Assay for codeine, morphine and ten potential urinary metabolites by gas chromatography-mass fragmentography

A mass fragmentography (MF) assay is described for ten potential, minor urinary metabolites of codeine (C) and morphine (M). Samples were hydrolyzed, extracted, derivatized with Tri-Sil Z and analyzed by methane chemical ionization (CI)-MF. The method is sensitive to ca. 0.01 μg/ml for all compounds with the exception of normorphine (NM) which was difficult to extract with chloroform. The sensitivity of the MF assay for NM was only ca. 0.10 μg/ml. Various solvent systems were investigated for optimization of extraction efficiency of all metabolites. A separate method for the extraction of NM is reported which utilizes a solid buffer-solvent combination, i.e., potassium carbonate-isopropanol. This latter method provided the best overall recovery of NM (39.0 ± 3.4%). Gas chromatographic (GC) retention times of C, M and metabolites are reported for three liquid phases (3%) on Gas-Chrom Q (100-120 mesh). Resolution of metabolites (as trisilyl derivatives) was best on Silar-5CP and this phase was used in metabolic studies of C and M. GC resolution was not complete for all compounds; however, selection of specific ions for monitoring by MF provided the required specificity for all compounds except the 6α- and 6β-hydroxy isomers. CI spectra for all metabolites are reported. The MF assay was used for urinary analysis of samples from guinea pigs that received single doses of C (15 mg/kg) or M (8 mg/kg). Following C administration 6α- and 6β-hydrocodol, 6α,β-hydromorphol (undifferentiated), HM and M were measured. Following M administration only 6α,β-hydromorphol was found. The amount of total metabolite as percent dose for each component was calculated as < 1%.