title: Stabilization and determination of a PPAR agonist in human urine using automated 96-well liquid-liquid extraction and liquid chromatography/tandem mass spectrometry.
abstract: Two stability challenges were encountered during development of an urine assay for a proliferator-activated receptor (PPAR) agonist, I (2-{[5,7-dipropyl-3-(trifluoromethyl)-1,2-benzisoxazol-6-yl]oxy}-2-methyl propionic acid), indicated for the treatment of Type II diabetes. First, the analyte was lost in urine samples due to adsorption on container surface which is a common problem during clinical sample handling. Secondly, the acylglucuronide metabolite (III), a major metabolite of I, displayed limited stability and effected the quantitation of parent drug due to the release of I through hydrolysis. Therefore, a clinical collection procedure was carefully established to stabilize I and its acylglucuronide metabolite, III, in human urine. The metabolite was not quantitated with this method. The urine samples are treated with bovine serum albumin (BSA) equal to 1.75% of the urine volume and formic acid equal to 1% of urine volume. Compound (I) and internal standard (II) were extracted from urine with 1 mL ethyl acetate using a fully automated liquid-liquid extraction in 96-well plate format. The analytes are separated by reverse phase high-performance liquid chromatography (HPLC) with tandem mass spectrometry in multiple-reaction-monitoring (MRM) mode used for detection. The urine method has a lower limit of quantitation (LLOQ) of 0.05 ng/mL with a linearity range of 0.05-20 ng/mL using 0.05 mL of urine. The method was validated and used to assay urine clinical samples.
authors.affiliation: Department of Drug Metabolism, Merck Research Laboratories, WP75A-303, West Point, PA 19486, USA. michelle_groff@merck.com
authors.forename: Michelle
authors.initials: M
authors.lastname: Groff
authors.forename: Kerry
authors.initials: K
authors.lastname: Riffel
authors.forename: Hengchang
authors.initials: H
authors.lastname: Song
authors.forename: Man-Wai
authors.initials: MW
authors.lastname: Lo
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007555:Isoxazoles / Q000378:metabolism / Q000494:pharmacology / Q000652:urine*; D013058:Mass Spectrometry / Q000379:methods*; D015394:Molecular Structure; D047492:Peroxisome Proliferator-Activated Receptors / Q000819:agonists*; D011422:Propionates / Q000378:metabolism / Q000494:pharmacology / Q000652:urine*; D015203:Reproducibility of Results
chemical_list: C503385:2-((5,7-dipropyl-3-(trifluoromethyl)-1,2-benzisoxazol-6-yl)oxy)-2-methyl propionic acid; D007555:Isoxazoles; D047492:Peroxisome Proliferator-Activated Receptors; D011422:Propionates