CN112834657A - 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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Abstract
The invention discloses a method for determining the concentration of amlodipine in blood plasma by liquid chromatography-mass spectrometry, which adopts a liquid chromatography-mass spectrometry system to determine, wherein a sample to be detected is firstly taken, a certain amount of mixed organic solvent is added for extraction, and after pretreatment, the sample is separated by a chromatographic column and is detected by a mass spectrometer. The method is rapid, accurate, high in sensitivity and simple and convenient to operate, and provides a basis for measuring the blood concentration of amlodipine; the linear range of the plasma standard curve of the method is 0.1-20 ng/mL, the precision RSD in batch and between batches is less than +/-15%, and the method is suitable for measuring the concentration of amlodipine in plasma.
Description
Technical Field
The invention belongs to the technical field of medicines, particularly relates to a method for determining a medicine, and particularly relates to a method for determining the concentration of amlodipine in blood plasma by liquid chromatography-mass spectrometry.
Background
Amlodipine, a 1, 4-dihydropyridine calcium antagonist, inhibits calcium ion influx through the "slow" channels of cardiac and vascular smooth muscle cells, and has become the main therapeutic approach for hypertension and ischemic heart diseases caused by high ionization. The plasma concentration peaks within 6-12 hours after a single oral therapeutic dose of amlodipine. The absolute bioavailability is 64-90%. The bioavailability of amlodipine is not affected by food. The distribution volume was about 21L/kg. In vitro studies of amlodipine have shown that about 93% of circulating drugs bind to plasma proteins in hypertensive patients. Amlodipine is widely (about 90%) metabolized in the liver to inactive substances. 10% of amlodipine prototypes and 60% of amlodipine metabolites are excreted via the urine. Amlodipine is eliminated from plasma in a biphasic manner with a terminal elimination half-life of about 30-50 hours and a steady state plasma concentration is reached after 7-8 days of continuous administration.
Some research evidence suggests that age changes, severe liver function impairment and severe renal function impairment may affect the pharmacokinetic properties of amlodipine, i.e. leading to higher plasma concentrations and longer half-lives. There is currently no evidence of pharmacokinetic interactions of amlodipine with other drugs. Amlodipine exhibits a dose-dependent linear pharmacokinetic profile. In the steady state, the plasma concentration fluctuates relatively little within 1 dosing interval.
At present, the speed, the precision, the sensitivity and the selectivity of the existing amlodipine measuring method need to be improved.
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 can improve the sensitivity, precision, selectivity and speed of detection.
In order to achieve the aim, 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:
(1) plasma sample pretreatment:
plasma with K2EDTA as anticoagulant, amlodipine-d 4 as internal standard; precisely adding 100 μ L of plasma sample into a 96-deep-well plate, adding 5 μ L of plasma sample at a volume ratio of 1: 1, adding 5 mu L of 0.04 ng/mu L of internal standard amlodipine-d 4 solution after uniformly mixing, adding 350 mu L of acetonitrile into a 96 deep-well plate after uniformly mixing, carrying out vortex mixing for 1min, centrifuging for 10min at 20 ℃ at 3000rpm, taking 200 mu L of supernatant into a 96 deep-well plate filled with 200 mu L of mixed organic solvent, wherein the mixed organic solvent is acetonitrile: water: formic acid: 1M ammonium acetate in a volume ratio of 70: 30: 0.5: 0.1, mixing the obtained mixture, uniformly mixing the mixture by vortex, centrifuging the mixture at the temperature of 20 ℃ and 3000rpm for 5min, and taking the mixture as a test sample to be detected;
(2) and (3) 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.
