Method for chiral separation and determination of levamlodipine in human plasma by LC-MS / MS (Liquid Chromatography-Mass Spectrometry / Mass Spectrometry) method

Abstract

The invention provides a method for measuring levamlodipine in human plasma through LC-MS / MS (liquid chromatography-mass spectrometry / mass spectrometry) chiral separation, which comprises the following steps: A, sample pretreatment: adding an internal standard working solution into to-be-measured plasma, then adding an extracting agent, carrying out vortex and centrifugation, then taking supernate, concentrating until the supernate is dry, and finally redissolving; b, preparing a standard curve and a quality control sample, namely preparing a levamlodipine standard curve sample and a quality control sample working solution, adding the working solution into blank plasma, uniformly mixing, and preparing the levamlodipine standard curve sample and the quality control sample with a series of concentration gradients. C, LC-MS / MS (liquid chromatography-tandem mass spectrometry) determination. According to the present invention, the chiral chromatographic column is adopted to perform the chromatographic separation of the enantiomer, and the LC-MS / MS detection method with characteristics of high specificity, strong specificity, rapid pretreatment, high efficiency, high reproducibility, strong operability, rapid analysis speed, low cost investment and wide application range is established.

Description

Technical Field

[0001] The invention relates to the field of medical biological detection, and in particular to a method for determining levamlodipine in human plasma by chiral separation using an LC-MS / MS method. Background Art

[0002] Amlodipine besylate (trade name: Norvasc) has the characteristics of mild action, stable blood pressure reduction, long-term safety, etc. At the same time, as a new calcium ion antagonist, amlodipine has become an important drug for the clinical treatment of angina pectoris and hypertension. Levamlodipine besylate (Levamlodipine besilate) is the first chiral separation optically pure drug in my country. The pharmacodynamic properties of amlodipine besylate are actually derived from levamlodipine, while side effects such as acroedema, headache, dizziness, and facial flushing come from dextroamlodipine.

[0003] Modern pharmacological studies have shown that the activity of L-amlodipine is nearly 1,000 times that of the D-amlodipine. Therefore, it is necessary to establish a method for determining the enantiomers of amlodipine in plasma in order to study the pharmacokinetic characteristics of S-amlodipine in vivo.

[0004] At present, there are many domestic literatures on racemic amlodipine, but there are relatively few detection methods for amlodipine levulinate in journals, magazines or patent literature, and there are generally a series of problems such as poor specificity, low reproducibility, difficult operation, and poor left-right rotation separation. Amlodipine has the characteristics of low dosage, high bioavailability, and long-lasting efficacy. After taking the medicine, due to its special nature, the blood concentration of amlodipine in the human body is very low. The Cmax of amlodipine levulinate in human plasma is only about 3 ng / mL at a dosage of 2.5 mg. According to the requirements of the Technical Guidelines for Bioavailability and Bioequivalence Studies of Chemical Drug Preparations, the quantitative lower limit of the general detection method should meet 1 / 10-1 / 20 of Cmax.

[0005] Prior art 1 (J. Academic Journal of Second Military Medical University, Jul. 2012, Vol. 33, No. 7) discloses a LC-MS / MS method for determining the blood concentration of amlodipine besylate and investigating the possibility of chiral transformation in the human body. However, the method uses an atmospheric pressure chemical ionization source (APCI), which has low sensitivity, and the plasma pretreatment uses a solid phase extraction column (HLB) followed by concentration, which has cumbersome steps and poor reproducibility. The use of solid phase extraction columns has greatly increased the cost of testing, which has invisibly increased the economic burden on enterprises and is not conducive to the promotion and application of testing technology. At the same time, in order to effectively separate chiral amlodipine, the single injection time is as long as 20 minutes. This detection method is far from meeting the needs of high-throughput detection of biological samples.

[0006] Prior art 2 (J.FENXI CESHI XUEBAO (Journal of Instrumetal Analysis) Sep. 2005, Vol. 24) discloses a LC-MS / MS method for chiral separation and determination of amlodipine in human plasma. The lower limit of quantification of the method is reduced to 0.100 ng / mL, which meets the requirements of the "Technical Guidelines for Bioavailability and Bioequivalence Studies of Chemical Drug Preparations" (the lower limit of quantification should meet 1 / 10-1 / 20 of Cmax), but the blood volume used is as high as 1mL, the operability is poor, and the single-needle analysis time is still 9 minutes. This detection method cannot fully meet the needs of high-throughput detection of biological samples.

