Quantitation of total homocysteine in human plasma by derivatization to its N(O,S)-propoxycarbonyl propyl ester and gas chromatography–mass spectrometry analysis

Abstract

Much evidence supports the hypothesis that mild or moderate hyperhomocysteinaemia represents an important and independent risk factor for occlusive vascular diseases. Therefore, the accurate and reliable determination of total plasma homocysteine has gained major importance for risk assessment. Furthermore, it can help in the detection of folate and vitamin B 12 deficiency. This has prompted us to develop a sensitive gas chromatography–mass spectrometry (GC–MS) method in order to quantify total homocysteine in human plasma. Prior to chromatography, reduced homocysteine was released from disulfide bonds by incubation with excess dithiothreitol and converted into its N(O,S)-propoxycarbonyl propyl ester by derivatization with n-propyl chloroformate. Aminoethylcysteine served as internal standard. The method proved to be highly linear over the entire concentration range examined (corresponding to 0–266 μ M homocysteine) and showed intra-assay and inter-assay variation (relative standard deviations) of approximately 5 and 5–10%, respectively. External quality control by comparison with duplicate analyses performed on a HPLC-based system revealed satisfactory correlation. The newly developed GC–MS based method provides simple, reliable and fast quantitation of total homocysteine and requires only inexpensive chemicals, which are easy to obtain.