e5
1.4e5
1.6e5
1.8e5
2.0e5
2.2e5
2.4e5
2.6e5
2.8e5
3.0e5
3.2e5
3.4e5
3.6e5
3.8e5
4.0e5
4.2e5
4.4e5
4.6e5
4.8e5
5.0e5
5.2e5
5.4e5
5.6e5
Intensity, cps
10.13
Patient IS
354 115 354 114
Comparison of dataComparison of data
Reproducibility Reproducibility (Inter(Inter--assay variation)assay variation)IECIEC <5% (CV) for most amino acids<5% (CV) for most amino acidsiTRAQiTRAQ/MSMS/MSMS <10% (CV) for most amino acids<10% (CV) for most amino acids
RecoveryRecoveryiTRAQiTRAQ/MSMS/MSMS Ranged from 80% up to 110%Ranged from 80% up to 110%
Linear Dynamic Range & Linear Dynamic Range & Low Limit of DetectionLow Limit of DetectionIECIEC 1 1 umolumol/L up to 4000 /L up to 4000 umolumol/L/LiTRAQiTRAQ/MSMS/MSMS 5 5 umolumol/L up to 700 /L up to 700 umolumol/L/L
AdvantagesiTRAQ reagent, LC-MS/MS method
Run Time (25 minutes)Run Time (25 minutes)
Although the initial sample preparation time is longer than Although the initial sample preparation time is longer than IEC methodIEC method
SpecificitySpecificity
Ability to create internal standards for any Ability to create internal standards for any analyteanalyte you wish to evaluateyou wish to evaluate
IECIEC--Urine Amino Acid Profile Urine Amino Acid Profile Drug InterferencesDrug Interferences
Minutes
0 10 20 30 40 50 60 70 80 90 100 110 120
mVo
lts
75
100
125
150
175
200
225
250
275
300
325
350
375
400
425
450
mVo
lts
175
200
225
250
275
300
325
350
375
400
425
450
475
500
525
550
Phse
r
Taur
Pea
Ure
a
Asp
Thr
Ser
Asn
Glu
Gln
(Sar
c) A
AA
A
Gly
Ala
Cit
(Aab
a)
Val
Cys
Met
Cysth
1Cy
sth 2
Ile
Leu
Tyr
b-al
a Ph
eA
aiba
(Hcy
s)(G
aba) Et
han
Am
m
Hyl
ys
ISO
rn
Lys
1-M
his
His
3-M
his
(Ans
)
Car
Arg
Hyp
ro
Pro
IS
570nmU1.09.005.300010 CRAWFORD BB
Name
440nmU1.09.005.300010 CRAWFORD BB
Name
IECIEC--Urine Amino Acid ProfileUrine Amino Acid Profile
Minutes
0 10 20 30 40 50 60 70 80 90 100 110 120
mV
olts
100
150
200
250
300
350
400
450
500
mV
olts
-50
0
50
100
150
200
250
300
350
400
(Hyp
ro)
(Pro
)
IS
Phse
r
Taur
Pea
U
rea
Asp
Thr
Ser
Asn
Glu
Gln
Sarc
AA
AA
Gly
Ala
(Cit)
aaba Val Cys
Met
(Cys
th 1
)C
ysth
2
(Ile)
Leu
Tyr
b-al
a
Phe
aaib
a
H
cys
Gab
a
Etha
n
Am
m
Hyl
ys
IS
Orn
Lys 1-M
his
His
3-M
his
(Ans
)
Car
Arg
440nmU16 08.364.122997 MALONEY B
Name
570nmU16 08.364.122997 MALONEY B
Name
Normal
XIC of +MRM (75 pairs): 557.3/114.1 amu Expected RT: 9.0 ID: Hcy_IS from Sample 5 (erndim MSUD) of 090205.wiff (Turbo Spray) Max. 9.9e4 cps.