[0007] Since the sample volume is often large in clinical pharmacokinetic testing, an analytical method is needed that can quickly separate chiral amlodipine with strong specificity, high sensitivity, simple operation and can fully meet the requirements of quantitative detection of biological samples. Summary of the invention

[0008] In order to solve the problem of long analysis time and large amount of blood used in single needle detection of levoamlodipine in the prior art, the present invention provides a liquid chromatography-mass spectrometry method with strong specificity, rapid separation of enantiomers, strong operability, fast and efficient pretreatment, small amount of blood used, higher sensitivity, lower detection cost, and more suitable for high-throughput detection, which is used for chiral separation and detection of levoamlodipine in human plasma; The present invention adopts the following technical scheme: A method for chiral separation and determination of levamlodipine in human plasma by LC-MS / MS method comprises the following steps: A. Sample pretreatment Add 10-50 μL of internal standard working solution to the plasma to be tested, then add the extractant, vortex, centrifuge, then take the supernatant and concentrate to dryness, and finally re-dissolve; B. Preparation of standard curve and quality control samples Prepare the working solution of levoamlodipine standard curve samples and quality control samples, add the working solution to blank plasma, mix well, and prepare a series of levoamlodipine standard curve samples and quality control samples with concentration gradients.

[0009] C. LC-MS / MS Determination Take the standard curve sample in step B for detection to establish the standard curve of amlodipine; The sample to be tested that has been processed in step A is taken for testing, and the concentration of levamlodipine in the sample to be tested is calculated using the established standard curve.

[0010] Preferably, the specific steps of the sample pretreatment are: Step 1: Take 50-200 μL of sample into a strip tube, add 10-50 μL of internal standard working solution to the sample, and add 10-50 μL of 75% methanol aqueous solution to the blank sample; Step 2: Add 200-800 μL of extraction agent to the above strip tubes and vortex for 1 min; Step 3: Centrifuge at 4°C and 3700 rpm for 10 min, take 150-600 μL of the supernatant into a strip tube, place it under a nitrogen stream, and blow dry; Step 4: Redissolve with 100-400 μL of mobile phase and vortex for 1 min to obtain the sample to be analyzed.

[0011] Preferably, the conditions for the LC-MS / MS determination are: The LC-MS system: AB Sciex Triple Quad™ 5500 LC-MS was used as the detection instrument for analysis and detection; Chromatographic column: chiral column; Mobile phase: organic reagent containing volatile salts; Elution mode: isocratic elution; Ion source type: ESI source; The ionization modes are: positive ion; Detection mode: Multiple reaction monitoring; The mass spectrometry parameters are shown in the following table:

[0012] Preferably, the internal standard working solution in step A is a 40.0 ng / mL amlodipine-d4 solution.

[0013] Preferably, the extractant in step A is an ethyl acetate solution containing 1% ammonia water.

[0014] Preferably, the preparation process of the standard curve sample and quality control sample working solution of levamlodipine in step B is: at room temperature and yellow light conditions, the levamlodipine stock solution of the test substance is serially diluted with the working solution diluent (75% methanol aqueous solution) to prepare the working solutions of the standard curve sample and the quality control sample. All working solutions are prepared in glass bottles and stored in a freezer.

[0015] Preferably, in step C, the chiral chromatographic column is selected from Phenomenex Lux 3 µm Cellulose-4 2×150 mm.

[0016] Preferably, in step C, mobile phase A is acetonitrile containing 0.05% ethanolamine, and mobile phase B is isopropanol.

[0017] Preferably, in step C, the volume ratio of mobile phase A to mobile phase B in isocratic elution is 95:5.

[0018] Preferably, in step C, the column temperature of the chromatographic column is 40° C., the temperature of the automatic sampler is 4° C., and the injection volume is 10 μL.

[0019] Preferably, in step C, the isocratic elution time is 3.5 min.

[0020] In summary, the beneficial effects of the present invention are: The present invention adopts a chiral chromatographic column Phenomenex Lux 3 µm Cellulose-4 2×150 mm for chromatographic separation of enantiomers, which has high specificity and strong specificity. Dextrorotatory amlodipine elutes at 1.26 min, and levorotatory amlodipine elutes at 1.64 min. The single-needle detection time is only 3.5 min, which greatly shortens the analysis time and meets the requirements of high-throughput detection of biological samples in clinical bioequivalence studies.