The liquid chromatography determination conditions were: the chromatographic column is Restek Allure PFPP, and the specification of the 5 mu m column is 50 multiplied by 2.1 mm; the temperature of the chromatographic column is 40 ℃; the mobile phase A is water: formic acid: 1M ammonium acetate in a volume ratio of 100: 0.5: 0.1 mixing the obtained mixture; the mobile phase B is acetonitrile: formic acid is mixed according to the volume ratio of 100: 0.5 mixing the obtained mixture; the washing liquid is methanol: water is mixed according to the volume ratio of 50: 50 mixing the obtained mixture; the autosampler temperature was 15 ℃; gradient elution with flow rate of 0.7mL/min, sample size of 10 μ L, and analysis time of 2.5 min;
the mass spectrometry conditions are as follows: the ion source is an electrospray ion source, the spraying voltage is 5500V, the atomizing temperature is 550 ℃, the spraying air pressure is 80Psi, the auxiliary heating air pressure is 5Psi, the air curtain air pressure is 20Psi, the collision air pressure is 8Psi, and the declustering voltages of amlodipine and the internal standard amlodipine-d 4 are both 30 eV; the entrance voltages of the collision chambers of the amlodipine and the internal standard amlodipine-d 4 are both 15 eV; the collision voltage of amlodipine and the internal standard amlodipine-d 4 is 18 eV; the outlet voltages of the collision chambers of the amlodipine and the internal standard amlodipine-d 4 are both 10 eV; detecting in a positive ion mode; the scanning mode is multiple reaction monitoring; the ion reactions for quantitative analysis were: m/z 408.8 → m/z 238.0, which is amlodipine; and m/z 412.9 → m/z 238.1, which is amlodipine-d 4.
Preferably, the gradient elution in step (2) is performed by the following procedure:
preferably, in the step (2), 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.
Preferably, the establishment of the standard curve equation comprises the following steps:
placing ten 100-microliter blank blood plasmas into a 96-deep-well plate, and sequentially naming the blank blood plasmas as a lowest quantitative lower limit sample, a standard sample 1, a standard sample 2, a standard sample 3, a standard sample 4, a standard sample 5, a standard sample 6, a highest quantitative upper limit sample, a zero-concentration sample and the blank sample to be ten samples in total, wherein the zero-concentration sample contains an internal standard amlodipine-d 4 solution and does not contain an amlodipine solution and is used for eliminating the interference of the internal standard amlodipine-d 4 solution on a detection result; the blank sample does not contain an amlodipine solution and an internal standard amlodipine-d 4 solution, and is used for eliminating the interference of the used blank plasma on the detection result;
adding 5 mul of amlodipine solution with the concentration of 0.002 ng/mul, 0.004 ng/mul, 0.01 ng/mul, 0.02 ng/mul, 0.04 ng/mul, 0.1 ng/mul, 0.24 ng/mul and 0.4 ng/mul to the lowest quantitative limit sample, the standard samples 1-6 and the highest quantitative limit sample in the form of stock solution, respectively adding 5 mul of amlodipine solution with the volume ratio of 1: 1, respectively mixing the ten samples, respectively adding 5 mu L of 0.04 ng/mu L of an internal standard amlodipine-d 4 solution into nine samples except for a blank sample, and adding 5 mu L of an internal standard amlodipine-d 4 solution into the blank sample according to the volume ratio of 1: 1, respectively and uniformly mixing the ten samples, respectively adding 350 mu L of acetonitrile into the ten samples, carrying out vortex mixing for 1min, centrifuging at 20 ℃ for 10min at 3000rpm, taking 200 mu L of supernatant of the supernatant into a 96 deep-hole plate filled with 200 mu L of mixed organic solvent, wherein the mixed organic solvent is acetonitrile: water: formic acid: 1M ammonium acetate in a volume ratio of 70: 30: 0.5: 0.1, mixing the obtained mixture, uniformly mixing the mixture by vortex, centrifuging the mixture at the temperature of 20 ℃ at 3000rpm for 5min, and taking the mixture as 10 parts of standard sample to be detected;
and respectively injecting 10 mu L of standard samples into a high performance liquid chromatography-tandem mass spectrometer, detecting chromatographic peaks of amlodipine and the internal standard amlodipine-d 4 in the samples, and obtaining a standard curve according to the chromatographic peaks so as to calculate the concentration of amlodipine in the plasma.
