title: A simple, sensitive and efficient assay for the determination of D- and L-lactic acid enantiomers in human plasma by high-performance liquid chromatography.
abstract: 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.
authors.affiliation: Department of Chemistry and Industrial Chemistry, University of Genoa, Via Dodecaneso 31, 16146 Genoa, Italy. giorgio.cevasco@unige.it
authors.forename: Giorgio
authors.initials: G
authors.lastname: Cevasco
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Piątek
authors.forename: Carlo
authors.initials: C
authors.lastname: Scapolla
authors.forename: Sergio
authors.initials: S
authors.lastname: Thea
mesh_terms: D000328:Adult; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase; D005260:Female; D006801:Humans; D019344:Lactic Acid / Q000097:blood* / Q000737:chemistry; D016014:Linear Models; D008297:Male; D008875:Middle Aged; D008968:Molecular Conformation; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013237:Stereoisomerism
chemical_list: D019344:Lactic Acid