[0021] The existing technologies all use atmospheric pressure chemical ionization (APCI) sources with high flow rates. The elements entering the triple quadrupole of the mass spectrometer in the same time are complex, which can easily cause contamination of the ion cavity and have an adverse effect on the life of the mass spectrometer. The present invention changes the APCI source of the mass spectrometer in the existing technology to an ESI source, and reduces the flow rate to 0.3 mL / min, which can effectively reduce the risk of contamination of the mass spectrometer cavity.

[0022] The blood sample pretreatment steps of the present invention are simplified, and optimally only 4 steps are required to achieve the same sample purification and concentration requirements for machine detection. It is not only highly operational and easy to use, but also can effectively reduce the probability of errors in the experimental process, greatly avoid the occurrence of repeated experiments, and greatly shorten the pretreatment time, thereby improving the detection efficiency of a single batch of samples.

[0023] The blood volume used in the present invention is reduced from 1000 μL in the prior art to 100 μL, and the lower limit of quantification is detected to 0.0500 ng / mL. The 10-fold reduction in the blood volume greatly reduces the volume of reagents for the supporting liquid-liquid extraction, greatly reduces the detection cost, and has higher sensitivity, which can better meet the detection of pharmacokinetic samples in the domestic bioequivalence experiment of levoamlodipine. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the mass spectrum of blank plasma of levamlodipine of the present invention; Figure 2 The mass spectrum of the quantitative lower limit of levamlodipine of the present invention; Figure 3 is the mass spectrum of chiral separation of enantiomers of the present invention; Figure 4 It is the horizon relationship diagram of left-handed ammonia chloride of the present invention. DETAILED DESCRIPTION

[0025] The following will be combined with the embodiments of the invention to clearly and completely describe the technical solutions in the embodiments of the invention. Obviously, the described embodiments are only part of the embodiments of the invention, not all of the embodiments. Based on the embodiments of the invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the invention.

[0026] Example 1 Method for detecting blood concentration of levamlodipine 1. Preparation of various solutions 1) Preparation of mobile phase A: Use a measuring cylinder to measure 1000 mL of acetonitrile and a pipette to transfer 500 L of ethanolamine into a glass bottle. Mix well and ultrasonicate for 20 min.

[0027] 2) Preparation of mobile phase B: Use a measuring cylinder to measure 1000 mL of isopropanol into a glass bottle and ultrasonicate for 20 min.

[0028] 3) Preparation of stock solution solvent: Use a measuring cylinder to measure 100 mL of methanol into a glass bottle.

[0029] 4) Preparation of working solution dilution: Use a measuring cylinder to measure 300 mL of methanol and 100 mL of water into a glass bottle.

[0030] 5) Preparation of extraction agent: Use a measuring cylinder to measure 500 mL of ethyl acetate into a glass bottle, add 5 mL of ammonia water, and mix well.

[0031] 6) Reconstitution solution: Use a measuring cylinder to measure 95 mL of mobile phase A and a pipette to transfer 5 mL of mobile phase B into a glass bottle and mix well.

[0032] 2. Preparation of stock solution 1) Preparation of stock solution of the test substance: Weigh about 10 mg of levamlodipine besylate reference substance and place it in a centrifuge tube. After correction by mass correction factor, pipette an appropriate amount of stock solution solvent to dissolve it and prepare a solution with a final concentration of 1 mg / mL.

[0033] 2) Preparation of internal standard stock solution (Stock-IS): Take one isotope internal standard levamlodipine besylate-d4 reference substance, pipette an appropriate amount of stock solution solvent to dissolve it, and prepare a solution with a final concentration of 0.1 mg / mL.

[0034] 3. Preparation of working solution 1) Working solution of standard curve and quality control samples: At room temperature and yellow light, serially dilute the analyte levoamlodipine stock solution with the working solution diluent to prepare working solutions of standard curve samples and quality control samples. All working solutions are prepared in glass bottles and stored in a freezer. The specific preparation process is shown in the table below.

