Analytical method for the detection of diazepam and its metabolites in human urine by LC-MS/MS

A technology of LC-MS and diazepam, which is applied in the field of analysis of diazepam and its metabolites in human urine samples, which can solve the problems of the specificity of recognition and other issues

Active Publication Date: 2016-10-05
SHANGHAI CRIMINAL SCI TECH RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the acid hydrolysis method is simpler, it will decompose benzodiazepines into benzophenones, which will affect the specificity of recognition

Method used

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  • Analytical method for the detection of diazepam and its metabolites in human urine by LC-MS/MS
  • Analytical method for the detection of diazepam and its metabolites in human urine by LC-MS/MS
  • Analytical method for the detection of diazepam and its metabolites in human urine by LC-MS/MS

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The preparation of embodiment 1 stock solution and working solution

[0031]Weigh an appropriate amount of oxazepam and diazepam, dissolve them in methanol, and prepare 0.5 mg / mL stock solution respectively. Temazepam and nordiazepam were diluted with methanol to 0.09mg / mL and 0.1mg / mL stock solutions, and the internal standard diazepam-d5 was diluted to 0.1mg / mL.

[0032] Diazepam, nordiazepam, temazepam, and oxazepam were prepared with methanol to make mixed working solutions with concentrations of 10 μg / mL, 10 μg / mL, 10 μg / mL, and 40 μg / mL, respectively, and oxazepam glucose Dilute glucuronide and temazepam glucuronide with methanol to 5 μg / mL, and use it to prepare solutions of various concentrations required for linear and quality control samples. Diazepam-d5 stock solution was diluted with methanol to 500ng / mL working solution.

Embodiment 2

[0033] Embodiment 2LC-MS chromatographic conditions

[0034] Waters Alliance 2695 HPLC system, Waters MS C18 column (2.1×150mm, 3.5μm), Waters MS C18 guard column (2.1×10 mm, 3.5 μm).

[0035] Mobile phase A (water with 2 mM ammonium formate and 0.05% formic acid) and B (acetonitrile with 2 mM ammonium formate and 0.05% formic acid). Gradient 10%B (0-0.5min), 10-20%B (0.5-2min), 20-50%B (2-6min), 50-70%B (6-13min), 70-95%B ( 13-13.5min), 95%B (13.5-16.5min), 95-10%B (16.5-17min), 10%B (17-21min), the flow rate is 0.20mL / min, the column temperature is 45℃, and the injection Volume 10 μL.

Embodiment 3

[0036] Embodiment 3MS mass spectrometry condition

[0037] Quattro Premier XE tandem mass spectrometer (Waters MS TechnoLogies, Manchester, UK), ESI source, positive ion mode, multiple reaction monitoring (MRM), electrospray voltage 3500V, nebulizer gas (nitrogen) flow rate 750L / h, temperature 450°C , flow rate of cone backflush gas (nitrogen) is 50L / h, ion source temperature is 120°C, collision gas is argon, and the pressure is maintained at 0.35Pa (3.5×10 -3 mBar).

[0038] For each compound, two precursor ions / product ions were selected to characterize the binding retention time, and the parameters are listed in Table 1. QuanLynx software equipped with MassLynx V4.1 was used for data processing.

[0039] Table 1 Mass spectrometry MRM parameters of diazepam, nordiazepam, oxazepam, oxazepam glucuronide, temazepam, temazepam glucuronide and internal standard

[0040]

[0041] NOTE: The underlined product ions are used for quantification.

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Abstract

The invention discloses an analysis method for detecting diazepam and its metabolites in human urine by LC-MS / MS. The samples were extracted by solid phase, and the chromatography was separated by C18 (Waters MS C18, 3.5μm×2.1×150mm) chromatographic column, gradient elution with mobile phase water (containing 2mM ammonium formate and 0.05% formic acid) and acetonitrile (containing 2mM ammonium formate and 0.05% formic acid); mass spectrometry using electrospray ionization (ESI ), multiple reaction monitoring (MRM) mode to detect target compounds. The method of the invention can directly detect the diazepam and metabolites in the urine sample without enzymatic hydrolysis or acid hydrolysis. And this method was used to test the urine samples of volunteers who took diazepam orally.

Description

technical field [0001] The invention belongs to the field of drug analysis, and relates to an analysis method for detecting diazepam and its metabolites in human urine samples by using LC-MS / MS method. Background technique [0002] Diazepam is one of the important drugs in the essential drug list promulgated by the World Health Organization, and is listed in the minimum medical needs list of the basic health care system. It is mainly used to treat insomnia, muscle relaxation, convulsions and other diseases. Diazepam, in addition to legitimate use, often exists in various criminal cases such as suicide, narcotic robbery, etc. caused by misuse or abuse of clinical and forensic toxicological drugs. Therefore, it is of great significance to detect trace amounts of diazepam in human samples. [0003] The in vivo metabolites of diazepam are mainly nordiazepam, temazepam, oxazepam, oxazepam glucuronide and temazepam glucuronide. As a typical drug of benzodiazepines, the content ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02G01N30/06
Inventor 张玉荣王鑫汪蓉饶渝兰梁晨叶海英曹芳琦
Owner SHANGHAI CRIMINAL SCI TECH RES INST
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