Determination of stable isotopically substituted histidine in human plasma by gas chromatography-mass spectrometry

A gas chromatographic-electron-impact mass spectrometric method for the determination of stable isotopically substituted histidine in human plasma has been developed. Histidine was derivatized to alpha N-trifluoroacetyl-imN-carbethoxyhistidine n-butyl ester (TCB derivative) by a three-step reaction: an initial esterification by 3 M n-butanolic hydrochloric acid, followed by trifluoroacetylation with trifluoroacetic anhydride and then ethoxycarbonylation with diethyl pyrocarbonate. Quantitation was carried out by selected-ion monitoring on the molecular ions (m/z 379, 383 and 385) of the respective TCB derivatives of histidine, [1-15N,5,beta,beta-2H3]histidine (histidine-[M + 4]) and [1,3-15N2,5,alpha,beta,beta-2H4]histidine (histidine-[M + 6]). The sensitivity, specificity, precision and accuracy of the method were demonstrated to be satisfactory for application to a pharmacokinetic study of histidine after administration of a trace amount of stable isotopically substituted histidine (histidine-[M + 4]) in humans.