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Online SPE LC-MS/MS (solid phase extraction liquid chromatography/mass spectrometry) analysis method for diazepam and metabolites thereof in human saliva

An analytical method and technology for diazepam, applied in the field of drug analysis, can solve the problems of cumbersome operation, time-consuming and labor-intensive off-line extraction technology, etc.

Inactive Publication Date: 2017-10-20
SHANGHAI CRIMINAL SCI TECH RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The object of the present invention is to provide a kind of online SPE LC-MS / MS analysis method of diazepam and its metabolites in people's saliva, set up a kind of fully automatic online solid phase extraction liquid mass spectrometry (online SPE LC-MS / MS) method for the quantitative detection of diazepam and its five metabolites (norazepam, oxazepam, temazepam, oxazepam glucuronide, temazepam glucuronide ), which overcomes the shortcomings of cumbersome, time-consuming and labor-intensive offline extraction technology, and reduces operational errors

Method used

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  • Online SPE LC-MS/MS (solid phase extraction liquid chromatography/mass spectrometry) analysis method for diazepam and metabolites thereof in human saliva
  • Online SPE LC-MS/MS (solid phase extraction liquid chromatography/mass spectrometry) analysis method for diazepam and metabolites thereof in human saliva
  • Online SPE LC-MS/MS (solid phase extraction liquid chromatography/mass spectrometry) analysis method for diazepam and metabolites thereof in human saliva

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] 1) Preparation of solution

[0057]1.1 Preparation of stock solution and working solution

[0058] Diazepam stock solution: Accurately weigh 1 mg of diazepam reference substance powder into a 10 mL volumetric flask, dissolve with methanol and dilute to the mark to obtain a diazepam stock solution with a concentration of 100 μg / mL.

[0059] Oxazepam stock solution: Accurately weigh 1 mg of oxazepam reference substance powder into a 10 mL volumetric flask, dissolve with methanol and dilute to the mark to obtain an oxazepam stock solution with a concentration of 100 μg / mL.

[0060] Norazepam stock solution: Precisely pipette 1 mL of norazepam reference solution into a 10 mL volumetric flask, and dilute to the mark with methanol to obtain a norazepam stock solution with a concentration of 100 μg / mL.

[0061] Temazepam stock solution: Precisely pipette 1 mL of temazepam reference solution into a 10 mL volumetric flask, and dilute to the mark with methanol to obtain a noraze...

Embodiment 2

[0107] 1) Selection of online solid phase extraction cartridges

[0108] Diazepam, norazepam, temazepam, and oxazepam are weakly basic compounds, and oxazepam glucuronide and temazepam glucuronide are weakly acidic compounds with very high polarity. Example Select three online extraction cartridges: HySphere Resin SH cartridge, HySphere C18HD cartridge and HySphere Resin GP cartridge to investigate the retention of each analyte on the cartridge.

[0109]The results show that in terms of peak area, the HySphere Resin SH small column has the strongest retention effect on each analyte, but each analyte is difficult to be eluted on the small column, especially diazepam, whose peak broadens for about 5 minutes , in addition, diazepam, norazepam and temazepam could not be completely separated; when the HySphere C18HD cartridge was used, the analytes could be separated well, but it showed relatively poor retention, especially for Oxazepam glucuronide and temazepam glucuronide, when ...

Embodiment 3

[0115] Embodiment 3 methodological verification

[0116] 1) Specificity

[0117] Take 6 blank saliva from different sources, replace the mixed internal standard working solution with the same volume of methanol, and pretreat the others according to the pretreatment method in Example 1, and then inject samples to investigate the specificity of the established method. For the chromatogram see figure 2 .

[0118] Depend on figure 2 It can be seen that there is no interference from endogenous impurities in the peak positions of each analyte and internal standard, so the specificity experiment meets the requirements.

[0119] 2) Sensitivity

[0120] Take blank saliva, add a certain amount of analyte standard, perform pretreatment according to the pretreatment method in Example 1, and then inject the sample. It is required that the signal-to-noise ratio of each analyte at the limit of detection (LOD) S / N≥3, and the signal-to-noise ratio at the limit of quantification (LOQ) S / ...

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Abstract

The invention provides an online PE LC-MS / MS (solid phase extraction liquid chromatography / mass spectrometry) analysis method for diazepam and metabolites thereof in human saliva. The analysis method comprises the steps as follows: a saliva sample is subjected to online solid phase extraction, and a HySphere Resin GP (10 mm x 2 mm) column is selected in the process of solid phase extraction; a C18 chromatographic column is used for chromatographic separation, and mobile-phase water (containing 2-mmol / L ammonium formate and 0.1 % formic acid) and mobile-phase acetonitrile (containing 2-mmol / L ammonium formate and 0.1 % formic acid) are used for gradient elution; ESI (electrospray ionization) and MRM (multi-reaction monitoring) are used for mass spectrum analysis for detecting target compounds. The simple, sensitive, stable and full-automatic online SPE LC-MS / MS analysis method is established for the first time to be used for quantitating diazepam and five metabolites (nordazepam, oxazepam, temazepam, oxazepam glucuronic acid conjugates and temazepam glucuronic acid conjugates) of diazepam in the human saliva.

Description

technical field [0001] The invention belongs to the field of drug analysis, in particular to an online SPELC-MS / MS analysis method for diazepam in human saliva and its metabolites, more specifically to a method for quantitatively detecting diazepam in human saliva and five metabolites thereof online SPELC-MS / MS analysis method. Background technique [0002] Most of human saliva is composed of water, others include organic matter (mucin, salivary amylase, lysozyme, immunoglobulin, urea, uric acid and free amino acids, etc.), inorganic matter (Na + 、K + , Ca 2+ , Cl - 、HCO 3- ) and cell debris. In recent years, saliva has been favored and welcomed more and more by forensic poison analysis workers, and it is used for the screening of drug poisons. Although the concentration of drug poison in saliva cannot be used to calculate the real concentration level in blood, it can reflect the recent drug use of individuals and still has very important analytical value. Compared wi...

Claims

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Application Information

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IPC IPC(8): G01N30/88
CPCG01N30/88
Inventor 张玉荣姜凤丽倪春芳梁晨汪蓉
Owner SHANGHAI CRIMINAL SCI TECH RES INST
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