CN113125613A - Quantitative analysis method of escitalopram in human plasma - Google Patents
Quantitative analysis method of escitalopram in human plasma Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention provides a quantitative analysis method for escitalopram in human plasma by applying a liquid chromatography-mass spectrometry (HPLC-MS/MS) technology. The method improves extraction recovery rate, reduces matrix effect, enhances selectivity, reduces interference on quantitative analysis of analytes and internal standards, and improves detection sensitivity.
Description
Technical Field
The invention belongs to the field of biological analysis, and particularly relates to a quantitative analysis method for escitalopram in human plasma by applying a liquid chromatography-mass spectrometry (HPLC-MS/MS) technology.
Background
The escitalopram serving as a novel antidepressant can effectively improve the curative effect of a patient, and has the advantages of low cost of defense generation, low incidence rate of adverse reactions in the cardiovascular system and the liver function, good curative effect and less adverse reactions.
In the evaluation of the imitation drug consistency, how to determine better and more stable bioequivalence evaluation is very important for the detection and analysis of the drug. The escitalopram biological sample has complex components and large matrix interference, and meanwhile, the sample is difficult to obtain and has small quantity, and the concentration of an analyte is usually very low, so the requirements on the technology for separating and analyzing the biological sample are higher and higher, and a high-sensitivity and high-selectivity escitalopram content analysis method is urgently needed to be established.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a quantitative analysis method of escitalopram in human plasma, starting from two aspects of sample pretreatment and detection technology.
The above object of the present invention is achieved by the following means.
A method for the quantitative analysis of escitalopram in human plasma comprising the steps of:
(1) preparation of standard working solution: weighing an escitalopram reference substance in a brown volumetric flask, adding a methanol solution to prepare the reference substance with the concentration of 50.00-500.0 mu g/mL-1The escitalopram stock solution is diluted into the concentration of 2.000-500.0 ng/mL by using a methanol-water solution with the volume ratio of 7:3-1Escitalopram standard curve working solution; repeating the steps once to prepare gradient concentration of 2.000-400.0 ng.mL-1The escitalopram quality control working solution; weighing a certain amount of internal standard escitalopram-d 4 into a glass bottle, and adding methanol to prepare the internal standard escitalopram-d 4 with the concentration of 10.00-50.00 mu g.mL-1The internal standard stock solution of (1) is diluted to 100.0 ng/mL by using a methanol-water solution with a volume ratio of 7:3-1The internal standard working solution of (4); storing all stock solutions and working solutions at 0-10 ℃ for later use;
respectively adding the working solution of the standard curve of escitalopram into blank normal plasma, uniformly mixing to prepare a standard curve with corresponding gradient, wherein the concentration range of the standard curve is 0.2000-50.00 ng.mL-1(ii) a Respectively adding the normal plasma of a blank person into the quality control working solution of escitalopram, uniformly mixing to prepare a quality control sample with a corresponding gradient, wherein the gradient concentration range is 0.2000-40.00 ng.mL-1；
(2) Sample treatment: taking a standard curve, a quality control sample, a test sample, blank human plasma and water which are equal in volume, respectively adding the internal standard working solution which is equal in volume into the standard curve, the quality control sample and the test sample, and respectively adding methanol aqueous solution which is equal in volume into the blank human plasma and the water; adding methanol into each mixed solution, mixing uniformly, centrifuging, transferring supernatant to a 96-well plate, and storing each extract;
(3) and (3) standard curve preparation: performing LC-MS/MS analysis and determination on the standard curve obtained in the step (2) and the extract of the quality control sample, and preparing a standard curve by taking the chromatographic peak area ratio of escitalopram and internal standard escitalopram-d 4 as an ordinate and the concentration of escitalopram in human plasma as an abscissa;
(4) quantitative analysis: and (4) processing the test sample according to the method in the step (3), and calculating according to the standard curve equation obtained in the step (3) to obtain the concentration of escitalopram in the test sample.
