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-beta-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 microM (PNP) and 2.5-100 microM (PNP-G and PNP-S). The instrumental limit of quantification for PNP-G and PNP-S was found to be 2.7 microM and 2.1 microM, respectively. The assay was applied for determination of PNP, PNP-G and PNP-S in rat small intestine perfusates.
Almási
Attila
A
Institute of Pharmaceutical Chemistry, University of Pécs, Rókus str. 2, H-7624 Pécs, Hungary.
Fischer
Emil
E
Perjési
Pál
P
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000706:statistics & numerical data; D020719:Glucuronides / Q000032:analysis; D007583:Jejunum / Q000378:metabolism; D009596:Nitrophenols / Q000032:analysis* / Q000737:chemistry / Q000493:pharmacokinetics; D010477:Perfusion; D051381:Rats; D015203:Reproducibility of Results; D013431:Sulfates / Q000032:analysis
D020719:Glucuronides; D009596:Nitrophenols; D013431:Sulfates; C024836:4-nitrophenol