An optimized HPLC-ECD detection method for the assay of homocysteine: an independent risk factor for vascular disease

An ion-paired reversed-phase high performance liquid chromatography with electrochemical detection (HPLC-ECD) was developed for the determination of total plasma homocysteine (tHcy). The method provids a simple, precise and reliable tHcy assay at low cost. The effect of each component of the mobile phase on homocysteine retention time in the HPLC-ECD system was studied, including the influences of buffer pH, buffer materials, buffer concentration, ion pair concentration, selectivity of the internal standard, and methanol concentration in the mobile phase. The specificity, extraction ratio, precision and accuracy of the assay were also studied. The optimal assay of homocysteine was achieved with a mobile phase of pH 2.6, 0.01 M monosodium phosphate buffer (containing 26 mM octane sulfonate sodium): methanol in a 81:19 (v/v) ratio with detecion at guard cell 800 mV, analytical cell El 500 mV, and E2 700 mV. The recovery of tHcy ranged from 96.7% to 106.2% at the concentration range of 0.5 μg/mL to 5 μg/mL in plasma. The mean correlation coefficients of intraassay (n = 6) and interassay (n = 6) calibration curves for tHcy were both 0.999 over a concentration range of 0.1 μg/mL to 10 μg/mL. The limit of detection for intraassay and interassay were 0.049 μg/mL (0.37 μM) and 0.088 μg/mL (0.65 μM), respectively and the limit of quantitation were 0.151 μg/mL (1.12 μM) and 0.266 μg/mL (1.97 μM), respectively. The assay is suitable for the determination of tHcy levels in clinical stroke patients.