Assay of trimethoprim, sulfamethoxazole and its N₄‐acetyl metabolite in biological fluids by high‐pressure liquid chromatography

Abstract

The most widely used method for the determination of SMZ and other sulfonamides is a colorimetric assay based upon the Bratton-Marshall reaction [13]. Rieder [14] modified this method in order to distinguish between total sulfonamide (free SMZ + SMZ N4-acetyl metabolite) and the "bacteriostatically active fraction" (free SMZ); other papers described densitometric [9] and chromatographic determinations [I 1,15,16].With the introduction of high-pressure liquid-chromatography (HPLC) to the biochemical field, some papers appeared concerning sulfonamide determination in drug products and biological fluids [17, 181, TMP [19]; two different chromatographic assays were suggested for mixtures of TMP and SMZ [20], leaving some unresolved problems for the determination of TMP.Recent papers on the HPLC assay of co-trimoxazole [21,22] suggested direct reversed-phase chromatography of serum and plasma following protein precipitation. This simplified sample preparation is preferred by many analysts because it is fairly fast and useful in special situations, such as are encountered in toxicological hospital laboratories and emergency rooms.Such a technique, according to our experience, is not suited for pharmacokinetic investigations where high sensitivity, good precision and accuracy are required; the lipids and other endogenous substances are also injected into the chromatographic column, and soon impair its efficiency and lead to shortened life. The low concentration of pertinent substances makes the sensitivity by this method [21] too low for pharmacokinetic studies. Bury's paper [22] describes a procedure requiring two different chromatographic determinations for SMZ and TMP respectively (excluding the SMZ N4-acetyl metabolite), necessitating two chromatographic instruments or extended analysis times. The methods in question do not use an internal analytical standard, reducing the precision.In this paper we describe a new method for reversedphase HPLC determination of TMP, SMZ, SMZ N4-acetyl metabolite in human plasma and urine. The drugs are extracted into an organic solvent with a single and rapid liquid-liquid extraction at pH 6.8. The assay is petformed in the presence of an internal standard.