CN102706972A - 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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- CN102706972A CN102706972A CN2012100116511A CN201210011651A CN102706972A CN 102706972 A CN102706972 A CN 102706972A CN 2012100116511 A CN2012100116511 A CN 2012100116511A CN 201210011651 A CN201210011651 A CN 201210011651A CN 102706972 A CN102706972 A CN 102706972A
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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 the hepatomicrosome and metabolic product 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 mainly is present in the endocytoplasmic reticulum of organs such as liver, intestines.CYP3A is as main hypotype in the liver CYP family, manyly exogenously all will pass through its metabolism with endogenous material, and the medicine that uses clinically has 60% metabolism through CYP3A, approximately like erythromycin, ketoconazole and statins.Research shows that some medicines and natural products exist interaction, can suppress the activity of CYP3A like grapefruit juice, makes that the bioavailability of medicament through the CYP3A metabolism improves.Therefore, the active right of CYP3A enzyme separated the medicine metabolism and interacts 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, through measuring midazolam and metabolite content thereof, calculates metabolic rate and can infer the activity of estimating the CYP3A enzyme.
At present, because high performance liquid chromatography has efficiently, quick and sensitive characteristics, thereby be widely used in the concentration of measuring midazolam and metabolic product thereof.Like (content of midazolam and the application in drug interaction research thereof in the HPLC method mensuration rat plasma such as Zhao Naping; Pharmaceutical Analysis magazine, 2007 the 27th phases) with a certain proportion of phosphate buffer and methyl alcohol as moving phase, the moving phase complicated preparation of this phosphoric acid salt buffer; And phosphate buffer is prone to separate out; Phosphate has certain infringement to instrument and pillar, and it adopts the external standard detection method, and operate miss can cause bigger influence to testing result.
Summary of the invention
The object of the present invention is to provide a kind of method that can measure midazolam in the hepatomicrosome and metabolic product 1-hydroxyl midazolam concentration thereof simultaneously, 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 the hepatomicrosome and metabolic product concentration thereof, comprises the steps:
1) sample pretreatment:
Get rat liver microsomes and add midazolam and carry out incubated in vitro, add the acetonitrile solution that contains the internal standard compound carbamazepine and 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 moving 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 detection:
Adopt PDAD, the detection wavelength is 230nm, and flow velocity is 1.0mLmin
-1, column temperature is 40 ℃, measures peak area, with the concentration of typical curve Equation for Calculating midazolam and metabolic product thereof.
Described metabolic product is a 1-hydroxyl midazolam.
Under the described chromatographic condition of step (2), 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 pre-service: adopt the acetonitrile precipitation method directly to handle sample, omitted the step of extracting, fast simple, albumen precipitation is effective, and can stop MC reaction well, is fit to the incubated in vitro experiment.
(2) moving phase: the mixed liquor of selecting water-acetonitrile-0.1% trifluoroacetic acid for use is as moving 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 influence that operate miss is brought testing result, and the retention time of carbamazepine is about 7～8min, and the analysis time of each sample is in 9 minutes.
Sample process of the present invention is easy, it is fast sensitive to detect, and can detect simultaneously the concentration of midazolam in the hepatomicrosome and 1-hydroxyl midazolam, can be applied to the evaluation study of CYP3A enzymatic activity.The range of linearity of midazolam is 0.06～12 μ gmL among the present invention
-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 the day to day precision standard deviation is all less than 10%.
Description of drawings
Fig. 1 is hatched the chromatogram of back sample for the midazolam hepatomicrosome.
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 be SB-C18 (4.6 * 12.5mm, 5 μ m, Agilent, USA); Moving 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
-1Sample size is 20 μ L; Detecting device is PDAD (DAD); Column temperature is 40 ℃; The detection wavelength is 230nm.
Hepatomicrosome preparation: adopt differential centrifugation to prepare hepatomicrosome.With the rat sacrificed by decapitation, take out liver rapidly, clean with ice-cold physiological saline, 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 once more 10, and the centrifugal 15min of 000g gets supernatant, and 100, the centrifugal 90min of 000g; Abandon supernatant, deposition is redissolved with the PBS of 10mL, and-80 ℃ of packing are preserved, and above all operations carries out in being lower than 4 ℃ of environment.With BCA method (dicinchonine acid system) mensuration microsomal protein content is 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; Behind 37 ℃ of water-bath preincubate 5min, add 20mMNADPH 10 μ L and start reaction, after continuing to hatch 5min; Place trash ice, the acetonitrile that adds 200 μ L is with cessation reaction.Add 5 μ gmL again
-1Carbamazepine in the mark working fluid, behind the vortex mixing 2min 13, the centrifugal 10min of 000rpm gets supernatant in the sample introduction 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
-1The microsome incubation system, by the operation of the disposal route 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), be horizontal ordinate (x with the corresponding each point concentration of MDZ
1) draw the typical curve of MDZ.With As
2/ Ai is ordinate (y
2), be horizontal ordinate (x with the corresponding each point concentration of 1-OHMDZ
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) the microsome incubation system, 6 parts of every kind of concentration are handled the back and are detected, and the peak area of record MDZ and 1-OHMDZ is the peak area of microsome incubation system.Other gets 6 of 1.5mL EP pipes, two one group; Final concentration and basic, normal, high three kinds of concentration are processed 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; The peak area of record 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, see table 1.
The extraction recovery result of table 1 hepatomicrosome incubation system MDZ and 1-OHMDZ (n=6, mean ± SD)
The precision of method and accuracy: (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) the 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, calculating precision and accuracy, the result sees table 2.
The precision of table 2 hepatomicrosome incubation system MDZ and 1-OHMDZ and accuracy result (n=6, mean ± SD)
Claims (4)
1. a method of measuring midazolam in the hepatomicrosome and metabolic product concentration thereof is characterized in that: comprise the steps:
1) sample pretreatment:
Get rat liver microsomes and add midazolam and carry out incubated in vitro, add the organic solvent that contains the internal standard compound carbamazepine and 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 moving 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 detection:
Adopt PDAD, the detection wavelength is 230nm, and flow velocity is 1.0mLmin
-1, column temperature is 40 ℃, measures peak area, with the concentration of typical curve Equation for Calculating midazolam and metabolic product thereof.
2. the method for midazolam and metabolic product concentration thereof in the mensuration hepatomicrosome according to claim 1 is characterized in that: described metabolic product is a 1-hydroxyl midazolam.
3. the method for midazolam and metabolic product concentration thereof in the mensuration hepatomicrosome according to claim 1 is characterized in that: the organic solvent in the said step (1) is an acetonitrile.
4. the method for midazolam and metabolic product concentration thereof in the mensuration hepatomicrosome according to claim 1 and 2; It is characterized in that: under the described chromatographic condition of step (2), 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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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| RU2701524C1 (en) * | 2019-06-04 | 2019-09-27 | федеральное государственное бюджетное учреждение "Национальный медицинский исследовательский центр имени академика Е.Н. Мешалкина" Министерства здравоохранения Российской Федерации (ФГБУ "НМИЦ им. ак. Е.Н. Мешалкина" Минздрава России) | Method for quantitative determination of disulphiram in biological media |
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| RU2701524C1 (en) * | 2019-06-04 | 2019-09-27 | федеральное государственное бюджетное учреждение "Национальный медицинский исследовательский центр имени академика Е.Н. Мешалкина" Министерства здравоохранения Российской Федерации (ФГБУ "НМИЦ им. ак. Е.Н. Мешалкина" Минздрава России) | Method for quantitative determination of disulphiram in biological media |
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