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Clinical Chemistry 47: 2157-2159, 2001;
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(Clinical Chemistry. 2001;47:2157-2159.)
© 2001 American Association for Clinical Chemistry, Inc.

Articles

Critical Role of pH for Derivatization of Homocysteine with Benzofurazanes

Anna Rita Bonfigli1, Gilberto Coppa2, Ivano Testa3, Anna Gambini2 and Roberto Testa1a

1 Department of Gerontological Research, Diabetology Unit, Italian National Research Centres on Aging, Via Birarelli 8, I-60121 Ancona, Italy;
2 Department of Clinical Pathology, General Hospital of Ancona, Ancona, Italy;
3 Institute of Internal Medicine, University of Ancona, Ancona, Italy

aauthor for correspondence: 39-071-2206106, e-mail r.testa@inrca.it


   Introduction
 
HPLC analysis combined with fluorescence detection is one of the most effective and widely used techniques for the specific determination of total homocysteine in plasma (tHCY) (1)(2)(3)(4). These methods often use benzofurazanes as derivatization reagents that are considered to be specific for the thiol group of HCY (5)(6)(7)(8)(9)(10)(11)(12)(13)(14). The specificity of benzofurazanes for thiols should be questioned because benzofurazanes have been used as NH2 derivatization reagents (15)(16). Because HCY possesses both -SH and -NH2 groups, we asked whether the widely reported pH of sample derivatization (from 8 to 9.5) (8)(13)(14) ensures thiol-selective derivatization. We performed sample derivatizations at pHs of 7.5–10.5 and an HPLC method (8) for tHCY determination with some modifications.

HCY, cysteamine, cysteine, cysteinylglycine, 7-fluoro-2,1,3-benzoxadiazole-4-sulfonamide (ABD-F), tris-carboxyethyl-phosphine, and other common reagents for the HPLC assay of tHCY were purchased from Sigma. Acetonitrile, isopropyl alcohol, and water, all . . . [Full Text of this Article]


   Acknowledgments
 

   References
 





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