Further, the liquid chromatography determination conditions in the step (3) further include: the volume of the syringe washing needle of the automatic sample injector is 500 mu L; the depth of a sample injection needle of the automatic sample injector is 45 mm; the cleaning speed of the automatic sample injector is 35 mu L/s; the sample injection speed of the automatic sample injector is 5 mu L/s; the soaking time is 5s when the sample injection needle of the automatic sample injector is cleaned; the automatic sample injector cleaning mode is before sample injection and after sample injection.
Compared with the prior art, the invention has the following advantages:
(1) the pretreatment method is simple and convenient, and is suitable for conventional determination;
(2) the specificity is strong: under the chromatographic conditions adopted in the experiment, the retention time of amlodipine is about 0.837min, the retention time of the internal standard amlodipine-d 4 is about 0.833min, the peaks of amlodipine and the internal standard amlodipine-d 4 are good, the measurement is free of the interference of miscellaneous peaks, and the base line is stable;
(3) the sensitivity is high: the minimum limit of quantitation of the plasma is 0.1ng/mL, the concentration of amlodipine in the plasma can be accurately determined, the sensitivity is high, and the specificity is strong;
(4) the method is rapid, accurate, high in sensitivity and simple and convenient to operate, and provides a basis for measuring the blood concentration of amlodipine. The linear range of the plasma standard curve of the method is 0.1-20 ng/mL, and the precision RSD in batch and between batches is less than +/-15%.
Drawings
FIG. 1 is a standard graph of amlodipine in human plasma as measured by HPLC-MS/MS;
FIG. 2 is a HPLC-MS/MS graph of human blank plasma;
FIG. 3 is a HPLC-MS/MS graph of human blank plasma added with amlodipine-d 4;
FIG. 4 is a HPLC-MS/MS graph of human blank plasma with amlodipine and amlodipine-d 4 added;
FIG. 5 is a HPLC-MS/MS graph of plasma samples with the addition of an internal standard amlodipine-d 4 after oral administration of amlodipine drug to healthy subjects.
Detailed Description
The present invention will be described in further detail with reference to examples.
Example (b): human K2Ammonia in EDTA plasmaDetermination of the concentration of amlodipine
First, experimental material and analytical equipment
Amlodipine (analyte) Toronto Research Chemicals or the same, higher-grade standard amlodipine-d 4 (internal standard) Toronto Research Chemicals or the same, higher-grade standard use reagents as given in Table 1 below:
TABLE 1 details of reagents
Note: the same or higher level of reagents may also be used
The analytical equipment used is shown in table 2 below:
TABLE 2 details of the devices used
| Assembly | Model number | Manufacturer(s) |
| Binary pump (Binary pump) | AC Pump | AB SCIEX |
| Degasser (deaerator) | Degasser | AB SCIEX |
| Column oven (constant temperature Column box) | AC Column oven | AB SCIEX |
| Autosampler (automatic sampler) | AC Autosampler | AB SCIEX |
| Sample rack | Rack Changer | AB SCIEX |
| Mass spectrometer | TRIPLE QUADTM6500+ | AB SCIEX |
| Data processor | Analyst 1.6.3(software) | AB SCIEX |
The same LC/MS system may also be used.
Second, liquid condition
1. Conditions of liquid chromatography
The chromatographic column is Restek Allure PFPP, 5 μm, and the specification of the column is 50X 2.1 mm; the temperature of the chromatographic column is 40 ℃; the mobile phase A is water: formic acid: 1M ammonium acetate in a volume ratio of 100: 0.5: 0.1 mixing the obtained mixture; the mobile phase B is acetonitrile: formic acid is mixed according to the volume ratio of 100: 0.5 mixing the obtained mixture; the washing liquid is methanol: water is mixed according to the volume ratio of 50: 50 mixing the obtained mixture; the autosampler temperature was 15 ℃; gradient elution, flow rate of 0.7mL/min, sample size of 10 μ L, analysis time of 2.5 min.
