Analysis method for rapidly determining concentration of lamidetan in human plasma

Abstract

The invention provides an analysis method for rapidly determining the concentration of lamidetan in human plasma. The method comprises the following steps: adding an internal standard labetalol solution and acetonitrile into a plasma sample, carrying out vortex, centrifuging, collecting a supernatant, adding an acetonitrile-water mixture into the supernatant, and carrying out vortex mixing to obtain a pretreated sample to be detected; the method comprises the following steps: preparing an electrospray ionization source for detection by adopting liquid chromatography-tandem mass spectrometry, carrying out isocratic elution by adopting a Venusil MP-C18 (2) chromatographic column, carrying out liquid chromatography separation on a to-be-detected sample, then carrying out mass spectrometry detection, drawing a standard curve based on a detection peak area ratio to obtain a regression equation, and finally calculating to obtain the lamidetan concentration in the to-be-detected sample. The method has the characteristics of simplicity and convenience in pretreatment operation, low plasma consumption and short analysis time, and is suitable for detection of lamidetan in plasma and clinical pharmacokinetic research.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and specifically relates to a method for rapidly determining selective 5-HT in human plasma. 1F Method for analyzing the concentration of the agonist lamivudine. Background Art

[0002] Lamiditan is a novel selective 5-HT 1F Agonist, and has central nervous system penetration. 1F The receptor has a high affinity and is presumed to stimulate 5-HT 1F Receptors, thereby inhibiting central and peripheral neuronal activity and the release of CGRP, achieving the effect of treating migraine. Lamiditan tablets were developed by Eli Lilly and Company of the United States and successfully obtained marketing approval from the U.S. Food and Drug Administration (FDA) on October 11, 2019. The trade name is: For the acute treatment of adult migraine patients with or without aura symptoms. It is worth noting that its mechanism of action is different from the currently widely used "triptan" migraine treatment drugs. Lamiditan does not have a vasoconstrictive effect, so it does not increase the risk of cardiovascular disease. It is safer for migraine patients with cardiovascular disease or at risk of cardiovascular disease. It is not only the first, but also the only triptan drug approved by the FDA for the acute treatment of adult migraine.

[0003] In order to promote the development of lamivudine generic drugs, it is necessary to monitor its concentration level in human plasma to obtain pharmacokinetic characteristics and then evaluate the bioequivalence of the original drug and generic drugs. However, there is currently no analytical method reported in any literature for the determination of lamivudine in human plasma. Summary of the invention

[0004] In order to fill the gap in the prior art, the object of the present invention is to provide a method for rapidly determining the concentration of lamivudine in human plasma.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] In one aspect, the present invention provides a method for rapidly determining the concentration of lamivudine in human plasma, comprising the following steps:

[0007] An internal standard solution and acetonitrile are added to the plasma sample, vortexed, and the supernatant is collected after centrifugation. An acetonitrile-water mixture is added to the supernatant, and vortexed to mix to obtain a pretreated sample to be tested;

[0008] Liquid chromatography-tandem mass spectrometry equipped with electrospray ionization source detection and isocratic elution on a Venusil MP-C18(2) column were used to separate the test samples by liquid chromatography and then detect them by mass spectrometry. A standard curve was plotted based on the detection peak area ratio to obtain a regression equation, and finally the concentration of lamiditan in the test samples was calculated.

[0009] In the above method, preferably, the internal standard solution is labetalol.

[0010] In the above method, preferably, the specific method of the pretreatment includes:

[0011] Add 50.0 μL of plasma sample to a 96-well plate, then add 50.0 μL of internal standard solution and 300 μL of acetonitrile, vortex mix and centrifuge to collect the supernatant, take 50.0 μL of supernatant to another clean 96-well plate, then add 150 μL of acetonitrile-water mixture, vortex mix to obtain the sample to be tested.

[0012] In the above method, preferably, the centrifugation time is 10 min, the centrifugation temperature is 4° C., and the centrifugation speed is 3900 rpm.

[0013] In the above method, preferably, the concentration of the internal standard solution is 500 ng / mL.

[0014] In the above method, preferably, the ratio of the acetonitrile-water mixture is 1:1 by volume.

[0015] In the above method, preferably, the chromatographic column used for liquid chromatography separation is: Venusil MP-C18 (2) chromatographic column, 5.0 μm, 4.6×100 mm; the mobile phase used is: phase A: an aqueous solution containing 0.2% formic acid and 5 mM ammonium acetate, phase B: acetonitrile.

