Gas chromatography-mass spectrometry of carboxylic acids in tissues as their tert.-butyldimethylsilyl derivatives.

Krebs cycle and related acids were isolated from liver, pancreas and stomach of rats by pulverizing frozen tissue samples in liquid nitrogen with an aqueous solution of perchloric acid and methoxylamine hydrochloride. Perchloric acid and basic and neutral compounds were removed by ion-exchange chromatography on DEAE-Sephadex and SP-Sephadex columns. Phosphate was removed without loss of citrate by partition chromatography on Sephadex G-25 in a butanol--water system. Loss of acids during evaporation of water was prevented by keeping the temperature low and by addition of ammonia and tetrabutyl-ammonium bromide. tert.-Butyldimethylsilyl derivatives were prepared, purified by chromatography on a Sephadex LH-20 column and separated by gas chromatography on a non-polar capillary column. Recoveries in the procedure were above 45% for all acids except acetoacetate (25%). The mass spectrometric fragmentation of the methoxime-tert.-butyldimethylsilyl derivatives was studied by analysis of the derivatives of deuterated acids. The intense ions at M-57 present in all spectra appeared to contain all original hydrogen atoms. Thus, the method should be suitable for quantitation by isotope dilution and in metabolic studies with deuterated compounds.