CN112461961A - Method for determining concentration of mizolastine in human plasma - Google Patents
Method for determining concentration of mizolastine in human plasma Download PDFInfo
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- PVLJETXTTWAYEW-UHFFFAOYSA-N Mizolastine Chemical compound N=1C=CC(=O)NC=1N(C)C(CC1)CCN1C1=NC2=CC=CC=C2N1CC1=CC=C(F)C=C1 PVLJETXTTWAYEW-UHFFFAOYSA-N 0.000 title claims abstract description 110
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- 238000000034 method Methods 0.000 title claims abstract description 67
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention belongs to the technical field of medicine detection, and particularly discloses a method for determining the concentration of mizolastine in human plasma.
Description
Technical Field
The invention relates to the technical field of medicine detection, in particular to a method for determining concentration of mizolastine in human plasma.
Background
Mizolastine (Mizolastine) is a long-acting, specific, highly selective sexual peripheral histamine H1 receptor antagonist, has antihistaminic and antiallergic activities, and has pharmacological effectsBesides the selective competitive inhibition of histamine H1 receptor and inhibition of histamine release from mast cells, it also has anti-inflammatory effects. Clinically used for treating seasonal allergic rhinitis (pollinosis), perennial allergic rhinitis and skin allergic diseases such as chronic urticaria, has the advantages of no anticholinergic effect and no central sedation, similar to other second generation antihistaminic H1 receptor antagonists. The product can be absorbed rapidly after oral administration, and has peak plasma concentration time (T)max) 1.5 hours, bioavailability of about 65%, pharmacokinetics is linear, mean elimination half-life is 13.0 hours, drug in plasma protein binding rate of about 98.4%. Therefore, the composition has the characteristics of quick response and lasting effect (more than 24 hours). The elimination of mizolastine is mainly metabolized in the liver by glucuronidation, and one of the other metabolic pathways is the formation of hydroxylated metabolites by cytochrome P4503a4 enzyme, none of which are pharmacologically active, and less than 0.5% of which are excreted in the urine as they are. Equilibrium studies with 14C-labeled mizolastine showed that 84% to 95% of the administered dose was excreted in the feces. Its main adverse reactions mainly include slight to moderate lethargy, hypodynamia, dry mouth, etc.
The method is a sensitive and rapid LC-MS/MS method established by the phylline tiger and the like (J.A.Med.Analyzer, 2016,36(11),2022-2028) and is used for rapidly detecting 7 antihistaminic chemical drugs, namely loratadine, diphenhydramine hydrochloride, cyproheptadine hydrochloride, cetirizine hydrochloride, promethazine hydrochloride, mizolastine and chlorphenamine maleate, which are added in a common traditional Chinese medicine preparation in the dermatology department. Proved by methodology, the method can be used as a detection method for detecting chemical drugs added with antihistamine in traditional Chinese medicine preparations in dermatology. The existing method for detecting the substance related to mizolastine adopts a liquid chromatography-mass spectrometer to detect the content of the mizolastine in the traditional Chinese medicine preparation, and the detection matrix is the traditional Chinese medicine preparation. The traditional Chinese medicine preparation has simple matrix composition, and the pretreatment method of the technology is not suitable for measuring the concentration of mizolastine in the plasma matrix and has certain limitation.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a method for determining the concentration of mizolastine in human plasma, which adopts an LC-MS/MS method to determine the concentration of mizolastine in human plasma, adopts a protein precipitation method to extract mizolastine from a plasma matrix, solves the problem of much interference of the plasma matrix, and has high sensitivity and accurate and reliable result.
In order to solve the technical problems, the invention adopts the following technical scheme:
in one aspect, the present invention provides a method for determining mizolastine concentration in human plasma, comprising the steps of: extracting mizolastine in blood plasma to obtain an extracting solution; and performing qualitative and quantitative determination on the extracting solution by adopting high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS).
Further, the liquid chromatography conditions of the high performance liquid chromatography tandem mass spectrometry comprise:
a chromatographic column: waters XSelect HSS C18The specification is 3.5 mu m and 3 multiplied by 50 mm; the mobile phase A is 0.1 percent formic acid aqueous solution; the mobile phase B is methanol; flow rate: 0.6 mL/min; gradient elution is adopted; stopping time: 4.0 min; washing the needle washing liquid: 15 s; sample introduction amount: 1 mu L of the solution; column temperature: 30 ℃; sample injector temperature: 4 ℃ is prepared.
Further, the needle washing liquid is 50% methanol water solution.
