MEASUREMENT OF THE ACTIVE LEFLUNOMIDE METABOLITE-(A77-1726) BY REVERSE-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

The immunosuppressive activity of leflunomide is expressed after conve rsion to its pharmacologically active metabolite A77 1726. Leflunomide is a potent immunosuppressant that inhibits both T-cell and B-cell ac tivity. To date, no pharmacokinetic data have been reported on lefluno mide or A77 1726, primarily because of lack of a suitable method for i ts analysis. We describe here the development and evaluation of a reve rse-phase high-performance liquid chromatographic (HPLC) method for th e analysis of A77 1726 in whole blood or plasma from humans or rabbits . In human blood, the method exhibited good analytic recoveries from 7 8 +/- 13.5% to 108 +/- 4.8% (mean +/- SD) for drug concentrations rang ing from 400 to 100,000 mu g/L. When using a sample volume of 0.25 ml the sensitivity of the method was found to be 400 mu g/L, with a worki ng standard range of up to 200,000 mu g/L. The sensitivity of the meth od can be increased to 40 mu g/L when 1.0 ml of sample is used. Betwee n-run coefficients of variation of 12.2 and 14.7% at A77 1726 mean con centrations of 1,006 and 8,146 mu g/L were found for this method. No s ignificant differences in recovery of drug were noted when either huma n 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.