[0035]

[0036] 2) Internal standard working solution: At room temperature and yellow light, dilute the internal standard stock solution with the working solution diluent to an internal standard working solution (WS-IS) with a concentration of 40.0 ng / mL. All working solutions are prepared in glass bottles and stored in a freezer. The specific preparation process is shown in the table below:

[0037] 4. Preparation of standard curve and quality control samples: 1) The standard curve samples of levamlodipine contain the following concentration levels: 1.00, 2.00, 5.00, 16.00, 40.0, 80.0, 160 and 200 ng / mL. They are arranged from low to high in terms of concentration and named STD1, STD2, ... STD8. STD0 represents the blank matrix sample with only the internal standard added, and DB represents the sample containing neither the analyte nor the internal standard.

[0038] 2) The quality control samples of levoamlodipine contain the following concentration levels: lower limit of quantitation quality control sample (LLOQ QC, 0.0500 ng / mL), low concentration quality control sample (LQC, 1.50 ng / mL), medium concentration quality control sample (MQC, 5.00 ng / mL), and high concentration quality control sample (HQC, 7.50 ng / mL). The specific preparation process is shown in the table below:

[0039] 5. Sample Pretreatment 1) Take 100 μL of samples into strip tubes, including blank samples, standard curve samples, quality control samples, and test samples; 2) Add 20 μL of 40.0 ng / mL amlodipine-d4 solution to the sample, and add 20 μL of 75% methanol aqueous solution to the blank sample; 3) Add 400 μL of ethyl acetate solution containing 1% ammonia to the above strip tubes and vortex for 1 min; 4) Centrifuge the solution obtained in step 3) at 4°C and 3700 rpm for 10 min, take the supernatant and place it in a strip tube, place it under a nitrogen flow, and blow dry; 5) The solid obtained in step 4) was re-dissolved in 200 μL of mobile phase, vortexed for 1 min, and placed in an automatic sampler at 4°C for injection and analysis.

[0040] 6. Liquid chromatography conditions Liquid chromatograph: ExionLC AD, SCIEX Chromatographic column: Phenomenex Lux 3µm Cellulose-4 2×150mm Column temperature: 40°C Autosampler temperature: 4°C Mobile phase A: acetonitrile containing 0.05% ethanolamine Mobile phase B: Isopropanol Isocratic elution: The volume ratio of mobile phase A to mobile phase B is 95:5 Elution time: 3.5 min Injection volume: 10 μL 7. Mass Spectrometry Conditions The ion source type is: ESI source; The ionization mode is: positive ion; Detection mode: Multiple reaction monitoring; The mass spectrometry parameters are as follows:

[0041] 8. Data processing Chromatograms and concentrations were collected and calculated in Analyst (1.6.3). Concentrations were calculated using the following regression equation (linear regression with a weighting factor of 1 / x2).

[0042] y = ax + b y is the peak area ratio of levamlodipine to levamlodipine-d4 x is the concentration ratio of levoamlodipine to levoamlodipine-d4.

[0043] 9. Testing of standard solutions and samples The standard curve samples are tested using the liquid chromatography and mass spectrometry conditions of step 6 and step 7, and the standard curve can be established by performing data processing according to step 8; the samples to be tested are tested, and the concentration of levamlodipine in plasma is determined using the established standard curve.

[0044] Example 2 Enantiomer separation and quantitative lower limit detection method 75% methanol aqueous solution was used to prepare 1.00, 2.00, 5.00, 16.00, 40.0, 80.0, 160 and 200 ng / mL levamlodipine solutions, and 10 μL of the above solution was added to 190 μL of blank plasma to prepare 0.0500, 0.100, 0.250, 0.800, 2.00, 4.00, 8.00 and 10.0 ng / mL levamlodipine standard curve samples, and the samples were treated according to the sample pretreatment steps in Example 1, and the following liquid chromatography and mass spectrometry conditions were used for sample loading: Column: Phenomenex Lux 3µm Cellulose-4 2×150mm; Mobile phase: Mobile phase A is acetonitrile containing 0.05% ethanolamine, and mobile phase B is isopropanol Elution method: isocratic elution with a volume ratio of mobile phase A to mobile phase B of 95:5 for 3.5 min; Ion source type: ESI source; Ionization mode: positive ion; Detection mode: Multiple reaction monitoring; The mass spectrometry parameters are shown in the following table:

[0045] The signal-to-noise ratio was recorded at the same time. The blank matrix chromatogram and the quantitative lower limit sample chromatogram were shown in Figure 1 and Figure 2 The results showed that when the lower limit of quantification of amlodipine was 0.0500 ng / mL, the signal-to-noise ratio was good and could meet the requirements of biological sample detection sensitivity.

