"High-pressure" liquid chromatography of sulfisoxazole and N4-acetylsulfisoxazole in body fluids

Abstract

We describe a simple, rapid chromatographic method for separating and quantitatively determining sulfisoxazole and its N4-acetyl metabolite in plasma and urine. A 100-micro L sample of plasma or urine is combined with 200 micro L of a solution containing 12 mg/L of the internal standard, N4-acetylsulfamethoxazole, in absolute methanol and centrifuged to obtain a clear supernatant solution. This solution is then eluted through a 10-micron microparticulate column with a mobile phase of 32/68 (by vol) methanol/sodium acetate buffer (0.01 mol/L, pH 4.7), at a flow rate of 1.2 mL/min. The eluted sompounds are detected by their absorption at 254 nm. We calculated concentration from the peak-height ratios of sulfisoxazole or N4-acetylsulfisoxazole to N4-acetylsulfamethoxazole. The peak-height ratio was linear with concentration in the range 0.05--200 mg/L for both drug and metabolite in plasma and urine. Because this assay can be completed within 30 min of obtaining a blood or urine sample, it should be a valuable tool in clinical drug monitoring and pharmacokinetic studies.