8.50 8.55 8.60 8.65 8.70 8.75 8.80 8.85 8.90 8.95 9.00 9.05 9.10 9.15 9.20 9.25Time, min
0.0
5000.0
1.0e4
1.5e4
2.0e4
2.5e4
3.0e4
3.5e4
4.0e4
4.5e4
5.0e4
5.5e4
6.0e4
6.5e4
7.0e4
7.5e4
8.0e4
8.5e4
9.0e4
9.5e4
9.9e4 8.89
InternalStandard
Patient
Minutes0 10 20 30 40 50 60 70 80 90 100 110 120
mV
olts
0
50
100
150
200
250
300
350
400
450
500
mV
olts
0
50
100
150
200
250
300
350
400
450
500
(Phs
er) Ta
ur(P
ea)
Ure
a
Asp
Thr Ser
Asn
Glu
Gln
(Sar
c)
Aaa
a
G
lyA
laC
it Aab
a
Val
Cys M
et
(Cys
th 1
)C
ysth
2
IleLe
u
Tyr
(B-a
la)
Phe
(Aai
ba)
(Hcy
s)
(Gab
a) (E
than
)
Am
m
(Hyl
ys)
ISO
rn
Lys
1-M
his His
(3-M
his)
(Ans
)
(Car
)
Arg
(Hyp
ro)
Pro
IS
570nmPL22.09.032.104796 WEBB C
Name
440nmPL22.09.032.104796 WEBB C
Name
IECIEC--Plasma Amino Acid ProfilePlasma Amino Acid Profile Maple Syrup Urine DiseaseMaple Syrup Urine Disease
: " " F ile : " 0 90 2 0 2 .w iff "s) : " 1 3 2. 1 /8 6 .1 D a "
5 .6 5 .7 5 .8 5 . 9 6 . 0 6. 1 6. 2 6 .3 6 .4 6 .5 6 .6 6 . 7 6 . 8 6. 9 7. 0 7 .1 7 .2 7 .3 7 .4 7 .5 7 . 6 7 . 7 7. 8 7 .93 4 4 2 4 9 5 5 6 3 70 79 8 7 9 4 1 0 2 1 1 1 1 1 9 1 2 8 13 8 14 7 1 57 1 6 7 1 7 5 1 8 4 1 9 2 2 0 0 2 0 8 21 6 2 24
T im e, m i n
0 .0
2 0 0 0. 0
4 0 0 0. 0
6 0 0 0. 0
8 0 0 0. 0
1 .0 e 4
1 .2 e 4
1 .4 e 4
1 .6 e 4
1 .8 e 4
2 .0 e 4
2 .2 e 4
2 .4 e 4
2 .6 e 4
2 .8 e 4
3 .0 e 4
3 .2 e 4
3 .4 e 4
3 .6 e 4
3 .8 e 4
4 .0 e 4
4 .2 e 4
4 .4 e 4
4 .6 e 4
4 .8 e 4
5 .0 e 4
5 .2 e 4
5 .4 e 4
5 .6 e 4
5 .8 e 4
6 .0 e 4
6 .2 e 4
6 .4 e 4
6 .6 e 4
6 .8 e 4
Intensity, cps
6 .7 6
6 .9 9
6 .4 9
Allo-isoleucine
Isoleucine
Leucine
Norleucine
Create internal standards Create internal standards for any for any analyteanalyte
you wish to evaluateyou wish to evaluate
Minutes0 10 20 30 40 50 60 70 80 90 100 110 120
mV
olts
100
150
200
250
300
350
400
450
500
mV
olts
100
150
200
250
300
350
400
450
500
Hyp
ro
Pro
IS
Phse
r
Taur
(Pea
)
Ure
a
Asp
Thr
Ser
Asn
Glu
Gln
(Sar
c) (A
aaa)
Gly
Ala
Cit
Aab
a
Val
Cys
Met
(Cys
th 1
)C
ysth
2
Ile
Leu
Tyr
(B-a
la)
Phe
(Aai
ba)
(Hcy
s)
(Gab
a)
Etha
n Am
m
(Hyl
ys)
IS
Orn
Lys
1-m
his
His
(3-m
his)
(Ans
)
(Car
) Arg
440nmPL147R ERNDIM (RABBIT DOWN)
Name
570nmPL147R ERNDIM (RABBIT DOWN)
Name
Create internal standards Create internal standards for any for any analyteanalyte
you wish to evaluateyou wish to evaluate
100 umol/L SaccharopineLabeling bufferiTRAQ
reagent 114
Hydroxylamine
Incubate 30 min
Dry the solutionResuspend with iTRAQ
114 internal standard
1.) Determination of the mass transition for Saccharopine2.) Determination of the retention time for Saccharopine3.) Optimize MS parameters for Saccharopine
Create internal standards Create internal standards for any for any analyteanalyte
you wish to evaluateyou wish to evaluate
