HPLC determination of d-3-hydroxybutyric acid by derivatization with a benzofurazan reagent and fluorescent detection: Application in the analysis of human plasma

A simple and sensitive new method for the determination of d-3-hydroxybutyric acid (D-3-HBA) in human plasma after derivatization is described. The proposed method is based on the reaction of (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) with D-3-HBA in the presence of O-(7-azobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) and N-ethyldiisopropylamine (DIEA) to produce a fluorescent derivative. The formed derivative was monitored fluorimetrically at λex = 489 nm and λem = 532 nm. The HPLC analysis was carried out by use of a C18 analytical column (Synergy Hydro 150 mm × 3 mm, i.d., 4 μm) with a binary gradient elution program of 0.1% aqueous trifluoroacetic acid versus methanol. The method showed satisfactory linearity (r2 = 0.9997) in the range from 20 to 500 μmol/L. The limit of detection (LOD) of the method was 7.7 μmol/L, while the limit of quantitation (LOQ) was 25.8 μmol/L. The analytical method was successfully applied to human plasma samples from normal healthy subjects.