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 KH(2)PO(4), 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 microl showed a good linearity in the range 0.01-2.5 mM with a correlation coefficient of 0.999. For the determination 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.
Yamato
Susumu
S
Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Japan. yamatos@niigata-pharm.ac.jp
Kobayashi
Kiyomi
K
Ebara
Keiko
K
Shimada
Kenji
K
Ohta
Shin
S
D000090:Acetoacetates / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods; D003979:Diazonium Compounds / Q000032:analysis* / Q000737:chemistry*
D000090:Acetoacetates; D003979:Diazonium Compounds; C016635:acetoacetic acid; C046321:4-nitrobenzene diazonium tetrafluoroborate