The volume of the syringe washing needle of the automatic sample injector is 500 mu L; the depth of a sample injection needle of the automatic sample injector is 45 mm; the cleaning speed of the automatic sample injector is 35 mu L/s; the sample injection speed of the automatic sample injector is 5 mu L/s; the soaking time is 5s when the sample injection needle of the automatic sample injector is cleaned; the automatic sample injector cleaning mode is before sample injection and after sample injection.
TABLE 3 gradient elution procedure
2. Conditions of Mass Spectrometry
The ion source is an electrospray ion source, the spraying voltage is 5500V, the atomizing temperature is 550 ℃, the spraying air pressure is 80Psi, the auxiliary heating air pressure is 5Psi, the air curtain air pressure is 20Psi, the collision air pressure is 8Psi, and the declustering voltages of amlodipine and the internal standard amlodipine-d 4 are both 30 eV; the entrance voltages of the collision chambers of the amlodipine and the internal standard amlodipine-d 4 are both 15 eV; the collision voltage of amlodipine and the internal standard amlodipine-d 4 is 18 eV; the outlet voltages of the collision chambers of the amlodipine and the internal standard amlodipine-d 4 are both 10 eV; detecting in a positive ion mode; the scanning mode is multiple reaction monitoring; the ion reactions for quantitative analysis were: m/z 408.8 → m/z 238.0, which is amlodipine; and m/z 412.9 → m/z 238.1, which is amlodipine-d 4.
Third, the experimental process
1. Preparation of amlodipine standard solution
The standard solution (containing stock solution and working solution) for the amlodipine standard curve is weighed and prepared by the following steps:
| weighing weight (mg) | Dissolved volume (μ l) | Final concentration (ng/. mu.l) |
| 1.030 | 10098 | 1000 |
Preparing 100 ng/muL amlodipine stock solution according to the preparation process, and then mixing the amlodipine stock solution with the volume ratio of 1: 1, sequentially diluting a methanol aqueous solution to prepare an amlodipine standard solution, wherein the specific dilution concentration is shown in the following table 4:
table 4 amlodipine standard solution preparation concentration
a: prepared directly from amlodipine (analyte)
The amlodipine standard solution is stored in a plastic container and a refrigerator (4 ℃) when not used, and the volume can be increased or reduced according to the proportion as required.
2. Preparation of amlodipine-d 4 internal standard solution
The weighing and preparation process of the standard solution of the amlodipine-d 4 internal standard is as follows:
| weighing weight (mg) | Dissolved volume (μ l) | Final concentration (ng/. mu.l) |
| 1.045 | 10067 | 100 |
Preparing 100 ng/mu L of amlodipine-d 4 stock solution according to the preparation process, and mixing the amlodipine-d 4 stock solution according to the volume ratio of 1: 1, diluting the methanol aqueous solution to prepare an amlodipine-d 4 internal standard solution with the concentration of 0.04 ng/. mu.L, wherein the specific dilution concentration is shown in the following table 5:
TABLE 5 preparation of standard solution of amlodipine-d 4
a: prepared directly from amlodipine-d 4 (internal standard)
b: for sample preparation procedures
The standard solution of amlodipine-d 4 internal standard is stored in plastic container and refrigerator (4 deg.C) when not in use, and the volume can be increased or decreased according to the proportion as required.
3. Linear test
Putting the blank plasma into a water bath at room temperature for unfreezing; transferring 10 parts of 100 mu L of blank plasma into a 96-deep-well plate (each standard curve sample, blank sample-00 and zero concentration sample-0), respectively and precisely adding 5 mu L of amlodipine standard solution or diluted solution with different concentrations according to the list in the following table 6 to prepare each sample, mixing uniformly to prepare drug-containing plasma with different concentrations, and carrying out the operation according to 'plasma sample pretreatment'. The ratio Y (Y As/Ai) of the amlodipine peak area As and the amlodipine-d 4 peak area Ai is calculated, and the blood concentration X is subjected to regression calculation by using the peak area ratio Y, and the result is shown in FIG. 1 and Table 7. The average ratio Y is used for carrying out regression calculation on the blood concentration X to obtain a regression equation Y which is 0.41X +0.00357, r is 0.9994, and a weight coefficient W is 1/X2The lowest quantitative limit of the amlodipine blood concentration measured by the method is as follows: 0.1 ng/mL.
