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 beta-hydroxybutyrate, as a highly sensitive derivative, from human plasma. Plasma spiked with beta-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-C-18 column with 40% methanol in water as mobile phase and 2,2'-dinitrobiphenyl as internal standard. Conditions affecting the extraction and derivatization of beta-hydroxybutyrate from spiked plasma were investigated. For beta-hydroxybutyrate determination the detection limit (S/N = 3; sample size, 10 A) 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%.