Method for simultaneously determining empagliflozin and metformin in human plasma

Abstract

The invention relates to the technical field of blood detection, in particular to the field of IPC G01N30, and more particularly relates to a method for simultaneously determining empagliflozin and metformin in human plasma. Comprising the following steps: S1, pretreating a to-be-detected sample by using a protein precipitation method; s2, carrying out liquid chromatography-tandem mass spectrometry detection on the pretreated sample; s3, a linear regression equation is obtained through fitting, y = ax + b, y is the area ratio of the to-be-detected substance to the internal standard peak of the to-be-detected substance, and x is the theoretical concentration of the standard curve sample of the to-be-detected substance. A protein precipitation method is adopted for sample pretreatment, and the method is rapid and reliable; meanwhile, the operation time of a single needle is as low as 2.3 min, two substances of empagliflozin and metformin can be detected at the same time, the detection efficiency is effectively improved, the dosage of a reagent is reduced, and the service life of an instrument is shortened.

Description

Technical Field

[0001] The present invention relates to the field of blood testing technology, in particular to the field of IPC G01N30, and more specifically to a method for simultaneously determining empagliflozin and metformin in human plasma. Background Art

[0002] The current goal of diabetes treatment is to achieve and maintain blood sugar levels as close to normal as possible to prevent long-term microvascular and macrovascular complications associated with elevated blood sugar. Oral treatments for the treatment of type 2 diabetes can be selected from known compounds: sulfonylureas, biguanides, thiazolidinediones, and α-glucosidase inhibitors, and each type of active drug is usually administered to the patient alone. However, when monotherapy becomes inappropriate, combination therapy is required. The chemical name of metformin hydrochloride is 1,1-dimethylbiguanide hydrochloride. Its mechanism of action is different from other types of oral anti-glycemic drugs. It can reduce the production of glycogen, reduce intestinal absorption of sugar, and can increase insulin sensitivity by increasing peripheral sugar uptake and utilization. It has become the first choice for patients with mild to moderate type 2 diabetes, especially obese type 2 diabetes patients. Empagliflozin is a sodium-glucose co-transporter 2 (SGLT2) inhibitor in the kidney. Its chemical formula is (1S)-1,5-anhydro-1-C-[4-chloro-3-[[4-[[(3S)-tetrahydro-3-furanyl]oxy]phenyl]methyl]phenyl]-D-glucitol. It can increase the excretion of glucose in urine and thus lower blood glucose levels independently of β-cell function. The simultaneous and efficient detection of empagliflozin and metformin in plasma is crucial for clinical monitoring.

[0003] The prior art does not involve a method for simultaneously detecting empagliflozin and metformin in plasma. For example, Chinese invention patent CN106706769A discloses a method for separating and determining empagliflozin and its optical isomers. The method uses high-performance liquid chromatography with a chiral column of amylose or cellulose derivatives as a chromatographic column to separate and determine empagliflozin and its optical isomers, which can be used for quality control of empagliflozin and pharmaceutical compositions containing empagliflozin. Summary of the invention

[0004] The present invention provides a method for simultaneously determining empagliflozin and metformin in human plasma, comprising the following steps:

[0005] S1, pre-preparation of samples using protein precipitation method;

[0006] S2, subjecting the pre-prepared samples to liquid chromatography-tandem mass spectrometry detection;

[0007] S3, fitting to obtain a linear regression equation, y=ax+b, to determine the contents of empagliflozin and metformin in the analyte, wherein y is the peak area ratio of the analyte and its internal standard, and x is the theoretical concentration of the standard curve sample of the analyte.

[0008] The step S1 specifically includes:

[0009] a. Mix the standard curve samples except the upper limit of quantitation, quality control samples, pre-equilibrium samples, double blank samples, pre-equilibrium treated double blank samples, SST (system suitability) samples and test samples with the internal standard sample respectively;

[0010] b, respectively mixing the double blank sample and the pre-equilibrated double blank sample with the methanol solution;

[0011] c, mix the standard curve sample of the upper limit of quantitation with the methanol solution;

[0012] d. Add acetonitrile to all samples and mix well.