Table 6 amlodipine standard curve formulating concentration
b: diluted solution of analyte: MeOH/H2O＝50/50
TABLE 7 standard curve of amlodipine in human plasma by HPLC-MS/MS method (n ═ 13)
4. Accuracy and precision
Putting the blank plasma into a water bath at room temperature for unfreezing; appropriate volumes of blank plasma were transferred to appropriate containers and amlodipine standard solutions were added to prepare 5 drug-containing plasma quality control samples (LLOQ, QL, QLM, QM, QH) of different concentrations and one associated standard curve, which were prepared as shown in table 8 below, according to the "plasma sample pretreatment" procedure. Making one batch and one following standard curve every day, continuously making 3 days for three batches, respectively making 6 samples for each concentration of the first batch and the second batch, respectively making 14 samples for each concentration of the third batch, 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 the standard curve on the day to obtain the actually measured concentration, calculating the precision between batches according to the actually measured concentration, and determining the ratio of the actually measured concentration to the added concentration As the accuracy, wherein the result is shown in Table 9. The result shows that the precision and the accuracy of the amlodipine blood plasma sample in batches and among batches are less than +/-15 percent and meet the requirements.
TABLE 8 quality control sample preparation concentration
a: final volume is source solution volume + plasma volume
Sufficient volume was dispensed into the labeled sample vials as required for each assay batch and stored at the theoretical temperature-80 ℃. The volume may be scaled up or down as desired.
TABLE 9 Intra-batch, inter-batch precision and accuracy of determination of amlodipine in plasma by HPLC-MS/MS method
5. Interference
Nine different blank plasma samples are respectively from different healthy human bodies, and the nine different blank plasma samples 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.
After the nine blank healthy human plasma samples from different sources were prepared and analyzed, the interference peak responses at the time points corresponding to the retention time of amlodipine were all lower than 20.0% of the response of amlodipine in the quantitative lower limit sample in the standard curve of the analysis batch, and the results are shown in table 10. The results show that the analysis method is specific to the analysis of amlodipine.
After nine blank healthy human plasma samples from different sources were prepared and analyzed, the interference peak responses at retention times consistent with the internal standard amlodipine-d 4 were all less than 5.0% of the internal standard amlodipine-d 4 response of the quantitative lower limit sample in the standard curve of the assay lot, see table 11 in the appendix. The results show that the analysis method has selectivity for the analysis of the internal standard amlodipine-d 4.
TABLE 10 comparison of interference data of blank healthy human plasma from nine different sources on amlodipine analyte
a: analyte peak area (selective sample)/analyte peak area (LLOQ of standard curve). times.100.0% to 20.0%
b: 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 in the sample".
TABLE 11 comparison of interference data of blank healthy human plasma from nine different sources against the internal standard amlodipine-d 4
a: the area of the peak of the analyte (selective sample)/the area of the peak of the internal standard (LLOQ of the standard curve) multiplied by 100.0 percent is less than or equal to 5.0 percent
b: 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 in the sample".
As can be seen from tables 10 and 11, 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.
6. Detection of human plasma samples
(1) Taking human blank plasma without amlodipine, precisely adding 100 mu L of plasma sample into a 96-deep-well plate, adding 10 mu L of plasma sample, and mixing the plasma sample with a volume ratio of 1: 1, adding 350 mu L of acetonitrile into a 96 deep-hole plate after uniformly mixing, carrying out vortex mixing for 1min, centrifuging for 10min at 20 ℃ at 3000rpm, taking 200 mu L of supernatant liquid into the 96 deep-hole plate filled with 200 mu L of mixed organic solvent, wherein the mixed organic solvent is acetonitrile: water: formic acid: 1M ammonium acetate in a volume ratio of 70: 30: 0.5: 0.1, vortex the resulting mixture, mix well, centrifuge at 3000rpm for 5min at 20 ℃ and then take 10 μ L sample for LC-MS/MS analysis, representative profile results are shown in figure 2.
