Evaluation of a LC-MS/MS method for quantitative amino acid analysis

Transcription

1 Evaluation of a LC-MS/MS method for quantitative amino acid analysis Patrice K. Held, Ph.D. Assistant Professor of Pathology University of Utah School of Medicine Assistant Medical Director, Biochemical Genetics Laboratory

2 Clinical Indications for Amino Acid Analysis Amino acid analysis should be considered in many clinical situations, including Lethargy, coma, seizures, or vomiting Hyperammonemia Metabolic acidosis or lactic acidemia Metabolic decompensation Unexplained developmental delay Abnormal amino acid results by newborn screening Diet monitoring in patients with known metabolic disorders

3 Ion Exchange Chromatography The GOLD standard for amino acid analysis

4 Ion Exchange Chromatography Inlet IE column Derivatization UV detector Sample Prep Injection Port Separation Detection

5 Plasma Sample Prep Ion Exchange Chromatography Samples are deproteinized with sulphosalycilic acid prior to injection CSF Samples are deproteinized with sulphosalycilic acid prior to injection Urine Creatinine is measured and samples are lyophilized and resuspended to a certain concentration per gram of creatinine Samples are deproteinized with sulphosalycilic acid prior to injection

6 Separation Ion Exchange Chromatography Matrix-SO 3- Li H 3 N-CHRCOOH Matrix-SO 3 -+ H 3 N-CHRCOOH + Li + Separation is affected by: 1) pka values of the side chains R 2) interaction of the side chains with the matrix 3) temperature 4) ionic strength

7 Detection Ion Exchange Chromatography Ninhydrin detection Eluting amino acids are mixed with ninhydrin and flow through a reaction coil. The high temperature in the coil facilitates the development of a purple color. The derivatized amino acids go through a UV detector. The concentration of the amino acids is proportional to the intensity of the purple color. 2 wavelengths are used: 570 nm (amino acids) and 440 nm (imino( acids, such as proline and hydroxyproline)

8 IEC-Plasma Amino Acid Profile Minutes mvolts mvolts Name 570nm PL DALTON L Name 440nm PL DALTON L Hypro IS Phser Pea Asp Asn Glu (Sarc) AAAA Pro Cit aaba Cys Taur Met (Cysth 1) (Cysth 2) (b-ala) Ile Phe (aaiba) (Hcys) (Gaba) (Ethan) Amm (Hylys) 1-Mhis Tyr Orn His 3-Mhis (Ans) (Car) Arg Thr Ser Gly Val Leu Lys Urea IS Ala Gln

9 Ion Exchange Chromatography Advantages Reproducibility High resolution Minimal sample preparation Large linear dynamic range 1 umol/l to 4000 umol/l approximately orders of magnitude Identification of atypical amino acids Disadvantages Run time (2.5 hours per sample) Interferences within urine samples Identification of atypical amino acids

10 Tandem Mass Spectrometry

11 Amino Acid Analysis by Tandem Mass Spectrometry Automated tandem mass spectrometry for mass newborn screening for r disorders in fatty acid, organic acid, and amino acid metabolism. Naylor EW, Chace DH. J Child Neurol Nov;14 Suppl 1:S4-8. Development of a method for rapid quantitation of amino acids by liquid chromatography-tandem tandem mass spectrometry (LC-MSMS) in plasma. Casetta B, Tagliacozzi D, Shushan B, Federici G. Clin Chem Lab Med May;38(5): Rapid comprehensive amino acid analysis by liquid chromatography/tandem mass spectrometry: comparison to cation exchange with post-column ninhydrin detection. Dietzen DJ, Weindel AL, Carayannopoulos MO, Landt M, Normansell ET, Reimschisel TE, Smith CH. Rapid Commun Mass Spectrom Nov;22(22):

12 itraq Reagent Methodology

13 itraq Reagent Reaction N N N O O O N O + N H 2 R O O H N O H N R O O H + HO O N O itraq Reagent Amino Acid Labeled Amino Acid Reporter group (mass 114 or 115) Balance group (mass 30 or 31) Amino acid Reactive group Courtesy of Applied Biosystems

