title: HPLC determination of D-3-hydroxybutyric acid by derivatization with a benzofurazan reagent and fluorescent detection: application in the analysis of human plasma.
abstract: 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.
authors: Department of Chemistry and Industrial Chemistry, University of Genoa, Via Dodecaneso, 31-16146 Genoa, Italy; CNR, Institute of Chemical Methodologies, Rome, Italy. Electronic address: giorgio.cevasco@unige.it.
authors: Giorgio
authors: G
authors: Cevasco
authors: Department of Chemistry and Industrial Chemistry, University of Genoa, Via Dodecaneso, 31-16146 Genoa, Italy. Electronic address: anna.piatek@chemessentia.it.
authors: Anna Maria
authors: AM
authors: Piątek
authors: Department of Chemistry and Industrial Chemistry, University of Genoa, Via Dodecaneso, 31-16146 Genoa, Italy. Electronic address: sergio.thea@unige.it.
authors: Sergio
authors: S
authors: Thea
mesh_terms: D020155:3-Hydroxybutyric Acid / Q000097:blood* / Q000737:chemistry* / Q000302:isolation & purification; D001583:Benzoxazoles / Q000737:chemistry*; D001774:Blood Chemical Analysis / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry; D057230:Limit of Detection; D010069:Oxadiazoles / Q000737:chemistry*; D010879:Piperazines / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013237:Stereoisomerism; D013997:Time Factors
chemical_list: C000591997:(2S)-2-amino-3-methyl-1-(4-(7-nitro-benzo-2,1,3-oxadiazol-4-yl)-piperazin-1-yl)-butan-1-one; D001583:Benzoxazoles; D007202:Indicators and Reagents; D010069:Oxadiazoles; D010879:Piperazines; C020160:benzofurazan; D020155:3-Hydroxybutyric Acid
keywords: Biological samples; Clinical/biomedical analysis; Fluorescence/luminescence; HPLC