(2) Taking human blank plasma without amlodipine, precisely adding 100 mu L of plasma sample into a 96-deep-well plate, adding 5 mu L of plasma sample, wherein the volume ratio is 1: 1, adding 5 mu L of 0.04 ng/mu L of internal standard amlodipine-d 4 solution after uniformly mixing, adding 350 mu L of acetonitrile into a 96 deep-well plate after uniformly mixing, carrying out vortex mixing for 1min, centrifuging for 10min at 20 ℃ at 3000rpm, taking 200 mu L of supernatant into a 96 deep-well plate filled with 200 mu L of mixed organic solvent, wherein the mixed organic solvent is acetonitrile: water: formic acid: 1M ammonium acetate in a volume ratio of 70: 30: 0.5: 0.1, vortex the resulting mixture, mix well, centrifuge at 3000rpm for 5min at 20 ℃ and take 10 μ L sample for LC-MS/MS analysis, representative profile results are shown in figure 3.
(3) Taking human blank plasma without amlodipine, precisely adding 100 mu L of blank plasma sample into a 96 deep-well plate, adding 5 mu L of amlodipine standard solution, adding 5 mu L of 0.04 ng/mu L of internal standard amlodipine-d 4 solution, uniformly mixing, adding 350 mu L of acetonitrile, vortex mixing for 1min, centrifuging at 20 ℃ at 3000rpm for 10min, taking 200 mu L of supernatant into the 96 deep-well plate filled with 200 mu L of mixed organic solvent, wherein the mixed organic solvent is acetonitrile: water: formic acid: 1M ammonium acetate in a volume ratio of 70: 30: 0.5: 0.1, vortex the resulting mixture, mix well, centrifuge at 3000rpm for 5min at 20 ℃ and then take 10 μ L sample for LC-MS/MS analysis, representative profile results are shown in figure 4.
(4) Collecting plasma of healthy subjects after oral administration of amlodipine or pharmaceutically acceptable salts thereof, precisely adding 100 mu L of collected human plasma samples into a 96-deep-well plate, and adding 5 mu L of the collected human plasma samples in a volume ratio of 1: 1, adding 5 mu L of 0.04 ng/mu L of internal standard amlodipine-d 4 solution after uniformly mixing, adding 350 mu L of acetonitrile into a 96 deep-well plate after uniformly mixing, carrying out vortex mixing for 1min, centrifuging for 10min at 20 ℃ at 3000rpm, taking 200 mu L of supernatant into a 96 deep-well plate filled with 200 mu L of mixed organic solvent, wherein the mixed organic solvent is acetonitrile: water: formic acid: 1M ammonium acetate in a volume ratio of 70: 30: 0.5: 0.1, vortex the resulting mixture, mix well, centrifuge at 3000rpm for 5min at 20 ℃ and then take 10 μ L sample for LC-MS/MS analysis, with representative profile results as shown in figure 5.
In conclusion, the invention provides a simple and convenient method for measuring the concentration of amlodipine in blood plasma by a pretreatment method, adopts a protein precipitation method, and is suitable for conventional measurement; meanwhile, under the chromatographic conditions adopted in the experiment, the retention time of amlodipine is about 0.837min, the retention time of the internal standard amlodipine-d 4 is about 0.833min, the peaks of amlodipine and the internal standard amlodipine-d 4 are good, the determination is free of the interference of miscellaneous peaks, and the base line is stable; the method has high specificity, can accurately measure the concentration of the amlodipine in the blood plasma, has high sensitivity, and has the minimum limit of quantitation of the blood plasma of 0.1 ng/mL; meanwhile, the method is rapid, accurate, high in sensitivity and simple and convenient to operate, and provides a basis for measuring the blood concentration of amlodipine. The linear range of the plasma standard curve of the method is 0.1-20 ng/mL, and the precision RSD in batch and between batches is less than +/-15%.
