Sensitive, automatic method for the determination of diazepam and its five metabolites in human oral fluid by online solid‐phase extraction and liquid chromatography with tandem mass spectrometry

Abstract

A novel and simple online solid-phase extraction liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous determination of diazepam and its five metabolites including nordazepam, oxazepam, temazepam, oxazepam glucuronide, and temazepam glucuronide in human oral fluid. Human oral fluid was obtained using the Salivette(®) collection device, and 100 μL of oral fluid samples were loaded onto HySphere Resin GP cartridge for extraction. Analytes were separated on a Waters Xterra C18 column and quantified by liquid chromatography with tandem mass spectrometry using the multiple reaction monitoring mode. The whole procedure was automatic, and the total run time was 21 min. The limit of detection was in the range of 0.05-0.1 ng/mL for all analytes. The linearity ranged from 0.25 to 250 ng/mL for oxazepam, and 0.1 to 100 ng/mL for the other five analytes. Intraday and interday precision for all analytes was 0.6-12.8 and 1.0-9.2%, respectively. Accuracy ranged from 95.6 to 114.7%. Method recoveries were in the range of 65.1-80.8%. This method was fully automated, simple, and sensitive. Authentic oral fluid samples collected from two volunteers after consuming a single oral dose of 10 mg diazepam were analyzed to demonstrate the applicability of this method.

Development of a simple and sensitive HPLC-MS/MS method for determination of diazepam in human plasma and its application to a bioequivalence study

“…In previous studies to develop a bioanalytical method to quantitate diazepam in human plasma, samples were prepared using the protein precipitation (PP) method or solid-phase extraction (SPE) method. [5][6][7][8] However, due to insufficient removal of impurities from the sample when using the PP method, the analytical method with samples prepared using the PP method may not be appropriate for the routine analysis of a large number of samples. [5,6] The SPE method is a complicating and timeconsuming sample preparation procedure.…”

“…[5,6] The SPE method is a complicating and timeconsuming sample preparation procedure. [7,8] Therefore, in this study, a liquid-liquid extraction (LLE) method was used to effectively remove endogenous substances and thus, improve the efficiency of analyzing a large number of samples for human pharmacokinetic research. Our developed method satisfied all of the MFDS and USFDA guidelines for validating a bioanalytical assay.…”

“…Although protein precipitation (PP) and solid phase extraction (SPE) methods have been previously used to extract the analyte, these methods are laborious and thus, may not be the best for routine analysis when processing a large number of plasma samples. [6][7][8] The extraction recoveries and matrix effects of diazepam following extraction at three QC concentrations were 81.92-86.63% and 88.15-93.54% (n = 6), respectively ( Table 3). The extraction recovery and absolute matrix effect of IS were 87.61% and 89.71%, respectively, at 200 ng/mL (Table 3).…”

“…[2] Previous studies have proposed several analytical methods using high-performance liquid chromatography (HPLC) with an ultraviolet (UV) [3,4] or mass spectrometry (MS) [5][6][7][8][9] detection to determine the concentrations of diazepam administered by various routes including nasal spray, rectal gel, intramuscular injection, and oral formulations. [10][11][12][13][14][15] However, these previously developed analytical methods have been associated with several limitations including inadequate sample preparation due to difficulty in removing impurities, [5] inadequate sensitivity (a lower limit of quantitation [LLOQ] up to 50 ng/mL), [6] labor-intensive sample preparation process, [7] the need for a relatively large volume of plasma (≥ 100 μL) for analysis, [8] and long retention times (≥ 8 min). [9] Therefore, a new bioanalytical method to quantitate diazepam has been needed to improve those limitations.…”

A Molecular Imprinted Polymer as a Flow-Through Optical Sensor for Oxazepam

“…Several analytical methods have been proposed for OXA determination: most of them use chromatographic techniques and others use fluorometry and capillary electrophoresis; also, some electrochemical sensors have been developed [ 4 – 13 ]. Since benzodiazepine metabolites are converted to benzophenone, a sensitive determination method could be performed using the hydrolyzed product [ 5 , 14 ].…”