A simple, sensitive and efficient assay for the determination of d- and l-lactic acid enantiomers in human plasma by high-performance liquid chromatography

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 λ(ex)=490 nm and λ(em)=532 nm. The separation was achieved by use of a C18 analytical column (Synergy Hydro 150 mm x 3 mm i.d., 4 μm). The calibration curve was linear over the on-column concentration range of 10-200 μmol/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 μmol/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.