High-performance liquid chromatography determination of ketone bodies in human plasma by precolumn derivatization with p-nitrobenzene diazonium fluoroborate.
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 (C(18)). 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-microl injection volume showed good linearity in the range of 1 to 400 microM 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 microM 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.
Yamato
Susumu
S
Department of Bioanalytical Chemistry, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata, Niigata 956-8603, Japan.
Shinohara
Kumiko
K
Nakagawa
Saori
S
Kubota
Ai
A
Inamura
Katsushi
K
Watanabe
Gen
G
Hirayama
Satoshi
S
Miida
Takashi
T
Ohta
Shin
S
D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003979:Diazonium Compounds / Q000737:chemistry*; D006801:Humans; D007657:Ketone Bodies / Q000097:blood*; D010666:Phenylpropionates / Q000737:chemistry
C118677:3-(2-hydroxyphenyl)propionic acid; D003979:Diazonium Compounds; D007657:Ketone Bodies; D010666:Phenylpropionates; C046321:4-nitrobenzene diazonium tetrafluoroborate