Analysis of pipecolic acid in biological fluids using capillary gas chromatography with electron-capture detection and [ 2H 11]pipecolic acid as internal standard

Abstract

A sensitive and accurate stable isotope dilution assay was developed for the measurement of pipecolic acid in body fluids using capillary gas chromatography with electron-capture detection. The method utilizes [ 2H 11]pipecolic acid as the internal standard. Sample preparation consisted of derivatization in aqueous solution (pH 11.5) of the amine moiety with methyl chloroformate to the N-methylcarbamate, followed by acidic ethyl acetate extraction at pH ≤ 2 and further derivatization of the carboxyl moiety with pentafluorobenzyl bromide, the excess of which was removed by solid-phase extraction. Control values have been determined in the plasma of at-term infants, age > 1 week ( n = 21, mean = 1.36 μ M, range = 0.47–3.27 μ M). The utility of the method was demonstrated by quantitating pipecolic acid in biological fluids derived from patients with peroxisomal disorders. The method was validated against an established electron-capture negative ion mass fragmentographic technique.