[0013] This application study found that the use of protein precipitation method to pre-prepare samples, and the simultaneous setting of standard curve samples, quality control samples, pre-equilibrium samples, double blank samples, pre-equilibrium treated double blank samples, and SST (system suitability) samples effectively improved the system suitability, and was able to process multiple samples at the same time, improving the experimental efficiency. Through specific solvents, certain groups in protein molecules can interact with each other, causing changes in the chemical structure of the protein, thereby causing it to lose its original solubility and stability. When the protein denatures and precipitates, it releases small molecules bound to it.

[0014] The standard curve samples include empagliflozin standard curve samples and metformin standard curve samples; the gradients of the empagliflozin standard curve samples and metformin standard curve samples are 8-12; and the concentration range of the standard curve samples is 3-4000 ng / mL.

[0015] The present application study found that the gradients of the standard curve samples of empagliflozin and metformin are 8-12; the concentration range of the standard curve samples is 3-4000ng / mL, which can take into account the detection accuracy, sensitivity and repeatability. The more gradients there are, that is, the more points there are in the standard curve, the smaller the uncertainty of the sample. This is because more gradient points can more accurately describe the functional relationship between the physical or chemical properties of the standard substance and the instrument response, thereby improving the accuracy of the experimental results. However, too many gradients may cause baseline drift and increased noise, thereby affecting accuracy. By using a concentration range of 3-4000ng / mL, the problem of reduced accuracy caused by misalignment and overlap of chromatographic peaks due to gradient lag and delay can be solved.

[0016] Preferably, the gradient of the empagliflozin standard curve samples and the metformin standard curve samples is 8-10.

[0017] Further preferably, the gradients of the empagliflozin standard curve samples and the metformin standard curve samples are 8.

[0018] The quality control samples include empagliflozin quality control samples and metformin quality control samples; the gradients of the empagliflozin quality control samples and metformin quality control samples are 3-6; and the concentration range of the standard curve samples is 3-3000 ng / mL.

[0019] Preferably, the gradient of the empagliflozin quality control sample and the metformin quality control sample is 3-4.

[0020] Further preferably, the gradients of the empagliflozin standard curve samples and the metformin standard curve samples are 4.

[0021] Preferably, the gradient of the empagliflozin quality control sample is: lower limit of quantitation (LLOQ QC, 3.00 ng / mL), low concentration (LQC, 9.00 ng / mL), medium concentration (GMQC, 42.0 ng / mL, MQC, 180 ng / mL), and high concentration (HQC, 450 ng / mL).

[0022] Preferably, the gradient of the metformin quality control sample is: lower limit of quantitation (LLOQ QC, 20.0 ng / mL), low concentration (LQC, 60.0 ng / mL), medium concentration (GMQC, 280 ng / mL, MQC, 1200 ng / mL), and high concentration (HQC, 3000 ng / mL).

[0023] Preferably, the gradient of the empagliflozin standard curve sample is: 3.00 ng / mL, 6.00 ng / mL, 15.0 ng / mL, 60.0 ng / mL, 120 ng / mL, 240 ng / mL, 480 ng / mL and 600 ng / mL, wherein 3.00 ng / mL is the lower limit of quantification (LLOQ) and 600 ng / mL is the upper limit of quantification (ULOQ).

[0024] Preferably, the gradient of the metformin standard curve sample is: 20.0 ng / mL, 40.0 ng / mL, 100 ng / mL, 400 ng / mL, 800 ng / mL, 1600 ng / mL, 3200 ng / mL and 4000 ng / mL; wherein 20.0 ng / mL is the lower limit of quantification (LLOQ) and 4000 ng / mL is the upper limit of quantification (ULOQ).

[0025] This application study found that by setting quality control samples and selecting specific gradients, the intra-batch and inter-batch precision and accuracy can be improved. The separation effect of quality control samples in liquid chromatography is affected by gradient changes. When the gradient changes too quickly, some components may not be fully separated, thereby affecting the accuracy of the quantitative results. On the contrary, if the gradient changes too slowly, although the separation can be improved, it may extend the analysis time and reduce the efficiency of the experiment. At the same time, the synergy between the specific gradient setting and the standard curve sample gradient can maintain the peak shape stability of the chromatographic peak. Avoid phenomena such as peak shape deformation, reduced peak height, reduced or increased peak area, etc., thereby affecting the accuracy of qualitative and quantitative results.