Escitalopram has the following structural formula:
preferably, the working conditions of the LC-MS/MS analytical determination process in step (3) are:
a. chromatographic conditions are as follows: the chromatographic column is Luna Omega Polar C18; mobile phase A: an aqueous solution containing 0.1% formic acid and 5mM ammonium formate; mobile phase B: methanol; temperature of the column oven: 40 ℃; flow rate: 0.3 mL/min; column pressure at equilibrium state of the column: 28.0-32.0 MPa; the elution mode is gradient elution;
b. mass spectrum conditions: the ion source is an ESI source and adopts a positive ion mode and a multi-reaction monitoring mode; electrospray voltage: 3000V; vortex ion spray temperature: 650 ℃; air curtain type: 35 psi; atomizing Gas species Gas 1: 45 psi; auxiliary Gas 2: 60 psi; data collection time: 3.6 min.
Preferably, the concentration of the escitalopram stock solution in the step (1) is 50.00-500.0 [ mu ] g/mL-1The concentration of the internal standard stock solution is 10.00-50.00 mu g/mL-1The gradient concentrations of the escitalopram working solution are respectively 2.000, 10.00, 20.00, 80.00, 200.0, 300.0 and 500.0 ng/mL-1The concentration gradient of the escitalopram quality control working solution is 2.000, 5.000, 100.0 and 400.0 ng.mL-1The concentration of the internal standard working solution is 100 ng-mL-1。
Preferably, the gradient concentration of the quality control sample in the step (1) is 0.2000, 0.5000, 10.00 and 40.00 ng/mL respectively-1Storing the quality control sample at-15 to-90 ℃; the gradient concentrations of the standard curve are respectively 0.1000, 1.000, 2.000, 8.000, 20.00, 30.00 and 50.00ng/mL-1The standard curve is freshly prepared.
Preferably, the centrifugation in step (2) is carried out at 17000 Xg for 15 min.
Preferably, the chromatographic conditions of step (3) further comprise: the sample injection volume is 2.00 mu L; the automatic sample injector cleaning solution is methanol; automatic injector wash needle volume: 200 mu L; pressure foot lifting amount: 48 mm; soaking time is 2s when the automatic sampling needle is cleaned; autosampler cleaning mode: cleaning before sample injection and cleaning after sample injection; autosampler temperature: 4.0 ℃; autosampler wash time: 0 s.
Preferably, the mass spectrometry conditions of step (3) further comprise: the inlet voltage is 10V and the collision cell outlet voltage is 8V.
Preferably, the monitored ion pair of escitalopram in the LC-MS/MS analysis and determination process in the step (3) is 325.300/109.100, the staying time of one ion pair in each scanning is 90MS, the declustering voltage is 121V, the collision energy is 34eV, and the staying time is 1.15 min; the internal standard escitalopram-d 4 is 329.300/113.100, the residence time of one ion pair in each scan is 90ms, the declustering voltage is 108V, the collision energy is 34eV, and the retention time is 1.15 min.
Compared with the prior art, the invention has the beneficial effects that: (1) the extraction recovery rate is improved and basically reaches more than 95 percent. (2) The matrix effect is reduced, and the absolute matrix factor reaches 0.97-1.05. (3) Enhances selectivity and reduces interference with quantitative analysis of the analyte and the internal standard. (4) The detection sensitivity is improved, and the quantitative range is determined, so that the expected quantitative lower limit sample meets the quantitative analysis requirement. The method for quantitatively detecting the escitalopram is used for determining the concentration of the escitalopram in human plasma by using a liquid chromatography-mass spectrometry (HPLC-MS/MS) technology and accurately quantifying the concentration of the escitalopram in the human plasma, and the method is feasible, simple and convenient to operate, high in automation degree of an instrument, high in repeatability, good in accuracy and strong in operability by reasonably selecting conditions such as chromatographic columns, dissolving solutions, mobile phases and mass spectrometry and optimizing process conditions such as flow rate, elution modes and mass spectrometry conditions, so that the method for quantitatively detecting the escitalopram is directly applied to sample detection and analysis in bioequivalence.