[0016] In the above method, preferably, the liquid chromatography separation and elution conditions are:

[0017] Isocratic elution: Phase A: Phase B = 50%: 50%

[0018] Elution time: 2.70min;

[0019] Flow rate: 0.650 mL / min;

[0020] Injection volume: 1.00 μL;

[0021] Autosampler temperature: 4 °C;

[0022] Column temperature: 40℃.

[0023] In the above method, preferably, the mass spectrometry conditions for mass spectrometry detection are:

[0024] Ion source: electrospray ionization (ESI);

[0025] Spray voltage: 5500V;

[0026] Spray gas (Gas1): 65psi;

[0027] Auxiliary gas (Gas2): 65psi;

[0028] Detection method: positive ion;

[0029] Ion source temperature: 500°C;

[0030] Collision-induced dissociation (CAD): 9;

[0031] Curtain Gas: 40psi;

[0032] Dwell time: 200ms.

[0033] In the above method, preferably, the mass spectrometry detection is performed using a quantitative analysis ion pair, and the quantitative analysis ion pair is:

[0034] Lamiditan m / z 378.4→360.2, collision energy (CE) 33 eV, declustering potential (DP) 80 V;

[0035] Labetalol m / z 329.1→161.9, collision energy (CE) 26 eV, declustering potential (DP) 80 V.

[0036] In the above method, preferably, the standard curve is specifically prepared as follows:

[0037] The linear regression equation was obtained by performing regression analysis with the theoretical concentration of the sample to be tested as the abscissa and the peak area ratio of the sample to be tested to the internal standard as the ordinate.

[0038] On the other hand, the present invention also provides the use of the above method in analyzing the concentration of lamiditan in a plasma sample; the lamiditan is used for the acute treatment of adult migraine patients with or without aura symptoms.

[0039] Beneficial effects of the present invention:

[0040] (1) There is no literature or patent report on the method of determining lamiditan in human plasma. Therefore, the LC-MS / MS method established in the present invention is the first of its kind and meets all the indicators of methodological validation.

[0041] (2) The present invention has the characteristics of simple pretreatment operation, and only requires one step of extraction and one step of dilution for analysis. The sample after extraction is easy to preserve and the analysis speed is fast, with the analysis time being only 2.7 minutes. Therefore, the present invention meets the requirements of high-throughput and large-volume biological sample detection.

[0042] (3) The sensitivity of the present invention is high, and the lower limit of quantification of lamiditan is 2.00 ng / mL. The method of the present invention has good accuracy, high precision, and good reproducibility, and can more accurately analyze the concentration of the drug.

[0043] (4) The LC-MS / MS method established by the present invention is used to determine the concentration of lamivudine in human plasma, which can accelerate the progress of the bioequivalence evaluation of the generic drug.

[0044] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0046] Figure 1 This is the mass spectrum of the product ion scan of lamiditan;

[0047] Figure 2 The MRM chromatograms of lamiditan (upper) and internal standard labetalol (lower) in blank plasma samples;

[0048] Figure 3 The MRM chromatograms of lamiditan (upper) and internal standard labetalol (lower) in the quantitative lower limit sample;

[0049] Figure 4 The MRM chromatograms of lamiditan (upper) and internal standard labetalol (lower) in the quantitative upper limit sample;

[0050] Figure 5 The drug concentration-time curve of lamiditan after a single oral administration of 100 mg lamiditan tablets to a healthy subject. DETAILED DESCRIPTION

[0051] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. The process, conditions, reagents, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and common common sense in the art, and the present invention does not particularly limit the contents.

[0052] Example

[0053] The method for rapidly determining the concentration of lamivudine in human plasma provided by the present invention can generally be divided into three parts, namely, extraction method (i.e., pretreatment method), liquid chromatography method and mass spectrometry method. In view of the shortcomings of the prior art, the present invention establishes an analytical method from the above three aspects.

[0054] 1. Preprocessing:

[0055] The amount of plasma used in the present invention is only 50.0 μL, which is suitable for clinical research bioanalysis. The extraction method in the present invention uses protein precipitation method, which has the advantages of simple operation, short extraction time, and no time-consuming concentration step. It is used with 96-well plates and is suitable for high-throughput sample pretreatment in clinical research.

[0056] The specific pre-processing steps are as follows:

[0057] 1. Add 50.0 μL of plasma sample, 50.0 μL of internal standard solution (labetalol concentration is 500 ng / mL), and 300 μL of acetonitrile to a 96-well plate;

[0058] 2. Vortex mix and centrifuge for 10 min (4°C, 3900 rpm);

[0059] 3. Take 50.0 μL of supernatant into another clean 96-well plate;

[0060] 4. Add 150 μL of acetonitrile-water (1:1, v / v) and vortex to mix.

