Abstract
An isocratic ion pair RP-HPLC method with UV-Vis detection has been developed and validated for simultaneous analysis of 4-nitrophenol (PNP), 4-nitrophenyl β-glucuronide (PNP-G), and 4-nitrophenyl sulfate (PNP-S) in rat bile samples using 4-ethylphenol (ETP) as internal standard. Chromatographic separation was achieved on a C18 column by isocratic elution with a mobile phase consisted of methanol-0.01 M citrate buffer pH 6.2 (47:53 v/v) containing 0.03 M TBAB. The flow rate was 1.0 ml min−1, the detection was affected at 290 nm. Calibration plots were generated over the concentration range 1–100 μM PNP, PNP-G, PNP-S with a common lower limit of quantification of 2.5 μM. Intra- and inter-day precision and repeatability were determined at six different concentrations. Results obtained by application of the method for determination of PNP, PNP-G and PNP-S in bile fractions collected during intestinal perfusion of PNP in hyperglycemic rats are presented.
Highlights
After oral administration of the phenolic compounds, the hepatic and intestinal glucuronic acid and sulfate conjugations have the most important roles as metabolic transformations [1]. 4-Nitrophenol (PNP) is excreted almost exclusively as its glucuronide (PNP-G) and sulfate (PNP-S) (Fig. 1) [2, 3]
The optimized reversed phase HPLC (RP-HPLC) assay using a mobile phase of methanol-0.01 M citrate buffer pH 6.2 (47:53, v/v) containing 0.03 M tetrabutyl-ammonium-bromide (TBAB) provided baseline separation of phase HPLC-UV assay to quantify 4-nitrophenol (PNP), PNP and both of its glucuronide (PNP-G), PNP-S and ETP in less than 14 min of chromatographic time (Figure 2)
In order to set up a simple, easy to perform assay to quantify PNP and its conjugates in the rat bile samples, we considered only isocratic RP-HPLC methods that can provide a straightforward and time-consuming separation of PNP and both of its glucuronide (PNP-G) and sulfate (PNP-S) conjugates
Summary
After oral administration of the phenolic compounds, the hepatic and intestinal glucuronic acid and sulfate conjugations have the most important roles as metabolic transformations [1]. 4-Nitrophenol (PNP) is excreted almost exclusively as its glucuronide (PNP-G) and sulfate (PNP-S) (Fig. 1) [2, 3]. As a continuation of our work in this field we have developed a modified isocratic ion-pair RP-HPLC-UV method using 4-ethylphenol (ETP) as internal standard to quantify PNP and its metabolites in the bile samples of the experimental animals. This work describes details (focusing on linearity, within-day precision and day-today precision) of the optimized easy to run reversed phase HPLC-UV assay to quantify 4-nitrophenol (PNP), 4-nitrophenyl β-glucuronide (PNP-G) and 4-nitrophenyl sulfate (PNP-S) in a large number of samples generated in animal experiments.
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