14 itraq Reagent Methodology AARG Amino Acid (patient) AARG Amino Acid (standard) Label Label Amino Acid (patient) Amino Acid (standard) 5.0E E E E E+04 Patient (transition ) Sample Standard (transition ) Area115 Amount = patient Area Amount 114 standard 0.0E Time (min) Courtesy of Applied Biosystems

15 Details of the Analysis Method API 4000 with Shimadzu HPLC Mobile Phase A-A 0.1% formic acid, 0.01% heptafluorobutyric acid in water Mobile Phase B-B 0.1% formic acid, 0.01% heptafluorobutyric acid in methanol C18 Column heated to 50 C Flow rate 0.8 ml/min

16 XIC of +MRM (26 pairs): 286.1/114.1 amu from Sample 2 (SST002) of wiff (Turbo Spray) Max cps. 8.0e5 7.5e5 7.0e5 6.5e5 6.0e5 Sample Name: "250" Sample ID: "" File: " wiff" Peak Name: "Phe" Mass(es): "310.2/115.1 amu" Comment: "" Annotation: "" Sample Index: 3 Sample Type: Standard Concentration: umol/l 1.5e6 Calculated Conc: 0.00 umol/l Acq. Date: 11/18/2008 Acq. Time: 1:52:07 PM 1.5e6 Patient Modified: No Proc. Algorithm: IntelliQuan - MQII Noise Percentage: 50 Base. Sub. Window: 1.00 min Peak-Split. Factor: 2 Report Largest Peak: No Min. Peak Height: cps Min. Peak Width: 0.00 sec Smoothing Width: 3 points RT Window: 240. sec Expected RT: 10.4 min Use Relative RT: No Int. Type: Valley Retention Time: 10.1 min Area: e+006 counts Height: 1.52e+006 cps Start Time: 9.90 min End Time: 10.6 min 1.4e6 1.4e6 1.3e6 1.3e6 1.2e6 1.2e6 1.1e6 1.1e6 1.0e6 9.5e5 9.0e Sample Name: "250" Sample ID: "" File: " wiff" Peak Name: "ISTD (Phe)(IS)" Mass(es): "310.2/114.1 amu" Comment: "" Annotation: "" Sample Index: 3 Sample Type: Standard Concentration: umol/l Calculated Conc: N/A 5.6e5 Acq. Date: 11/18/2008 Acq. Time: IS 1:52:07 PM 5.4e5 Modified: No Proc. Algorithm: IntelliQuan - MQII 5.2e5 Noise Percentage: 50 Base. Sub. Window: 1.00 min Peak-Split. Factor: 2 5.0e5 Report Largest Peak: No Min. Peak Height: cps 4.8e5 Min. Peak Width: 0.00 sec Smoothing Width: 3 points RT Window: 240. sec 4.6e5 Expected RT: 10.4 min Use Relative RT: No 4.4e5 Int. Type: Valley Retention Time: 10.1 min 4.2e5 Area: e+006 counts Height: 5.75e+005 cps 4.0e5 Start Time: 9.90 min End Time: 10.5 min 3.8e5 3.6e5 3.4e e5 3.2e5 5.5e5 Int ensity, cps 8.0e5 7.5e5 Intensity, cps 3.0e5 2.8e5 7.0e5 2.6e5 5.0e5 6.5e5 6.0e5 5.5e5 2.4e5 2.2e5 2.0e5 5.0e5 1.8e5 4.5e5 4.5e5 4.0e5 3.5e5 1.6e5 1.4e5 4.0e5 3.5e5 3.0e5 1.2e5 3.0e5 1.0e5 2.5e5 8.0e4 2.0e e4 1.5e5 4.0e4 1.0e5 2.0e4 5.0e Time, min Time, min 2.5e5 2.0e5 1.5e5 1.0e5 5.0e Time, min

