Simple and simultaneous determination of sulphapyridine and acetylsulphapyridine in human serum by column-switching high-performance liquid chromatography

Summary Objective: A high‐performance liquid chromatography (HPLC) with an automated on‐line column‐switching system was used for the simultaneous determination of sulphapyridine and acetylsulphapyridine, two major active metabolites related to the adverse effects of sulphasalazine, in human serum. Methods: Serum samples were directly injected into the HPLC, with the valve automatically switched on to remove serum proteins and other hydrophilic components remaining in the pre‐column after elution of sulphapyridine and acetylsulphapyridine to the analytical column. Results: Serum proteins did not interfere with the analysis of either compound. The recoveries of SLP and Ac‐SLP from drug‐free human serum were 93·03–99·18% and CV were 2·88–4·34%. The within‐run reproducibility of assays was excellent with relative standard deviations (RSD) of 1·01–3·90% (SLP) and 0·77–5·56% (Ac‐SLP). The limit of quantification of sulphapyridine and acetylsulphapyridine was 3·13 μg/mL and 0·50 μg/mL, respectively. The serum concentrations in a patient with ulcerative colitis, who took 1·0 g sulphasalazine twice daily, were 31·20 μg/mL for sulphapyridine and 14·64 μg/mL for acetylsulphapyridine at 7 h after ingestion. Conclusion: The present simple and reproducible assay was useful for the monitoring of serum sulphapyridine and acetylsulphapyridine.