The invention selects a chromatographic column: waters XSelect HSS C18The specification is 3.5 mu m and 3 multiplied by 50 mm; the response value to mizolastine in plasma is highest, the peak shape is good, the peak shape is symmetrical and not trailing, and the peak-off time is not too far forward or too far backward and is just proper.
Considering the influence of the flow rate on the drug peak-out time and the influence of the peak-out effect caused by overlarge sample injection amount, the flow rate is selected to be 0.6mL/min, and the sample injection amount is 1 mu L, so that the drug peak-out effect and the response value can be ensured, the drug can be completely eluted and tightened, the pollution to a chromatographic column is avoided, and the recovery rate and the sensitivity of subsequent mass spectrometry detection are improved.
Further, the procedure of the gradient elution is:
the invention adopts a gradient elution mode to detect mizolastine in plasma, the elution proportion of a mobile phase is changed at different time, and the problems of plasma matrix effect and the like are comprehensively considered, so that the peak shape of a drug is symmetrical and perfect, the drug can be sufficiently eluted, the recovery rate is high, and the interference is small.
Further, the mass spectrum conditions of the high performance liquid chromatography tandem mass spectrum comprise:
ESI source is adopted, ionization mode: electrospray ionization positive ion mode (ESI); the scanning mode is as follows: multiple Reaction Monitoring (MRM);
Name | Q1Mass(Da) | Q3Mass(Da) | Dwell(msec) | DP | CE | Polarity |
MZST | 433.3 | 308.1 | 100 | 140 | 30 | Positive |
MZST-13C-d3 | 437.3 | 308.2 | 100 | 140 | 31 | Positive |
the mass spectrum switching valve is set as follows:
TotalTime(min) | |
|
1 | 1.0 | B |
2 | 2.7 | A |
the ion source parameters were set as follows:
the mizolastine in the blood plasma can be effectively separated and identified through the strong component identification capability of the mass spectrometer, and the quantitative detection is carried out.
Further, the preparation process of the extracting solution is as follows: taking a whole blood sample to be detected, adding an anticoagulant K2EDTA, centrifuging to obtain a plasma sample to be detected, adding an internal standard working solution, adding an extraction solvent, performing vortex, centrifuging, and taking supernate to obtain an extracting solution.
Further, the extraction solvent is methanol. The diluted solution was 50% aqueous methanol.
Further, the internal standard in the internal standard working solution is mizolastine-d3。
Further, the preparation process of the internal standard working solution is as follows: weighing mizolastine-13C-d3Dissolving in methanol in a volumetric flask to prepare 10 ug/mL-1An internal standard control stock solution;
getting mizolastine-d3Placing 50 μ L of internal standard reference substance stock solution in 50mL volumetric flask, adding methanol to constant volume to scale mark, and shaking to obtain solution with concentration of 100ng/mL-1The internal standard working solution of (4).
Further, the conditions of the vortex centrifugation are as follows: vortex for 3min, centrifuge 2450g, 4 deg.C, 10 min.
Further, the qualitative and quantitative method comprises the following steps: the qualitative method is that the retention time of the drug to be detected is consistent with that of the drug standard, and the drug is determined as the target drug by combining the qualitative ion pair and the quantitative ion pair in the mass spectrum parameter; the quantitative method comprises the following steps: and (3) performing linear least square regression calculation on the theoretical concentration of the analyte in the standard curve by comparing the peak area of the analyte with the peak area of the internal standard, and calculating the actually measured concentration of the analyte in the sample by using the obtained regression equation.
Further, the preparation process of the standard curve is as follows: taking an mizolastine standard working solution, diluting the mizolastine standard working solution into blank plasma, and preparing a mizolastine standard curve plasma sample; taking a standard curve plasma sample, adding an internal standard working solution, carrying out vortex centrifugation, taking supernate, determining by adopting high performance liquid chromatography tandem mass spectrometry (LC-MS/MS), recording the peak area corresponding to mizolastine at each concentration, and preparing a standard curve regression equation by taking the peak area ratio of the mizolastine and the internal standard as a vertical coordinate and the concentration of the mizolastine as a horizontal coordinate;
further, the concentration range of the standard series of mizolastine working solutions is 100-16000 ng/mL; preferred concentrations are 16000, 12800, 4000, 2000, 1000, 400, 200, 100 ng/mL.
Further, the concentration range of the mizolastine standard curve plasma sample is 5-800 ng/mL, preferably 5, 10, 20, 50, 100, 200, 640, 800 ng/mL.
Further, the preparation of the mizolastine standard working solution comprises the following steps: weighing mizolastine in a brown wide-mouth glass bottle, dissolving with methanol to prepare 1 mg/mL-1Stock solutions of standard controls; and diluting the standard reference substance stock solution with 50% methanol water to prepare a standard series of mizolastine working solution.
