HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC DETERMINATION OF N-[2(S)-(MERCAPTOMETHYL)-3-(2-METHYLPHENYL)-1-OXO-PROPYL]-L-METHIONINE, THE ACTIVE PLASMA METABOLITE OF A PRODRUG ATRIOPEPTIDASE INHIBITOR (SCH-42495), USING A THIOL SELECTIVE (AU/HG) AMPEROMETRIC DETECTOR

A high-performance liquid chromatographic assay for the determination of N-[2(S)-(mercaptomethyl)-3-(2-methylphenyl)-1-oxopropyl]-L-methionine (SCH 42354; II), the active metabolite of the atriopeptidase inhibitor prodrug, N-[2(S)-(acetylthiomethyl)-3-(2-methylphenyl)-1-oxopropyl]-L-Methionine ethyl ester (SCH 42495; I), in human plasma was validated for use in clinical pharmacokinetic studies. Plasma (200-mu-l) was processed by protein precipitation with acetone containing the internal standard, N-[2(S)-(mercaptomethyl)-3-(2-methylphenyl)-1-oxopropyl]-L-ethionine (III). Compound II was recovered (ca. 90%) in the supernatant after centrifugation and prepared for injection by the addition of 0.15 M monochloroacetic acid containing 0.2 mM EDTA. Separation of II and III was accomplished on commercially available reversed-phase C. columns designed for the separation of basic compounds. Both compounds were detected using amperometric detection (+0.125 V versus Ag/AgCl) on a thin-layer Au/Hg amalgam electrode. The lower limit of quantitation was 10 ng/ml, where the inter-assay precision (coefficient of variation) was +/-11.4% and the inter-assay accuracy (bias) was + 1.0%. No endogenous interferences were observed in the extracts obtained from drug-free plasma. The detector response (using either peak area or height ratios of II to III) was linear from 0.01 to 1.0-mu-g/ml. Compound II was stable in plasma supplemented with EDTA and sodium hydrogensulfite for at least 3 months when stored frozen at -78-degrees-C; no significant decomposition of II was observed following three freeze-thaw cycles. The feasibility of this liquid chromatographic assay with electrochemical detection was demonstrated with plasma samples from hypertensive subjects administered 100 mg of compound I.