An HPLC assay for carbamazepine phase I metabolites and their glucuronidesin urine

A binary gradient HPLC assay was developed for the separation and quantitat ion of carbamazepine (CBZ), carbamazepine-epoxide (CBZ-ep), carbamazepine-1 0,11-transdiol (CBZ-diol), carbamazepine-2-hydroxide (CBZ-OH), carbamazepin e-3-hydroxide (CBZ-3OH) and carbamazepine-acridan (CBZ-acr) extracted from patient urine. The internal standard was 10-methoxy-carbamazepine. The init ial phase was 70:15:15 phosphate buffer-methanol-acetonitrile followed by a linear gradient commencing at 13 minutes to 50:35:15 phosphate buffer-meth anol-acetonitrile at 24 minutes. The flow rate was constant at 2 mL/min. Th e UV absorbance detector wavelength was changed at 18 minutes from 240 nm t o 280 nm. Typical retention times for CBZ-diol, CBZ-2OH, CBZ-ep, CBZ-3OH, C BZ-acr, CBZ and internal standard were 5.26, 8.36, 10.46, 12.51, 14.2, 23 a nd 27.53 minutes respectively. The minimum quantifiable limit (MQL) for all of the analytes was 0.2 mu g/mL except for CBZ-2OH where the MQL was 2 mu g/mL. Precision and accuracy of the assay was 1.3 to 19.4% for the 6 analyt es. Liquid-liquid extraction with ethyl acetate resulted in recovery of the ana lytes from urine greater than 74% except for CBZ-diol where recovery was 40 %. The concentrations of the glucuronides of CBZ and metabolites were calcu lated by measuring their concentrations before and after hydrolysis.