CN102706972B - Determining method of concentrations of midazolam and metabolic product thereof in liver microsomes - Google Patents
Determining method of concentrations of midazolam and metabolic product thereof in liver microsomes Download PDFInfo
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- CN102706972B CN102706972B CN201210011651.1A CN201210011651A CN102706972B CN 102706972 B CN102706972 B CN 102706972B CN 201210011651 A CN201210011651 A CN 201210011651A CN 102706972 B CN102706972 B CN 102706972B
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Abstract
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
The present invention relates to the detection method of a kind of midazolam and metabolic product thereof, relate in particular to the method for measuring midazolam in hepatomicrosome and Metabolites Concentration thereof, belong to field of medical examination.
Background technology
Cytochrome P450 (CYP450) is the medicine important enzyme system of metabolism in vivo, in drug metabolism and biotransformation, plays an important role, and is mainly present in the endocytoplasmic reticulum of the organs such as liver, intestines.CYP3A is as main hypotype in liver CYP family, and many exogenous and endogenous material all will pass through its metabolism, and the medicine using clinically approximately has 60% metabolism of passing through CYP3A, as erythromycin, ketoconazole and statins.Research shows, some medicines and natural products exist interaction, as grapefruit juice can suppress the activity of CYP3A, makes to improve through the bioavilability of the medicine of CYP3A metabolism.Therefore, the right solution drug metabolism of the activity of CYP3A enzyme and interaction are very important.Midazolam (MDZ) is as the probe medicine of CYP3A, be widely used for the research of inside and outside, 1-hydroxyl midazolam (1-OHMDZ) is its main metabolic product, by measuring midazolam and metabolite content thereof, calculates metabolic rate and can infer the activity of evaluating CYP3A enzyme.
At present, because high performance liquid chromatography has efficient, fast and sensitive, thereby be widely used in the concentration of measuring midazolam and metabolic product thereof.As (content of midazolam and the application in drug interaction research thereof in HPLC method mensuration rat plasma such as Zhao Naping, Pharmaceutical Analysis magazine, the 27th phase in 2007) using a certain proportion of phosphate buffer and methyl alcohol as mobile phase, the mobile phase preparation of this phosphate-containing damping fluid is loaded down with trivial details, and phosphate buffer is easily separated out, phosphate has certain infringement to instrument and pillar, and it adopts external standard detection method, and operate miss can cause larger impact to testing result.
Summary of the invention
The object of the present invention is to provide a kind of method that can simultaneously measure midazolam in hepatomicrosome and metabolic product 1-hydroxyl midazolam concentration thereof, thereby be applied to the evaluation of CYP3A enzymatic activity.
To achieve these goals, technical scheme of the present invention has adopted a kind of method of measuring midazolam in hepatomicrosome and Metabolites Concentration thereof, comprises the steps:
1) sample pretreatment:
Get rat liver microsomes and add midazolam to carry out incubated in vitro, add the acetonitrile solution containing internal standard compound carbamazepine to carry out protein precipitation, get supernatant 20 μ L sample introductions after centrifugal;
2) chromatographic column is separated:
Adopt universal C18 chromatographic column, filler is Agilent Zorbax SB-C18, and mobile phase is the mixed liquor of water-acetonitrile-0.1% trifluoroacetic acid, and the volume ratio of this mixed liquor is 44～54: 26～36: 15～25;
3) ultraviolet detects:
Adopt diode array detector, detection wavelength is 230nm, and flow velocity is 1.0mLmin
-1, column temperature is 40 ℃, measures peak area, calculates the concentration of midazolam and metabolic product thereof with typical curve equation.
Described metabolic product is 1-hydroxyl midazolam.
Under chromatographic condition step (2) Suo Shu, the retention time of 1-hydroxyl midazolam, midazolam and interior mark carbamazepine is respectively 3～4min, 4～5min, 7～8min.
Adopt method of the present invention to have the following advantages:
(1) sample preprocessing: adopt the direct processing sample of acetonitrile precipitation method, omitted the step of extracting, fast simple, albumen precipitation is effective, and can stop well MC reaction, is applicable to incubated in vitro experiment.
(2) mobile phase: select the mixed liquor of water-acetonitrile-0.1% trifluoroacetic acid as mobile phase, the use of ion-pairing agent trifluoroacetic acid can improve that 1-hydroxyl midazolam, midazolam and carbamazepine chromatographic peak peak shape are asymmetric, peak broadening and conditions of streaking, obtained good separating effect, theoretical cam curve reaches more than 10000, can detect the concentration of midazolam and metabolic product thereof simultaneously.
(3) internal standard method: using carbamazepine is interior mark, has reduced the impact that operate miss is brought testing result, the retention time of carbamazepine is in 7～8min left and right, and the analysis time of each sample is in 9 minutes.
Sample process of the present invention is easy, detect and sensitively to the concentration of midazolam in hepatomicrosome and 1-hydroxyl midazolam, can detect fast simultaneously, can be applied to the evaluation study of CYP3A enzymatic activity.In the present invention, the range of linearity of midazolam is 0.06～12 μ gmL
-1, the range of linearity of 1-hydroxyl midazolam is 0.06～3 μ gmL
-1, extraction recovery is more than 95%, and in a few days day to day precision standard deviation is all less than 10%.
Accompanying drawing explanation
Fig. 1 is the chromatogram of sample after midazolam liver microsomes incubation.
Wherein, 1 is 1-hydroxyl midazolam, and 2 is midazolam, and 3 is carbamazepine (internal standard compound).
