CN114384170A - Method for determining concentration of amlodipine in blood plasma by liquid chromatography-mass spectrometry - Google Patents
Method for determining concentration of amlodipine in blood plasma by liquid chromatography-mass spectrometry Download PDFInfo
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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
- G01N30/02—Column chromatography
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- G01—MEASURING; TESTING
- 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
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G01N30/02—Column chromatography
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- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8624—Detection of slopes or peaks; baseline correction
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- G01N30/8634—Peak quality criteria
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/02—Column chromatography
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- G01N2030/042—Standards
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Abstract
The invention discloses a method for determining the concentration of amlodipine in blood plasma by liquid chromatography-mass spectrometry, which comprises the steps of taking amlodipine-d 4 as an internal standard, adding a precipitator for protein precipitation, taking supernate, adding a diluent, pretreating, separating by a chromatographic column, and detecting by a mass spectrometer. The invention establishes a method for determining the concentration of amlodipine in human plasma by a high-throughput and high-resolution high performance liquid chromatography-tandem mass spectrometry method; the linear range of the plasma standard curve of the method is 80.000 pg/mL-8000.000 pg/mL, and the precision CV% in batches and among batches is less than +/-15%.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a method for determining the concentration of amlodipine in blood plasma by liquid chromatography-mass spectrometry.
Background
Amlodipine (Amlodipine, AML) is a nifedipine calcium antagonist. The effect of inhibiting calcium-induced aortic contraction is 2 times that of nifedipine. Amlodipine is characterized by slow speed of binding and dissociation with receptors, so that the drug action is delayed and the maintenance time is long. The selective effect on vascular smooth muscle is greater than that of nifedipine. It can increase cardiac output and coronary flow, increase myocardial oxygen supply, reduce oxygen consumption, and improve exercise ability for patients with myocardial ischemia. In addition, the product can activate LDL receptor, reduce accumulation of fat in artery wall and inhibit collagen synthesis, thus having anti-arteriosclerosis effect.
The oral preparation is slowly absorbed by oral administration, the peak reaching time is 6-8 h, the bioavailability is 64%, and the apparent distribution volume is 21L/kg. Most of the metabolites are metabolized in the liver, and the metabolites have no calcium antagonism. The clearance rate is 7 ml/kg.min, and the elimination half-reduction period is 36 h; in the elderly and patients with hypohepatia, the product has slow elimination speed, and half-life of elimination is prolonged to 48h and 60h respectively. Eating does not affect the pharmacokinetics of the product.
The existing analysis method for detecting amlodipine in human plasma is not uniform, and the flux, selectivity and sensitivity of the existing analysis method need to be discussed.
Disclosure of Invention
The invention aims to provide a method for determining the concentration of amlodipine in blood plasma by liquid chromatography-mass spectrometry, which is used for solving the technical problems in the background art.
The technical scheme of the invention provides a method for determining the concentration of amlodipine in blood plasma by liquid chromatography-mass spectrometry, wherein a blood plasma sample is pretreated and then the concentration of the blood plasma sample is detected by high performance liquid chromatography-tandem mass spectrometry, and the method comprises the following steps:
s1 pretreatment of plasma samples: with K2EDTA as anticoagulant, amlodipine-d 4 as internal standard; adding 50 mu L of sample into a well of a 96-well plate, adding 50 mu L of internal standard amlodipine-d 4 working solution with the concentration of 1.000ng/mL, uniformly mixing, and adding 250 mu L of acetonitrile with the volume ratio of 100:0.2 into each sample well: mixing the mixed solution of acetic acid and sealing plate, mixing uniformly for 5min, and centrifuging the sample at 2623g for 5min at 4 ℃; taking 100. mu.L of centrifuged supernatant to another 96-well collection plate, adding 200. mu.L of methanol at a volume ratio of 50:50: 0.5: water: mixing formic acid solution, sealing plate, and mixing; the sample was centrifuged at 2623g for 5min at 4 ℃ untilSample introduction, wherein the processes are all operated under the condition of room temperature yellow light;
s2 sample measurement: and (3) injecting 10 mu L of test sample into a high performance liquid chromatography tandem mass spectrometer, detecting chromatographic peaks of amlodipine and the internal standard amlodipine-d 4 in the sample, and calculating the concentration of amlodipine in the plasma sample according to the chromatographic peaks.