Sample Name: "erndim_sacc" Sample ID: "" Fi le: "090526.wiff"Peak Name: "Sacc" Mass(es): "421.4/115.1 Da"Comment: "" Annotation: ""
Sample Index: 5 Sample Type: Unknown Concentration: N/A Calculated Conc: 0.00 Acq. Date: 5/26/2009 Acq. Time: 4:34:59 PM Modified: Yes Proc. Algorithm: Specify Parameters - MQ III Noise Percentage: 50 Base. Sub. Window: 1.00 minPeak-Split. Factor: 4 Report Largest Peak: No Min. Peak Height: 200.00 cpsMin. Peak Width: 0.00 secSmoothing Width: 3 pointsRT Window: 10.0 secExpected RT: 4.35 minUse Relative RT: No Int. Type: Base To Base Retention Time: 4.35 minArea: 5.74e+004 countsHeight: 8.90e+003 cpsStart Time: 4.21 minEnd Time: 4.66 min
2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.82 14 30 44 57 70 81 93 104 115 126 137 149 161 173 186 198 210 221 232
Time, min
0.0
2000.0
4000.0
6000.0
8000.0
1.0e4
1.2e4
1.4e4
1.6e4
1.8e4
2.0e4
2.2e4
2.4e4
2.6e4
2.8e4
3.0e4
3.2e4
3.4e4
3.6e4
3.8e4
4.0e4
4.2e4
4.4e4
4.6e4
4.8e4
5.0e4
5.2e4
5.4e4
5.6e4
5.8e4
6.0e4
6.2e4
6.4e4
6.6e4
6.8e4
7.0e4
7.2e4
7.4e4
7.6e4
7.8e4
8.0e4
8.2e4
8.4e4
8.6e4
Intensity, cps
6.48
4.35
Sample Name: "erndim_sacc" Sample ID: "" Fi le: "090526.wiff"Peak Name: "Sacc_IS(IS)" Mass(es): "421.4/114.1 Da"Comment: "" Annotation: ""
Sample Index: 5 Sample Type: Unknown Concentration: 100. Calculated Conc: N/A Acq. Date: 5/26/2009 Acq. Time: 4:34:59 PM Modified: Yes Proc. Algorithm: Specify Parameters - MQ III Noise Percentage: 50 Base. Sub. Window: 1.00 minPeak-Split. Factor: 4 Report Largest Peak: No Min. Peak Height: 500.00 cpsMin. Peak Width: 0.00 secSmoothing Width: 3 pointsRT Window: 300. secExpected RT: 4.35 minUse Relative RT: No Int. Type: Base To Base Retention Time: 4.35 minArea: 1.42e+005 countsHeight: 2.34e+004 cpsStart Time: 4.21 minEnd Time: 4.68 min
2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.82 14 25 37 48 59 70 81 93 105 117 130 142 154 165 176
Time, min
0.0
1000.0
2000.0
3000.0
4000.0
5000.0
6000.0
7000.0
8000.0
9000.0
1.0e4
1.1e4
1.2e4
1.3e4
1.4e4
1.5e4
1.6e4
1.7e4
1.8e4
1.9e4
2.0e4
2.1e4
2.2e4
2.3e4
2.4e4
2.5e4
2.6e4
2.7e4
2.8e4
2.9e4
3.0e4
3.1e4
3.2e4
3.3e4
3.4e4
3.5e4
3.6e4
3.7e4
3.8e4
3.9e4
4.0e4
4.1e4
4.2e4
4.3e4
4.4e4
4.5e4
4.6e4
4.7e4
4.8e4
4.9e4
Intensity, cps
6.47
4.35
Internal StandardSaccharopine
Patient SampleSaccharopine
SaccharopineConcentration
IEC
52 umol/LiTRAQ
45 umol/L
Linear Dynamic RangeLinear Dynamic RangeLow Limit of DetectionLow Limit of Detection
Internal Standards Internal Standards A Challenge for all MS/MS applicationsA Challenge for all MS/MS applications……
ChallengesChallenges iTRAQiTRAQ reagent, LCreagent, LC--MS/MS methodMS/MS method