Further, the standard curve regression equation is obtained as follows: taking 50 μ L of each standard curve plasma sample, adding 50 μ L of internal standard working solution, vortexing for 3min, centrifuging 2450g, and performing centrifugation at 4 deg.C for 10 min; 1 mu L of sample injection LC-MS/MS system to obtain a chromatogram; and calculating the concentration of mizolastine in the plasma according to the ratio of the chromatographic peak area of the mizolastine to the internal standard peak area in the chromatogram, and making a standard curve regression equation of the mizolastine.
Furthermore, after the LC-MS/MS system detects, mizolastine generates a peak within about 1.74min, the peak shape is good, and no impurity peak in human plasma interferes with the determination of the compound, which indicates that the method has strong specificity.
Further, the regression equation of the standard curve is as follows:
the regression equation of the measured standard curve of mizolastine in human plasma is that y is 0.00695x +0.000268, and R is 0.9991; the linear range of the mizolastine is 5 ng/mL-800 ng/mL, and the lowest quantitative lower limit of the blood concentration of the mizolastine is 5 ng/mL.
The method can obtain high recovery rate, effectively reduce the interference of impurity peaks, avoid the influence of the traditional method caused by solvent effect, and avoid the situations of complexity and uncontrolled conditions of the traditional method.
The invention adopts mizolastine-13C-d3As an internal standard substance, the determination of the concentration of mizolastine in plasma has good reproducibility and high accuracy;
the method has system applicability, no selectivity to blank plasma from different sources, specific determination of mizolastine and good separation degree.
The method has small dosage, only 50 mu L of blood plasma sample is needed for each test, only 1 mu L of sample is needed for sample injection, and the compound can be accurately determined.
Compared with the prior art, the invention has the following beneficial effects:
the invention establishes a human plasma collecting, separating and analyzing method based on an LC-MS-MS method, and is successfully applied to the concentration detection of mizolastine in human plasma. The method has the advantages that the problem of much interference of the plasma matrix is solved by adopting a protein precipitation method to extract the mizolastine from the plasma matrix and adopting an LC-MS/MS method to measure the mizolastine concentration in human plasma, the sensitivity is high, the result is accurate and reliable, the matrix effect, the hyperlipemia matrix effect and the hemolysis matrix effect are all free of interference, the accuracy and the precision are high, the dosage is small, and the method can be used for the analysis requirements of clinical large-batch samples.
The invention provides a simple method for determining the concentration of mizolastine in human plasma by a pretreatment method, which adopts a protein precipitation method and is suitable for conventional determination; meanwhile, under the chromatographic condition adopted in the experiment, the mizolastine retention time is about 1.74min, the peak shape is good, the measurement is free of the interference of the miscellaneous peak, and the base line is stable; the method has high specificity, and can accurately determine mizolastine in blood plasma3The concentration and the sensitivity are higher, and the minimum limit of plasma quantification of the mizolastine is 5 ng/mL; the method is rapid, accurate, high in sensitivity and simple and convenient to operate, and provides a basis for measuring the blood concentration of mizolastine. The linear range of the plasma standard curve of mizolastine in the method is 5-800 ng/mL, and the precision RSD in batch and between batches is less than +/-15%. The method has good reproducibility and high accuracy, is not interfered by a substrate, a hyperlipemia substrate and a hemolysis substrate, and has no interference phenomenon of the blood plasma of different crowds to the method.
The method can meet the pharmacokinetic requirement of human bodies and can be applied to clinical pharmacokinetic research.
Drawings
FIG. 1 is a standard graph of mizolastine in human plasma measured by LC-MS/MS;
FIG. 2 LC-MS/MS method for detecting mizolastine and mizolastine-13C-d3Liquid mass diagram of (1);
FIG. 3 is a scanning mass spectrum of mizolastine positive ion multi-reaction detection;
FIG. 4 Imidazostine-13C-d3And (3) detecting and scanning a mass spectrogram by positive ion multi-reaction.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention.