In a preferred embodiment, the liquid chromatography conditions are: a chromatographic column: thermo Scientific Hypersil GOLD 100X 2.1mm 1.9 μm; temperature of the chromatographic column: 40 ℃; mobile phase A: the volume percentage of water/acetic acid is 100/0.05; mobile phase B: pure acetonitrile; needle washing liquid: the volume percentage of water/methanol/formic acid is 20/80/0.1; plunger cleaning solution: 90/10% water/methanol by volume; the temperature of the autosampler is 4 ℃; gradient elution with flow rate of 0.4mL/min, sample size of 10 μ L, and analysis time of 5 min; amlodipine and amlodipine-d 4 the expected retention time is around 2.70 min.
In a preferred embodiment, the mass spectrometry conditions are: the ion source is an electrospray ion source, the temperature of an ion transmission pipe is 325 ℃, and the temperature of steam is 350 ℃. The collision voltage of amlodipine and amlodipine-d 4 is both 10V, and detection is carried out in a positive ion mode; the scanning mode is multiple reaction monitoring. The ion reactions for quantitative analysis were: amlodipine: m/z 409.1 → 238.0 and amlodipine-d 4: m/z 413.3 → 238.0.
In a preferred embodiment, the concentration of amlodipine in the plasma sample is calculated by taking the peak area ratio of amlodipine and the internal standard amlodipine-d 4 into a standard curve equation by using an internal standard method in S2, and the standard curve equation is verified by quality control samples.
In a preferred embodiment, the establishment of the standard curve equation comprises the following steps:
a1, putting 190 mu L of blank plasma into a polypropylene tube, and respectively adding 10 mu L of amlodipine working solutions with the concentrations of 1.600, 3.200, 8.000, 16.000, 32.000, 64.000, 128.000 and 160.000ng/mL in the form of working solution;
a2, after mixing uniformly, respectively taking 50 mu L of standard sample 1, standard sample 2, standard sample 3, standard sample 4, standard sample 5, standard sample 6, standard sample 7, standard sample 8 and zero-concentration sample, adding 50 mu L of 1.000ng/mL internal standard amlodipine-d 4 solution, and adding 50 mu L of methanol aqueous solution with volume fraction of 50% into the double blank sample;
a3 mixing well, adding 250 μ L acetonitrile with volume ratio of 100: 0.2: mixing the mixed solution of acetic acid and sealing plate, mixing uniformly for 5min, and centrifuging the sample at 2623g for 5min at 4 ℃;
a4. mu.L of centrifuged supernatant was transferred to another 96-well collection plate, and 200. mu.L of methanol at a volume ratio of 50:50: 0.5: water: mixing formic acid solution, sealing plate, and mixing; the sample was centrifuged at 2623g for 5min at 4 ℃ for injection. The above processes are all operated under the condition of room temperature yellow light;
and A5, respectively injecting 10 mu L of standard samples into a high performance liquid chromatography tandem mass spectrometer, detecting chromatographic peaks of amlodipine and an internal standard amlodipine-d 4 in the samples, and obtaining a standard curve according to the chromatographic peaks for calculating the concentration of amlodipine in the plasma sample.
In a preferred embodiment, the establishment of the quality control comprises the steps of:
b1, preparing a quality control sample working solution with amlodipine concentrations of 1.600, 4.800, 24.000, 48.000 and 120.000 ng/mL;
b2, putting 380 mu L of blank plasma in a polypropylene tube, respectively adding 20 mu L of quality control working solution, uniformly mixing, respectively taking 50 mu L of quality control sample, and adding 50 mu L of 1.000ng/mL internal standard amlodipine-d 4 solution;
b3 mixing, adding 250 mu L acetonitrile with the volume ratio of 100:0.2 into each sample hole: mixing the mixed solution of acetic acid and sealing plate, mixing uniformly for 5min, and centrifuging the sample at 2623g for 5min at 4 ℃;
b4. mu.L of centrifuged supernatant was taken and put into another 96-well collection plate, 200. mu.L of methanol at a volume ratio of 50:50: 0.5: water: mixing formic acid solution, sealing plate, and mixing; centrifuging the sample at 2623g at 4 deg.C for 5min, and performing the above steps under room temperature and yellow light condition;
and B5, respectively injecting 10 mu L of standard sample into a high performance liquid chromatography tandem mass spectrometer, detecting the chromatographic peaks of amlodipine and the internal standard amlodipine-d 4 in the quality control sample, and calculating the determination concentration of the quality control sample according to the standard curve.