[0046] Mass spectra of chiral separation of enantiomers are shown in Figure 3 , dextro-amlodipine eluted at 1.26 min, and levo-amlodipine eluted at 1.64 min. The results showed that the chiral chromatographic column Phenomenex Lux 3µm Cellulose-4 2×150mm was used for chromatographic separation of enantiomers, with high specificity and strong specificity. The single-needle gradient elution time was only 3.5 min, which perfectly matched high-throughput detection and met the requirements for rapid detection of biological samples in clinical practice.

[0047] Example 3 Linear relationship detection method Preparation of standard curve working solution: Use a 1 / 10000 balance to accurately weigh 1 mg of levoamlodipine, add 1 mL of organic reagent methanol to dissolve it, and obtain a standard solution with a concentration of 1 mg / mL (named: stock-1), and then dilute it to 1.00, 2.00, 5.00, 16.00, 40.0, 80.0, 160 and 200 ng / mL with 75% methanol aqueous solution according to the dilution steps in Table 3 to obtain the standard curve working solution.

[0048] The specific dilution steps of the standard curve working solution are as follows:

[0049] Preparation of standard curve samples: 10 μL of a series of standard curve working solutions was added to 190 μL of blank plasma matrix to obtain standard curve samples with concentrations of 0.0500, 0.100, 0.250, 0.800, 2.00, 4.00, 8.00 and 10.0 ng / mL.

[0050] The samples were treated according to the sample pretreatment steps in Example 1, and the sample concentration of the standard curve of levamlodipine was used as the horizontal coordinate, and the peak area ratio of levamlodipine and the internal standard was used as the vertical coordinate. The least square method was used for regression calculation to obtain the linear regression equation, which was the standard curve. The linear relationship diagram is shown in FIG. Figure 4 The linear correlation coefficient was calculated to be r=0.9989, indicating that the detection method of the present invention has a good linear relationship within the concentration range of 0.0500-10.0 ng / mL for levamlodipine, meeting the requirement of the guiding principle of r>0.9900.

[0051] Example 4 Precision and Accuracy Test To ensure the precision and accuracy of the detection method described in the present invention, this embodiment tested four quality control concentrations, namely, the quantitative lower limit concentration, the low quality control concentration, the medium quality control concentration and the high quality control concentration, for a total of 4 concentrations of quality control samples.

[0052] The preparation of quality control working solution and quality control samples is the same as in Example 1.

[0053] The samples were processed according to the sample pretreatment steps in Example 1, and each quality control sample was paralleled with 6 copies to evaluate the precision and accuracy within the batch. The precision was evaluated by calculating the coefficient of variation (%CV) of the detection concentration, and the mean and deviation of the detection concentration were calculated to evaluate the accuracy. The results are detailed in the table below.

[0054] Intra-batch precision and accuracy

[0055] According to the current version of the Chinese Pharmacopoeia, Part IV, General Rules 9012, Validation Guidelines for Quantitative Analysis Methods of Biological Samples and ICHM10 Guidelines, the mean of intra-batch accuracy should generally be within ±15% of the labeled value of the quality control sample, and the accuracy of the lower limit of quantification should be within ±20% of the labeled value; the coefficient of variation of intra-batch precision should generally not exceed 15%, and the coefficient of variation of the lower limit of quantification should not exceed 20%. It can be seen from the results that the precision and accuracy of each quality control concentration sample are within the range required by the guidelines, indicating that the detection method of the present invention has good precision and accuracy.

[0056] Example 5 BE sample determination The present invention uses LC-MS / MS method to quantitatively detect the concentration of levoamlodipine in EDTA-K2 anticoagulated human plasma samples in the "randomized, open, double-period, double-crossover design bioequivalence study of levoamlodipine tablets in healthy human fasting / postprandial state". A total of 2304 fasting / postprandial samples were tested. The Cmax of amlodipine (1.71 ng / mL~5.08 ng / mL) in all individuals met the requirement that the lower limit of quantification (0.0500 ng / mL) was lower than 1 / 10-1 / 20 of Cmax, indicating that the linear range setting of this method was reasonable; a total of 180 samples were selected for biological sample reanalysis, and the percentage deviation (%D) of all samples was between -14.0% and 8.0%, which met the requirements of the current edition of the "Chinese Pharmacopoeia" General Chapter 9012 Validation Guidelines for Quantitative Analysis Methods of Biological Samples and the ICH M10 Guidelines: at least 67% of the reanalyzed samples had %D within the acceptance standard of ±20.0%, proving that the invented method is robust and reliable.

