Development and Evaluation of an Isotope Dilution ... - ACS Publications

Jan 9, 2003 - Elevated plasma homocysteine has been identified as a strong and independent risk factor for cardiovascular diseases, and recently, it h...
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Anal. Chem. 2003, 75, 775-784

Development and Evaluation of an Isotope Dilution LC/MS Method for the Determination of Total Homocysteine in Human Plasma Bryant C. Nelson,*,§ Christine M. Pfeiffer,‡ Lorna T. Sniegoski,† and Mary B. Satterfield†

Analytical Chemistry Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899-0001, and Division of Laboratory Sciences, Centers for Disease Control and Prevention, Atlanta, Georgia 30341-3724

Elevated plasma homocysteine has been identified as a strong and independent risk factor for cardiovascular diseases, and recently, it has been associated with the development of dementia in older adults. Selected ionmonitoring isotope-dilution LC/MS (electrospray) has been developed and evaluated as a reference method for the accurate determination of total homocysteine in human plasma. Homocysteine is quantitatively isolated from plasma via the use of anion-exchange resins and then detected and quantified in stabilized plasma extracts with selected ion-monitoring LC/MS. This method is shown to be highly comparable to LC/MS/MS determinations in terms of its analytical accuracy and precision, yet this alternative measurement approach does not necessitate the enhanced instrumentation or added expense required of tandem MS/MS determinations. LC/MS detection of homocysteine was linear (standard error of the estimate for the regression line was 0.0323) over 3 orders of magnitude, and the calculated limits of detection and quantification were 0.06 µmol/L (0.12 ng on column) and 0.6 µmol/L (1.2 ng on column), respectively. Independent calibration curves showed excellent linearity (r2 g 0.996) between 0 and 25 µmol/L homocysteine over a 3-day period. The accuracy and precision of total homocysteine measurements for patient samples and quality control pools using LC/MS were compared to total homocysteine measurements using LC/MS/MS, GC/MS, FPIA, and LC-FD. LC/MS performed well in relation to the other homocysteine methods in terms of its capability to accurately quantify plasma homocysteine over the normal range (5-15 µmol/L). Homocysteine (Hcy) is a sulfur-containing amino acid that is biosynthesized from the essential amino acid methionine. The increased export of Hcy from cells, whether due to faulty metabolism or to a genetic defect, results in the buildup of Hcy in the peripheral blood system (plasma) where it circulates predominantly conjugated to proteins. Elevated levels of Hcy in plasma are strongly correlated with an increased risk of * To whom correspondence should be addressed. E-mail: bryant.nelson@ genzyme.com. † National Institute of Standards and Technology. ‡ Centers for Disease Control and Prevention. § Current address: Genzyme Corporation, One Mountain Road, Framingham, MA 01701. 10.1021/ac0204799 Not subject to U.S. Copyright. Publ. 2003 Am. Chem. Soc.