Drawings
FIG. 1 is a standard curve of the method for the quantitative analysis of escitalopram in human plasma according to the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
The quantitative analysis method of escitalopram in human plasma established by the invention comprises the following steps:
(1) preparation of standard working solution: weighing an escitalopram reference substance in a brown volumetric flask, adding a methanol solution to prepare the reference substance with the concentration of 50.00-500.0 mu g/mL-1The escitalopram stock solution is diluted into the concentration of 2.000-500.0 ng/mL by using a methanol-water solution with the volume ratio of 7:3-1Escitalopram standard curve working solution; repeating the steps once to prepare gradient concentration of 2.000-400.0 ng.mL-1The escitalopram quality control working solution; weighing a certain amount of internal standard escitalopram-d 4 into a glass bottle, and adding methanol to prepare the internal standard escitalopram-d 4 with the concentration of 10.00-50.00 mu g.mL-1The internal standard stock solution of (1) is diluted to 100.0 ng/mL by using a methanol-water solution with a volume ratio of 7:3-1The internal standard working solution of (4); storing all stock solutions and working solutions at 0-10 ℃ for later use;
respectively adding the working solution of the standard curve of escitalopram into blank normal plasma, uniformly mixing, and preparing a standard curve with corresponding gradient, wherein the concentration range is 2000-50.00 ng.mL-1(ii) a Respectively adding the normal plasma of a blank person into the quality control working solution of escitalopram, uniformly mixing to prepare a quality control sample with a corresponding gradient, wherein the gradient concentration range is 0.2000-40.00 ng.mL-1；
(2) Sample treatment: taking a standard curve, a quality control sample, a test sample, blank human plasma and water which are equal in volume, respectively adding the internal standard working solution which is equal in volume into the standard curve, the quality control sample and the test sample, and respectively adding methanol aqueous solution which is equal in volume into the blank human plasma and the water; adding methanol into each mixed solution, mixing uniformly, centrifuging, transferring supernatant to a 96-well plate, and storing each extract;
(3) and (3) standard curve preparation: performing LC-MS/MS analysis and determination on the standard curve obtained in the step (2) and the extract of the quality control sample, and preparing a standard curve by taking the chromatographic peak area ratio of escitalopram and internal standard escitalopram-d 4 as an ordinate and the concentration of escitalopram in human plasma as an abscissa;
(4) quantitative analysis: and (4) processing the test sample according to the method in the step (3), and calculating according to the standard curve equation obtained in the step (3) to obtain the concentration of escitalopram in the test sample.
The process of the present invention will be further described below by taking specific examples of the conditions for carrying out the process.
EXAMPLE 1 preparation of test solutions and working solutions
Mobile phase A: approximately 500mL of ultrapure water is weighed into a 1L volumetric flask, 1000. mu.L of formic acid and 2000. mu.L of 2.5M ammonium formate solution are added, diluted with ultrapure water to a constant volume to a scale, and ultrasonically degassed for later use. Storage conditions were as follows: and (4) room temperature. The validity period is as follows: and 2 days.
Mobile phase B: mass spectrometric grade methanol solution. Storage conditions were as follows: and (4) room temperature. The validity period is as follows: 3 months.
Methanol: water (30:70, v: v) [ denoted diluent 1 ]: a glass bottle was charged with 300mL of methanol and 700mL of water, and mixed by shaking. Storage conditions were as follows: 4 ℃ is prepared. The validity period is as follows: 3 months.
The stock solutions of escitalopram were prepared separately by two independent weighings. All prepared stock solutions should be stored in a refrigerator at 0-10 ℃. Before obtaining the long-term storage stability data of the stock solution, the stock solution is considered to be stable in the storage time period, and the measured actual stability data is taken as the standard. The working solutions for preparing the standard curve and quality control samples must be from two independently weighed stock solutions.