[0057] Comparative Example 1: Prior Art 1 1. Sample pretreatment steps: Step 1: Accurately measure 1000μL of sample plasma and place it in a 1.5mL centrifuge tube. The sample includes blank samples, standard curve samples, quality control samples, and test samples; Step 2: Accurately add 10 μL of internal standard working solution (except blank sample); Step 3: Add 20 μL of 5% ammonium acetate aqueous solution, vortex mix for 1 min, centrifuge at 7000 g for 10 min, and draw all the supernatant for solid phase extraction; Step 4: Activation of solid phase cartridge: First, activate the solid phase extraction cartridge (HLB) with 1 mL of methanol and 1.5 mL of water; Step 5: Loading the solid phase column: The supernatant of the centrifuged plasma sample is passed through the column, and then washed with 1 mL of water, 1 mL of 20% formic acid aqueous solution, and 1 mL of 70% methanol aqueous solution to elute and remove the plasma protein components; Step 6: Washing the solid phase column: Wash the solid phase extraction column with 1 mL of water, 1 mL of 20% formic acid aqueous solution, and 1 mL of 70% methanol aqueous solution to remove plasma protein components; Step 7: Elution from solid phase column: Finally, elute the target compound with 1 mL of methanol solution containing 2.5% ammonia water, and collect the eluate; Step 8: Concentration after solid phase extraction: The eluate was blown dry in a nitrogen stream in a 30°C water bath; Step 9: Reconstitute and sample after concentration: Add 50 μL of mobile phase to the residue and vortex to mix for 5 minutes. Centrifuge at 16000g for 10 minutes and take 25 μL for injection and analysis.

[0058] 2. Chromatographic conditions: The chiral enantiomers of amlodipine racemate were separated using a CHIRAL-AGP column (150 mm × 4 mm, 5 μm). The mobile phase was 10 mmol / L ammonium acetate buffer (pH 4.38)-isopropanol (98:2, V / V). The flow rate was 0.7 mL / min, and the separation was isocratic. The single injection time was 20 min (the elution times of dextro-amlodipine and levo-amlodipine were 12.3 min and 16.9 min, respectively). The injection volume was 25 μL, and the analysis was performed at room temperature.

[0059] 3. Mass spectrometry conditions: Atmospheric pressure chemical ionization source (APCI), corona discharge current: 12μA, heated capillary temperature: 200℃, vaporization chamber temperature: 450℃, sheath gas (N2) pressure: 0.36 Mpa, auxiliary gas (N2) flow: 3L / min, collider argon pressure: 0.24pa, positive ion detection, multiple ion selected reaction monitoring (MRM) scanning analysis, ion reaction used for quantitative analysis: m / z409.0 / 237.9, linear range of 0.1031-20.62ng / mL.

[0060] Comparative Example 2: Prior Art 2 1. Sample pre-treatment steps are as follows: Step 1: Under light-proof conditions, accurately measure 1000 μL of sample plasma and place it in a glass test tube. The sample includes blank samples, standard curve samples, quality control samples, and test samples; Step 2: Accurately add 10 μL of internal standard working solution (except blank sample); Step 3: Add 100 μL of mobile phase, 100 μL of internal standard solution and 200 μL of alkalizing agent (1 mol / L sodium carbonate aqueous solution) in sequence and mix well; Step 4: Add 4 mL of ether, vortex mix for 1 min, and centrifuge at 3500 r / min for 10 min; Step 5: Take the upper ether layer into another dry test tube and blow dry it in a nitrogen stream in a 40°C water bath; Step 6: The residue was dissolved in 200 μL of mobile phase and analyzed by LC-MS / MS.

[0061] 2. Chromatographic conditions: The chiral enantiomers of amlodipine racemate were separated using an Ultron ES-OVM chiral column (150 mm × 4.6 mm, 5 μm). The mobile phase was phosphate buffer at 1 mL / min, the column temperature was 35 °C, the single needle elution time was 9 min, the injection volume was 25 μL, and the analysis was performed at room temperature.

[0062] Mass spectrometry conditions: atmospheric pressure chemical ionization source (APCI), ionization voltage 4000 V, vaporization chamber temperature: 450 ° C, curtain gas, nebulizer gas and collision gas are all nitrogen, with flow rates of 20, 35 and 4 L / min respectively, positive ion detection, multiple ion selected reaction monitoring (MRM) scanning analysis, the ion reaction used for quantitative analysis is m / z 409 / 238, and the lower limit of quantification is 0.100 ng / mL.