[0061] 5. The injection volume is 1.0 μL.

[0062] 2. Chromatographic analysis:

[0063] Chromatography: The sample to be tested was separated by liquid chromatography using a Venusil MP-C18(2) column and isocratic elution [50% phase A: 50% phase B]. Mobile phase A was an aqueous solution containing 0.2% formic acid and 5 mM ammonium acetate, and mobile phase B was acetonitrile.

[0064] Lamiditan has a small molecular weight and is easily interfered by other endogenous substances in plasma. The chromatographic separation of the present invention uses a Venusil MP-C18 (2) chromatographic column, which has good retention for both the analyte and the internal standard, and the peak shape is symmetrical. In this mode, the instrument analysis throughput is high, the chromatographic running time is only 2.7 minutes, the detection is fast, and it is suitable for large-scale sample analysis in clinical research.

[0065] 3. Mass spectrometry analysis:

[0066] An electrospray ion source and positive ion detection were used, with a spray voltage of 5500 V, gas 1 (Gas1) 65 psi, gas 2 (Gas2) 65 psi, curtain gas (Curtain Gas) 40 psi, ion source temperature of 500°C, collision induced dissociation 9, dwell time of 200 ms, the quantitative analysis ion pair of lamiditan was m / z 378.4→360.2, collision energy (CE) 33 eV, and declustering voltage (DP) 80 V, and the quantitative analysis ion pair of internal standard labetalol was m / z 329.1→161.9, collision energy (CE) 26 eV, and declustering voltage (DP) 80 V.

[0067] The present invention is described in detail below through specific embodiments:

[0068] Embodiment 1:

[0069] Abbreviation Description:

[0070]

[0071] 1 Materials

[0072] 1.1 Instruments

[0073] Chromatograph: LC-30AD fast liquid chromatography system, Shimadzu Corporation, Japan.

[0074] Mass spectrometer: Model 5500 triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source (Turbo Ion Spray) from Sciex, Canada.

[0075] Data processing software used: Analyst (version 1.6.3), Sciex, Canada.

[0076] Centrifuge: 5810R multifunctional desktop centrifuge, Eppendorf, Germany.

[0077] Analytical balance: CPA225D analytical balance, Beijing Sartorius Instrument Co., Ltd.

[0078] 1.2 Reference substances and reagents

[0079] Lamiditan (content 98.9%) was purchased from CATO, and labetalol hydrochloride (content 99.8%) was purchased from China Food and Drug Inspection Institute. Methanol (HPLC grade) was purchased from Sigma, USA. Formic acid (HPLC grade) was purchased from TCI. Ammonium acetate (HPLC grade) was purchased from ROE. Deionized water (18.2 mΩ, TOC ≤ 50 ppb) was prepared by Milli-Q ultrapure water system.

[0080] 2 Methods

[0081] 2.1 Solution and sample preparation

[0082] Standard series samples: Accurately weigh an appropriate amount of reference substance, dissolve it in methanol and make up to volume to prepare a stock solution with a concentration of about 1.00 mg / mL of lamiditan. Accurately pipette an appropriate amount of each stock solution and dilute it stepwise with human blank plasma to obtain a mixed standard series of samples, with a lamiditan concentration range of 2.00 -500 ng / mL.

[0083] Quality control samples: Four mixed quality control samples of lamiditan were prepared in a similar manner to the standard series samples. The concentration of low quality control (LQC) was 5.00 ng / mL, the concentration of accessory middle quality control (AMQC) was 40.0 ng / mL, the concentration of medium quality control (MQC) was 180 ng / mL, and the concentration of high quality control (HQC) was 400 ng / mL.

[0084] Internal standard solution: Accurately weigh the labetalol hydrochloride reference substance, dissolve it in methanol and make up to volume to prepare an internal standard stock solution with a concentration of about 1.00 mg / mL. Accurately pipette an appropriate amount of the above internal standard stock solution, dilute it with methanol, and obtain an internal standard solution with a labetalol concentration of 500 ng / mL.

[0085] 2.2 Plasma sample processing

[0086]

[0087]

[0088] 2.3 Chromatographic and mass spectrometry conditions Chromatographic conditions:

[0089]

[0090] Mass spectrometry conditions:

[0091]

[0092] 3. Verification of the Method in This Example

[0093] The LC-MS / MS method validation of lamivudine provided in this example includes selectivity, standard curve and lower limit of quantification, precision and accuracy, matrix effect, recovery, residue and stability, etc., which complies with the requirements of ICH M10 guidelines.

