A simple and rapid ion-pair HPLC method for simultaneous quantitation of 4-nitrophenol and its glucuronide and sulfate conjugates

Because of its simple and well characterized metabolic profile, 4-nitrophenol is widely used as a model substrate to investigate the influence of drug therapy, disease, nutrient deficiencies and other physiologically altered conditions on conjugative drug metabolism in animal studies. For simultaneous determination of 4-nitrophenol (PNP), 4-nitrophenyl-β-d-glucuronide (PNP-G) and 4-nitrophenyl-sulfate (PNP-S) in samples generated in rat small intestine luminal perfusion experiments, an ion-pair HPLC assay coupled with UV detection was set up. The RP-HPLC separation was achieved with a methanol–water mixture (50:50, v/v) containing 0.01 M tetrabutyl-ammonium-bromide with UV detection of the analytes at 290 nm. The isocratic system was operated at ambient temperature and required less than 7 min of chromatographic time. The method provided good enough within-day precision, between-day precision and linearity in the target concentration ranges of 6–1200 μM (PNP) and 2.5–100 μM (PNP-G and PNP-S). The instrumental limit of quantification for PNP-G and PNP-S was found to be 2.7 μM and 2.1 μM, respectively. The assay was applied for determination of PNP, PNP-G and PNP-S in rat small intestine perfusates.