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标题：High Performance Liquid Chromatographic Determination of Acetoacetate by Post-Column Derivatization with p-Nitrobenzene Diazonium Fluoroborate
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作者：Susumu Yamato ; Kiyomi Kobayashi ; Keiko Ebara 等
期刊名称：Biological and Pharmaceutical Bulletin
印刷版ISSN：0918-6158
电子版ISSN：1347-5215
出版年度：2003
卷号：26
期号：4
页码：397-400
DOI：10.1248/bpb.26.397
出版社：The Pharmaceutical Society of Japan
摘要：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 m M KH₂PO₄, 4 m M 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 μl showed a good linearity in the range 0.01—2.5 m M 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.
关键词：acetoacetate;diazo reagent;high performance liquid chromatography (HPLC);3-hydroxybutyrate;ketone body