The technical scheme of the invention has the beneficial effects that:
(1) an isotope internal standard is used to ensure the elution consistency of the object to be detected and the internal standard;
(2) the pretreatment method is simple and convenient and is suitable for high-throughput measurement;
(3) the specificity is strong: under the chromatographic conditions used in the experiment, the retention time of amlodipine is about 2.70min, and the retention time of the internal standard amlodipine-d 4 is about 2.70 min. The amlodipine and the internal standard amlodipine-d 4 have good peak shapes, no miscellaneous peak obviously interferes the measurement, and the base line is stable;
(4) the method is rapid, accurate, strong in specificity and low in sensitivity, and provides a basis for measuring the blood concentration of amlodipine; the linear range of the plasma standard curve of the method is 80.000-8000.000 pg/mL.
Drawings
FIG. 1 is an ion scanning mass spectrum of a product of amlodipine in an LC-MS/MS detection method of amlodipine in human plasma;
FIG. 2 is an ion scanning mass spectrum of the product of amlodipine-d 4 in the LC-MS/MS detection method of amlodipine in human plasma;
FIG. 3 is a LC-MS/MS graph of human blank plasma;
FIG. 4 is a LC-MS/MS graph of human blank plasma supplemented with amlodipine and amlodipine-d 4;
FIG. 5 is a standard curve diagram of amlodipine in human plasma measured by LC-MS/MS method.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments. The embodiments of the present invention have been presented for purposes of illustration and description, and are not intended to be exhaustive or limited to the invention in the form disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiment was chosen and described in order to best explain the principles of the invention and the practical application, and to enable others of ordinary skill in the art to understand the invention for various embodiments with various modifications as are suited to the particular use contemplated.
The technical scheme of the invention provides a method for determining the concentration of amlodipine in blood plasma by liquid chromatography-mass spectrometry, which comprises the following specific determination processes:
first, experimental material and analytical equipment
Amlodipine calcium (analyte): chinese food and drug testing research institute or standard substance with same or higher grade
Amlodipine-d 4 sodium salt (internal standard): the reagents used by Toronto Research Chemicals or equivalent, higher-grade standards are shown in Table 1 below:
TABLE 1 details of reagents
Note: the same or higher levels of reagents may be used.
The analytical equipment used is shown in table 2 below:
TABLE 2 details of the devices used
Second, liquid phase chromatographic conditions
1. Condition of liquid-phase color spectrum
A chromatographic column: thermo Scientific Hypersil GOLD 100X 2.1mm 1.9 μm; temperature of the chromatographic column: 40 ℃; mobile phase A: the volume percentage of water/acetic acid is 100/0.05; mobile phase B: pure acetonitrile; needle washing liquid: the volume percentage of water/methanol/formic acid is 20/80/0.1; plunger cleaning solution: 90/10% water/methanol by volume; the temperature of the autosampler is 4 ℃; gradient elution with flow rate of 0.4mL/min, sample size of 10 μ L, and analysis time of 5 min; amlodipine and amlodipine-d 4 the expected retention time is around 2.70 min. Specific gradient elution procedures are shown in table 3;
TABLE 3 gradient elution procedure
2. Mass spectrum conditions:
the ion source is an electrospray ion source, the temperature of an ion transmission pipe is 325 ℃, and the temperature of steam is 350 ℃. The collision voltage of amlodipine and amlodipine-d 4 is both 10V, and detection is carried out in a positive ion mode; the scanning mode is multiple reaction monitoring. The ion reactions for quantitative analysis were: amlodipine: m/z 409.1 → 238.0 and amlodipine-d 4: m/z 413.3 → 238.0.
Third, the experimental process
1. Preparation of amlodipine standard curve working solution
Preparing an amlodipine standard curve working solution: accurately weighing a proper amount of amlodipine standard substance, and dissolving the amlodipine standard substance in methanol after correcting the quality correction coefficient to obtain a stock solution with the final concentration of 0.500 mg/mL. Then, the amlodipine working solution is prepared by sequentially diluting with 50% methanol, and the specific dilution concentration is shown in the following table 4:
table 4 amlodipine working solution formulation concentration
SS denotes stock solution, WS denotes working solution. The amlodipine standard curve working solution is prepared in a brown glass bottle and stored at the temperature of-20 ℃, and the volume can be increased or reduced according to the proportion as required.
