APPLICATION OF ELECTROSPRAY MASS-SPECTROMETRY IN THE IDENTIFICATION OF INTACT GLUCURONIDE AND SULFATE CONJUGATES OF CLOZAPINE IN RAT

1. The phase II metabolites in the bile, urine and faeces of rat dosed with clozapine were investigated by means of electrospray mass spectr ometry (ESMS) in both positive and negative ion modes. 2. When operate d at a cone voltage of 45 V, this soft ionization technique permitted the detection of quasi molecular ions of both sulphate and glucuronide conjugates of hydroxylated phase I metabolites of clozapine. With the cone voltage set at 90 V, however, the ESMS also contained highly dia gnostic ions resulting from the loss of 80 Daltons (sulphur trioxide) or 176 Daltons (the glucuronide moiety) from sulphates and O-glucuroni des respectively. 3. A sufficient quantity of one metabolite was isola ted from rat bile to permit further analysis by H-1-nmr. This metaboli te, which was also found in rat urine, was proved to be 7-O-glucuronyl -7-hydroxyclozapine. The analogous sulphate metabolite was also identi fied in bile by ESMS. 4. Corresponding glucuronide and sulphate conjug ates of a hydroxylated N-desmethyl clozapine were similarly detected i n rat bile. There was insufficient material to permit analysis by H-1- NMR, but it appears likely that conjugation was also at the 7-position of N-desmethylclozapine. 5. Finally, the sulphate conjugate of a hydr oxy dechlorinated derivative of clozapine was identified by ESMS in bo th urine and bile. By analogy with a previous report of a similar meta bolite in man, the metabolite was tentatively identified as 8-hydroxy- 8-deschloroclozapine.