Gas chromatographic-mass spectrometric determination of alpha-ketoisocaproic acid and [2H7]alpha-ketoisocaproic acid in plasma after derivatization with N-phenyl-1,2-phenylenediamine.
A method for determination of alpha-ketoisocaproic acid (KIC) and [4,5,5,5,6,6,6-2H7]alpha-ketoisocaproic acid ([2H7]KIC) in rat plasma was developed using gas chromatography-mass spectrometry-selected ion monitoring (GC-MS-SIM). [5,5,5-2H3]alpha-Ketoisocaproic acid ([2H3]KIC) was used as an analytical internal standard to account for losses associated with the extraction, derivatization and chromatography. The keto acids were extracted by cation-exchange chromatography using BondElut SCX cartridge and derivatized with N-phenyl-1,2-phenylenediamine to form N-phenylquinoxalinone derivatives. Quantitation was performed by SIM of the respective molecular ions at m/z 278, 281 and 285 for the derivatives of KIC, [2H3]KIC and [2H7]KIC on the electron impact method. The limit of detection was found to be 70 fmol per injection (S/N=3) and the limit of quantitation for [2H7]KIC was around 50 nM in rat plasma. Endogenous KIC concentrations in 50 microl of rat plasma were measured with relative intra- and inter-day precision of 4.0% and 3.3%, respectively. The intra- and inter-day precision for [2H7]KIC spiked to rat plasma in the range of 0.1 to 10 microM gave good reproducibility with relative standard deviation (RSD) of 6.5% and 5.4%, respectively. The intra- and inter-day relative errors (RE) for [2H7]KIC were less than 6.4% and 3.8%, respectively. The method was applied to determine the plasma concentration of [2H7]KIC after an intravenous administration of [2H7]KIC in rat.
Department of Pathophysiology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Hachioji, Japan.
T
T
Matsukawa
H
H
Hasegawa
Y
Y
Shinohara
T
T
Hashimoto
D000818:Animals; D002138:Calibration; D003903:Deuterium; D008401:Gas Chromatography-Mass Spectrometry; D007536:Isomerism; D007651:Keto Acids / Q000097:blood* / Q000493:pharmacokinetics; D010655:Phenylenediamines / Q000737:chemistry*; D051381:Rats; D017207:Rats, Sprague-Dawley
D007651:Keto Acids; C427843:N-phenyl-1,2-phenylenediamine; D010655:Phenylenediamines; C013082:alpha-ketoisocaproic acid; D003903:Deuterium