Add time:08/15/2019         Source:sciencedirect.com

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 μl) 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 C8 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 μg/ml. Compound II was stable in plasma supplemented with EDTA and sodium hydrogensulfite for at least 3 months when stored frozen at −78°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.

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