High-performance liquid chromatographic method for the direct determination of 4-methylumbelliferone and its glucuronide and sulfate conjugates: Application to studies in the single-pass in situ perfused rat intestine—liver preparation

A direct high-performance liquid chromatographic (HPLC) assay was developed for the separation and determination of 4-methylumbelliferone (4MU) and its glucuronide (MUG) and sulfate (MUS) conjugates in the cell-free perfusate (“plasma”) from in situ perfused rat intestine—liver preparation. In addition, a procedure was developed to extract and determine 4MU in the whole blood perfusate. Perfusate plasma containing an internal standard (umbelliferone) was precipitated with methanol (1:4, v/v), and injected into a reversed-phase HPLC system with gradient elution. 4MU and the same internal standard were also extracted directly from the whole blood perfusate with ethyl acetate and injected into a reversed-phase HPLC system with isocratic elution. Inter- and intra-day precision studies ( n = 5 for each) for both the plasma and whole blood procedures demonstrated relative standard deviations of less than 10% at all concentrations studied. The compounds were stable in either the plasma or blood extracts at room temperature for up to 72 h. The procedures were successfully used to analyze perfusate samples obtained from the single-pass in situ perfusion of rat intestine—liver system with either trace (0.95 n M) or 32.3 μ M concentrations of 4MU. The intestine was responsible for the formation of most of the MUG formed by the intestine—liver preparation during steady-state perfusion with either input concentration of 4MU.