Assay methods for the measurement of total homocyst(e)ine in plasma

Analytical methods to measure plasma total homocyst(e)ine (tHcy) concentrations are reviewed. High-pressure liquid chromatography (HPLC) with fluorometric detection is the most widely used method to determine plasma tHcy concentrations. Both monobromobimane and ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (SBD-F) are popular thiol-specific fluorogenic agents suitable for tHcy analysis. Monobromobimane has the advantage that it is highly reactive towards thiols, but its hydrolysis products are also fluorogenic, thus necessitating complex chromatography to obtain satisfactory separation between the compounds of interest and interferents. SBD-F does not show fluorescence, thus allowing isocratic separation of SBD-F derivatized thiols. SBD-thiol adducts are light sensitive and require protection against light to ensure reliable results. HPLC with electrochemical detection is also often used and has the advantage that no derivatization of thiols is required prior to detection. A recently reported liquid chromatography electrospray tandem mass spectrometric assay has the potential to become the reference method for plasma tHcy analysis. Other methods to measure plasma tHcy concentrations include gas-liquid chromatography, capillary electrophoresis, and immunoassays. Fluorescence polarization immunoassay compares well with gas chromatography-mass spectrometry and may become the method of choice in routine diagnostic clinical chemistry laboratories. Instability of tHcy in whole blood as well as postprandial and orthostatic variation are preanalytical factors that should be accounted for in plasma tHcy analysis. Between-method and between-laboratory variations in serum tHcy analysis are not yet satisfactory; certified reference material and standardization of the plasma tHcy assay will be essential to reduce between-laboratory bias.