[0063] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for chiral separation and determination of levamlodipine in human plasma by LC-MS / MS, characterized in that: The following steps are involved: A. Pretreatment of the sample to be tested Add 10-50 μL of internal standard working solution to the plasma to be tested, then add the extractant, vortex, centrifuge, then take the supernatant and concentrate to dryness, and finally re-dissolve; B. Preparation of Standard Curve and Quality Control Samples Prepare the working solution of levamlodipine standard curve samples and quality control samples, add the working solution to the blank plasma, mix well, and prepare a series of levamlodipine standard curve samples and quality control samples with a concentration gradient; C. LC-MS / MS determination Take the standard curve sample in step B for detection to establish a standard curve for amlodipine; The sample to be tested after the treatment in step A is taken for testing, and the concentration of levamlodipine in the sample to be tested is calculated using the established standard curve; The specific steps of the sample pretreatment are as follows: Step 1: Take 50-200 μL of sample into a strip tube, add 10-50 μL of internal standard working solution to the sample, and add 10-50 μL of 75% methanol in water to the blank sample; Step 2: Add 200-800 μL of extraction reagent to the above tube strips and vortex for 1 min; Step 3: Centrifuge at 4°C and 3700 rpm for 10 min. Transfer 150-600 μL of the supernatant to a strip tube and dry under a stream of nitrogen. Step 4: Redissolve with 100-400 μL of mobile phase and vortex for 1 minute to obtain the sample to be analyzed.

2. The method for chiral separation and determination of levamlodipine in human plasma by LC-MS / MS according to claim 1, characterized in that: The chromatographic conditions for the LC-MS / MS assay are: Liquid chromatography-mass spectrometry (LC-MS) was used as the detection instrument for analysis and detection: AB Sciex Triple Quad™ 5500 LC-MS was used as the detection instrument; Chromatographic column: Chiral column Phenomenex Lux 3 µm Cellulose-4 2×150 mm; Mobile phase: Mobile phase A is acetonitrile containing 0.05% ethanolamine, and mobile phase B is isopropanol; Elution mode: isocratic elution.

3. The method for chiral separation and determination of levamlodipine in human plasma by LC-MS / MS according to claim 1, characterized in that: The mass spectrometry conditions for the LC-MS / MS determination are: Ion source type: ESI source; Ionization mode: positive ion; Detection mode: Multiple reaction monitoring; The mass spectrometry parameters are shown in the following table: 。 4. The method for chiral separation and determination of levamlodipine in human plasma by LC-MS / MS according to claim 1, characterized in that: The internal standard working solution described in step A is a 40.0 ng / mL levamlodipine-d4 solution.

5. The method for chiral separation and determination of levamlodipine in human plasma by LC-MS / MS according to claim 1, characterized in that: The extractant in step A is an ethyl acetate solution containing 1% ammonia water.

6. The method for chiral separation and determination of levamlodipine in human plasma by LC-MS / MS according to claim 1, characterized in that: The preparation process of the working solutions of the standard curve samples and quality control samples of levamlodipine in step B is as follows: under room temperature and yellow light conditions, the stock solution of the analyte levamlodipine is serially diluted with the working solution diluent (75% methanol in water) to prepare the working solutions of the standard curve samples and quality control samples. All working solutions are prepared in glass bottles and stored in a freezer.

7. The method for chiral separation and determination of levamlodipine in human plasma by LC-MS / MS according to claim 1, characterized in that: In step C, the volume ratio of mobile phase A to mobile phase B in the isocratic elution is 95:

5.

8. The method for chiral separation and determination of levamlodipine in human plasma by LC-MS / MS according to claim 1, characterized in that: In step C, the column temperature of the chromatographic column was 40° C., the temperature of the automatic sampler was 4° C., and the injection volume was 10 μL.

9. The method for chiral separation and determination of levamlodipine in human plasma by LC-MS / MS according to claim 1, characterized in that: In step C, the isocratic elution time is 3.5 min.

10. The method for chiral separation and determination of levamlodipine in human plasma by LC-MS / MS according to claim 1, characterized in that: The concentration range of the levamlodipine standard curve samples is 0.0500-10.0 ng / mL.

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