ISOLATION, IDENTIFICATION AND DETERMINATION OF SULFAMETHOXAZOLE AND ITS KNOWN METABOLITES IN HUMAN PLASMA AND URINE BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

From human urine the following metabolites of sulfamethoxazole (S) wer e isolated by preparative HPLC: 5-methylhydroxysulfamethoxazole (SOH), N-4-acetyl-5-methylhydroxysulfamethoxazole (N4SOH) and sulfamethoxazo le-N-1-glucuronide (SgIuc). The compounds were identified by NMR, mass spectrometry, infrared spectrometry, hydrolysis by beta-glucuronidase and ratio of capacity factors. The analysis of S and the metabolites N-4-acetylsulfamethoxazole (N-4), SOH, N-4-hydroxysulfamethoxazole (N4 OH), N4SOH, and Sgluc in human plasma and urine samples was performed with reversed-phase gradient HPLC with UV detection. In plasma, S and N-4 could be detected in high concentrations, while the other metaboli tes were present in only minute concentrations. In urine, S and the me tabolites and conjugates were present. The quantitation limit of the c ompounds in plasma are respectively: S and N-4 0.10 mu g/ml; N4SOH 0.1 3 mu g/ml; N4OH 0.18 mu g/ml; SOH 0.20 mu g/ml; and Sgluc 0.39 mu g/ml , In urine the quantitation limits are: N-4 and N4OH 1.4 mu g/ml; S 1. 5 mu g/ml; N4SOH 1.9 mu g/ml; SOH 3.5 mu g/ml; and Sgluc 4.1 mu g/ml. The method was applied to studies with healthy subjects and HIV positi ve patients.