Escitalopram stock solution (denoted S-ASXTPL)]，78.28μg·mL-1: a certain amount of escitalopram is weighed and placed in two glass bottles respectively, and the weights are recorded and marked as S-ASXTPL-STD and S-ASXTPL-QC respectively. Calculating the true weight of the analyte according to the purity, moisture, presence of salt formation and form of escitalopram, adding appropriate amount of methanol to obtain final concentration of 78.28. mu.g.mL-1(ii) escitalopram stock solution, vortex mixing; the compound may be dissolved with ultrasound if necessary.
Internal standard stock solution [ marked as S-IS-ASXTPL]，38.65μg·mL-1: an amount of escitalopram-d 4 was placed in a glass vial and the weight was recorded and marked as S-IS-ASXTPL. The true weight of the internal standard was calculated from the purity, moisture, salification and form of escitalopram-d 4. Adding a proper amount of methanol to obtain a final concentration of 38.65 mug. multidot.mL-1The escitalopram-d 4 internal standard stock solution is mixed by vortex; the compound may be dissolved with ultrasound if necessary.
Internal standard working solution (100ng/mL) [ denoted as W-IS-ASXTPL ]: adding 3365 mu L of diluent 1 into a polypropylene centrifuge tube, then adding 500.0 mu L of escitalopram-d 4 internal standard stock solution S-IS-ASXTPL, and vortex mixing to obtain a secondary internal standard stock solution. Then 49.00mL of diluent 1 was added to the volumetric flask, and 1000.0. mu.L of the second internal standard stock solution was added to the flask by pipette and mixed by vortexing. The W-IS-ASXTPL IS stored in a refrigerator at 0-10 ℃, and IS considered to be stable for a long time before long-term storage stability data of the working fluid are obtained.
Preparation of analyte working solution: in a polypropylene centrifuge tube, diluent 1 was added in the volume specified in the table below and vortex mixed. All prepared working solutions are stored in a refrigerator at 0-10 ℃. The working solutions were considered stable for the duration of storage until long term storage stability data were obtained.
Note: S-ASXTPL-STD is escitalopram stock solution, and is subsequently used for preparing a standard curve; W-ASXTPL-STD 1-7 represents standard curve working solution.
Preparation of a standard curve: in a polypropylene centrifuge tube, as shown in the following table, a suitable amount of blank normal plasma was pipetted, the corresponding working solution was added, vortexed and mixed (approximately 60 seconds) to prepare the corresponding standard curve. In each assay batch of the method validation, the standard curve should be freshly formulated.
Note: the formulation volume of the standard curve can be adjusted according to actual needs, but the final target concentration must be kept consistent and faithfully recorded.
Preparing a quality control working solution: in a polypropylene centrifuge tube, diluent 1 was added in the volume specified in the table below and vortex mixed. All prepared working solutions are stored in a refrigerator at 0-10 ℃. The working fluid was considered stable for the duration of storage until long term storage stability data was obtained for the working fluid.
Note: S-ASXTPL-QC is escitalopram quality control stock solution, and is subsequently used for preparing a quality control sample; W-ASXTPL-HQC/MQC/LQC/LLOQQC represents quality control working solution.
Preparing a quality control sample: in a polypropylene centrifuge tube, as shown in the following table, a proper amount of blank normal plasma was added by a pipette, and then, the corresponding working solution was added, and vortexed and mixed (about 60 seconds) to prepare a corresponding quality control sample. In each assay batch of the process validation, the quality control samples should be freshly prepared.
Note: the preparation volume of the quality control sample can be adjusted according to actual needs, but the final target concentration must be kept consistent and recorded correctly.
Example 2
An LC-MS/MS quantitative analysis method for escitalopram in human plasma, which comprises the following steps:
(1) preparation of standard working solution: the gradient concentrations of escitalopram working solution are respectively 2.000, 10.00, 20.00, 80.00, 200.0, 300.0 and 500.0 ng.mL-1The concentration gradient of the escitalopram quality control working solution is 2.000, 5.000, 100.0 and 400.0 ng.mL-1(ii) a The gradient concentrations of the standard curve are 0.1000, 1.000, 2.000, 8.000, 20.00, 30.00, 50.00ng/mL respectively-1The concentration of the quality control sample is respectively 0.2000, 0.5000, 10.00 and 40.00 ng/mL-1；
The internal standard working solution is 100.0 ng.mL-1；
The solutions may be prepared by the methods described in example 1, or by other methods, as long as the final concentration is ensured.
