Liquid chromatographic-tandem mass spectrometric urine assay for a highly metabolized cyclic ureidobenzenesulfonamide: issues concerning assay specificity and quality control preparation

An LC-MS-MS method was validated for the quantitation of a beta (3) agonist (A) in human urine to support Phase I studies. A was designed to accelerat e metabolism for weight reduction. During assay development a significant l oss of A was apparent from frozen urine quality control samples. The additi on of 0.75% bovine serum albumin (BSA) in urine (v/v) was required to maxim ize the recovery of A from urine. Urine samples were basified and extracted into methyl t-butyl ether-isopropyl alcohol (90:10, v/v). The organic laye r was washed, evaporated, reconstituted, and injected onto a 5 cm, CS HPLC column prior to MS-MS analysis. The standard curve was linear from 5 to 500 ng/ml. Intraday precision for peak area ratios from BSA urine samples at s even separate concentrations over a range of 5-500 ng/ml (n = 5) was <4.0% and calculated concentrations were within 91-115% of nominal concentrations . Interday precision for BSA urine quality control (QC) samples at four sep arate concentrations (n = 10 of each) was <5.0% and individual calculated c oncentrations were within 90-111% of nominal concentrations, This work emph asizes that potential metabolites and quality control standards should be p repared and assayed as early as possible in method development., especially before the sample collection section of the clinical protocol is prepared. The methods described here have wide utility to other compounds containing basic benzene sulfonamides and to beta (3) agonist candidates, (C) 2001 El sevier Science B.V. All rights reserved.