Published on Web 01/09/2003

coronary artery disease (CAD), cerebrovascular disease (CVD), and peripheral vascular disease (PVD).1-6 Recent clinical studies have demonstrated that plasma Hcy levels correlate better than cholesterol levels with an increased risk of arteriosclerosis.7 A meta-analysis of 27 case-controlled clinical studies estimated that a 5 µmol/L increase in plasma Hcy increased the risk for CAD and CVD by factors (odds ratios) of 1.7 and 1.5, respectively.8 Elevated plasma Hcy has been associated with the development of dementia (Alzheimer’s disease)9,10 and nutritional cofactor deficiency,11-15 and it is a contributing factor in the pathogenesis of neural tube defects.16-18 Hyperhomocysteinemia, as this condition is medically described, is currently indicated/diagnosed when plasma Hcy attains values g10 µmol/L.19-21 Plasma Hcy is analytically determined as total Hcy (tHcy), which refers to the sum of interchangeable reduced (free homocysteine), oxidized (homocystine, homocysteine-cysteine disulfide), and proteinbound (homocysteine-albumin disulfide) Hcy forms that exist in plasma.22 (1) McCully, K. S. Am. J. Pathol. 1969, 56, 111-128. (2) Clarke, R.; Daly, L.; Robinson, K.; Naughten, E.; Cahalane, S.; Fowler, B. G. N. Engl. J. Med. 1991, 324, 1149-1155. (3) Refsum, H.; Ueland, P. M.; Nygard, O.; Vollset, S. E. Annu. Rev. Med. 1998, 49, 31-62. (4) Welch, G. N.; Loscaizo, J. N. Engl. J. Med. 1998, 338, 1009-1015. (5) Guba, S. C.; Fink, L. M.; Fonseca, V. Am. J. Clin. Pathol. 1996, 105, 709-722. (6) de Jong, S. Clin. Chem. Lab. Med. 2001, 39, 714-716. (7) Cramer, D. A. Lab. Med. 1998, 29, 410-417. (8) Boushey, C. J.; Beresford, S. A.; Omenn, G. S.; Motulsky, A. G. J. Am. Med. Assoc. 1995, 274, 1049-1057. (9) Loscalzo, J. N. Engl. J. Med. 2002, 346, 466-468. (10) Seshadri, S.; Beiser, A.; Selhub, J.; Jacques, P. F.; Rosenberg, I. H.; D’Agostino, R. B.; Wilson, P. W. F.; Wolf, P. A. N. Engl. J. Med. 2002, 346, 476-483. (11) Dudman, N. P.; Wilcken, D. E.; Wang, J.; Lynch, J. F.; Macey, D.; Lundberg, P. Arterioscler. Thromb., Vasc. Biol. 1993, 13, 1253-1260. (12) Stabler, S. P.; Lindenbaum, J.; Allen, R. H. J. Nutr. 1996, 126, 1266S1272S. (13) Flynn, M. A.; Herbert, V.; Nolph, G. B.; Krause, G. J. Am. Coll. Nutr. 1997, 16, 258-267. (14) Kang, S. S. J. Nutr. 1996, 126, 1273S-1275S. (15) Pietrzik, K.; Bronstrup, A. Eur. J. Pediatr. 1998, 157, S135-S138. (16) Nelen, W. L. Clin. Chem. Lab. Med. 2001, 39, 758-763. (17) Herrmann, W. Clin. Chem. Lab. Med. 2001, 39, 666-674. (18) Refsum, H. Brit. J. Nutr. 2001, 85, S109-S113. (19) Sainato, D. Clin. Lab. News 2001, 27, 1. (20) Medina, M. A.; Amores-Sanchez, M. I. Eur. J. Clin. Invest. 2000, 30, 754762. (21) Selhub, J.; Jacques, P. F.; Rosenberg, I. H.; Rogers, G.; Bowman, B. A.; Gunter, E. W.; Wright, J. D.; Johnson, C. L. Ann. Intern. Med. 1999, 131, 331-339.

