Metabolic studies with phenobarbitone, primidone and their N-alkyl derivatives: quantification of substrates and metabolites using chemical ionization gas chromatography-mass spectrometry.
Metabolic studies with phenobarbitone, primidone and some of their N-alkyl derivatives required the concurrent assay of any mixture of these substrates (twelve compounds) and their major metabolites (an additional twenty-two compounds) in urine. The method described in the present report met this requirement by incorporating two complementary derivatization techniques into a gas chromatographic-mass spectrometric (GC-MS) assay procedure. Following hydrolysis of conjugates with beta-glucuronidase, urine samples were extracted with ethyl acetate (3 X 5 ml). The combined extracts were dried over sodium sulphate, divided into two equal portions, and the solvent was removed. One residue was derivatized by propylation using 1-iodopropane with base catalysis. The other residue was silylated using methyl-N-(tert.-butyldimethylsilyl)trifluoroacetamide. The derivatives in each case were analysed by GC-MS, using temperature-programmed packed-column GC and chemical ionization MS. Mass spectra were acquired over an appropriate mass range, and peak areas for the compounds of interest were determined from specific mass chromatograms. Satisfactory precision, accuracy, specificity and sensitivity were obtained for all analytes. All compounds produced satisfactory derivatives by at least one procedure; twelve compounds could be analysed by both techniques. The method illustrates the utility of chemical ionization GC-MS for the simultaneous quantitative analysis of multiple related analytes in complex biological samples.
Treston
A M
AM
Department of Biochemistry, University of Queensland, Royal Brisbane Hospital, Herston, Australia.
Hooper
W D
WD
D000284:Administration, Oral; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D008297:Male; D010634:Phenobarbital / Q000008:administration & dosage / Q000378:metabolism / Q000652:urine*; D011324:Primidone / Q000008:administration & dosage / Q000378:metabolism / Q000652:urine*
D011324:Primidone; D010634:Phenobarbital