17 Comparison of data Reproducibility (Inter IEC itraq/msms Recovery (Inter-assay variation) <5% (CV) for most amino acids <10% (CV) for most amino acids itraq/msms Ranged from 80% up to 110% Linear Dynamic Range & Low Limit of Detection IEC itraq/msms 1 umol/l up to 4000 umol/l 5 umol/l up to 700 umol/l

18 Advantages itraq reagent, LC-MS/MS method Run Time (25 minutes) Although the initial sample preparation time is longer than IEC method Specificity Ability to create internal standards for any analyte you wish to evaluate

19 IEC-Urine Amino Acid Profile Drug Interferences Minutes mvolts mvolts 570nm U CRAWFORD BB Name Name 440nm U CRAWFORD BB Urea Gly Amm Hypro IS Pro Asp Asn Glu (Sarc) Phser Gln AAAA Cit (Aaba) Val Met Pea Cysth 1 Cysth 2 Ile Leu b-ala Phe (Hcys) (Gaba) Cys Tyr Ethan Hylys 1-Mhis 3-Mhis (Ans) Car Arg Ala Thr His IS Orn Ser Lys Aaiba Taur

20 IEC-Urine Amino Acid Profile nm U MALONEY B Name 570nm U MALONEY B Name XIC of +MRM (75 pairs): 557.3/114.1 amu Expected RT: 9.0 ID: Hcy_IS from Sample 5 (erndim MSUD) of wiff (Turbo Spray) e4 Max. 9.9e4 cps. Amm 250 mvolts 300 Taur 9.5e4 9.0e4 8.5e4 8.0e4 7.5e4 Gly Normal 200 mvolts Phser Pea 7.0e4 6.5e4 6.0e4 5.5e4 5.0e4 4.5e4 4.0e4 3.5e4 Internal Standard Ser Asp (Hypro) Thr Asn Glu Gln Sarc AAAA (Pro) Ala (Cit) aaba Val Cys Met (Cysth 1) Cysth 2 (Ile) Leu Tyr b-ala Phe aaiba Hcys Gaba Ethan Hylys IS IS Orn Lys 1-Mhis His 3-Mhis (Ans) Car Arg e e e4 1.5e4 1.0e4 Patient Urea Time, min Minutes

21 IEC-Plasma Amino Acid Profile Maple Syrup Urine Disease nm PL WEBB C Name 440nm PL WEBB C Name 500 : " " F ile : " w iff " s) : "132.1/86.1 Da" e4 6.6e4 Leucine e e4 6.0e4 5.8e4 5.6e e4 5.2e4 5.0e4 4.8e e4 4.4e4 4.2e4 Int ensity, cps mvolts 4.0e4 3.8e4 3.6e4 3.4e4 3.2e4 3.0e4 2.8e4 2.6e4 2.4e (Phser) Taur (Pea) Isoleucine Urea 6.49 Asp Thr Ser Norleucine 6.99 Gln Asn Glu (Sarc) Aaaa Gly Ala Cit Aaba Val Cys Met (Cysth 1) 2 Ile Leu Tyr (B-ala) Phe (Aaiba) (Hcys) (Gaba) (Ethan) Amm (Hylys) IS Orn Lys 1-Mhis His (3-Mhis) (Ans) (Car) Arg mvolts 2.2e4 2.0e4 1.8e4 Pro 1.6e e4 1.2e4 (Hypro) IS 1.0e Allo-isoleucine Tim e, min Minutes

22 Create internal standards Create internal standards for any analyte you wish to evaluate Minutes mvolts mvolts Hypro Pro IS Phser Taur (Pea) Urea Thr Ser (Sarc) (Aaaa) Cit Aaba Val Cys Asn (Cysth 1) Cysth 2 Tyr (B-ala) Ile (Aaiba) (Hcys) (Gaba) Ethan (Hylys) Amm 1-mhis (3-mhis) (Ans) (Car) Asp Glu Ala His IS Gly Gln Orn Met Phe Lys Leu Arg Name 440nm PL147R ERNDIM (RABBIT DOWN) Name 570nm PL147R ERNDIM (RABBIT DOWN)

