Rapid and Accurate HPLC Assay for Plasma Total Homocysteine and Cysteine in a Clinical Laboratory Setting

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Abstract
Although several approaches for measuring plasma total homocysteine by HPLC have been described during the last few years (1)(2)(3)(4), none combines all the desired features for a rapid, user-friendly, and robust assay: (a) a stable, efficient, and nonhazardous reducing agent; (b) incorporation of a suitable internal standard; and (c) rapid, isocratic separation of the thiols of interest, using a mobile phase of mild pH. We have therefore modified the method of Vester and Rasmussen (5) by using tris(2-carboxyethyl)phosphine (TCEP), a newer stable, water-soluble phosphine derivative introduced by Gilfix et al. (6), as the reducing agent, cystamine as the internalnd isocratic separation of the thiols extracted from only 50 μL of pla standard, asma within 6 min.