Claims (5)
1. A method for determining the concentration of amlodipine in blood plasma by liquid chromatography-mass spectrometry is characterized in that: the plasma sample is pretreated and then the concentration of the plasma sample is detected by high performance liquid chromatography-tandem mass spectrometry, and the specific method comprises the following steps:
(1) plasma sample pretreatment:
plasma with K2EDTA as anticoagulant, amlodipine-d 4 as internal standard; precisely adding 100 μ L of plasma sample into a 96-deep-well plate, adding 5 μ L of plasma sample at a volume ratio of 1: 1, adding 5 mu L of 0.04 ng/mu L of internal standard amlodipine-d 4 solution after uniformly mixing, adding 350 mu L of acetonitrile into a 96 deep-well plate after uniformly mixing, carrying out vortex mixing for 1min, centrifuging for 10min at 20 ℃ at 3000rpm, taking 200 mu L of supernatant into a 96 deep-well plate filled with 200 mu L of mixed organic solvent, wherein the mixed organic solvent is acetonitrile: water: formic acid: 1M ammonium acetate in a volume ratio of 70: 30: 0.5: 0.1, mixing the obtained mixture, uniformly mixing the mixture by vortex, centrifuging the mixture at the temperature of 20 ℃ and 3000rpm for 5min, and taking the mixture as a test sample to be detected;
(2) and (3) sample measurement:
injecting 10 mu L of test sample into a high performance liquid chromatography-tandem mass spectrometer, detecting chromatographic peaks of amlodipine and an internal standard amlodipine-d 4 in the sample, and calculating the concentration of amlodipine in the plasma sample according to the chromatographic peaks;
the liquid chromatography determination conditions were: the chromatographic column is RestekAllure PFPP with the diameter of 5 mu m and the specification of the column is 50 multiplied by 2.1 mm; the temperature of the chromatographic column is 40 ℃; the mobile phase A is water: formic acid: 1M ammonium acetate in a volume ratio of 100: 0.5: 0.1 mixing the obtained mixture; the mobile phase B is acetonitrile: formic acid is mixed according to the volume ratio of 100: 0.5 mixing the obtained mixture; the washing liquid is methanol: water is mixed according to the volume ratio of 50: 50 mixing the obtained mixture; the autosampler temperature was 15 ℃; gradient elution with flow rate of 0.7mL/min, sample size of 10 μ L, and analysis time of 2.5 min;
the mass spectrometry conditions are as follows: the ion source is an electrospray ion source, the spraying voltage is 5500V, the atomizing temperature is 550 ℃, the spraying air pressure is 80Psi, the auxiliary heating air pressure is 5Psi, the air curtain air pressure is 20Psi, the collision air pressure is 8Psi, and the declustering voltages of amlodipine and the internal standard amlodipine-d 4 are both 30 eV; the entrance voltages of the collision chambers of the amlodipine and the internal standard amlodipine-d 4 are both 15 eV; the collision voltage of amlodipine and the internal standard amlodipine-d 4 is 18 eV; the outlet voltages of the collision chambers of the amlodipine and the internal standard amlodipine-d 4 are both 10 eV; detecting in a positive ion mode; the scanning mode is multiple reaction monitoring; the ion reactions for quantitative analysis were: m/z 408.8 → m/z 238.0, which is amlodipine; and m/z 412.9 → m/z 238.1, which is amlodipine-d 4.
2. The method for determining amlodipine concentration in blood plasma by LC-MS according to claim 1, wherein: the gradient elution procedure in the step (2) is as follows:
3. the method for determining amlodipine concentration in blood plasma according to claim 1 or 2, wherein: in the step (2), an internal standard method is adopted, and the concentration of amlodipine in the plasma sample is calculated by substituting the peak area ratio of amlodipine and an internal standard amlodipine-d 4 into a standard curve equation.
