UPLC/MS/MS detection method of 1-hydroxymidazolam concentration in liver microsome

A hydroxymidazolam and detection method technology, which is applied in the field of drug detection, can solve the problems of long time consumption, large injection volume, unsuitable concentration of 1-hydroxymidazolam, etc., and achieve short analysis time and high sample injection efficiency. The effect of small volumes and high intra- and inter-assay accuracy and precision

Inactive Publication Date: 2015-12-09
成都华西海圻医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the gradient elution procedure of this method lasts for 8 minutes, takes a long time, and the injection volume is large, so it is not suitable for detecting the concentration of 1-hydroxymidazolam alone

Method used

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  • UPLC/MS/MS detection method of 1-hydroxymidazolam concentration in liver microsome
  • UPLC/MS/MS detection method of 1-hydroxymidazolam concentration in liver microsome
  • UPLC/MS/MS detection method of 1-hydroxymidazolam concentration in liver microsome

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The reagents and instruments are all from commercially available products.

[0044] 1. Reference substance and internal standard

[0045] 1-Hydroxymidazolam (OH-MDZ): molecular weight 341.77, purity 99.4%, batch number 1385527 (sigma-aldrich);

[0046] O-tolylhydramine citrate (positive ion internal standard): purity 99%, batch number 048F0510V (sigma-aldrich).

[0047] 2. Main instruments

[0048]

[0049] 3. Main software used in the test

[0050] UNIFI1.6.1: UPLC / MS / MS data acquisition and concentration calculation.

[0051] 4. Detection method

[0052] ((1) Establish a temperature incubation system for liver microsomes, add internal standard substances and serial concentrations of 1-hydroxymidazolam reference substance, and prepare standard curve samples;

[0053] (2) Inject the standard curve sample into the UPLC / MS / MS LC / MS instrument for detection; the detection conditions are as follows:

[0054] Column: ACQUITYUPLC-BEHC18, 1.7μm, 2.1×50mm (Waters company)

[0055] Mobile phase...

Embodiment 2

[0121] Example 2 Application of the method of the present invention

[0122] 1. Enzyme kinetics test

[0123] When determining enzyme kinetic parameters, the concentration of the probe substrate is usually selected between 1 / 3Km and 3Km, the metabolic rate should be linear with the reaction time, and the substrate metabolized should be less than 10%. In the case of quantifiable metabolites, the lowest possible protein concentration should be used to reduce non-specific binding. Combined with the Km reported in the literature and the results of the preliminary test, the concentration of liver microsomal protein was selected as 0.2 mg / mL, and the concentration of the probe substrate midazolam was 0.5, 0.6, 0.8, 1, 1.5, 5, 20, and 50 μM, respectively.

[0124] The incubation system medium is 0.1M phosphate buffered saline (PBS), including probe substrate, liver microsomes (final concentration 0.2mg / mL) and NADP regeneration system (final concentration is 10mMG-6-P, 1U / mLG-6 -PDH, 10mM...

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Abstract

The invention discloses a UPLC/MS/MS detection method of the 1-hydroxymidazolam concentration in a liver microsome. The UPLC/MS/MS detection method comprises the following steps that 1, a liver microsome incubation system is established, serial concentrations of 1-hydroxymidazolam comparison products are added to prepare a standard curve sample; 2, the standard curve sample is injected into a UPLC/MS/MS liquid chromatograph/mass spectrometer for detection, and a standard 1-hydroxymidazolam curve is obtained according to the detection result; 3, a sample to be detected is prepared by adopting the same method as the step 1 and is injected into the UPLC/MS/MS liquid chromatograph/mass spectrometer, detection is performed under the same conditions as the step 2, and the 1-hydroxymidazolam concentration in the sample to be detected is obtained according to the standard curve. The UPLC/MS/MS detection method is short in analysis time, high in sensitivity and small in sample size, meets the methodological validation requirement, is suitable for determination of the 1-hydroxymidazolam concentration in the liver microsome incubation sample and has wide application prospect.

Description

Technical field [0001] The invention relates to a method for detecting drugs, in particular to a UPLC / MS / MS method for detecting the concentration of 1-hydroxymidazolam in liver microsomes. Background technique [0002] CYP450 enzymes are the main enzymes involved in drug metabolism, including CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 and other isoenzymes closely related to drug metabolism. Studies have shown that most drug-drug interactions are related to the induction and inhibition of CYP450 enzyme activity, which is mainly caused by enzyme inhibition. The use of in vitro methods for drug metabolism studies can effectively shorten the development cycle of new drugs, directly observe the selective metabolism of substrates by enzymes and the induction and inhibition of enzymes by substrates, determine drug metabolism pathways, and predict potential drug interactions in vivo. At present, the commonly used in vitro method is the probe drug method, which establish...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/88G01N30/06
Inventor 赖力岑小波肖媛媛龚梅曾强
Owner 成都华西海圻医药科技有限公司
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