1. Main instrument
API 5500 triple quadrupole LC MS (AB Sciex corporation) with electrospray ionization source and Analyst (Version1.6.3) data processing system with Agilent1290 autosampler, liquid chromatograph, column oven (Agilent, USA)
Mettleteledo XPR 2/type a millionth (METTLER, switzerland);
2. chromatographic conditions
A chromatographic column: waters XSelect HSS C183.5μm(3×50mm)
Mobile phase A: 0.1% aqueous formic acid; mobile phase B: methanol
Flow rate: 0.6 mL/min; stopping time: 4.0min
Washing the needle washing liquid: 15 s; sample introduction amount: 1 mu L of the solution; column temperature: 30 ℃; sample injector temperature: 4 deg.C
The mobile phase ratio was set as follows:
3. conditions of Mass Spectrometry
ESI source is adopted, ionization mode: electrospray ionization positive ion mode (ESI); the scanning mode is as follows: multiple Reaction Monitoring (MRM);
Name | Q1Mass(Da) | Q3Mass(Da) | Dwell(msec) | DP | CE | Polarity |
MZST | 433.3 | 308.1 | 100 | 140 | 30 | Positive |
MZST-13C-d3 | 437.3 | 308.2 | 100 | 140 | 31 | Positive |
the mass spectrum switching valve is set as follows:
TotalTime(min) | |
|
1 | 1.0 | B |
2 | 2.7 | A |
the ion source parameters were set as follows:
Parameter | Value |
CUR | 40 |
CAD | Medium |
IS | 5500 |
|
500 |
GS1 | 60 |
GS2 | 60 |
EP | 12 |
CXP | 12 |
4. anticoagulant: k2EDTA
5. Internal standard: mizolastine-13C-d3
6. Data processing
Chromatographic retention time and chromatographic peak area were collected and processed by Analyst (Version1.6.3) and quantified. And (3) performing linear least square regression calculation according to the ratio of the peak area of the analyte to the peak area of the internal standard to the ratio of the concentration of the analyte to the concentration of the internal standard in the standard curve, and calculating the actually measured concentration of the analyte in the sample according to the obtained regression equation.
7. The pretreatment method before the detection of the sample comprises the following steps:
taking whole blood, adding anticoagulant K2EDTA, centrifuging to obtain blank plasma, refrigerating for sample dilution or preparation, and thawing before use.
50 mul of standard curve plasma sample is added with 50 mul of internal standard working solution before use, 300 mul of methanol is added, vortex is carried out for 3min, centrifugation is carried out, 2450g, 4 ℃,10 min, and sample injection is carried out.
50 mul SST is used, 50 mul internal standard working solution is added, 300 mul methanol is added, vortex is carried out for 3min, centrifugation is carried out, 2450g, 4 ℃,10 min, and sample injection is carried out.
Adding 50 μ L of internal standard working solution before 50 μ L of quality control plasma sample, adding 300 μ L of methanol, vortexing for 3min, centrifuging, 2450g, 4 deg.C, 10min, and waiting for sample injection.
mu.L of methanol was added to 50. mu.L of blank plasma before use, 300. mu.L of methanol was added, vortexed for 3min, centrifuged, 2450g, 4 ℃,10 min, and after injection, recorded as: either a Doubleblank or Carryover sample.
Or 50 μ L blank plasma is used by adding 50 μ L internal standard working solution, adding 300 μ L methanol, vortexing for 3min, centrifuging, 2450g, 4 deg.C, 10min, and recording as: blank sample.
Adding 50 μ L of methanol before 50 μ L of ultrapure water is used, adding 300 μ L of methanol, vortexing for 3min, centrifuging, 2450g, 4 ℃,10 min, and recording as: reagent and materials blank samples.
Remarking: double blank samples for Doubleblank, double blank samples for residual investigation for carryover, single blank samples with blank added only with internal standard, and blank samples for acceptance of Reagent consumables for Reagent and Materials blank.
Example 1
1. Solution preparation
Mobile phase A: 1000mL of ultrapure water (0.1% formic acid aqueous solution) was weighed, 1mL of formic acid was added, and the mixture was mixed and sonicated.
Mobile phase B: (methanol) 1000mL of methanol (Merck) was measured and transferred to an appropriate solvent flask and sonicated.
Diluted solution (50% aqueous methanol): 50mL of methanol and 50mL of ultrapure water were transferred to an appropriate solvent bottle and mixed.
Needle washing solution preparation (50% methanol water): 500mL of methanol and 500mL of ultrapure water were transferred to an appropriate solvent bottle and mixed.
2. Preparing a standard solution:
preparation of Imidamustine Standard control Stock solution (MZST STD Stock,1.000mg/mL)
Weighing a certain amount of mizolastine reference substance, placing the mizolastine reference substance in a self-made cup-shaped aluminum foil paper, recording the weight, placing the aluminum foil paper in a 20mL brown wide-mouth glass bottle, calculating the actual weight of an analyte according to the actual weighing value and the mizolastine content, adding a proper amount of methanol, finally preparing the mizolastine with the concentration of 1.000mg/mL, and shaking up. Storing at-20 deg.C.
