The development and validation of a sensitive and specific HPLC method for SDZ WAG 994 (I) in dog, monkey and rat blood is described. Sample preparation entailed double solid phase extraction (SPE) of I and the internal standard from 0.5 ml of animal blood using a phenyl and propyl sulfonic acid cation exchange column, sequentially. Chromatographic separation was achieved on a YMC Basic C-8 narrowbore HPLC column and the eluates were detected by UV absorption at 266 nm. The method has a linear response up to at least 1800 ng/ml with a limit of quantification of 1 ng/ml across all species. Analysis of 'blinded' quality control dog and monkey blood samples over 3 or 4 days produced median precisions of 2.89 and 4.77%, and median reproducibilities of 4.86 and 10.9%, respectively. Curve fitting of variability estimates indicated that concentration independent error contributed 3-9% of the total method error for the tandem SPE procedure. Extracted endogenous material from blood matrices, several potential metabolites and cyclohexyladenosine were well resolved from the peaks of interest. The stability of I in dog blood stored at -20 degrees C is at least 6 months. The overall absolute and relative recovery of I using the tandem SPE procedure was 85.5 +/- 5.1% and 96.5 +/- 5.0%, respectively. The ruggedness of the method has been demonstrated by multiple analyses, from several toxicokinetic studies, performed by different analysts using comparable instrumentation.
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