[0094] [Selectivity]

[0095] The peak area of ​​the chromatographic coeluting interferent at the retention time of each analyte and internal standard must be less than 20% of the LLOQ peak area (analyte) and less than 5% of the peak area (internal standard) by analyzing blank plasma and LLOQ samples from 6 different sources.

[0096]

Standard curve and lower limit of quantitation

[0097] Theoretical concentration of the analyte is taken as the horizontal axis (x), and the peak area ratio of the analyte to the internal standard is taken as the vertical axis (y). The linear regression operation is performed using the weighted (W=1 / x2) least square method, and the linear regression equation obtained is the standard curve. At least 75% of the standard curve samples and at least 6 concentration levels should meet the accuracy deviation of the calculated concentration within ±15.0% (within ±20.0% of the lower limit of quantification concentration level) to be accepted.

[0098] Accuracy and precision

[0099] At least 4 concentrations of quality control samples (low, sub-medium, medium, and high concentrations) are performed for each analysis batch, and 6 samples are analyzed for each concentration. Inter-batch accuracy and precision need to be evaluated on at least 3 analysis batches. The intra-batch and inter-batch accuracy of the quantitative lower limit should be within ±20.0%, and the precision should be within 20% to be acceptable. The intra-batch and inter-batch accuracy of the remaining concentration levels of quality control samples should be within ±15%, and the precision should be within 15.0%.

[0100]

Recovery rate

[0101] The treatment recovery was calculated by comparing the peak area ratios of the two treatment methods obtained by adding the quality control sample working solution before and after extraction.

[0102]

Matrix effect

[0103] Blank plasma from 6 different sources was used to prepare two quality control plasma samples with low and high concentrations. Three samples were analyzed for each concentration. The average accuracy of the measured values ​​of the quality control samples of each source and each concentration was within ±15.0% of the labeled value.

[0104]

Residue

[0105] After the ULOQ sample analysis, the blank plasma sample was analyzed immediately. The chromatographic peak areas at the retention times of the analyte and internal standard in the blank plasma sample and the LLOQ sample were compared to assess whether the carryover affected the accuracy of the analysis.

[0106]

stability

[0107] When investigating the stability of the analyte in plasma samples, the plasma samples at LQC and HQC concentration levels were placed in different temperatures and environments. A total of four placement conditions were investigated, namely:

[0108] 1) Short-term stability: Place at room temperature for 17 hours,

[0109] 2) Place in the autosampler at 4°C for 99 hours,

[0110] 3) undergo 5 freeze-thaw cycles (from -80°C to room temperature),

[0111] 4) Place at -80℃ for 40 days.

[0112] Stability samples at each concentration level under each storage condition were considered stable if the accuracy RE% did not exceed ±15.0%.

[0113] 4 Results of the method verification of this embodiment

[0114] Selectivity of method

[0115] The product ion scanning mass spectrum of lamiditan is shown in Figure 1 As shown in Figure 2, the retention times of lamiditan and internal standard labetalol are approximately 2.12 min and 1.87 min, respectively. Figures 2 to 4 As shown, no interference was observed at any retention time.

[0116] Standard curve and limit of quantitation

[0117] The linear range of the determination of lamiditan in plasma samples from the clinical study of lamiditan is 2.00-500ng / mL, and the typical linear regression equation of the standard curve of the analyte is:

[0118] Lamiditan: y = 0.0153x + 0.000402 (r 2 =0.9986)

[0119] The concentration of lamivudine in the quantitative lower limit sample was 2.00 ng / mL. The intra- and inter-batch accuracy of the quantitative lower limit was between -4.4% and 2.9%, and the intra- and inter-batch precision was within 4.9%, both meeting the acceptance criteria.

[0120] Precision and accuracy of the method

[0121] The intra-batch and inter-batch accuracy of lamivudine was between -1.1% and 10.6%, and the intra-batch and inter-batch precision was within 4.7%, both meeting the acceptance criteria. The results are shown in Table 1.

[0122] Table 1: Precision and accuracy of lamivudine in human plasma

[0123]

[0124] Treatment recovery rate

[0125] At the LQC, MQC and HQC concentration levels, the extraction recoveries of lamiditan were 97.6%, 101.4% and 100.1% respectively, and the recovery of internal standard labetalol was 104.2%, meeting the requirements of analytical testing.

[0126] Matrix Effects

[0127] The average accuracy RE of the quality control samples from different sources was between -3.4% and 2.8% of the labeled value. The above results show that the matrix effect does not interfere with the accuracy of the analysis of the analyte.