Preparation of standard series working solution:
taking a proper amount of standard reference substance stock solution, diluting with methanol to obtain an mizolastine working solution: 100000, 16000, 12800, 4000, 2000, 1000, 400, 200, 100 ng/mL.
Preparation of Standard Curve plasma samples
10 μ L of each of the standard series of working solutions was diluted into blank plasma to a total volume of 0.2mL, and standard curve plasma samples were prepared with mizolastine concentration values of 5, 10, 20, 50, 100, 200, 640, 800 ng/mL.
SST samples: 10 μ L of the standard series working solution was taken and diluted into blank plasma to give a total volume of 0.2mL and an mizolastine concentration value of 5ng/mL in the prepared SST samples.
3. Preparation of internal standard solution
Mizolastine-13C-d3Control stock solution formulation (MZST-13C-d3 IS Stock,10.00mg/mL)
Adding 992 μ L methanol directly, dissolving 10mg reference substance, shaking to obtain 10.00mg/mL reference substance stock solution, and storing at-20 deg.C.
Mizolastine-13C-d3Internal standard working solution preparation (IS Spike,100ng/mL)
Taking MZST-13C-d3IS Stock 100. mu.L to 900. mu.L methanol, and mixing well to obtain 1.000mg/mL IS Spike-1.
Mixing IS Spike-1100 μ L to 900 μ L methanol to obtain 100.0 μ g/mL IS Spike-2.
Transferring IS Spike-250 μ L into a 50mL volumetric flask, adding methanol to a constant volume to a scale, and mixing to obtain IS Spike (100.0 ng/mL).
4. Preparation of quality control standard solution
Preparation of mizolastine quality control reference Stock solution (MZST QC Stock,1.000mg/mL)
Weighing a certain amount of mizolastine reference substance, placing the mizolastine reference substance in a self-made cup-shaped aluminum foil paper, recording the weight, placing the aluminum foil paper in a 20mL brown wide-mouth glass bottle, calculating the actual weight of an analyte according to the actual weighing value and the mizolastine content, adding a proper amount of methanol, finally preparing the mizolastine with the concentration of 1.000mg/mL, and shaking up. Storing at-20 deg.C.
Preparing a quality control working solution:
taking a quality control reference substance stock solution, diluting the stock solution into methanol to prepare the mizolastine quality control working solution with the concentration of 120000, 12000, 3000, 300 and 100ng/mL
Preparing a quality control plasma sample:
taking a quality control working solution, diluting the quality control working solution into blank plasma, and preparing an mizolastine quality control plasma sample solution with the concentration of 6000, 600, 150, 15 and 5 ng/mL; corresponding to AQL, HOQ QC, MOQ QC, LOQ QC, LLOQ.
5. Methodology validation
5.1 Linear Range and lower quantitative limits
10 μ L of each of the standard series of working solutions was diluted into blank plasma to a total volume of 0.2mL, and standard curve plasma samples were prepared with mizolastine concentration values of 5, 10, 20, 50, 100, 200, 640, 800 ng/mL.
Taking 50 μ L of standard curve plasma sample, Blank plasma Double Blank sample, carryover sample; taking 50 mu L of ultrapure water as an RB sample; 50 μ L of IS Spike diluent (methanol) was taken to RB, Double blank, carryover sample wells and 50 μ L of IS Spike was taken to the other corresponding sample wells.
Take 300. mu.L of methanol to the corresponding sample well.
Plate sealed, vortex for 3 min.
Centrifugation at 4 ℃ and 2450g for 10 min.
mu.L of the sample was introduced into a chromatographic system for analysis.
And (4) conclusion: the linear curve chart of the obtained standard curve sample is shown in 1, wherein the standard curve equation of mizolastine in human plasma measured by the LC-MS/MS method is as follows: 0.00695x +0.000268, R is 0.9991; the linear range of mizolastine is 5 ng/mL-800 ng/mL, the average determination is 6 times, the SD of R is 0.0008, and the CV is 0.1%; the slope SD was 0.0008, the CV was 11.1%, and the lowest quantitative lower limit of the plasma concentration of mizolastine was 5ng/mL, which is a good linear relationship in the linear range.
5.2 precision and accuracy
Taking four quality control plasma samples with different concentrations, wherein the concentration of the mizolastine quality control plasma sample liquid is 600 ng/mL, 150 ng/mL, 15 ng/mL or 5 ng/mL; corresponding to HOQ QC, MOQ QC, LOQ QC and LLOQ concentration, and detecting the concentrations. As a review of accuracy and precision within and between batches. Each concentration was prepared in 6 replicates and tested at least three times. The mean values were used to assess batch accuracy and precision. The results are given in the following table:
table 1: LC-MS/MS method for determining precision and accuracy of mizolastine in plasma in batch and between batches
The results show that: the plasma sample precision and accuracy deviation of mizolastine are less than 15%. The measured values for the low, medium and high concentration levels of the standard plasma sample should be within 15% of the indicated values, with an intra-and inter-batch precision (RSD) of less than 15%. The method has good precision and accuracy in detecting the concentration of mizolastine in the plasma.