2. Preparation of amlodipine quality control working solution
Preparing an amlodipine working solution: accurately weighing a proper amount of amlodipine standard, after correcting the quality correction coefficient, dissolving the amlodipine standard in methanol to obtain stock solution with the final concentration of 0.500mg/mL, and sequentially diluting with 50% methanol to prepare amlodipine working solution, wherein the specific dilution concentration is shown in the following table 5:
TABLE 5 amlodipine quality control working solution formulation concentration
SS denotes stock solution, WS denotes working solution. The amlodipine working solution is prepared in a brown glass bottle and stored at the temperature of-20 ℃, and the volume can be increased or reduced according to the proportion as required.
3. Preparation of amlodipine-d 4 internal standard working solution
Preparing an amlodipine-d 4 internal standard working solution: taking one amlodipine-d 4 standard substance, correcting by a mass correction coefficient, and dissolving the amlodipine-d 4 standard substance in 80% methanol to obtain a stock solution with the final concentration of 0.500 mg/mL. Then methanol aqueous solution with volume fraction of 50% is used for dissolving and diluting to prepare 20.000ng/mL internal standard amlodipine-d 4 working solution, and the specific dilution concentration is shown in the following table 6:
TABLE 6 working solution formulation concentration of amlodipine-d 4
The amlodipine-d 4 internal standard working solution is prepared in a brown glass bottle and stored at the temperature of-20 ℃, and the volume can be increased or decreased according to the proportion as required.
4. Linear experiment
Unfreezing blank plasma at room temperature; transferring 8 parts of 190 mu L blank plasma into a polypropylene tube (each standard curve sample), respectively and precisely adding 10 mu L of amlodipine working solution with different concentrations to prepare each sample, uniformly mixing to prepare drug-containing plasma with different concentrations, and carrying out pretreatment on the plasma samples. Calculating the ratio Y (Y is As/Ai) of the amlodipine peak area As and the peak area Ai of the internal standard amlodipine-d 4, and performing regression calculation on the blood concentration X by using the peak area ratio Y. The average ratio Y is used for carrying out regression calculation on the blood concentration X, and the lowest quantitative limit of the amlodipine blood concentration measured by the method is as follows: 80.000 pg/mL. The standard curve parameters of amlodipine in human plasma measured by LC-MS/MS method are shown in Table 7:
TABLE 7 Standard Curve (pg/mL) of amlodipine in human plasma by LC-MS/MS method
5. Accuracy and precision
Unfreezing blank plasma at room temperature; transferring blank plasma with proper volume into a polypropylene tube, adding amlodipine quality control working solution to prepare 5 drug-containing plasma quality control samples (LLOQ, LQC, M1QC, M2QC and HQC) with different concentrations and a following standard curve, and performing the operation according to 'plasma sample pretreatment'. And (3) in at least two days, completing in at least three analysis batches, calculating the ratio Y of the amlodipine peak area As and the internal standard amlodipine-d 4 peak area Ai, substituting the ratio Y into a standard curve on the same day to obtain the measured concentration, calculating the precision between batches according to the measured concentration, and determining the ratio of the measured concentration to the added concentration As the accuracy, wherein the result is shown in Table 8. The result shows that the precision and accuracy of the amlodipine blood plasma sample in batches and among batches are less than +/-15 percent and meet the requirements.
As required for each assay batch, a sufficient volume was selected for dispensing into labeled polypropylene tubes and stored at-70 ℃. The volume may be scaled up or down as desired.
TABLE 8 LC-MS/MS method for determining the accuracy and precision of amlodipine in plasma
6. Matrix effect
The six different blank plasma samples are respectively from different healthy human bodies, and are prepared and analyzed in the same analysis batch according to the sample preparation steps to evaluate the interference of the different blank plasma on the amlodipine analyte and the internal standard amlodipine-d 4. Wherein the CV percent of the internal standard normalized matrix factor is less than or equal to 5.3 percent.
TABLE 9 matrix Effect of blank healthy human plasma from six different sources
Note: the area peak area is considered zero when "no significant peak can be integrated (or no peak)" or "the retention time of the peak area does not match the retention time of the analyte or internal standard in the sample".