(2) Sample treatment: respectively adding 100.0 mu L of blank matrix (blank human plasma), water, test samples, and the standard curve and the quality control sample obtained in the step (1) into a polypropylene centrifugal tube by using a pipettor, and respectively marking the blank matrix, the water, the test samples, the standard curve and the quality control sample as a double blank group, a sample group, a label group and a quality control group; adding 20.0 mu L of diluent 1 into the double blank group, adding 10.0 mu L of diluent 1 and 10.0 mu L of internal standard working solution W-IS-ASXTPL into the sample group, and respectively adding 10.0 mu L of internal standard working solution W-IS-ASXTPL into the standard curve group and the quality control group by using a pipettor;
adding 600 μ L of methanol to each sample using a pipette, vortex mixing for 1min, and centrifuging at 17000 × g for 15min in a 4 ℃ centrifuge; transferring 200/300 mu L of supernatant into a 96-well plate by using a pipettor, and carrying out vortex mixing to prepare for sample injection analysis; before sample injection analysis, all the extracts after sample treatment are stored in an autosampler at the temperature or in a refrigerator at 0-10 ℃.
(3) And (3) standard curve preparation: and (3) performing the following LC-MS/MS determination analysis on the standard curve group and the quality control group extracts obtained in the step (2), preparing a standard curve consisting of 7 concentration level standard curves for each analysis batch, and detecting by using a quality control sample.
Chromatographic conditions are as follows:
a chromatographic column: luna Omega 1.6 μm Polar C18, 50X 2.1mm Column, Phenomenex
Mobile phase A: an aqueous solution containing 0.1% formic acid and 5mM ammonium formate;
mobile phase B: methanol;
flow rate: 0.3 mL/min;
sample introduction volume: 2.00 mu L;
approximate column pressure at column equilibrium: 30.0 Mpa;
autosampler wash solution: methanol;
automatic injector wash needle volume: 200 mu L;
pressure foot lifting amount: 48 mm;
soaking time when cleaning the sample injection needle of the automatic sample injector: 2 s;
moving sample injector cleaning mode: before and after sample introduction;
automatic injector washs the setting: soaking after flushing;
autosampler wash time: 0 s;
autosampler temperature: 4 ℃;
temperature of the column oven: at 40 ℃.
Chromatographic gradient:
| Time(min) | A(％) | B(％) | flow rate (mL/min) |
| 0.00 | 55.0 | 45.0 | 0.3000 |
| 0.20 | 25.0 | 75.0 | 0.3000 |
| 1.80 | 5.0 | 95.0 | 0.3000 |
| 2.50 | 5.0 | 95.0 | 0.3000 |
| 2.51 | 55.0 | 45.0 | 0.3000 |
| 3.60 | 55.0 | 45.0 | 0.3000 |
Note: cycle Time analysis of single sample: about 3.6 minutes (time difference from the start of one sample to the start of the next sample).
Mass spectrum conditions:
an ion source: ESI;
ionization mode: a positive ion mode;
detection mode: monitoring multiple reactions;
electrospray voltage: 3000V;
vortex ion spray temperature: 650 deg.C
Air curtain type: 35 psi;
atomizing Gas species (Gas 1): 45 psi;
kind of auxiliary Gas (Gas 2): 60 psi;
inlet voltage: 10 v;
collision cell exit voltage: 8 v;
data collection time: 3.6 min.
Injecting: the dwell time refers to the time during which an ion pair is being monitored, one ion pair at a time.