5.3 System applicability:
taking a pretreated mizolastine quality control plasma sample with the concentration of 5 ng/mL; 1 microliter sample injection detection, and repeated analysis for 5 times;
and recording the chromatographic peak area value and the retention time of the object to be detected and the internal standard, and calculating the chromatographic peak area ratio of the object to be detected and the internal standard and the variation coefficient of the chromatographic peak retention time. The system suitability results for each batch are shown in table 2 below:
table 2: systemic applicability of mizolastine
And (4) conclusion: the signal to noise ratio (S/N is not less than 5) of the substance to be detected, and the variation coefficient of the ratio of the area of the substance to be detected to the area of the internal standard chromatographic peak obtained by 5 times of repeated analysis is less than 5%; the variation coefficient of the retention time of the sample to be tested and the internal standard chromatographic peak obtained by 5 times of repeated analysis is less than 15%. The retention time variation coefficient of the substance to be detected mizolastine is 0.1-0.4%, the retention time variation coefficient of the internal standard is 0.0-0.4%, and the variation coefficient of the area ratio is 0.5-2.6%; the method is applicable to the detection method of the medicine in the blood plasma for determining the concentration of mizolastine.
5.4 Selectivity
Selectivity refers to the ability to distinguish interference in a biological matrix when an analyte is measured by chromatographic methods. Before the method is validated, at least 6 different batches of blank substrates need to be screened for method validation.
Taking 50 mu L of blank plasma from different sources of 6 Chinese population, pretreating to obtain Double blank sample, and then injecting 1 mu L of blank plasma into a chromatographic system for analysis.
Taking 50 mu L of 6 blank plasma from different sources, preparing the concentration of the prepared mizolastine quality control plasma sample solution to be 5ng/mL, and after pretreatment, injecting 1 mu L of the sample solution into a chromatographic system for analysis;
the results of the recording of each blank sample and the quantification of the lower concentration level of the standard plasma sample are given in the following table.
Table 3: comparison table for selective investigation of analytes, internal standards from blank healthy human plasma from six different sources (mizolastine)
Remarking: the interference between the retention time of the analyte at 1.76min + -0.1 min and the retention time of the internal standard at 1.76min + -0.1 min is represented by 0.
And (4) conclusion: the peak area of the chromatographic peak at the retention time of the analyte in at least 6 blank matrix samples from different sources is lower than 20.0% of the peak area of the to-be-measured substance at the corresponding blank matrix LLOQ concentration from different sources, and the peak area of the internal standard at the retention time of the internal standard is lower than 5.0% of the peak area of the internal standard at the corresponding blank matrix LLOQ concentration from different sources. The measured value of the LLOQ concentration prepared by 6 blank matrixes from different sources is in the range of 80.0-120.0% of the theoretical value. The method provided by the invention aims at the interference range of the analytes in the blank matrix samples from different sources to be 0.0-1.8%, and the internal standard interference response value is 0.0-0.1%. . Therefore, blank plasma of different human bodies does not interfere the detection result of mizolastine, the method can be used for detecting mizolastine in different human body plasma, and plasma of different people has no influence on the detection method.
5.5 recovery
5.5.1 extraction recovery of analytes
Analyte recovery sample
Taking quality control plasma sample liquid of mizolastine with the concentration of 15.00, 150.0 and 600.0ng/mL, and preparing 6 parts in parallel;
addition of an internal standard MZST-13C-d3Working solution with the concentration of 100.0ng/mL is pretreated, and 1 mu L of working solution is injected into a chromatographic system for analysis.
Recovery reference sample:
the extraction procedure is identical to the extraction procedure for extracting the recovery samples, but the analyte and internal standard solutions do not participate in the extraction procedure. The concentrations of the extract recovery sample and the recovery reference sample were the same in this assay batch.
5.5.2 extraction recovery of internal standard
The internal standard extraction recovery rate is investigated and performed simultaneously with the analyte, and the operation is consistent
And (4) conclusion: 1) the extraction recovery rate of the imidazole statin to be detected in the high, medium and low concentration ranges is 109.2%, 109.9% and 101.3%, and the corresponding coefficient of variation CV values are 1.8%, 3.2% and 1.7%; the total recovery rate is 106.8%, and the total recovery rate variation CV value is 4.4%.