[0128] Residue

[0129] After the ULOQ sample analysis, the blank plasma sample was analyzed immediately. The peak areas of the analyte and internal standard retention time in the blank plasma sample were less than 20% of the LLOQ analyte peak area and less than 5% of the internal standard peak area, indicating that there was no residual effect under this method.

[0130] Plasma stability study

[0131] The results of the plasma stability test are shown in Table 2, which show that lamiditan is stable under the conditions investigated.

[0132] Table 2 Stability of lamiditan in human plasma under different conditions

[0133]

[0134] Example 2: Application of the method of Example 1 in human pharmacokinetic studies

[0135] The validated method was used to analyze lamiditan in human plasma to evaluate the pharmacokinetic characteristics of lamiditan. A healthy subject took a single oral dose of 100 mg lamiditan tablets, and the plasma drug concentration-time curve was shown in Figure 5 The sensitivity of the detection method can fully describe the pharmacokinetic characteristics of lamiditan, and the method has good reproducibility and high measurement accuracy.

[0136] The present invention uses specific embodiments to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, for those skilled in the art, according to the idea of ​​the present invention, there will be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as a limitation on the present invention.

Claims

1. A method for rapidly determining the concentration of lamivudine in human plasma, comprising the following steps: Adding internal standard solution and acetonitrile to the plasma sample, vortexing, collecting the supernatant after centrifugation, adding acetonitrile-water to the supernatant, vortexing and mixing to obtain the pretreated sample to be tested; Liquid chromatography-tandem mass spectrometry equipped with electrospray ionization source detection and isocratic elution on a Venusil MP-C18(2) column were used to separate the test samples by liquid chromatography and then detect them by mass spectrometry. A standard curve was plotted based on the detection peak area ratio to obtain a regression equation, and finally the concentration of lamiditan in the test samples was calculated.

2. The method according to claim 1, wherein: The stable isotope internal standard solution is labetalol.

3. The method according to claim 1, wherein: The specific method of the pretreatment includes: Add 50.0 μL of plasma sample to a 96-well plate, then add 50.0 μL of internal standard solution and 300 μL of acetonitrile, vortex mix and centrifuge to collect the supernatant, take 50.0 μL of supernatant to another clean 96-well plate, then add 150 μL of acetonitrile-water mixture, vortex mix to obtain the sample to be tested. In the above method, preferably, the centrifugation time is 10 min, the centrifugation temperature is 4° C., and the centrifugation speed is 3900 rpm.

4. The method according to claims 1 to 3, wherein: The concentration of the internal standard solution is 500 ng / mL, and the ratio of the acetonitrile-water mixture is 1:1 by volume.

5. The method according to claim 1, wherein: The chromatographic column used for liquid chromatography separation is: Venusil MP-C18 (2) chromatographic column, 5.0 μm, 4.6×100 mm; the mobile phases used are: Phase A: an aqueous solution containing 0.2% formic acid and 5 mM ammonium acetate, Phase B: acetonitrile.

6. The method according to claim 5, wherein: The elution conditions for liquid chromatography separation are: Isocratic elution: Phase A: Phase B = 50%: 50% Elution time: 2.70min; Flow rate: 0.650 mL / min; Injection volume: 1.00 μL; Autosampler temperature: 4 °C; Column temperature: 40℃.

7. The method according to claim 1, wherein: The mass spectrometry conditions for mass spectrometry detection were: Ion source: electrospray ionization (ESI); Spray voltage: 5500V; Spray gas (Gas1): 65psi; Auxiliary gas (Gas2): 65psi; Detection method: positive ion; Ion source temperature: 500°C; Collision-induced dissociation (CAD): 9; Curtain Gas: 40psi; Dwell time: 200ms.

8. The method according to claim 1 or 6, wherein: The mass spectrometry detection was performed using a quantitative analysis ion pair, wherein the quantitative analysis ion pair is: Lamiditan m / z 378.4→360.2, collision energy (CE) 33 eV, declustering potential (DP) 80 V; Labetalol m / z 329.1→161.9, collision energy (CE) 26 eV, declustering potential (DP) 80 V.

9. The method according to claim 1, wherein: The standard curve is specifically made as follows: The linear regression equation was obtained by performing regression analysis with the theoretical concentration of the sample to be tested as the abscissa and the peak area ratio of the sample to be tested to the internal standard as the ordinate.

10. Use of the method according to any one of claims 1 to 9 in analyzing the concentration of lamiditan in a plasma sample; the lamiditan is used for the acute treatment of adult migraine patients with or without aura symptoms.

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