Majallah-i Dānishgāh-i ̒Ulūm-i Pizishkī-i Bābul (Apr 2005)
Measurement of total plasma Homocysteine with reverse phase high performance liquid chromatography (RP-HPLC) method without internal standard
Abstract
Background and Objective: Homocysteine is an amino acid with a free thiol (Sulphydryl) group and is an intermediate formed during the metabolism of methionine to cysteine. Measurement of total plasma Homocysteine may be of value in several clinical conditions including homocysteinuria, atherosclerosis, thrombophilia and folate/vitamin B12 deficiency. The purpose of this study was to measure total plasma Homocysteine using RP-HPLC with fluorescence detection method. Methods: Total plasma Homocysteine was measured with reverse-phase HPLC with fluorescence detection after reduction of Homocysteine with tris (2-carboxyetile) phosphine (TCEP) and derivatization of plasma thiols with ammonium 7-fluorobenzo-2-oxa 1,3-diazole-4-sulfonate (SBD-F). Findings: The limit of detection for Homocysteine was 0.2 µmol/l. The within day coefficient of variation (CV) was 2.67% to 4.56% and the between day coefficient of variation was 5.43% to 8.17%. The mean recovery of Homocysteine was 93% to 103.4%. Conclusion: The results show that the HPLC method with fluorescence detection for measurement of total plasma Homocysteine, is sensitive (The limit of detection 0.2 µmol/l), accurate (CV between 2.67% and 8.17%) and it has acceptable recovery (93% to 103.4%). Therefore, it is confident method especially for investigate application.