2) Internal standard MZST-13C-d3The extraction recovery rate of (2) was 90.8%, and the recovery rate CV value was 3.3%.
5.6 matrix Effect
1) Matrix effects refer to the inhibition or enhancement of the ionization of an analyte by a component present in a biological matrix. Taking blank human plasma of 6 different batches, preparing one part for each batch, adding a standard solution and an internal standard solution with the same concentration as the processed low-concentration and high-concentration quality control samples after pretreatment, and analyzing.
The analysis was carried out in 6 replicates of pure solutions corresponding to the concentrations of the low-concentration and high-concentration quality control samples, in the absence of matrix.
2) Preparation of hemolysis matrix: taking 100 mu L of blank whole blood, carrying out ultrasonic destruction on blood cells, taking 20 mu L of blank whole blood, adding 980 mu L of normal blank plasma, and uniformly mixing to obtain 2% of hemolyzed plasma subjected to ultrasonic destruction, wherein severe hemolysis is considered.
Two samples of the analyte at two concentration levels (LOQ QC, HOQ QC) were prepared 6 times using simulated hemolyzed plasma, processed and then injected into a chromatographic system for analysis.
3) The method comprises the steps of using a simulated hyperlipidemia plasma sample for evaluation (preparing a 5.0% hyperlipidemia plasma sample of medium-long chain fat emulsion injection), using the simulated hyperlipidemia plasma sample to prepare 6 parts of low and high concentration level (LOQ QC and HOQ QC) samples of an object to be detected, processing the samples, and injecting the processed samples into a chromatographic system for analysis.
And (4) conclusion: coefficient of variation CV% of matrix factor normalized by internal standard: 1.4-2.1; mean deviation in accuracy of the lysomatrix effect range: 3.3-5.0%; mean accuracy deviation range for the hyperlipidemia matrix effect: 6.9-7.8%; the method of the invention has no obvious matrix effect, and has no obvious hemolysis and hyperlipemia matrix effect.
5.7 stability
And (3) carrying out stability investigation on human plasma quality control samples with low and high concentrations (LOQ QC and HOQ QC) of the object to be detected under the following conditions:
1) repeated freeze thawing at-70 deg.C for 5 times.
2) Standing at room temperature for 24h for stabilization.
3) It is stable at-20 deg.C for 44 days.
4) Whole blood stability: and (3) respectively inspecting the change of the ratio of peak areas of analytes containing low and high concentration levels (LOQ QC and HOQ QC) placed in ice-water bath in whole blood for (0h) and (0.5-2 h), after placing corresponding inspection time points, centrifugally separating the whole blood sample into plasma, adding an internal standard, and processing according to a corresponding plasma sample processing operation method, wherein each concentration level is parallel to 6 parts.
The samples after stability investigation and the samples prepared now are tested simultaneously, and the comparison result of each concentration is as follows:
table 4 stability at room temperature for 24 h-short term stability results:
table 5-20 ℃ conditions 44d stabilization-long term stability results:
table 6-70 ℃ conditions 44d stabilization-long term stability results:
table 7-70 ℃ repeated freeze-thaw 5 times stability results:
table 8 whole blood stability results:
the CV value of the concentration of each drug in the plasma detected by the method of the invention under the above investigation conditions is less than 10%, which indicates that the method of the invention has stronger stability for detecting mizolastine in whole blood and plasma under the above investigation conditions.
5.8 human plasma sample detection
50uL of mizolastine plasma sample was precisely added to a 96-well plate, 50uL of IS Spike was added, 300uL of methanol was added to the 96-well plate, vortex mixed for 3min, centrifuged at 2450g for 10min at 4 ℃, and 1uL was injected into the chromatographic system for LC-MS/MS analysis, the results are shown in FIG. 2.
And (4) conclusion: proved by methodology, the method has the advantages of high sensitivity, strong applicability, good precision and accuracy and good stability, the extraction recovery rate of the medicine is within an acceptable range, no obvious matrix effect is seen, and the method also has the advantages of high speed and high flux.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for determining the concentration of mizolastine in human plasma is characterized by comprising the following steps: extracting mizolastine in blood plasma to obtain an extracting solution; performing qualitative and quantitative determination on the extracting solution by adopting a high performance liquid chromatography-tandem mass spectrum;
the liquid chromatography conditions of the high performance liquid chromatography tandem mass spectrum comprise:
a chromatographic column: waters XSelect HSS C18The specification is 3.5 mu m and 3 multiplied by 50 mm; the mobile phase A is 0.1 percent formic acid aqueous solution; the mobile phase B is methanol; flow rate: 0.6 mL/min; gradient elution is adopted; stopping time: 4.0 min; washing the needle washing liquid: 15 s; sample introduction amount: 1 mu L of the solution; column temperature: 30 ℃; sample injector temperature: 4 ℃ is prepared.
