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A Liquid Chromatography ? Mass Spectrometry Method for the Determination of Alpha-Ketoglutaric Acid in Human Plasma.

Gundoju Suresh, PE Thirugnanam, and Atmakuru Ramesh

Alpha-ketoglutaric acid (A-KG) is currently under investigation as promising cancer cell damaging agent and cyanide antidote. A simple, specific liquid chromatography-mass spectrometry (LC-MS/MS) method was developed for the quantitative determination of A-KG in human plasma. Derivatization of A-KG was achieved by using N-methyl imidazole in presence of trifluroacetic anhydrate. Several parameters, which affected the yield of the derivatization reaction, such as reaction temperature and time were optimized. An imidazole derivative of A-KG was extracted using methyl tertiary butyl ether and quantified in positive electron spry ionization mode (mass-to-charge ratio (m/z):197). The method was fully validated using the parameters such as specificity, linearity, limit of detection and quantification, precision, accuracy, recovery, robustness and ruggedness. Linearity was demonstrated over the concentration rage of 100-2500 ng mL-1 with a correlation coefficient (R2) 0.9991. The limit of detection and quantification were found to be 40 and 100 ng mL-1 respectively. Intra- and inter-day accuracy and precision were within the acceptable limit (<8%).The robustness and ruggedness was evaluated using Plackett-Burman experimental design.