Rapid simultaneous determination of metabolic clearance of multiple compounds catalyzed in vitro by recombinant human UDP-glucuronosyltransferases

The purpose of this study was to test the applicability of n-in-one (cocktail) incubations in the determination of intrinsic clearance (Clint) as the slope of the linear portion of the Michaelis-Menten curve (velocity V vs. substrate concentration [S]) where substrate concentrations were low. A rapid, sensitive, and selective liquid chromatography tandem mass spectrometry (LC/MS/MS) method was developed for the analysis of samples produced by single-substrate and n-in-one (seven substrates: entacapone, 17β-estriol, umbelliferone, 4-methylumbelliferone, tolcapone, hydroxyquinoline, and paracetamol) incubations conducted in 96-well plates with different recombinant UDP-glucuronosyltransferases (UGTs). The Clint values obtained with n-in-one incubations were compared with those obtained in single-compound incubations and with Vmax/Km values determined by estimating the enzyme kinetic parameters Vmax and Km from the Michaelis-Menten curve. When substrate concentrations were well below their Km values, Clint values determined as the slope of the linear part of the Michaelis-Menten fitting correlated well with the values determined as Vmax/Km ratios from the Michaelis-Menten curve. The correlation between Clint values determined in single-substrate and n-in-one incubations was high as well. Together, the n-in-one incubations, the determination of Clint values as the slope of the linear part of the Michaelis-Menten fitting, and LC/MS/MS as an analytical method proved to be effective approaches for increasing throughput in the first-phase screening of metabolic properties.