Determining method of concentrations of midazolam and metabolic product thereof in liver microsomes
A technology for midazolam and metabolites, which is applied in the field of medical testing, can solve the problems of cumbersome preparation of a mobile phase containing phosphate buffer, easy precipitation of phosphate buffer, influence of test results, etc., and achieves good separation effect and protein precipitation. Good results and easy sample handling
Inactive Publication Date: 2014-03-05
HENAN UNIV OF SCI & TECH
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For example, Zhao Naping et al. (Determining the content of midazolam in rat plasma by HPLC and its application in drug interaction research, Journal of Drug Analysis, 2007 No. 27) with a certain proportion of phosphate buffer and methanol as the flow Phase, the preparation of the mobile phase containing phosphate buffer is tedious, and the phosphate buffer is easy to precipitate, and phosphate has certain damage to the instrument and column. It adopts the external standard detection method, and the operation error will cause a large impact on the detection results. influences
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[0021] Chromatographic conditions: the chromatographic column is Agilent Zorbax SB-C18 (4.6mm x 150mm 5μm); the guard column is SB-C18 (4.6×12.5mm, 5μm, Agilent, USA); the mobile phase is water-acetonitrile-0.1% trifluoroacetic acid The mixed solution, the volume ratio of the mixed solution is 49:31:20, the flow rate is 1.0ml·min -1 ; The injection volume was 20 μL; the detector was a diode array detector (DAD); the column temperature was 40° C.; the detection wavelength was 230 nm.
[0022] Liver microsome preparation: Liver microsomes were prepared by differential centrifugation. The rats were decapitated, and the liver was quickly taken out, washed with ice-cold saline, blotted dry with filter paper and weighed. Add 25 mL of sucrose PBS buffer (phosphate buffer saline) per 10 g of liver, and fully homogenize in an ice bath. Take the homogenate and centrifuge at 10,000g for 15min, take the supernatant and centrifuge again at 10,000g for 15min, take the supernatant, and cent...
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The invention relates to a determining method of concentrations of midazolam and metabolic products thereof in liver microsomes. The method comprises the following steps: 1) pre-treating a sample; 2) separating with a chromatographic column, wherein a universal C18 chromatographic column is adopted, Agilent Zorbax SB-C18 is adopted as filler, and mixed solution of water, methyl cyanide and 0.1 percent trifluoroacetic acid in a volume ratio of (44-54):(26-36):(15-25) is adopted as a mobile phase; and 3) detecting with violet rays, wherein a diode array detector is adopted, a detection wavelength is 230nm, the flowing speed is 1.0mL.min<-1>, the column temperature is 40 DEG C; and detecting the peak area, and calculating concentrations of midazolam and metabolic products by using a standard curve equation. The method is easy in sample treatment and sensitive and quick in detection, can be used for detecting concentrations of the midazolam and 1-hydroxyl midazolam in the liver microsomes simultaneously, and can be applied to evaluation study of CYP3A enzyme activity. According to method, the linear range of the midazolam is between 0.06 and 12mu g.mL<-1>, the linear range of the 1-hydroxyl midazolam is between 0.06 and 3mu g.mL<-1>, the extraction recycling rate is above 95 percent, and within-day and day-to-day precision standard deviations are both less than 10 percent.
Description
technical field [0001] The invention relates to a method for detecting midazolam and its metabolites, in particular to a method for measuring the concentration of midazolam and its metabolites in liver microsomes, belonging to the field of medical testing. Background technique [0002] Cytochrome P450 (CYP450) is an important enzyme system for drug metabolism in vivo, plays an important role in the process of drug metabolism and biotransformation, and mainly exists in the endoplasmic reticulum of liver, intestine and other organs. CYP3A is the main subtype of the liver CYP family, and many exogenous and endogenous substances are metabolized by it. About 60% of clinically used drugs are metabolized by CYP3A, such as erythromycin, ketoconazole and Statins. Studies have shown that some drugs interact with natural products. For example, grapefruit juice can inhibit the activity of CYP3A, which increases the bioavailability of drugs metabolized by CYP3A. Therefore, the activity...
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IPC IPC(8): G01N30/02
Inventor王勇邱相君王哲徐涛胡国新
OwnerHENAN UNIV OF SCI & TECH