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A variety of different methods have been developed for the measurement of plasma tHcy, and the most commonly used approaches have been based on the use of liquid chromatography with either fluorescence detection (LC-FD)23-25 or electrochemical detection (LC-ED),26-28 fluorescence polarization immunoassay (FPIA),29,30 and gas chromatography coupled with mass spectrometry (GC/MS).31-36 Comprehensive reviews of these traditional methods have been published.37-39 Most recently, LC coupled with isotope-dilution tandem mass spectrometry (LC/MS/ MS) has been effectively utilized to measure plasma tHcy.40-42 The recognition and acceptance of elevated tHcy as a valid cardiovascular disease risk factor and the resulting increase in demand for routine tHcy measurements has stimulated the need for well-established primary reference methods.13,19,39,43 However, none of the published methods are currently recognized as primary reference methods within the Hcy research community. Of the published methods, perhaps the measurement approaches based on the use of isotope-dilution LC/MS/MS have the greatest potential of being accepted and utilized as dedicated Hcy reference methods. MS coupled with a suitable separations technique, that is, LC, has the capacity for unambiguous analyte identification/ confirmation. Isotope-dilution LC/MS or LC/MS/MS methods have the additional capacity for highly accurate analyte quantification at trace levels. NIST has investigated the development and use of liquid chromatography coupled with isotope-dilution electrospray-ionization mass spectrometry (LC/ESI-MS) in selected ion-monitoring (SIM) mode to address the need for tHcy primary reference methods. Briefly, a plasma sample is spiked with the Hcy analogue ([2H4]-Hcy), reduced with alkaline dithiothreitol (DTT) and extracted with a solid-phase anion-exchange resin. The (22) Ueland, P. M.; Refsum, H.; Stabler, S. P.; Malinow, M. R.; Andersson, A.; Allen, R. H. Clin. Chem. 1993, 39, 1764-1779. (23) Jacobsen, D. W.; Gatautis, V. J.; Green, R.; Robinson, K.; Savon, S. R.; Secic, M.; Ji, J.; Otto, J. M.; Taylor, L. M. Clin. Chem. 1994, 40, 873-881. (24) Pfeiffer, C. M.; Huff, D. L.; Gunter, E. W. Clin. Chem. 1999, 45, 290-292. (25) Ubbink, J. B.; Vermaak, W. J. H.; Bissbort, S. J. Chromatogr. 1991, 565, 441-446. (26) Malinow, M. R.; Kang, S. S.; Taylor, L. M.; Wong, P. W. K.; Coull, B.; Inahara, T.; Mukerjee, D.; Sexton, G.; Upson, B. Circulation 1989, 79, 1180-1188. (27) Houze, P.; Gamra, S.; Madelaine, I.; Bousquet, B.; Gourmel, B. J. Clin. Lab. Anal. 2001, 15, 144-153. (28) Rabenstein, D. L.; Yamashita, G. T. Anal. Biochem. 1989, 180, 259-263. (29) Shipchandler, M. T.; Moore, E. G. Clin. Chem. 1995, 41, 991-994. (30) Marangon, K.; O’Byrne, D.; Devaraj, S.; Jialal, I. Am. J. Clin. Pathol. 1999, 112, 757-762. (31) Stabler, S. P.; Marcell, P. D.; Podell, E. R.; Allen, R. H. Anal. Biochem. 1987, 162, 185-196. (32) Stabler, S. P.; Lindenbaum, J.; Savage, D. G.; Allen, R. H. Blood 1993, 81, 3404-3413. (33) Shinohara, Y.; Hasegawa, H. T., K.; Hashimoto, T. J. Chromatogr., B 2001, 758, 283-288. (34) MacCoss, M. J.; Fukagawa, N. K.; Matthews, D. E. Anal. Chem. 1999, 71, 4527-4533. (35) Ducros, V.; Schmitt, D.; Pernod, G.; Faure, H.; Polack, B.; Favier, A. J. Chromatogr., B 1999, 729, 333-339. (36) Pietzsch, J.; Julius, U.; Hanefeld, M. Clin. Chem. 1997, 43, 2001-2004. (37) Amores-Sanchez, M. I.; Medina, M. A. Clin. Chem. Lab. Med. 2000, 38, 199-204. (38) Rasmussen, K.; Moller, J. Ann. Clin. Biochem. 2000, 37, 627-648. (39) Ubbink, J. B. Semin. Thromb. Hemostasis 2001, 26, 233-241. (40) Gempel, K.; Gerbitz, K. D.; Casetta, B.; Bauer, M. Clin. Chem. 2000, 46, 122-123. (41) Magera, M. J.; Lacey, J. M.; Casetta, B.; Rinaldo, P. Clin. Chem. 1999, 45, 1517-1522. (42) Chan, T. M.; Casetta, B.; Shushan, B. Clin. Chem. 1998, 44S, A125. (43) Tripodi, A.; Chantarangkul, V.; Lombardi, R.; Lecchi, A.; Mannucci, P. M.; Cattaneo, M. Thromb. Haemostasis 2001, 85, 291-295.