Detecting standard curve and quality control sample according to the above chromatogram and mass spectrum conditions, processing chromatogram collection and chromatogram peak integration by software Analyst 1.6.3(AB Sciex), taking the chromatographic peak area ratio of escitalopram and internal standard escitalopram-d 4 as ordinate, taking the concentration of escitalopram in human plasma as abscissa, and weighting (W is 1/x)2) The least squares method performs a linear regression with the ratio (Y) of the concentration (X) to the peak area of escitalopram in plasmaThe regression equation (Y ═ a + bX) is a standard curve, specifically, Y ═ 0.125413x-0.000371994(r ═ 0.125413 x-0.000371994)20.9987) in ng/mL, showing that escitalopram is linear well within the range of 0.1000 to 50.00 ng/mL.
(4) Quantitative analysis: extracting escitalopram from human plasma by a protein precipitation treatment mode, performing vortex centrifugation, taking out supernatant, and performing LC-MS/MS (liquid chromatography-mass spectrometry) analysis according to the conditions in the step (3); the volume of test sample used was 1.0mL and the sample-treated plasma anticoagulant was Gs-Na. And (4) calculating according to the standard curve equation obtained in the step (3) to obtain the concentration of escitalopram in the test sample.
Example 3
This example evaluates the accuracy and precision within and between batches of the LC-MS/MS method used in example 2 for the determination of escitalopram concentration in human plasma. Operating according to the method, preparing the quality control samples with the concentrations of LLOQQC, LQC, MQC and HQC, measuring 6 samples with each concentration in three continuous analysis batches, repeating the measurement for 3 times, calculating the precision, and obtaining the results shown in the table
Example 4
This example evaluates the long term/short term stability of stock solutions and the long term/short term stability of working solutions for determining escitalopram concentration in human plasma by the LC-MS/MS method in example 1. Solution preparation: long-term and short-term S-ASXTPL-STD, S-ISASXTPL stock solution and W-ASXTPL-STD7, W-ASXTPL-STD1 and W-IS-ASXTPL working solution are added into a polypropylene centrifuge tube respectively, then a proper amount of specified dilution solution IS added, the solution IS diluted to a stability evaluation solution with the concentration of STD1, and vortex mixing IS carried out. The average stability was calculated by repeating 6 samples per solution operating as described above, and the results are shown in the following table
| Name of solution | Short term conditions | Average stability | Long term conditions | Average stability |
| S-ASXTPL-STD | Room temperature, 6 hours | 101.63％ | At 4 ℃ for 43 days | 98.73％ |
| S-ISASXTPL | Room temperature, 6 hours | 95.47％ | 4 ℃ for 57 days | 98.85％ |
| W-ASXTPL-STD7 | Room temperature, 6 hours | 101.22％ | At 4 ℃ for 43 days | 101.01％ |
| W-ASXTPL-STD1 | Room temperature, 6 hours | 101.75％ | At 4 ℃ for 43 days | 99.46％ |
| W-IS-ASXTPL | Room temperature, 6 hours | 94.20％ | 4 ℃ for 49 days | 97.97％ |
The LC-MS/MS detection method for the content of escitalopram in a human plasma sample, which is established by the invention, has the extraction recovery rate of more than 90 percent; the matrix effect is reduced, and the absolute matrix factor reaches 0.95-1.10; the selectivity is enhanced, and the interference on the quantitative analysis of the analyte and the internal standard is reduced; the method has the advantages of high efficiency, stability, sensitivity and strong operability, has important significance for the research of the equivalence of the escitalopram, can be directly applied to sample detection and analysis in bioequivalence, and has wide application prospect in the evaluation of the pharmaceutical imitation consistency of the escitalopram.
