An improved method for simultaneous analysis of aminothiols in human plasma by high performance liquid chromatography with fluorescence detection

A new method for the simultaneous determination of d- and l-lactic acid in human plasma has been developed using high-performance liquid chromatography (HPLC) with fluorescence detection. This method is based on the reaction of lactic acid with (2S)-2-amino-3-methyl-1-[4-(7-nitro-benzo-2,1,3-oxadiazol-4-yl)-piperazin-1-yl]-butan-1-one (NBD-PZ-Val) in the presence of O-(7-azobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) and N-ethyldiisopropylamine (DIEA) to produce fluorescent diastereomeric derivatives that were easily monitored fluorimetrically at ex = 490nm and em = 532 nm. The separation was achieved by use of a C18 analytical column (Synergy Hydro 150mm×3mm i.d., 4m). The calibration curve was linear over the on-column concentration range of 10–200 mol/L for d-lactic acid and 0.5–4.0 mmol/L for l-lactic acid. The sensitivity was good with a limit of detection of 5.24 mol/L for d-lactic acid and 0.15 mmol/L for l-lactic acid. The analytical method was successfully applied to human plasma samples from normal healthy subjects.