A method for the determination of a prostaglandin D(2) receptor antagonist (I, a compound being evaluated for the prevention of niacin induced flushing) and its acyl glucuronide metabolite (II) in human plasma is presented. The method utilized high performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection using an atmospheric pressure chemical ionization (APCI) interface operated in the positive ionization mode. The product ion was a radical cation generated via a homolytic bond cleavage. A chemical analog of the drug was used as internal standard (III). The acyl glucuronide metabolite (II) was detected using the same precursor-to-product ion transition used for the parent compound after chromatographic separation of I and II. Drug and metabolite were extracted using semi-automated, 96-well format solid phase extraction (SPE), and chromatography was performed using a reverse phase analytical column with an isocratic mobile phase. The chromatographic retention factor (k') of II was found to be highly sensitive to mobile phase formic acid concentration. An adjustment in mobile phase formic acid concentration improved the chromatographic separation between II and a mono-hydroxylated metabolite after an unexpected lack of MS/MS selectivity between the two molecules was observed. The dependence of retention factor on formic acid concentration (k' increased as formic acid concentration decreased) was thought to indicate polar interactions between II and the stationary phase. The stability of II in spiked human plasma was determined. The rate of hydrolysis back to parent compound was relatively low (approximately 0.1 and 0.5% per hour at room temperature and 4 degrees C, respectively) indicating that significant changes in analyte concentrations did not occur during sample processing. The concentration range of the assay was 10-2500 ng/mL for both drug and glucuronide metabolite.