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

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. A mixture of 50 μL of plasma, 25 μL of internal standard, and 25 μL of phosphate-buffered saline (PBS, pH 7.4) was incubated with 10 μL of 100 g/L TCEP (Pierce Chemical Co.) for 30 min at room temperature to reduce and release protein-bound thiols, after which 90 μL of 100 g/L trichloroacetic acid containing 1 mmol/L EDTA was added for deproteinization. After the sample was centrifuged for 10 min at 13000 g , 50 μL of the supernatant was added to an autosampler vial containing 10 μL of 1.55 mol/L NaOH; 125 μL of 0.125 mol/L borate buffer containing 4 mmol/L EDTA, pH 9.5; and 50 μL of 1 g/L SBD-F (Wako Chemicals) in the borate buffer. The sample was then incubated for 60 min at 60 °C. HPLC was carried out on a 2690 Alliance solvent delivery system …