High-performance liquid chromatography determination of ketone bodies in human plasma by precolumn derivatization with p-nitrobenzene diazonium fluoroborate |
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Authors: | Yamato Susumu Shinohara Kumiko Nakagawa Saori Kubota Ai Inamura Katsushi Watanabe Gen Hirayama Satoshi Miida Takashi Ohta Shin |
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Institution: | a Department of Bioanalytical Chemistry, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata, Niigata 956-8603, Japan b Department of Pharmacy, Toyama Rousai Hospital, Uodu, Toyama 937-0042, Japan c Department of Pharmacy, Tsubame Rousai Hospital, Tsubame, Niigata 959-1228, Japan d Division of Endocrinology and Metabolism, Department of Homeostatic Regulation and Development, Niigata University Graduate School of Medical and Dental Sciences, Chuo-ku, Niigata 951-8510, Japan e Department of Clinical Laboratory Medicine, Juntendo University School of Medicine, Bunkyo-ku, Tokyo 113-8421, Japan f Department of Pharmaceutical Health Care and Sciences, Faculty of Pharmaceutical Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192-0392, Japan |
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Abstract: | We developed and validated a sensitive and convenient high-performance liquid chromatography (HPLC) method for the specific determination of ketone bodies (acetoacetate and d-3-hydroxybutyrate) in human plasma. p-Nitrobenzene diazonium fluoroborate (diazo reagent) was used as a precolumn derivatization agent, and 3-(2-hydroxyphenyl) propionic acid was used as an internal standard. After the reaction, excess diazo reagent and plasma proteins were removed by passing through a solid-phase cartridge (C18). The derivatives retained on the cartridge were eluted with methanol, introduced into the HPLC system, and then detected with UV at 380 nm. A calibration curve for acetoacetate standard solution with a 20-μl injection volume showed good linearity in the range of 1 to 400 μM with a 0.9997 correlation coefficient. For the determination of d-3-hydroxybutyrate, it was converted to acetoacetate before reaction with the diazo reagent by an enzymatic coupling method using d-3-hydroxybutyrate dehydrogenase and lactate dehydrogenase. A calibration curve for d-3-hydroxybutyrate standard solution also showed good linearity in the range of 1.5 to 2000 μM with a 0.9988 correlation coefficient. Analytical recoveries of acetoacetate and d-3-hydroxybutyrate in human plasma were satisfactory. The method was successfully applied to samples from diabetic patients, and results were consistent with those obtained using the thio-NAD enzymatic cycling method used in clinical laboratories. |
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Keywords: | Acetoacetate d-3-Hydroxybutyrate" target="_blank">d-3-Hydroxybutyrate p-Nitrobenzene diazonium fluoroborate HPLC UV detection Human plasma Diabetes mellitus |
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