Embodiment
Chromatographic condition: chromatographic column is Agilent Zorbax SB-C18 (4.6mm x 150mm 5 μ m); Guard column is SB-C18 (4.6 * 12.5mm, 5 μ m, Agilent, USA); Mobile phase is the mixed liquor of water-acetonitrile-0.1% trifluoroacetic acid, and the volume ratio of this mixed liquor is 49: 31: 20, and flow velocity is 1.0mlmin
-1; Sample size is 20 μ L; Detecting device is diode array detector (DAD); Column temperature is 40 ℃; Detection wavelength is 230nm.
Liver microsome preparation: adopt differential centrifugation to prepare hepatomicrosome.By rat sacrificed by decapitation, take out rapidly liver, with ice-cold physiological saline, clean, after blotting, filter paper weighs, and every 10g liver adds 25mL sucrose PBS damping fluid (phosphate buffer), fully homogenate in ice bath.Get homogenate 10, the centrifugal 15min of 000g, gets supernatant again 10, and the centrifugal 15min of 000g, gets supernatant, the centrifugal 90min of 100,000g; Abandon supernatant, precipitation is redissolved with the PBS of 10mL, and-80 ℃ of packing are preserved, and above all operations carries out in lower than 4 ℃ of environment.With BCA method (dicinchonine acid system) mensuration microsomal protein content, be 20mgmL
-1.
The processing of hepatomicrosome incubated in vitro and sample: draw microsome 10 μ L in the EP of 1.5mL pipe, add MDZ titer 25 μ L and PBS damping fluid 215 μ L, after 37 ℃ of water-bath preincubate 5min, add 20mMNADPH 10 μ L to start reaction, continue to hatch after 5min, be placed in trash ice, add the acetonitrile of 200 μ L with cessation reaction.Add again 5 μ gmL
-1carbamazepine in mark working fluid, vortex mixes after 2min 13, the centrifugal 10min of 000rpm, gets supernatant in the sample injection bottle of automatic sampler, sets 20 μ L sample introductions.
Typical curve: preparation MDZ concentration is respectively 0.06,0.12,0.3,0.6,1.2,3,6,12 μ gmL
-1with 1-OHMDZ concentration be 0.06,0.12,0.2,0.3,0.6,1.2,2,3 μ gmL
-1microsome incubation system, by the disposal route operation of above-mentioned sample, measure MDZ area A s
1, 1-OHMDZ area A s
2, interior mark peak area Ai.With As
1/ Ai is ordinate (y
1), the corresponding each point concentration of the MDZ of take is horizontal ordinate (x
1) draw the typical curve of MDZ.With As
2/ Ai is ordinate (y
2), the corresponding each point concentration of the 1-OHMDZ of take is horizontal ordinate (x
2) draw the typical curve of 1-OHMDZ.The typical curve regression equation of MDZ is: y
1=0.9157x
1-0.1157 (r=0.9996); The typical curve regression equation of 1-OHMDZ is: y
2=1.2251x
2-0.0691 (r=0.9996).
Extraction recovery: (MDZ concentration is 0.12,0.6,6.00 μ gmL to prepare basic, normal, high three kinds of concentration
-1with 1-OHMDZ concentration be 0.12,0.3,2.00 μ gmL
-1) microsome incubation system, 6 parts of every kind of concentration, detect after processing, and record the peak area of MDZ and 1-OHMDZ, are the peak area of microsome incubation system.Separately get 6 of 1.5mL EP pipes, two one group; Final concentration and basic, normal, high three kinds of concentration are made in every assembly, and (MDZ concentration is 0.12,0.6,6.00 μ gmL
-1with 1-OHMDZ concentration be 0.12,0.3,2.00 μ gmL
-1) identical standard solution, 20 μ L sample detection; Recording the peak area of variable concentrations MDZ and 1-OHMDZ, is pure target peak area.Calculate the peak area of microsome incubation system and the ratio of pure target peak area, be extraction recovery, in Table 1.
The extraction recovery result of table 1 liver microsomes incubation system MDZ and 1-OHMDZ (n=6, mean ± SD)
The preci-sion and accuracy of method: (MDZ concentration is 0.12,0.6,10.8 μ gmL to prepare basic, normal, high three kinds of concentration
-1with 1-OHMDZ concentration be 0.12,0.3,2.7 μ gmL
-1) microsome incubation system, each concentration is carried out 6 sample analysis, measure for three days on end, according to the same day typical curve separately calculate the concentration of MDZ and 1-OHMDZ, calculate preci-sion and accuracy, the results are shown in Table 2.
The preci-sion and accuracy result of table 2 liver microsomes incubation system MDZ and 1-OHMDZ (n=6, mean ± SD)
Claims (1)
1. a method of measuring midazolam in hepatomicrosome and metabolic product 1-hydroxyl midazolam concentration thereof, is characterized in that: comprise the steps:
(1) sample pretreatment:
Get rat liver microsomes and add midazolam to carry out incubated in vitro, add the organic solvent containing internal standard compound carbamazepine to carry out protein precipitation, get supernatant 20 μ L sample introductions after centrifugal;
(2) chromatographic column is separated:
Adopt universal C18 chromatographic column, filler is Agilent Zorbax SB-C18, and mobile phase is the mixed liquor of water-acetonitrile-0.1% trifluoroacetic acid, and the volume ratio of this mixed liquor is 44～54:26～36:15～25;
(3) ultraviolet detects:
Adopt diode array detector, detection wavelength is 230nm, and flow velocity is 1.0mLmin
-1, column temperature is 40 ℃, measures peak area, calculates the concentration of midazolam and metabolic product thereof with typical curve equation;
Organic solvent in described step (1) is acetonitrile;
Under chromatographic condition step (2) Suo Shu, the retention time of metabolic product 1-hydroxyl midazolam, midazolam and interior mark carbamazepine is respectively 3～4min, 4～5min, 7～8min.
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