Mehmet Kemal ERBİL, Emin Özgür AKGÜL, Halil YAMAN, Cumhur BİLGİ, Ömer ÖZCAN, Erdinç ÇAKIR

A Comparison of Three High Performance Liquid Chromatographic (HPLC) Methods for Measurement of Plasma Total Homocysteine

Homocysteine (tHcy) has been in routine use in many centers after this amino acid was reported as an independent risk factor for cardiovascular, cerebrovascular and peripheral vascular diseases. Many high-performance liquid chromatographic (HPLC) methods including a commercially available kit (Chromsystems) were reported for measuring tHcy in biologic samples. Although the commercial kits are believed to have higher precision, sensitivity and accuracy as well as practical and fast, it may cause high prices by increased test ordering. In this study we aimed to compare two different low-cost methods which had similar advantages with commercial kit as a reference method. In the first method the sample was derivatized with 7-fluoro-2,1,3-benzoxa sulfonamide (ABD-F) following the reduction with tris-(2-carboxylethyl) phosphine (TCEP). Homocysteine peak was obtained at 4.7 min and total run time was 7 min. In the second method, samples were derivatized with 7-Fluorobenzo-2-oxa-1,3-diazole-4-sulfonic acid (SBD-F). Homocysteine peak was obtained at 2.9 min and total run time was 5 min. In both methods cysteamine hydrochloride was added as an internal standard and C18 (ODS) (150 X 4.6 mm, 5 µm) column was used. Intraassay CV's of SBD-F and ABD-F methods was 0.98% and 1.83%, respectively. Interassay CV values were 2.22% and 2.49%, respectively. Both methods show linearity up to 200 µmol /L. Recovery was 99.37% and 98.10% in SBD-F method after additions of 7-14 µmol / L homocysteine standard. Similarly, for ABD-F method, recovery was 99.7% and 100.7% following the addition of homocysteine standard (6.7-13.4 µmol/L). A significant correlation was found between both methods and reference method (P < 0.001). In conclusion, SBD-F and ABD-F methods were reproducible, rapid, and easy to use for the quantification of plasma tHcy, with similar advantages of the commercially available kit. Moreover, the SBD-F method may be the method of choice among these assays because of the lowest cost per test and better precision.

Anahtar Kelimeler:

Homocysteine, HPLC, Method, SBDF, ABDF

A Comparison of Three High Performance Liquid Chromatographic (HPLC) Methods for Measurement of Plasma Total Homocysteine

Keywords:

Homocysteine, HPLC, Method, SBDF, ABDF,

PDF

___

Mudd SH, Finkelstein JD, Refsum H et al. Homocysteine and its disulfide derivatives: a suggested consensus terminology. Arterioscler Thromb Vasc Biol 20: 1704-6, 2000. 2. Graham IM, Daly LE, Refsum HM et al. Plasma homocysteine as a risk factor for vascular disease. JAMA 277: 1775 –81, 1997.
Pahor M, Elam MB, Garrison RJ et al. Emerging noninvasive biochemical measures to predict cardiovascular risk. Arch Intern Med 159: 237-45, 1999.
Clarke R, Daly L, Robinson K et al. Hyperhomocysteinemia: an independent risk factor for vascular disease. N Engl J Med 324: 1149-55, 1991.
Boushey CJ, Beresford SA, Omenn GS et al. A quantitative assessment of plasma homocysteine as a risk factor for vascular disease: Probable benefits of increasing folic acid intakes. JAMA 274: 1049-57, 1995.
Finkelstein JD, Martin JJ. Homocysteine. Int J Biochem Cell Biol 32: 385-9, 2000.
Ueland PM, Refsum H, Stapler SP et al. Total homocysteine in plasma or serum: methods and clinical applications. Clin Chem 39: 1764-79, 1993. 8. Bald E, Chwatko G, Glowacki R et al. Analysis of plasma thiols by high-performance liquid chromatography with ultraviolet detection. J Chromatogr A 1032: 109-15, 2004.
Ubbink JB. Assay methods for the measurement of total
homocyst(e)ine. Semin Thromb Hemost 26: 233-41, 2000.
Febriani AD, Sakamoto A, Ono H et al. Determination of total homocysteine in dried blood spots using high performance liquid chromatography for homocystinuria newborn screening. Pediatr Int 46: 5-9, 2004.
Ducros V, Demuth K, Sauvant MP et al. Methods for homocysteine analysis and biological relevance of the results. J Chromatogr B 781: 207-26, 2002.
Hoffer LJ. Methods for measuring sulfur amino acid metabolism. Curr Opin Clin Nutr Metab Care 5: 511-7, 2002.
Fickerstrand T, Refsum H, Kvalheim K et al. Homocysteine and other thiols in plasma and urine: automated determination and sample stability. Clin Chem 39: 263-71, 1993.
Fermo I, De Vecchi E, Arcelloni C et al. Methodological aspects of total plasma homocysteine measurement. Haematologica 82: 246-50, 1997.
Dias VC, Bamforth FJ, Tesanovic M et al. Evaluation and intermethod comparison of the Bio-Rad high-performance liquid chromatographic method for plasma total homocysteine. Clin Chem 44: 2199-201, 1998.
Ubbink JB, Hayward Vermaak WJ, Bissbort S. Rapid high- performance liquid chromatographic assay for total homocysteine levels in human serum. J Chromatogr. 565: 441-6, 1991.
Gilfix BM, Blank DW, Rosenblatt DS. Rapid high-performance liquid chromatographic assay for total homocysteine levels in human serum. Clin Chem 43: 687-8, 1997.
Kuo K, Still R, Cale S et al. Standardization (external and internal) of HPLC assays for plasma homocysteine. Clin. Chem. 43: 1653- 5, 1997.
Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1: 307-10, 1986.
Daskalakis I, Lucock MD, Anderson A et al. Determination of plasma total homocysteine and cysteine using HPLC with fluorescence detection and an ammonium 7-fluoro-2, 1, 3- benzoxadiazole-4-sulphonate (SBD-F) derivatization protocol optimized for antioxidant concentration, derivatization reagent concentration, temperature and matrix pH. Biomed
Chromatogr 10: 205-12, 1996. (2.) Pfeiffer CM, Huff DL, Gunter EW. Rapid and accurate HPLC assay for plasma total homo-cysteine and cysteine in a clinical laboratory setting. Clin Chem 45: 290-2, 1999.
Reddy MN, Behnke C. A rapid and simple assay to determine total homocysteine and other thiols in pediatric samples by HPLC and fluorescence detection. J Liq Chromatogr Relat Technol 20: 1391-1408, 1997.