23 Create internal standards for any analyte you wish to evaluate 100 umol/l Saccharopine Labeling buffer itraq reagent 114 Incubate 30 min Hydroxylamine Dry the solution Resuspend with itraq 114 internal standard 1.) Determination of the mass transition for Saccharopine 2.) Determination of the retention time for Saccharopine 3.) Optimize MS parameters for Saccharopine

24 Create internal standards for any analyte you wish to evaluate Sample Name: "erndim_sacc" Sample ID: "" Fi le: " 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/ e4 Acq. Time: 4:34:59 PM 8.4e4 Modified: Yes Proc. Algorithm: Specify Parameters - MQ III 8.2e4 Noise Percentage: 50 Base. Sub. Window: 1.00 min 8.0e4 Peak-Split. Factor: 4 Report Largest Peak: No 7.8e4 Min. Peak Height: cps Min. Peak Width: 0.00 sec 7.6e4 Smoothing Width: 3 points RT Window: 10.0 sec 7.4e4 Expected RT: 4.35 min Use Relative RT: No 7.2e4 Patient Sample Saccharopine 6.48 Sample Name: "erndim_sacc" Sample ID: "" Fi le: " wiff" Peak Name: "Sacc_IS(IS)" Mass(es): "421.4/114.1 Da" Comment: "" Annotation: "" Sample Index: 5 Sample Type: Unknown Concentration: e4 Calculated Conc: N/A Acq. Date: 5/26/ e4 Acq. Time: 4:34:59 PM 4.7e4 Modified: Yes Proc. Algorithm: Specify Parameters - MQ III 4.6e4 Noise Percentage: 50 Base. Sub. Window: 1.00 min 4.5e4 Peak-Split. Factor: 4 4.4e4 Report Largest Peak: No Min. Peak Height: cps 4.3e4 Min. Peak Width: 0.00 sec Smoothing Width: 3 points 4.2e4 RT Window: 300. sec Expected RT: 4.35 min 4.1e4 Use Relative RT: No 4.0e4 Internal Standard Saccharopine 6.47 Int. Type: Base To Base Retention Time: 4.35 min Area: 5.74e+004 counts Height: 8.90e+003 cps Start Time: 4.21 min End Time: 4.66 min 7.0e4 6.8e4 6.6e4 6.4e4 Int. Type: Base To Base Retention Time: 4.35 min Area: 1.42e+005 counts Height: 2.34e+004 cps Start Time: 4.21 min End Time: 4.68 min 3.9e4 3.8e4 3.7e4 3.6e4 6.2e4 3.5e4 IEC itraq 6.0e4 Saccharopine 5.8e4 5.6e4 Concentration 5.4e4 5.2e4 5.0e e4 umol/l 4.6e4 4.4e4 45 umol/l 4.2e4 4.0e4 3.8e4 Intensity, cps Intensity, cps 3.4e4 3.3e4 3.2e4 3.1e4 3.0e4 2.9e4 2.8e4 2.7e4 2.6e4 2.5e4 2.4e4 2.3e4 2.2e4 2.1e e4 2.0e4 3.4e4 1.9e4 3.2e4 1.8e4 3.0e4 1.7e4 2.8e4 1.6e4 2.6e4 2.4e4 2.2e4 1.5e4 1.4e4 1.3e4 1.2e4 2.0e4 1.1e4 1.8e4 1.0e4 1.6e e e e Time, mi n Time, mi n

25 Challenges itraq reagent, LC-MS/MS method Linear Dynamic Range Low Limit of Detection Internal Standards A Challenge for all MS/MS applications

26 Linear Dynamic Range _linearity.rdb (Phe): "Linear" Regression ("No" weighting): y = 1.03 x (r = ) 7.9 Phenylalanine orders of magnitude 7.0 Approximately 5umol/L up to 700 umol/l Analyte Conc. / IS Conc.