Linear Dynamic RangeLinear Dynamic Range090318_linearity.rdb (Phe): "Linear" Regression ("No" weighting): y = 1.03 x + 0.0389 (r = 0.9999)
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5Analyte Conc. / IS Conc.
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
7.9 Phenylalanine2-3 orders of magnitudeApproximately 5umol/L up to 700 umol/L
Low Limit of DetectionLow Limit of Detection
Labeled Amino Acid
N
N
O
Mass of 114 or 115
Mass of 30 or 31
Amino Acid
Mass TransitionsParent ion 114 (internal standard)
115 (patient sample)
Blank Sample ISSample Name: "blank_LB" Sample ID: "" File: "090318.wiff"Peak Name: "Arg" Mass(es): "319.2/115.1 Da"Comment: "" Annotation: ""
Sample Index: 15 Sample Type: Standard Concentration: 0.00 uM Calculated Conc: 0.00 uM Acq. Date: 3/18/2009 Acq. Time: 6:32:37 PM Modified: Yes
4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.219 36 54 69 83 96 109 123 137 153 169 186 207 227 247
Time, min
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
2000
2100
2200
2300
2400
2500
2600
2700
2800
2900
3000
3100
3200
3300
3400
3500
3600
3700
3800
3900
4000
Intensity, cps
7.16
6.61
5.25
4.45
4.76
Sample Name: "blank_LB" Sample ID: "" File: "090318.wiff"Peak Name: "Arg_IS(IS)" Mass(es): "319.2/114.1 Da"Comment: "" Annotation: ""
Sample Index: 15 Sample Type: Standard Concentration: 100. uM Calculated Conc: N/A Acq. Date: 3/18/2009 Acq. Time: 6:32:37 PM Modified: Yes Proc. Algorithm: Specify Parameters - MQ III Noise Percentage: 50 Base. Sub. Window: 1.00 minPeak-Split. Factor: 4 Report Largest Peak: No Min. Peak Height: 500.00 cpsMin. Peak Width: 0.00 secSmoothing Width: 3 pointsRT Window: 300. secExpected RT: 5.60 minUse Relative RT: No Int. Type: Base To Base Retention Time: 5.25 minArea: 8.54e+005 countsHeight: 1.56e+005 cpsStart Time: 5.02 minEnd Time: 5.59 min
4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.240 58 73 87 100 113 127 141 157 173 190 211 231 251
Time, min
0.0
5000.0
1.0e4
1.5e4
2.0e4
2.5e4
3.0e4
3.5e4
4.0e4
4.5e4
5.0e4
5.5e4
6.0e4
6.5e4
7.0e4
7.5e4
8.0e4
8.5e4
9.0e4
9.5e4
1.0e5
1.1e5
1.1e5
1.2e5
1.2e5
1.3e5
1.3e5
1.4e5
1.4e5
1.5e5
1.5e5
1.6e5
Intensity, cps
5.25Internal StandardWater Blank
Minutes
0 10 20 30 40 50 60 70 80 90 100 110 120 130
mV
olts
125
150
175
200
225
250
275
300
325
350
375
400
425
450
mV
olts
125
150
175
200
225
250
275
300
325
350
375
400
425
450
Phs
er
Taur
Pea
U
rea
Asp
Thr
Ser
Asn
Glu
Gln
(Sar
c)
AA
AA
Gly
Ala
Citr
aaba
Val
C
ys
Met
(Cys
th 1
)C
ysth
2
Ile
Leu
Tyr
b-al
a
Phe
aaib
a
Hom
ocys
Gab
a Eth
an
Am
m
Hyl
ys
ISO
rn
Lys
1-M
his
His
3-M
his
(Ans
)
Car
Arg
(H
ypro
)
Pro
IS
570nmU8.08.196.20434 ANDERSON R
Name
440nmU8.08.196.20434 ANDERSON R
Name