The implementation of the present invention has been described in detail, however, the present invention is not limited to the specific details of the above-described embodiments, and various simple modifications can be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
Claims (8)
1. A method for the quantitative analysis of escitalopram in human plasma, characterized in that it comprises the following steps:
(1) preparation of standard working solution: weighing an escitalopram reference substance in a brown volumetric flask, adding a methanol solution to prepare the reference substance with the concentration of 50.00-500.0 mu gmL-1The escitalopram stock solution is diluted into the concentration of 2.000-500.0 ng/mL by using a methanol-water solution with the volume ratio of 7:3-1Escitalopram standard curve working solution; repeating the steps once to prepare gradient concentration of 2.000-400.0 ng.mL-1The escitalopram quality control working solution; weighing a certain amount of internal standard escitalopram-d 4 into a glass bottle, and adding methanol to prepare the internal standard escitalopram-d 4 with the concentration of 10.00-50.00 mu g.mL-1The internal standard stock solution of (1) is diluted to 100.0 ng/mL by using a methanol-water solution with a volume ratio of 7:3-1The internal standard working solution of (4); storing all stock solutions and working solutions at 0-10 ℃ for later use;
respectively adding the working solution of the standard curve of escitalopram into blank normal plasma, uniformly mixing to prepare a standard curve with corresponding gradient, wherein the concentration range of the standard curve is 0.2000-50.00 ng.mL-1(ii) a Respectively adding the normal plasma of a blank person into the quality control working solution of escitalopram, uniformly mixing to prepare a quality control sample with a corresponding gradient, wherein the gradient concentration range is 0.2000-40.00 ng.mL-1；
(2) Sample treatment: taking a standard curve, a quality control sample, a test sample, blank human plasma and water which are equal in volume, respectively adding the internal standard working solution which is equal in volume into the standard curve, the quality control sample and the test sample, and respectively adding methanol aqueous solution which is equal in volume into the blank human plasma and the water; adding methanol into each mixed solution, mixing uniformly, centrifuging, transferring supernatant to a 96-well plate, and storing each extract;
(3) and (3) standard curve preparation: performing LC-MS/MS analysis and determination on the standard curve obtained in the step (2) and the extract of the quality control sample, and preparing a standard curve by taking the chromatographic peak area ratio of escitalopram and internal standard escitalopram-d 4 as an ordinate and the concentration of escitalopram in human plasma as an abscissa;
(4) quantitative analysis: and (4) processing the test sample according to the method in the step (3), and calculating according to the standard curve equation obtained in the step (3) to obtain the concentration of escitalopram in the test sample.
2. The method for the quantitative analysis of escitalopram in human plasma according to claim 1, wherein the working conditions of the LC-MS/MS analytical determination process in step (3) are as follows:
a. chromatographic conditions are as follows: the chromatographic column is Luna Omega Polar C18; mobile phase A: an aqueous solution containing 0.1% formic acid and 5mM ammonium formate; mobile phase B: methanol; temperature of the column oven: 40 ℃; flow rate: 0.3 mL/min; column pressure at equilibrium state of the column: 28.0-32.0 MPa; the elution mode is gradient elution;
b. mass spectrum conditions: the ion source is an ESI source and adopts a positive ion mode and a multi-reaction monitoring mode; electrospray voltage: 3000V; vortex ion spray temperature: 650 ℃; air curtain type: 35 psi; atomizing Gas species Gas 1: 45 psi; auxiliary Gas 2: 60 psi; data collection time: 3.6 min.
3. The method according to claim 1, wherein the method for the quantitative analysis of escitalopram in human plasma comprises the following steps: the concentration of the escitalopram stock solution in the step (1) is 50.00-500.0 mug.mL-1The concentration of the internal standard stock solution is 10.00-50.00 mu g/mL-1The gradient concentrations of the escitalopram working solution are respectively 2.000, 10.00, 20.00, 80.00, 200.0, 300.0 and 500.0 ng/mL-1The concentration gradient of the escitalopram quality control working solution is 2.000, 5.000, 100.0 and 400.0 ng.mL-1The concentration of the internal standard working solution is 100 ng-mL-1。
4. The method according to claim 1, wherein the gradient concentrations of the quality control sample in step (1) are 0.2000, 0.5000, 10.00 and 40.00 ng-mL respectively-1Storing the quality control sample at-15 to-90 ℃; the gradient concentrations of the standard curve are respectively 0.1000, 1.000, 2.000, 8.000, 20.00, 30.00 and 50.00ng/mL-1The standard curve is freshly prepared.