3. the method for determining mizolastine concentration in human plasma according to claim 1, wherein the mass spectrometric conditions of high performance liquid chromatography tandem mass spectrometry comprise:
ESI source is adopted, ionization mode: electrospray ionization positive ion mode (ESI); the scanning mode is as follows: multiple Reaction Monitoring (MRM);
the mass spectrum switching valve is set as follows:
the ion source parameters were set as follows:
。
4. the method for determining mizolastine concentration in human plasma according to claim 1, wherein the extract is prepared by the following steps: taking a whole blood sample to be detected, adding an anticoagulant K2EDTA, centrifuging to obtain a plasma sample to be detected, adding an internal standard working solution, adding an extraction solvent, carrying out vortex centrifugation, and taking supernatant to obtain an extracting solution.
5. The method for determining mizolastine concentration in human plasma according to claim 4, wherein the internal standard working solution is prepared by the following steps: weighing mizolastine-13C-d3Dissolving in methanol in a volumetric flask to prepare 10 ug/mL-1An internal standard control stock solution;
getting mizolastine-d3Placing 50 μ L of internal standard reference substance stock solution in 50mL volumetric flask, adding methanol to constant volume to scale mark, and shaking to obtain solution with concentration of 100ng/mL-1The internal standard working solution of (4).
6. The method for determining mizolastine concentration in human plasma according to claim 4, wherein the conditions of vortex centrifugation are as follows: vortex for 3min, centrifuge 2450g, 4 deg.C, 10 min.
7. The method for determining the concentration of mizolastine in human plasma according to claim 1, wherein the qualitative and quantitative method comprises the following steps: the qualitative method is that the retention time of the drug to be detected is consistent with that of the drug standard, and the drug is determined as the target drug by combining the qualitative ion pair and the quantitative ion pair in the mass spectrum parameter; the quantitative method comprises the following steps: and (3) performing linear least square regression calculation on the theoretical concentration of the analyte in the standard curve by comparing the peak area of the analyte with the peak area of the internal standard, and calculating the actually measured concentration of the analyte in the sample by using the obtained regression equation.
8. The method for determining mizolastine concentration in human plasma according to claim 7, wherein the standard curve is prepared by the following steps: taking an mizolastine standard working solution, diluting the mizolastine standard working solution into blank plasma, and preparing a mizolastine standard curve plasma sample; taking a standard curve plasma sample, adding an internal standard working solution, carrying out vortex centrifugation, taking supernate, determining by adopting high performance liquid chromatography tandem mass spectrometry, recording the peak area corresponding to mizolastine at each concentration, and preparing a standard curve regression equation by taking the peak area ratio of the mizolastine and the internal standard as a vertical coordinate and the concentration of the mizolastine as a horizontal coordinate;
the concentration range of the standard series of mizolastine working solutions is 100-16000 ng/mL;
the concentration range of the mizolastine standard curve plasma sample is 5-800 ng/mL.
9. The method for determining mizolastine concentration in human plasma according to claim 8, wherein the standard curve regression equation is obtained by the following steps: taking 50 μ L of each standard curve plasma sample, adding 50 μ L of internal standard working solution, vortexing for 3min, centrifuging 2450g, and performing centrifugation at 4 deg.C for 10 min; 1 mu L of sample injection LC-MS/MS system to obtain a chromatogram; and calculating the concentration of mizolastine in the plasma according to the ratio of the chromatographic peak area of the mizolastine to the internal standard peak area in the chromatogram, and making a standard curve regression equation of the mizolastine.
10. The method for determining mizolastine concentration in human plasma according to claim 9, wherein the regression equation of the standard curve is as follows:
the regression equation of the measured standard curve of mizolastine in human plasma is that y is 0.00695x +0.000268, and R is 0.9991; the linear range of the mizolastine is 5 ng/mL-800 ng/mL, and the lowest quantitative lower limit of the blood concentration of the mizolastine is 5 ng/mL.
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CN114814018A (en) * | 2022-04-18 | 2022-07-29 | 合肥创新医药技术有限公司 | Method for determining doxylamine in human plasma through LC-MS/MS |
CN114814018B (en) * | 2022-04-18 | 2024-05-24 | 合肥创新医药技术有限公司 | Method for determining doxylamine in human plasma by LC-MS/MS |
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