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extracted Hcy is then eluted from the resin, vacuum-concentrated and quantified by LC/MS. The method was developed to be used by the clinical and biomedical research community to validate/ corroborate plasma tHcy measurements that are typically determined via more routine tHcy measurement procedures, that is, LC-FD, FPIA, etc., and to value assign tHcy in Standard Reference Materials (SRMs). It is envisioned that this new method could best be used to periodically check the accuracy and performance of routine tHcy methods or to check the validity of suspect (excessively high or excessively low) data points. For comparative purposes, the optimized LC/MS method was modified to permit the detection and quantification of the extracted Hcy via multiple reaction-monitoring (MRM) LC/MS/MS analysis. In addition, an established capillary GC/MS method based on selected-ion monitoring of esterified Hcy was adapted from the literature 31,32 and modified for use in the NIST laboratories. These three mass spectrometry methods (LC/MS, LC/MS/MS and GC/MS), as well as two clinical methods (LC-FD, FPIA), were subsequently used to assess tHcy levels in human plasma samples. To our knowledge, this is the first comparative study of mass-spectrometrybased methods for measurement of Hcy. In general, the mass spectrometric results were highly comparable to each other and to the clinical method results. The increased specificity provided by the use of anion-exchange resins, as well as the characteristically reliable performance of stable isotopically labeled internal standards, allows LC/MS to be an effective quantitative approach for the accurate determination of Hcy in plasma. The development and assay features of this new selected-ion-monitoring isotopedilution LC/MS method are reported herein. EXPERIMENTAL SECTION Reagents and Materials. DL-Homocystine (purity g99%), DTT, human serum albumin, human hemoglobin, L-methionine, and L-cysteine were obtained from Sigma Chemical Company (St. Louis, MO). DL-[2H8]-Homocystine was obtained from C.D.N. Isotopes (Pointe-Claire, Quebec). DL-[2H8]-Homocystine contains eight stable 2H isotopes (3, 3, 3′, 3′, 4, 4, 4′, 4′-d8) incorporated into the methylene groups, and the supplier’s listed purity was >99.3 atom % deuterium. The purity of both DL-homocystine and DL-[2H8]-homocystine were confirmed by LC and mass spectrometry analysis. The derivatizing reagent, [N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide] (MTBSTFA), was obtained from Pierce Chemical Company (Rockford, IL). Citrate-stabilized pooled human plasma was obtained from Interstate Blood Bank (Memphis, TN). Human plasma patient samples and quality control (QC) human plasma pools were obtained from the Centers for Disease Control and Prevention (CDC) in Atlanta, GA. Poly-Prep prefilled chromatographic anion exchange columns (AG 1-X8) were purchased from Bio-Rad (Hercules, CA). Each anion-exchange column held an anion-exchange resin bed volume of 2 mL, 100200 mesh, in the chloride form. The 2 mL bed volume had an ion exchange capacity of 2.4 meq. Purified water (18 MΩ), prepared using a Millipore Milli-Q purification system, was used to prepare all samples and standards. All other chemical reagents and solvents were ACS reagent grade unless stated otherwise. Note: When not in use, crystalline homocystine standards were stored at room temperature and homocysteine stock/standard solutions were stored at -80 °C/-20 °C. Reagent concentrations given in terms of percent (%) are to be considered as mass

fractions (g/g) in all listed procedures, except where noted otherwise. Preparation of analyte stocks or standards and plasma samples were performed gravimetrically in all listed procedures, except where noted otherwise. LC/MS and LC/MS/MS. (1) Preparation of Hcy Stock Solutions and Calibration Standards. Homocysteine (Hcy) and deuterated homocysteine ([2H4]-Hcy) stock solutions (∼7400 µmol/L) were prepared by chemically reducing gravimetrically prepared homocystine and deuterated homocystine ([2H8]-homocystine) stock solutions, respectively. Briefly, dried homocystine or deuterated homocystine powder (∼10 mg) was weighed into a glass vial containing a known amount (∼10 g) of 0.1 mol/L HCl. The homocystine stock solution was then neutralized (pH ∼ 7) by the addition of a small portion (100 µL) of 10 mol/L NaOH. An appropriate amount (100 mg) of solid dithiothreitol (DTT) was added to the stock solution so that the concentration of DTT in solution was 1%. The final concentration of the Hcy stock solution was calculated upon the basis of the relationship that 1 mole of homocystine is chemically reduced to 2 mol of Hcy by the action of DTT. The reduced and stabilized Hcy stock solutions were subdivided into 1-mL aliquots and stored in 2-mL Eppendorf sample tubes at -80 °C until use. Hcy and [2H4]-Hcy standards (µmol/L) were prepared by weighing known amounts of the appropriate stock solution into weighed amounts of 1.5% aqueous DTT as required. (2) Preparation of Aqueous Linearity Standards. An internal standard stock solution (10 mL) containing ∼10 µmol/L [2H4]-Hcy in 1.5% DTT was prepared in a glass vial. An aqueous stock solution (1 mL) containing ∼500 µmol/L Hcy was prepared using the 10 µmol/L [2H4]-Hcy solution as the diluent. A set of 17 volumetric serial dilutions was prepared from the aqueous stock solution covering the range from 0.01 to 500 µmol/L Hcy using the 10 µmol/L [2H4]-Hcy stock solution as diluent. Each aqueous standard was injected (15 µL) in duplicate onto the LC/MS system to estimate the method’s linear dynamic range, limit of detection (LOD), and limit of quantification (LOQ). (3) Preparation of Samples and Calibrants. Approximately 30 µL of ∼70 µmol/L [2H4]-Hcy internal standard stock solution was added to a citrate-stabilized plasma sample (200 µL) in a 2-mL microcentrifuge tube (the final concentration of [2H4]-Hcy in the sample was ∼10 µmol/L). The tube was vortex-mixed for 1 min, 400 µL of 1.5% DTT in 0.15 mol/L NaOH was added, and the tube was vortex-mixed again for 1 min. The sample was then incubated for g15 min in a water bath held at 40 °C ( 1 °C to release and to reduce the protein-bound and oxidized forms of Hcy, respectively. Following the incubation period, the sample was kept cold (on ice) until anion exchange extraction of the total free Hcy could be performed. Plasma calibrants (200 µL) were prepared in an identical manner as the samples, except that sequentially increasing amounts of a ∼70 µmol/L Hcy stock solution were added to samples that contained a constant amount (∼30 µL) of ∼70 µmol/L [2H4]-Hcy internal standard stock solution. The sequential Hcy concentrations corresponded to 0 (calibrant blank), 5, 10, 15, 20, 25, and 50 µmol/L Hcy, and the [2H4]-Hcy concentration corresponded to a constant value of 10 µmol/L in the respective calibrants. Each calibrant tube was prepared and incubated as described previously and kept on ice until anion exchange extraction.

