High Performance Liquid Chromatographic Determination of Acetoacetate by Post-Column Derivatization with p-Nitrobenzene Diazonium Fluoroborate

We have applied a color-developing reagent, p-nitrobenzene diazonium fluoroborate (diazo reagent) as a post-column derivatization tool for the specific determination of acetoacetate (AcAc) in high performance liquid chromatography (HPLC). A mobile phase consisting of 50 mM KH2PO4, 4 mM tetra-n-butylammonium phosphate (TBAP) as an ion-pair reagent and 2 v/v% methanol, pH 3.5, diazo reagent solution with 0.2% triton X-100, and alkaline solution of 1.5 mol/l NaOH were pumped using three independent pumps. Specific color development on-line was monitored at 645 nm. A calibration curve for ACAC standard solution with an injection volume of 20 pl showed a good linearity in the range 0.01-2.5 mM with a correlation coefficient of 0.999. For the de-termination of 3-hydroxybutyrate (3-HOBA), 3-HOBA was converted to ACAC by an enzymatic-coupling method using 3-HOBA dehydrogenase and lactate dehydrogenase. Analytical recoveries of ACAC and 3-HOBA added to serum and urine were satisfactory.