4. The method for determining amlodipine concentration in blood plasma by LC-MS according to claim 3, wherein: the establishment of the standard curve equation comprises the following steps:
placing ten 100-microliter blank blood plasmas into a 96-deep-well plate, and sequentially naming the blank blood plasmas as a lowest quantitative lower limit sample, a standard sample 1, a standard sample 2, a standard sample 3, a standard sample 4, a standard sample 5, a standard sample 6, a highest quantitative upper limit sample, a zero-concentration sample and the blank sample to be ten samples in total, wherein the zero-concentration sample contains an internal standard amlodipine-d 4 solution and does not contain an amlodipine solution and is used for eliminating the interference of the internal standard amlodipine-d 4 solution on a detection result; the blank sample does not contain an amlodipine solution and an internal standard amlodipine-d 4 solution, and is used for eliminating the interference of the used blank plasma on the detection result;
adding 5 mul of amlodipine solution with the concentration of 0.002 ng/mul, 0.004 ng/mul, 0.01 ng/mul, 0.02 ng/mul, 0.04 ng/mul, 0.1 ng/mul, 0.24 ng/mul and 0.4 ng/mul to the lowest quantitative limit sample, the standard samples 1-6 and the highest quantitative limit sample in the form of stock solution, respectively adding 5 mul of amlodipine solution with the volume ratio of 1: 1, respectively mixing the ten samples, respectively adding 5 mu L of 0.04 ng/mu L of an internal standard amlodipine-d 4 solution into nine samples except for a blank sample, and adding 5 mu L of an internal standard amlodipine-d 4 solution into the blank sample according to the volume ratio of 1: 1, respectively and uniformly mixing the ten samples, respectively adding 350 mu L of acetonitrile into the ten samples, carrying out vortex mixing for 1min, centrifuging at 20 ℃ for 10min at 3000rpm, taking 200 mu L of supernatant of the supernatant into a 96 deep-hole plate filled with 200 mu L of mixed organic solvent, wherein the mixed organic solvent is acetonitrile: water: formic acid: 1M ammonium acetate in a volume ratio of 70: 30: 0.5: 0.1, mixing the obtained mixture, uniformly mixing the mixture by vortex, centrifuging the mixture at the temperature of 20 ℃ at 3000rpm for 5min, and taking the mixture as 10 parts of standard sample to be detected;
and respectively injecting 10 mu L of standard samples into a high performance liquid chromatography-tandem mass spectrometer, detecting chromatographic peaks of amlodipine and the internal standard amlodipine-d 4 in the samples, and obtaining a standard curve according to the chromatographic peaks so as to calculate the concentration of amlodipine in the plasma.
5. The method for determining amlodipine concentration in blood plasma according to claim 1 or 2, wherein: the conditions for the liquid chromatography determination in the step (2) further include: the volume of the syringe washing needle of the automatic sample injector is 500 mu L; the depth of a sample injection needle of the automatic sample injector is 45 mm; the cleaning speed of the automatic sample injector is 35 mu L/s; the sample injection speed of the automatic sample injector is 5 mu L/s; the soaking time is 5s when the sample injection needle of the automatic sample injector is cleaned; the automatic sample injector cleaning mode is before sample injection and after sample injection.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114384170A (en) * | 2021-12-15 | 2022-04-22 | 安徽万邦医药科技股份有限公司 | Method for determining concentration of amlodipine in blood plasma by liquid chromatography-mass spectrometry |
| CN114609287A (en) * | 2022-03-18 | 2022-06-10 | 四川尚锐分析检测有限公司 | Method for detecting amlodipine, losartan and losartan metabolite thereof in blood plasma by LC-MS (liquid chromatography-mass spectrometry) method |
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2021
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114384170A (en) * | 2021-12-15 | 2022-04-22 | 安徽万邦医药科技股份有限公司 | Method for determining concentration of amlodipine in blood plasma by liquid chromatography-mass spectrometry |
| CN114609287A (en) * | 2022-03-18 | 2022-06-10 | 四川尚锐分析检测有限公司 | Method for detecting amlodipine, losartan and losartan metabolite thereof in blood plasma by LC-MS (liquid chromatography-mass spectrometry) method |
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