Hcy was extracted from plasma samples/calibrants using the Poly-Prep anion-exchange columns as follows (all extractions were completed utilizing a vacuum manifold system): (1) The column was preconditioned by rinsing with 3 mL of each of the following in sequence: methanol, water; (2) the sample was applied to the column and allowed to equilibrate for 1-2 min; (3) the column was washed with 9 mL of water, followed by 3 mL of methanol; (4) and finally, Hcy was eluted from the column by rinsing the column with 1 mL of methanol containing 0.4 mol/L of glacial acetic acid. The sample was evaporated (30 min with heat) to dryness using a Dry-Vac spin vacuum system and reconstituted with 200 µL of 1.5% aqueous DTT. Each sample/calibrant extract was injected (15 µL) in triplicate onto the LC/MS system. The Hcy calibration equations resulting from analysis of the plasma calibrants were corrected for the presence of endogenous Hcy by subtracting the endogenous Hcy concentration from each calibration point. (4) Optimization of Hcy Reduction (DTT Concentration Study). Pooled plasma was spiked to contain 10 µmol/L [2H8]homocystine. An aqueous standard of 10 µmol/L [2H8]-homocystine was also prepared for comparison. The plasma was divided into six 250-µL aliquots and prepared as described previously (section 3, above), except that the aliquots were incubated with 500 µL of either 0, 0.5, 1, 1.5, 2, or 2.5% DTT in 0.15 mol/L NaOH prior to anion exchange extraction and LC/MS analysis. The aqueous standard of [2H8]-homocystine was analyzed by LC/MS directly. Responses for [2H8]-homocystine ([M + H]+ ) m/z 277), [2H4]-Hcy ([M + H]+ ) m/z 140), and Hcy ([M + H]+ ) m/z 136) were simultaneously recorded (SIM mode) for each sample. Complete reduction of [2H8]-homocystine was indicated by loss of the response for m/z 277 and increase in the response for m/z 140. Complete release of protein-bound Hcy was indicated by increase and final stabilization of the response area ratio for m/z 136/m/z 140. (5) Optimization of Hcy Reduction (Heating Time Study). Pooled plasma was spiked to contain 10 µmol/L [2H4]-Hcy internal standard and then divided into six 250-µL aliquots. Each aliquot was prepared as described previously (section 3, above), except that the individual aliquots were subjected to sequentially increasing periods (0, 15, 30, 45, 60, and 75 min) of incubation (40 °C water bath) prior to anion exchange extraction and LC/MS analysis. The responses for [2H4]-Hcy ([M + H]+ ) m/z 140) and Hcy ([M + H]+ ) m/z 136) were simultaneously recorded (SIM mode) for each sample. (6) Optimization of Hcy Extraction (Extraction/Recovery Study). Low level (5 µmol/L), mid level (11 µmol/L) and high level (20 µmol/L) extraction/recovery samples were prepared in pooled plasma by adding known masses of Hcy stock solution (∼70 µmol/L) to weighed aliquots of plasma. Each aliquot was then spiked with a constant mass of [2H4]-Hcy stock solution (∼70 µmol/L) and extracted and analyzed by LC/MS as described previously (section 3, above). The resulting peak area ratios, along with independently prepared calibration plots, were used to determine the efficiency of the extraction step. The contribution from endogenous Hcy (7.9 µmol/L) in the plasma was subtracted from all Hcy values before computing final extraction efficiencies. (7) LC/MS and LC/MS/MS Instrumentation and Methods. LC/MS analyses were performed on a Waters 2795 LC Analytical Chemistry, Vol. 75, No. 4, February 15, 2003