27 Low Limit of Detection Mass Transitions Parent ion 114 (internal standard) 115 (patient sample) Sample Name: "blank_lb" Sample I D: "" File: " wiff" Peak Name: "Arg" Mass(es): "319.2/115.1 Da" Comment: "" Annotation: "" Sample Index: 15 Sample Type: Stand ard Concentration: um Calculated Conc : um 4000 Acq. Date: 3/18/ 2009 Acq. Time: 6:32: 37 PM 3900 Blank Water Sample Blank 7.16 Sample Name: "blank_lb" Sample ID: "" File: " wiff" Peak Name: "Arg_IS(IS)" Mass(es): "319.2/114.1 Da" Comment: "" Annotation: "" Sample I ndex: 15 Sample T ype: Standard Concentr ation: 100. um Calculat ed Conc : N/A Acq. Dat e: 3/18/2009 Acq. Tim e: 6:32:37 PM Internal Standard e5 IS 1.5e5 Mass of 114 or 115 N Modified: Ye s Modified : Yes Proc. Al gorithm : Specify Parameters - MQ I II Noise Pe rcentag e: 50 Base. Su b. Wind ow: 1.00 min Peak-Spl it. Fac tor: 4 Report L argest Peak: No Min. Peak Height: cps Min. Peak Width : 0.00 sec Smoothing Width : 3 points RT Window: 300. sec Expected RT: 5.60 min Use Rela tive RT : No Int. Typ e: Base To Base Retention Time: 5.25 min Area: 8.54e+005 counts Height: 1.56e+005 cps Start Time: 5.02 min End Time : 5.59 min 1.5e5 1.4e5 1.4e5 1.3e5 1.3e5 1.2e5 1.2e5 1.1e e e5 Mass of 30 or 31 N O Amino Acid Intensity, cps Intensity, cps 9.5e4 9.0e4 8.5e4 8.0e4 7.5e4 7.0e4 6.5e4 6.0e4 5.5e e e4 Labeled Amino Acid e4 3.5e4 3.0e e e e e Time, min Time, mi n

28 Minutes mvolts mvolts Ion Exchange Chromatography Abnormal Urine Amino Acid Profile Name 570nm U ANDERSON R Name 440nm U ANDERSON R Urea Gly Cys Lys Arg (Hypro) Pro IS Phser Taur Pea Glu (Sarc) Asn AAAA Citr aaba Val Met (Cysth 21) Ile Leu b-ala Phe aaiba Tyr Homocys Gaba (Ans) Car Ethan Hylys Asp 3-Mhis Gln Ser Ala Thr 1-Mhis Amm IS His Orn Concentration (umol/gram creatinine) Cystine 2150 Ornithine 1949 Arginine 5761 Lysine 7430 Citrulline 14 Valine 121

29 Linear Dynamic Range & Low limit of detection The lower limit of quantitation is approximately 5 umol/l for some amino acids and the upper range of linearity is only approximately 700 umol/l You have a small range for where you can accurately quantitate an amino acid Dilutions may be necessary to achieve an accurate concentration May require several injections per sample

30 Internal Standards _linearity.rdb (Asn): "Linear" Regression ("No" weighting): y = 1.35 x (r = ) Analyte area/is area Asparagine Y=1.35X Area115 Amount = patient Area Amount Analyte Conc. / IS Conc. 114 standard Analyte conc/is conc

31 Internal Standards The concentration of the internal standards may not be exactly 100 umol/l and may vary from lot to lot Good laboratory practice to verify the concentrations of the internal standards by a different laboratory method

32 Conclusions Ion Exchange Chromatography itraq reagents & LC-MS/MS Sample Prep Sample Run Time Reproducibility 15 minutes 120 minutes 150 minutes 25 minutes <5% (CV) <10% (CV) Specificity Linear Range & Low Limit of Detection Good 1 umol/l up to 4000 umol/l Better 5 umol/l up to 700 umol/l

33 Acknowledgements Applied Biosystems Bruno Casetta Scott Daniels Seyed Sadjadi Lisa Sapp Babu Purkayastha ARUP Institute for Clinical & Experimental Pathology