As can be seen from table 9, blank plasma of different human bodies did not interfere with the detection result of amlodipine. Therefore, the method can be used for detecting the concentration of amlodipine in the plasma of different human bodies.
7. Dilution reliability
The sample preparation method for inspecting the reliability of 2 times of sample dilution comprises the following steps: taking an amlodipine stock solution with the concentration of 0.500mg/mL, and taking 50% methanol as a diluent to prepare a working solution with the amlodipine concentration of 240.000 ng/mL.
Unfreezing blank plasma at room temperature, transferring 380 mu L of blank plasma to a polypropylene tube, adding 20 mu L of the working solution to prepare a drug-containing plasma dilution quality control sample (the concentration is 12000.000pg/mL), vortexing and mixing uniformly to prepare a sample higher than the upper limit of quantitation, and then taking 50 mu L of the sample to a new polypropylene tube, adding 50 mu L of the blank plasma to dilute 2 times to obtain a diluted 2-fold sample (the concentration is 6000.000 pg/mL). Then, the operation of "pretreatment of plasma sample" was performed in parallel for 6 times. The results are shown in Table 10. The accuracy deviation is-0.7% -3.8%, which shows that the dilution reliability meets the acceptance standard.
TABLE 10 dilution reliability examination results
8. Recovery rate
The extraction recovery rate is determined by comparing the analyte/internal standard response in the treated spiked sample with the response value of the spiked analyte/internal standard in the treated blank matrix. Samples of LQC, M2QC, HQC3 concentration levels were prepared, 6 replicates per concentration level. Standards were accepted for the analyte of each concentration level of sample and internal standards for all concentration levels: the peak area of the normal extracted sample, the peak area of the sample after the substrate is extracted, and the CV percent of the recovery rate of the sample at each concentration level are 1.3 to 2.8 percent. Amlodipine and amlodipine-d 4 both meet the acceptance criteria and the results are shown in tables 11 and 12.
TABLE 11 recovery rate of extraction of analyte
Table 12 internal standard recovery
9. Stability of
(1) Freeze thaw stability
The preparation method of the sample is the same as that of the quality control sample, the sample is respectively placed in a refrigerator at minus 70 ℃ or a refrigerator at minus 20 ℃ for storage after the preparation is finished, then the sample is taken out and placed for melting under the condition of room temperature, each sample is frozen and thawed for 5 times, each group is parallel to 6 parts, then 10 mu L of test sample is taken and injected into the high performance liquid chromatography tandem mass spectrometer according to the operation of 'plasma sample pretreatment', and the result is shown in tables 13 and 14. The deviation of the measured concentration and the standard value of the high/low concentration sample is in the range of-11.3% -3.3% under the condition of 5 times of freezing and thawing at-70 ℃, and the deviation of the measured concentration and the standard value of the high/low concentration sample is in the range of-6.6% -4.9% under the condition of 5 times of freezing and thawing at-20 ℃, which indicates that the sample is stable after 5 times of freezing and thawing treatment in a refrigerator at-70 ℃ or-20 ℃.
TABLE 13-70 ℃ stability test results of 5 times of freezing and thawing at room temperature
TABLE 14-20 ℃ stability test results of 5 times of freezing and thawing at room temperature
(2) Long term stability
The preparation method of the sample is the same as that of the quality control sample, the sample is placed in a refrigerator with the temperature of-70 ℃ for storage for 52 days after the preparation is finished, then the sample is taken out and placed at room temperature for melting, 6 parts of each group are parallel, then 10 mu L of the test sample is taken and injected into the high performance liquid chromatography tandem mass spectrometer according to the operation of 'pretreatment of plasma samples', and the result is shown in Table 15.
After the high/low concentration samples were stored at-70 ℃ for 52 days, the deviation in accuracy between the measured concentration and the indicated value was in the range of-3.6% to 6.0%, indicating that the samples were stable when stored in a refrigerator at-70 ℃ for 52 days.