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separations module coupled to a Waters-Micromass Quattro Ultima triple-quadrupole MS system with an installed ESI source. The MS system was operated as a stand-alone single-stage quadrupole mass analyzer during the development and evaluation of the selected ion-monitoring LC/MS method. The LC system was outfitted with a quaternary pump, a photodiode array (PDA) detector, a temperature-controlled (10 °C) autosampler, and an in-line mobile phase vacuum degasser. Samples were analyzed using a Supelcosil LC-CN cyano analytical column (4.6 mm × 250 mm, 5 µm particle size) with an attached Supelcosil LC-CN guard column (3 × 20 mm, 5-µm particle size). In all instances, the column temperature was thermostated at 30 °C. The LC elution conditions were as follows (all solvent percentages are volume fractions): buffer A ) 0.1% formic acid in water; buffer B ) 0.1% formic acid in methanol; time program ) 0 min, 96% A/4% B; 8.0 min, 96% A/4% B; 9 min, 0% A/100% B; 10 min, 0% A/100% B; 11 min, 96% A/4% B; 15 min, 96% A/4% B; flow rate ) 500 µL/ min. Scanning, selected-ion-monitoring (SIM), and multiplereaction-monitoring (MRM) mode mass spectra of Hcy and [2H4]Hcy were obtained and optimized via positive ion ESI. Tuning and mass calibration were performed by infusing a solution of RbCsI. The following instrumental parameters were used for LC/MS detection and quantification of Hcy and [2H4]-Hcy in SIM mode: dwell time ) 0.5 s, interchannel delay ) 0.1 s, interscan delay ) 0.05 s, capillary voltage ) 2.71 kV, cone voltage ) 30 V, hex 1 ) 16.6 V, hex 2 ) 0 V, aperture ) 0.5 V, source temperature ) 120 °C, desolvation temperature ) 400 °C, cone gas flow ) 155 L/h, desolvation gas flow ) 540 L/h, ion energy ) 0.5 V, and multiplier ) 650 V. The relevant SIM ions were m/z 136 for Hcy and m/z 140 for [2H4]-Hcy. The following instrumental parameters were used for LC/MS/MS detection and quantification of Hcy and [2H4]Hcy in MRM mode: dwell time ) 1 s, interchannel delay ) 0.03 s, interscan delay ) 0.02 s, capillary voltage ) 3.00 kV, cone voltage ) 40 V, hex 1 ) 14.6 V, hex 2 ) 0.5 V, aperture ) 0.0 V, source temperature ) 120 °C, desolvation temperature ) 400 °C, cone gas flow ) 155 L/h, desolvation gas flow ) 540 L/h, ion energy ) 0.5 V, multiplier ) 650 V, entrance lens ) -6 V, exit lens ) 5 V, collision energy ) 9 V, collision gas (argon) pressure ) ∼3.3 × 10-4 kPa. The relevant MRM mass transitions were m/z 136 f m/z 90 for Hcy and m/z 140 f m/z 94 for [2H4]-Hcy. GC/MS. (1) Preparation of Samples and Calibrants. The GC/MS method used in this work was based on the method of Stabler et al.31,32 Briefly, 1 mg of homocystine and 1 mg of [2H8]-homocystine were weighed into separate 100-mL volumetric flasks, and each flask was diluted to volume with distilled water. The concentration in each flask was approximately 0.037 mmol homocystine/kg solution. To a 2-mL microcentrifuge tube, a weighed aliquot (70 µL for calibrants and 35 µL for samples) of the [2H8]-homocystine stock solution was added. In this way, the [2H8]-homocystine solution was added to all calibrant and sample tubes. For calibrants, aliquots of the homocystine solution were added to give ratios of homocystine:[2H8]-homocystine of ∼0.3:1-2:1. For samples, 125 µL of plasma was weighed into each tube. To each sample and calibrant tube, 0.1 mL of 1% DTT in 1 mol/L NaOH was added, plus sufficient distilled water to bring the total volume to ∼1.1 mL. The tubes were placed in a heating block at 40 °C for 1 h. After 1 h, the samples were extracted with the anion778

