Improved HPLC method for total plasma homocysteine detection and quantification.

Abstract

Recent clinical research has pointed at hyperhomocysteinemia as an independent risk factor in a number of cardiovascular and neurological diseases. We have improved a chromatographic method of total plasma homocysteine measurements in order to obtain higher sensitivity, reliability and reproducibility. The method demonstrates excellent linearity (R=0.999), range (<2-100 microM), precision (instrumental RSD 0.06 and method RSD 1.17), accuracy (recovery of 99.92 and RSD 1.27), reproducibility, quantification limit and ruggedness (e.g. pH from 2.0 to 2.5). Because even a small increase in homocysteine level can be a significant risk factor of cardiovascular diseases, such a precise method is required. The constructed method allows the measurement of plasma pyridoxal phosphate, PLP, the co-enzyme form of vitamin B(6), on the same column and similar reagents. The developed method has been successfully applied to measure both total plasma and serum homocysteine in a group of acute stroke patients.