TABLE 15-70 ℃ long-term storage stability test results
10. Human plasma sample detection
Samples thawed at room temperature or newly formulated samples were vortexed and mixed. Adding 50 mu L of sample (standard curve, quality control sample, system applicability sample or biological sample to be detected, and adding 50 mu L of blank matrix sample for double blank sample or zero sample) into the hole of a 96-well plate; for the double blank sample or ULOQ Without IS sample, 50. mu.L of 50% methanol solution was added, for the other samples 50. mu.L of internal standard working solution (1.000ng/mL) was added, and after mixing, 250. mu.L of acetonitrile at a volume ratio of 100:0.2 was added to each sample well: mixing the mixed solution of acetic acid and sealing plate, mixing uniformly for 5min, and centrifuging the sample at 2623g for 5min at 4 ℃; take 100. mu.L of centrifuged supernatant to another 96-well collection plate, add 200. mu.L of methanol at a volume ratio of 50: 0.5: water: mixing formic acid solution, sealing plate, and mixing; the sample was centrifuged at 2623g for 5min at 4 ℃ for injection. The above processes are all carried out under room temperature yellow light.
FIG. 1 is an ion scanning mass spectrum of a product of amlodipine in an LC-MS/MS detection method of amlodipine in human plasma; FIG. 2 is an ion scanning mass spectrum of the product of amlodipine-d 4 in the LC-MS/MS detection method of amlodipine in human plasma; FIG. 3 is a LC-MS/MS graph of human blank plasma; FIG. 4 is a LC-MS/MS graph of human blank plasma supplemented with amlodipine and amlodipine-d 4; FIG. 5 is a standard curve diagram of amlodipine in human plasma measured by LC-MS/MS method.
The method for determining the concentration of amlodipine in blood plasma provided by the invention adopts simple protein precipitation treatment, and is suitable for conventional determination; from the above analysis, under the chromatographic conditions adopted in the experiment, the retention time of amlodipine is about 2.70min, the retention time of the internal standard amlodipine-d 4 is about 2.70min, the peaks of amlodipine and the internal standard amlodipine-d 4 are good, no obvious peak interference is generated in the determination, and the baseline is stable. The linear range of the plasma standard curve of the method is 80.000 pg/mL-8000.000 pg/mL, and the precision CV% in batches and among batches are both less than +/-15%; the method has high sensitivity, specificity and stability, is convenient, controllable and can accurately measure the concentration of the amlodipine in the blood plasma; meanwhile, the method is accurate and has good reproducibility, and a basis is provided for the blood concentration determination of amlodipine.
It is to be understood that the described embodiments are merely a few embodiments of the invention, and not all embodiments. All other embodiments, which can be derived by one of ordinary skill in the art and related arts based on the embodiments of the present invention without any creative effort, shall fall within the protection scope of the present invention. Structures, devices, and methods of operation not specifically described or illustrated herein are generally practiced in the art without specific recitation or limitation.
Claims (6)
1. The method for determining the concentration of amlodipine in blood plasma by liquid chromatography-mass spectrometry is characterized in that a blood plasma sample is pretreated and then the concentration of the blood plasma sample is detected by high performance liquid chromatography-tandem mass spectrometry, and comprises the following steps:
s1 pretreatment of plasma samples: with K2EDTA as anticoagulant, amlodipine-d 4 as internal standard; adding 50 mu L of sample into a well of a 96-well plate, adding 50 mu L of internal standard amlodipine-d 4 working solution with the concentration of 1.000ng/mL, uniformly mixing, and adding 250 mu L of acetonitrile with the volume ratio of 100:0.2 into each sample well: mixing the mixed solution of acetic acid and sealing plate, mixing uniformly for 5min, and centrifuging the sample at 2623g for 5min at 4 ℃; taking 100. mu.L of centrifuged supernatant to another 96-well collection plate, adding 200. mu.L of supernatantMethanol in a ratio of 50:50: 0.5: water: mixing formic acid solution, sealing plate, and mixing; centrifuging the sample at 2623g at 4 deg.C for 5min, and performing the above steps under room temperature and yellow light condition;
s2 sample measurement: and (3) injecting 10 mu L of test sample into a high performance liquid chromatography tandem mass spectrometer, detecting chromatographic peaks of amlodipine and the internal standard amlodipine-d 4 in the sample, and calculating the concentration of amlodipine in the plasma sample according to the chromatographic peaks.
2. The method for determining amlodipine concentration in blood plasma according to claim 1, wherein the liquid chromatography conditions are: a chromatographic column: thermo Scientific Hypersil GOLD 100X 2.1mm 1.9 μm; temperature of the chromatographic column: 40 ℃; mobile phase A: the volume percentage of water/acetic acid is 100/0.05; mobile phase B: pure acetonitrile; needle washing liquid: the volume percentage of water/methanol/formic acid is 20/80/0.1; plunger cleaning solution: 90/10% water/methanol by volume; the temperature of the autosampler is 4 ℃; gradient elution with flow rate of 0.4mL/min, sample size of 10 μ L, and analysis time of 5 min; amlodipine and amlodipine-d 4 the expected retention time is around 2.70 min.
