Measurement of the Active Leflunomide Metabolite (A77 1726) by Reverse-Phase High-Performance Liquid Chromatography

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Abstract
The immunosuppressive activity of leflunomide is expressed after conversion to its pharmacologically active metabolite A77 1726. Leflunomide is a potent immunosuppressant that inhibits both T-cell and B-cell activity. To date, no pharmacokinetic data have been reported on leflunomide or A77 1726, primarily because of lack of a suitable method for its analysis. We describe here the development and evaluation of a reverse-phase high-performance liquid chromatographic (HPLC) method for the analysis of A77 1726 in whole blood or plasma from humans or rabbits. In human blood, the method exhibited good analytic recoveries from 78 +/- 13.5% to 108 +/- 4.8% (mean +/- SD) for drug concentrations ranging from 400 to 100,000 micrograms/L. When using a sample volume of 0.25 ml the sensitivity of the method was found to be 400 micrograms/L, with a working standard range of up to 200,000 micrograms/L. The sensitivity of the method can be increased to 40 micrograms/L when 1.0 ml of sample is used. Between-run coefficients of variation of 12.2 and 14.7% at A77 1726 mean concentrations of 1,006 and 8,146 micrograms/L were found for this method. No significant differences in recovery of drug were noted when either human or rabbit plasma or whole blood was used as the medium of analysis. In whole-blood specimens, A77 1726 was found to be stable for up to 10 days at -20 or -70 degrees C.

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