LC-MS-MS with Post-Column Reagent Addition for the Determination of Zolpidem and its Metabolite Zolpidem Phenyl-4-carboxylic Acid in Oral Fluid after a Single Dose

A rapid, selective and sensitive LC-MS-MS method with a post-column addition of acetonitrile was developed and fully validated for the quantitative determination of zolpidem and its major metabolite, zolpidem phenyl-4-carboxylic acid (ZPCA), in oral fluid. Preliminary sample treatment was limited to a simple dilution of 1 mL oral fluid specimen aliquots with methanol. Chromatography was performed on a Capcell Pak C-18 MGII column (250 x 2.0 mm, 5 mu m i.d., Shiseido, Tokyo, Japan) with isocratic elution using a water-acetonitrile mobile phase with 0.1% formic acid, 5% acetonitrile and 20 mM ammonium acetate in an aqueous phase at a flow rate of 0.4 mL/min. Acetonitrile was added post-column at a flow rate of 0.4 mL/min to enhance ionization of the analytes in the MS source. Detection was carried out on a QTrap(TM) 6500 mass spectrometer in positive ionization mode. Good linearities were generated over the range of 0.05-200 ng/mL for zolpidem and 0.1-200 ng/mL for ZPCA. Limit of detection for zolpidem and ZPCA were 0.01 ng/mL and 0.05 ng/mL, respectively, whereas LLOQs were 0.05 ng/mL and 0.1 ng/mL, respectively. This method meets the required criteria for bioanalytical analyses to be used for clinical and forensic purposes. Application of this method was demonstrated by testing authentic samples collected after a single oral dose to obtain insights into the general detectability and detection windows of zolpidem and ZPCA in oral fluid.