Direct determination of diastereomeric 13C-labeled ketoprofen glucuronides in human urine by nuclear magnetic resonance spectroscopy

A new method based on the 13C-labeling and nuclear magnetic resonance (NMR) spectroscopy was developed to study the human urinary excretion of diastereomeric acylglucuronides after the oral administration of 100 mg racemic [3-13C]ketoprofen (KP). The urinary excretion of diastereomeric [3-13C]KP glucuronides formed from [3-13C]KP was followed by proton-decoupled 13C NMR spectroscopy (9.4 T, 10min accumulation time) without any separation procedures such as extraction and chromatography. The C3 resonances due to the diastereomers were satisfactorily separated from each other by the addition of methyl-β-cyclodextrin as an achiral shift reagent through the formation of inclusion complexes. The C3 resonances were detected with an acceptable signal-to-noise ratio over 8 h at a therapeutic dosage. The concentrations of [13C]KP glucuronides were calculated from the inverse gated decoupling and proton-decoupling experiments using [2-13C]glycine as an internal standard. The urinary excretion of diastereomeric [3-13C]KP glucuronides over 8 h amounted to 29% (downfield position) and 25% (upfield position) of the administered dose, respectively, and the former was presumed to have S-configuration. The elimination rate constants of both glucuronides were virtually the same (K = 0.19 h−1). In addition, the susceptibility of the glucuronides to acyl migration as well as hydrolysis was suggested under the physiological conditions (37 °C, pH 7.3). The present direct approach is simple and convenient, and has the potential for wide application to the analysis of the labile acylglucuronides of 2-arylpropionic acids in biofluids.