Simultaneous Determination of Sulfasalazine and Its Metabolites Sulfapyridine and N-Acetylsulfapyridine in Human Serum by Ion-Pair High-Performance Liquid Chromatography Using a Polymer-Based Column

An HPLC method is described for the simultaneous and rapid determination of sulfasalazine (salicylazosulfapyridine) and two of its metabolites, sulfapyridine and N-acetylsulfapyridine, in human serum. The range of quantitation is 0.1 to 12 μg/mL for sulfasalazine and sulfapyridine and 0.25 to 12 μg/mL for W-acetylsulfapyridine. Serum is mixed with acetonitrile containing the internal standard sulfamethazine and the ion-pairing agent tetraethylammonium chloride. The acetonitrile extract is concentrated and analyzed by HPLC, using a new polymer- based column, and detected by UV spectroscopy at 270nm. This paper is the first both to describe the simultaneous analysis of all three of the compounds from serum and to present sulfasalazine concentration-time data following oral administration to humans.