Trace analysis of β-hydroxybutyrate in human plasma by derivatization and high-performance liquid chromatography

A simple and sensitive liquid chromatographic method is described for determination of β-hydroxybutyrate, as a highly sensitive derivative, from human plasma. Plasma spiked with β-hydroxybutyrate was extracted with acetonitrile and derivatized with 4-bromomethyl-7-methoxycoumarin, in a homogeneous system, using 15-crown-5 as activator. The resulting derivative was separated on a LiChroCART Rp-C18 column with 40% methanol in water as mobile phase and 2,2′-dinitrobiphenyl as internal standard. Conditions affecting the extraction and derivatization of β-hydroxybutyrate from spiked plasma were investigated. For β-hydroxybutyrate determination the detection limit (S/N=3; sample size, 10 μL) was approximately 5 nmol mL−1; the relative standard deviation was <9% for intra-day assay (n=6) and <10% for inter-day assay (n=6). All recoveries were >98%.