Development of a HPLC-DAD Method for the Simultaneous Quantification of 5-O-β-D-galactopyranosyl-7-methoxy-3’,4’-dihydroxy-4-phenylcoumarin and its Aglycone in Feces

A sensitive and specific HPLC-DAD method was devel-oped and validated for the simultaneous quantification of 5-O-β-D-ga-lactopyranosyl-7-methoxy-3’,4’-dihydroxy-4-phenylcoumarin (4-PC) and its aglycone in rat feces. The 4-phenylcoumarins are important antidiabetic and gastroprotective bioactive metabolites of a highly commercialized medicinal plant complex in Mexico, the Copalchi complex. Both the sample preparation and the quantification method were developed; the methodology allows, for the first time ever, the simultaneous determination of the 4-PC (Rt= 2.4 min) and its metabo-lized aglycone (Rt= 7.5 min), aimed to their pharmacokinetic analysis, specially their elimination. Linearity was determined in the range, 0.09–4.5 µg/mL for both compounds (R2= 0.9999). Accuracy was <15.0% for 4-PC and aglycone with an interassay precision of maxi-mum %RSD of 6.1% for 4-PC and 2.7% for the aglycone. The intraassay precision was %RSD < 7.5 for 4-PC and < 3.5% for the aglycone. Analyte recovery from spiked samples was always > 98.02% for 4-PC and > 96.61% for its aglycone. The method was successfully applied to a single-dose preclinical pharmacokinetics preliminary study in rats.