5. The method according to claim 1, wherein the centrifugation in step (2) is carried out at 17000 Xg for 15 min.
6. The method for the quantitative analysis of escitalopram in human plasma according to claim 2, wherein said chromatographic conditions of step (3) further comprise: the sample injection volume is 2.00 mu L; the automatic sample injector cleaning solution is methanol; automatic injector wash needle volume: 200 mu L; pressure foot lifting amount: 48 mm; soaking time is 2s when the automatic sampling needle is cleaned; autosampler cleaning mode: cleaning before sample injection and cleaning after sample injection; autosampler temperature: 4.0 ℃; autosampler wash time: 0 s.
7. The method for the quantitative analysis of escitalopram in human plasma according to claim 2, wherein said mass spectrometric conditions of step (3) further comprise: the inlet voltage is 10V and the collision cell outlet voltage is 8V.
8. The method for the quantitative analysis of escitalopram in plasma according to claim 2, wherein said LC-MS/MS analysis in step (3) determines the ion pair for escitalopram monitoring to be 325.300/109.100, the retention time of each ion pair scanning is 90MS, the declustering voltage is 121V, the collision energy is 34eV, and the retention time is 1.15 min; the internal standard escitalopram-d 4 is 329.300/113.100, the residence time of one ion pair in each scan is 90ms, the declustering voltage is 108V, the collision energy is 34eV, and the retention time is 1.15 min.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107941962A (en) * | 2017-12-28 | 2018-04-20 | 北京和合医学诊断技术股份有限公司 | Detect the liquid phase chromatography analytical method of escitalopram medicament contg in blood |
| CN109085264A (en) * | 2018-08-03 | 2018-12-25 | 杭州佰勤医疗器械有限公司 | Liquid chromatography tandem mass spectrometry detects the kit of antidepressant and its application in serum plasma |
| WO2018236990A1 (en) * | 2017-06-20 | 2018-12-27 | Seattle Gummy Company | Gelatin gummy compostion and methods of making and using thereof |
| CN110455945A (en) * | 2019-08-06 | 2019-11-15 | 北京回龙观医院(北京心理危机研究与干预中心) | The method and kit of 5 kinds of psychotropic agents and its main metabolites in a kind of detection blood |
-
2019
- 2019-12-31 CN CN201911402564.7A patent/CN113125613A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018236990A1 (en) * | 2017-06-20 | 2018-12-27 | Seattle Gummy Company | Gelatin gummy compostion and methods of making and using thereof |
| CN107941962A (en) * | 2017-12-28 | 2018-04-20 | 北京和合医学诊断技术股份有限公司 | Detect the liquid phase chromatography analytical method of escitalopram medicament contg in blood |
| CN109085264A (en) * | 2018-08-03 | 2018-12-25 | 杭州佰勤医疗器械有限公司 | Liquid chromatography tandem mass spectrometry detects the kit of antidepressant and its application in serum plasma |
| CN110455945A (en) * | 2019-08-06 | 2019-11-15 | 北京回龙观医院(北京心理危机研究与干预中心) | The method and kit of 5 kinds of psychotropic agents and its main metabolites in a kind of detection blood |
Non-Patent Citations (3)
| Title |
|---|
| CARLOS E.M. DE SOUSA等: "Determination of Escitalopram in Human Plasma by High Performance Liquid Chromatography-Tandem Mass Spectrometry", LATIN AMERICAN JOURNAL OF PHARMACY * |
| SEMIN PARK等: "Development and validation of a high performance liquid chromatography–tandem mass spectrometric method for simultaneous determination of bupropion, quetiapine and escitalopram in human plasma", BIOMED. CHROMATOGR. * |
| 陈珺等: "艾司西酞普兰在健康人体的药动学", 中国新药与临床杂志 * |
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