Ion Exchange ChromatographyIon Exchange Chromatography Abnormal Urine Amino Acid ProfileAbnormal Urine Amino Acid Profile
Concentration (umol/gram creatinine)
Cystine
2150Ornithine
1949Arginine
5761Lysine 7430Citrulline
14Valine
121
The lower limit of The lower limit of quantitationquantitation is approximately 5 is approximately 5 umolumol/L for some amino acids and the upper /L for some amino acids and the upper range of linearity is only approximately 700 range of linearity is only approximately 700 umolumol/L/L
You have a small range for where you can You have a small range for where you can accurately accurately quantitatequantitate an amino acidan amino acid
Dilutions may be necessary to achieve an accurate Dilutions may be necessary to achieve an accurate concentration concentration
May require several injections per sampleMay require several injections per sample
Linear Dynamic Range &Linear Dynamic Range & Low limit of detectionLow limit of detection
Internal StandardsInternal Standards
090318_linearity.rdb (Asn): "Linear" Regression ("No" weighting): y = 1.35 x + 0.00536 (r = 0.9997)
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0Analyte Conc. / IS Conc.
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
10.5
11.0
11.5
12.0
12.5
13.0
Asparagine
Ana
lyte
area
/IS a
rea
Analyte
conc/IS conc
Y=1.35X + 0.00536
×114
standardAmountAreaAreaAmount ×
patient×= 115
The concentration of the internal standards The concentration of the internal standards may not be exactly 100 may not be exactly 100 umolumol/L and may /L and may vary from lot to lotvary from lot to lot
Good laboratory practice to verify the Good laboratory practice to verify the concentrations of the internal standards by concentrations of the internal standards by a different laboratory method a different laboratory method
Internal StandardsInternal Standards
ConclusionsConclusions
Ion ExchangeIon ExchangeChromatographyChromatography
iTRAQiTRAQ
reagents reagents & LC& LC--MS/MSMS/MS
Sample PrepSample Prep 15 minutes15 minutes 120 minutes120 minutes
Sample Run TimeSample Run Time 150 minutes150 minutes 25 minutes25 minutes
ReproducibilityReproducibility <5% (CV) <5% (CV) <10% (CV)<10% (CV)
SpecificitySpecificity GoodGood BetterBetter
Linear Range &Linear Range &Low Limit of Low Limit of
DetectionDetection
1 1 umolumol/L up to /L up to 4000 4000 umolumol/L/L
5 5 umolumol/L up to /L up to 700 700 umolumol/L/L
AcknowledgementsAcknowledgements
Applied Applied BiosystemsBiosystemsBruno CasettaBruno CasettaScott DanielsScott DanielsSeyed SadjadiSeyed SadjadiLisa SappLisa SappBabuBabu PurkayasthaPurkayastha
ARUP Institute for Clinical & ARUP Institute for Clinical & Experimental Pathology Experimental Pathology ®®