Development and validation of ultra-high-performance liquid chromatography-mass spectrometry method for the determination of raloxifene and its phase II metabolites in plasma: Application to pharmacokinetic studies in rats.

The aim of this study is to establish a reliable liquid chromatography-mass spectrometry method to simultaneously quantitate raloxifene, and its major metabolites, raloxifene-6-glucuronide, raloxifene-4'-glucuronide, and raloxifene-6-sulfate in rat plasma samples for pharmacokinetic studies. The separation of the analytes was achieved on a Waters BEH C18 column. Water (0.1% formic acid) and acetonitrile were used as the mobile phases for elution. A one-step protein precipitation using a mixture solvent was applied for plasma sample preparation. The method was validated following the FDA guidance. The results showed that the linear range were 1.95-1000 nM for raloxifene-6-glucuronide, and raloxifene-4'-glucuronide, 0.195-100 nM for raloxifene-6-sulfate, and 0.195-200 nM for raloxifene, respectively. The lower limit of quantification was 1.95, 1.95, 0.195, and 0.195 nM for raloxifene-6-glucuronide, raloxifene-4'-glucuronide, raloxifene-6-sulfate, and raloxifene, respectively. Only 20 µl of plasma sample was required since the method is sensitive. The intra- and interday variance is <15% and the accuracy is within 85-115%. The variance of matrix effect and recovery were <15%. The method was successfully applied in a pharmacokinetic study in rats with oral administration of raloxifene.

Glucuronides Hydrolysis by Intestinal Microbial ß-Glucuronidases (GUS) Is Affected by Sampling, Enzyme Preparation, Buffer pH, and Species.

Glucuronides hydrolysis by intestinal microbial ß-Glucuronidases (GUS) is an important procedure for many endogenous and exogenous compounds. The purpose of this study is to determine the impact of experimental conditions on glucuronide hydrolysis by intestinal microbial GUS. Standard probe 4-Nitrophenyl ß-D-glucopyranoside (pNPG) and a natural glucuronide wogonoside were used as the model compounds. Feces collection time, buffer conditions, interindividual, and species variations were evaluated by incubating the substrates with enzymes. The relative reaction activity of pNPG, reaction rates, and reaction kinetics for wogonoside were calculated. Fresh feces showed the highest hydrolysis activities. Sonication increased total protein yield during enzyme preparation. The pH of the reaction system increased the activity in 0.69-1.32-fold, 2.9-12.9-fold, and 0.28-1.56-fold for mouse, rat, and human at three different concentrations of wogonoside, respectively. The Vmax for wogonoside hydrolysis was 2.37 ± 0.06, 4.48 ± 0.11, and 5.17 ± 0.16 µmol/min/mg and Km was 6.51 ± 0.71, 3.04 ± 0.34, and 0.34 ± 0.047 µM for mouse, rat, and human, respectively. The inter-individual difference was significant (4-6-fold) using inbred rats as the model animal. Fresh feces should be used to avoid activity loss and sonication should be utilized in enzyme preparation to increase hydrolysis activity. The buffer pH should be appropriate according to the species. Inter-individual and species variations were significant.