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exchange resins and eluted with 3 mL of 0.4 M glacial acetic acid in methanol. The extracts were blown down under nitrogen in a water bath at 40 °C and then transferred to 1-mL reactivials. To each calibrant, 50 µL of MTBSTFA and 100 µL of acetonitrile were added; to each sample, 25 µL and 50 µL, respectively, were added. Samples and calibrants were heated at 90 °C for 30 min in a heating block and then transferred to autosampler vials for GC/ MS analysis. (2) GC/MS Instrumentation and Methods. GC/MS analyses were performed on an Agilent 5890 series II GC/MS system; the MS system utilized an electron ionization source. Samples were introduced via autosampler through the GC equipped with a splitless injector and a 30 m nonpolar capillary column (DB-5 MS). The injector and detector temperatures were 250 °C and 280 °C, respectively. The temperature program was 150 °C (initial), 0.5 min (hold time), 30 °C/min (heating rate) to 200 °C, 15 °C/ min (heating rate) to 290 °C (final temperature), 2 min (hold time), for a total time of 10.2 min. The electron multiplier voltage was tune voltage + 518. The injection volume was 1 µL. The monitored masses, beginning at 5 min, corresponded to the derivatized molecular ion for Hcy minus the butyl group, [M - butyl, m/z 420]+, and the derivatized molecular ion for [2H4]-Hcy minus the butyl group, [M - butyl, m/z 424]+. The retention time of the Hcy derivative under these analysis conditions was ∼7.7 min. Calibrants containing known mass ratios of unlabeled to labeled material were analyzed along with samples. The resulting area ratios for each calibrant, along with the known mass ratios, were subjected to linear regression analysis to produce a calibration equation. The calibration equation was then used to calculate the level of tHcy in plasma samples. RESULTS AND DISCUSSION Optimization of Hcy Reduction. The majority (∼99%) of circulating plasma Hcy exists as oxidized Hcy (homocystine, homocysteine-cysteine dimer, and protein-bound homocysteine), which consists mainly of Hcy bound to albumin.20 For accurate quantification of tHcy, it is critical that all of the oxidized forms of Hcy are converted to reduced Hcy (free Hcy); otherwise, low and inaccurate values for tHcy will result. Pooled plasma was spiked to contain 10 µmol/L [2H8]-homocystine instead of [2H4]Hcy to determine how much DTT was required to completely convert [2H8]-homocystine (oxidized) to [2H4]-Hcy (reduced) while simultaneously releasing protein-bound Hcy (details found in Experimental). After spiking exogenous [2H8]-homocystine into plasma and incubating aliquots of the plasma with increasing concentrations of alkaline DTT, the optimal concentration of DTT was determined to be ∼1.5%. By using an incubation medium containing 1.5% DTT in 0.15 mol/L NaOH, the response for [2H4]Hcy and the response area ratio for Hcy/[2H4]-Hcy were maximized. Further increases in DTT concentration did not yield the release of additional Hcy, and in fact, appeared to cause deterioration of the chromatographic profile. An additional experiment was performed in which a time-controlled equilibration study was conducted with pooled plasma to determine the effect of heating time on the complete release of protein-bound Hcy from plasma proteins. Plasma aliquots, spiked with 10 µmol/L [2H4]-Hcy internal standard, were subjected to increasing periods of equilibration in a heated water bath (details found in the Experimental Section). Results from this study indicated that (1) the [2H4]-Hcy

internal standard completely equilibrates within the plasma matrix in