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 ChromatographyIon Exchange Chromatography

“The GOLD standard for amino acid analysis”

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

Tandem Mass SpectrometryTandem Mass Spectrometry

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.

iTRAQiTRAQ®®

Reagent MethodologyReagent Methodology

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 ®®