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

Abstract

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 C18 MGII column (250 × 2.0 mm, 5 μm 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 QTrapTM 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.

Increased levels of acidic free-N-glycans, including multi-antennary and fucosylated structures, in the urine of cancer patients

“…The following conditions were used: column, Shim-pack Scepter C18-120 (3 μm, 2.1 × 150 mm; Shimadzu), solvent A: 0.2% formic acid, solvent B: 0.2% (v/v) formic acid in 10% (v/v) acetonitrile, 40% (v/v) methanol, flow rate 0.25 mL/min, column temperature, 45°C. For stabilizing the ionization, 0.2 mL/min of acetonitrile was added post-column via a microvolume Tee-connector (Valco Instruments, Houston, TX, USA) by an additional LC-20AD pump [ 45 – 47 ]. Samples were measured in a randomized order.…”

Zolpidem and zolpidem phenyl‐4‐carboxylic acid pharmacokinetics in oral fluid after a single dose

“…A post‐column addition of acetonitrile was achieved with a separate isocratic pump of the UltiMate ® 3000 Intelligent LC series (Thermo Scientific Dionex, Idstein, Germany). Instrument parameters were as described previously . Two calibration curves, using concentrations from 0.05 ng/mL to 200 ng/mL for zolpidem and from 0.1 ng/mL to 200 ng/mL for ZPCA, were employed to extend the assay's dynamic range.…”

“…Eligible volunteers received a single oral dose of zolpidem tartrate tablets (10 mg, Sanofi-Aventis Minsheng Pharmaceutical Co., Ltd, Hangzhou, China, Product lot: 9T034). Oral fluids were sampled before and at 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,24,30,36,42,48,54,60,66, and 72 hours after dosing.…”

“…A simultaneous sensitive quantification for zolpidem and ZPCA was obtained according to our previously published analytical method. 9 Briefly, 100 μL of an internal zolpidem-d 6 standard and 300 μL of methanol were added to 100 μL of oral fluid. After mixing and centrifugation, the supernatant was decanted into a new vial.…”

“…Instrument parameters were as described previously. 9 Two calibration curves, using concentrations from 0.05 ng/ mL to 200 ng/mL for zolpidem and from 0.1 ng/mL to 200 ng/mL for ZPCA, were employed to extend the assay's dynamic range. The lower limits of quantification (LLOQs) were 0.05 ng/mL and 0.1 ng/ mL for zolpidem and ZPCA, respectively.…”