3. The method for determining amlodipine concentration in plasma by LC-MS according to claim 1, wherein the mass spectrometric conditions are: the ion source is an electrospray ion source, the temperature of an ion transmission pipe is 325 ℃, and the temperature of steam is 350 ℃. The collision voltage of amlodipine and amlodipine-d 4 is both 10V, and detection is carried out in a positive ion mode; the scanning mode is multiple reaction monitoring. The ion reactions for quantitative analysis were: amlodipine: m/z 409.1 → 238.0 and amlodipine-d 4: m/z 413.3 → 238.0.
4. The method for determining concentration of amlodipine in blood plasma according to claim 1, wherein an internal standard method is adopted in S2, and the concentration of amlodipine in the blood plasma sample is calculated by substituting the peak area ratio of amlodipine and an internal standard amlodipine-d 4 into a standard curve equation, wherein the standard curve equation is verified by quality control samples.
5. The method for determining amlodipine concentration in plasma according to claim 4, wherein said standard curve equation is established by the following steps:
a1, putting 190 mu L of blank plasma into a polypropylene tube, and respectively adding 10 mu L of amlodipine working solutions with the concentrations of 1.600, 3.200, 8.000, 16.000, 32.000, 64.000, 128.000 and 160.000ng/mL in the form of working solution;
a2, after mixing uniformly, respectively taking 50 mu L of standard sample 1, standard sample 2, standard sample 3, standard sample 4, standard sample 5, standard sample 6, standard sample 7, standard sample 8 and zero-concentration sample, adding 50 mu L of 1.000ng/mL internal standard amlodipine-d 4 solution, and adding 50 mu L of methanol aqueous solution with volume fraction of 50% into the double blank sample;
a3 mixing well, adding 250 μ L acetonitrile with volume ratio of 100: 0.2: mixing the mixed solution of acetic acid and sealing plate, mixing uniformly for 5min, and centrifuging the sample at 2623g for 5min at 4 ℃;
a4. mu.L of centrifuged supernatant was transferred to another 96-well collection plate, and 200. mu.L of methanol at a volume ratio of 50:50: 0.5: water: mixing formic acid solution, sealing plate, and mixing; the sample was centrifuged at 2623g for 5min at 4 ℃ for injection. The above processes are all operated under the condition of room temperature yellow light;
and A5, respectively injecting 10 mu L of standard samples into a high performance liquid chromatography tandem mass spectrometer, detecting chromatographic peaks of amlodipine and an internal standard amlodipine-d 4 in the samples, and obtaining a standard curve according to the chromatographic peaks for calculating the concentration of amlodipine in the plasma sample.
6. The method for determining amlodipine concentration in blood plasma according to claim 4, wherein said quality control is established by the steps of:
b1, preparing a quality control sample working solution with amlodipine concentrations of 1.600, 4.800, 24.000, 48.000 and 120.000 ng/mL;
b2, putting 380 mu L of blank plasma in a polypropylene tube, respectively adding 20 mu L of quality control working solution, uniformly mixing, respectively taking 50 mu L of quality control sample, and adding 50 mu L of 1.000ng/mL internal standard amlodipine-d 4 solution;
b3 mixing, adding 250 mu L acetonitrile with the volume ratio of 100:0.2 into each sample hole: mixing the mixed solution of acetic acid and sealing plate, mixing uniformly for 5min, and centrifuging the sample at 2623g for 5min at 4 ℃;
b4. mu.L of centrifuged supernatant was taken and put into another 96-well collection plate, 200. mu.L of methanol at a volume ratio of 50:50: 0.5: water: mixing formic acid solution, sealing plate, and mixing; centrifuging the sample at 2623g at 4 deg.C for 5min, and performing the above steps under room temperature and yellow light condition;
and B5, respectively injecting 10 mu L of standard sample into a high performance liquid chromatography tandem mass spectrometer, detecting the chromatographic peaks of amlodipine and the internal standard amlodipine-d 4 in the quality control sample, and calculating the determination concentration of the quality control sample according to the standard curve.
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