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 λ(ex)=489 nm and λ(em)=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 (r(2)=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.

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.

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

Altered levels of aminothiols in biological fluids are thought to be an important risk indicator for several diseases, and reliable methods for the accurate determination of aminothiols concentrations in plasma are thus required. In this paper ammonium 5-bromo-7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (SBD-BF) is proposed as a convenient fluorogenic derivatizating reagent for the determination of aminothiols (cysteine, cysteinylglycine, homocysteine and glutathione) by HPLC with fluorescence detection. The reactions of SBD-BF with aminothiols at room temperature are about three-times faster than those of ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (the most frequently employed reagent) at 60 degrees C. The derivatives of SBD-BF with cysteine, cysteinylglycine, homocysteine and glutathione are easily separated by HPLC and their calibration curves show excellent linearity over the range 0.05-20 micromol/L with excellent r(2) values for all analytes. SBD-BF reacts with thiols under mild conditions, i.e. at 25 degrees C over about 30 min, and is proposed as a suitable fluorogenic reagent for thiol derivatization to be introduced in analytical clinical chemistry. The detection limits of Cys, Cys-Gly, Hcy and GSH at a signal-to-noise ratio of 5 were 0.1 microM for Cys, 0.01 microM for Cys-Gly and Hcy, and 0.02 microM for GSH. Furthermore, validation parameters of the proposed method are quite satisfactory. As an application of this method the determination of thiol derivatives in human plasma was carried out on a number of samples.