LIPOPHILIC BENZAMIDE AND ANILIDE DERIVATIVES AS HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY INTERNAL STANDARDS - APPLICATION TO SIROLIMUS (RAPAMYCIN) DETERMINATION

Finding a suitable internal standard in reversed-phase high-performanc e liquid chromatography is often difficult. A general approach for sel ecting and synthesizing the proper internal standard is presented and applied to a validated method for quantitation of sirolimus in several biological matrices. A series of fifteen N-alkylbenzamides, N-alkylto luamides and N-alkanoylanilines with a log P range of 3.51 to 6.68 wer e synthesized as internal standards; N-undecyl-o-toluamide was evaluat ed most extensively. Sirolimus quantitation involves a simple sample c lean-up procedure followed by isocratic chromatography on a heated C-1 8 analytical column with an 70% methanol-water mobile phase and ultrav iolet detection at 278 nm. This method was linear from 2.5 to 200 ng w ith a limit of quantitation of 2.5 ng using a 1-ml blood sample. Sirol imus recovery was above 72.1%. The intra-day and inter-day coefficient s of variation were less than 11.7%. Several drugs and sirolimus metab olites do not interfere with the analysis. This method was used to mea sure sirolimus in blood from rats, rabbits and humans. (C) 1997 Elsevi er Science B.V.