Determination of plasma total homocysteine and cysteine using HPLC with fluorescence detection and an ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulphonate (SBD-F) derivatization protocol optimized for antioxidant concentration, derivatization reagent concentration, temperature and matrix pH

A sensitive HPLC-fluorescence method for determining total endogenous plasma homocysteine (Hey), cysteine (Cys) and cysteinylglycine (Cys-Gly) following derivatization with ammonium 7-fluoro 2,1,3-benzoxodiazole-4-sulphonate (SBD-F) is described. Quantitation utilizes an internal standard, 2-mercaptoethylamine. The derivatization procedure has been optimized for concentration of SBD-F, reducing agent (tributylphosphine) and temperature. Findings indicate that values for plasma determinations vary according to the nature of the matrix in which calibration standards are made up. If quantitation is based on a peak height ratio, then standards should be made up in either pH 7.4 phosphate buffered saline or plasma taking into account the endogenous thiol concentration. These findings are based on calibration data, and 30 plasma samples quantified using thiol standards made up in plasma, pH 7.4 and pH 9.5 buffers. By defining how this matrix/pH effect influences thiol quantitation, it should be possible to make a more meaningful comparison of Hey measurements between laboratories. The chromatographic separation was investigated at several mobile-phase pH values with the following conditions ascertained to be optimal: a mobile phase consisting of 5% (v/v) acetonitrile in 0.1 M KH2PO4, pH 2.15 was run at a flow rate of 0.5 mL/min. It was used in conjunction with a Supelco LC-18 base deactivated analytical column (150 x 4.6 cm i.d. 3 mu M bonded silica). The internal standard and thiols were measured by fluorescence detection at 385 nm excitation and 515 nm emmission. Plasma levels are easily measured in a 100 mu L volume. Storage for 2 months at -20 degrees C resulted in no deterioration of thiols. Furthermore, no difference in thiol levels was observed between bloods collected in lithium heparin and EDTA. Collected blood should, however, be separated as soon as possible to avoid red cell metabolism of Hey which was observed in a case of hyperhomocysteinemia. Once derivatized, thiols are stable for at least one week at +4 degrees C.