[0026] The internal standard sample includes a solution prepared by metformin-d6 and empagliflozin-d4.

[0027] The pre-balanced samples, double blank samples, pre-balanced double blank samples, and SST samples are all K2EDTA (dipotassium ethylenediaminetetraacetate) human plasma.

[0028] The parameters of the chromatographic column of the liquid chromatography are: inner diameter 1-3 mm, length 50-150 mm, particle size 1-5 μm, pore size

[0029] Preferably, the parameters of the chromatographic column of the liquid chromatography are: inner diameter 2-3 mm, length 50-150 mm, particle size 1-5 μm, pore size

[0030] Further preferably, the parameters of the chromatographic column of the liquid chromatography are: inner diameter 2-3 mm, length 50-100 mm, particle size 1-3 μm, pore size

[0031] More preferably, the parameters of the chromatographic column of the liquid chromatography are: inner diameter 2.1 mm, length 50 mm, particle size 1.8 μm, pore size

[0032] The column temperature of the liquid chromatography is 35-45°C, and the temperature of the automatic sample injector is 5-12°C.

[0033] Preferably, the column temperature of the liquid chromatography is 35-40°C, and the temperature of the automatic sample injector is 5-10°C.

[0034] Furthermore, the column temperature of the liquid chromatography was 40°C and the temperature of the automatic sample injector was 8°C.

[0035] The mobile phase of the liquid chromatography comprises a mobile phase A and a mobile phase B, wherein the mobile phase A comprises an aqueous solution containing 0.01-0.5 wt % acetic acid, and the mobile phase B comprises acetonitrile or an acetonitrile aqueous solution.

[0036] Preferably, the mobile phase A comprises an aqueous solution containing 0.01-0.2 wt % acetic acid, and the mobile phase B comprises acetonitrile or an acetonitrile aqueous solution.

[0037] Further preferably, the mobile phase A comprises an aqueous solution containing 0.01-0.15 wt % acetic acid, and the mobile phase B comprises acetonitrile.

[0038] More preferably, the mobile phase A comprises an aqueous solution containing 0.1 wt % acetic acid.

[0039] The liquid chromatography adopts gradient elution, and the procedure of the gradient elution includes: 0-1.2min, the volume ratio of mobile phase B to mobile phase is 3-10wt%; 1.2-1.8min, the volume ratio of mobile phase B to mobile phase is 80-95wt%; after 1.8-2min, the volume ratio of mobile phase B to mobile phase is 3-10wt%.

[0040] Preferably, the liquid chromatography adopts gradient elution, and the gradient elution procedure includes: 0-1.2min, the volume ratio of mobile phase B to mobile phase is 3-5wt%; 1.2-1.8min, the volume ratio of mobile phase B to mobile phase is 80-90wt%; after 1.8-2min, the volume ratio of mobile phase B to mobile phase is 3-5wt%.

[0041] Further preferably, the liquid chromatography adopts gradient elution, and the gradient elution procedure includes: 0-1.2min, the volume ratio of mobile phase B to mobile phase is 5wt%; 1.2-1.8min, the volume ratio of mobile phase B to mobile phase is 90wt%; after 1.8-2min, the volume ratio of mobile phase B to mobile phase is 5wt%.

[0042] The gradient elution procedure also includes: 0-0.8min, flow rate is 0.1-0.3mL / min, 0.8-2.3min, flow rate is 0.2-0.6mL / min, and after 2.3min, the flow rate is 0.1-0.3mL / min.

[0043] Preferably, the gradient elution procedure further includes: 0-0.8 min, flow rate of 0.1-0.2 mL / min, 0.8-2.3 min, flow rate of 0.2-0.4 mL / min, and after 2.3 min, flow rate of 0.1-0.2 mL / min.

[0044] Further preferably, the gradient elution procedure also includes: 0-0.8 min, flow rate is 0.2 mL / min, 0.8-2.3 min, flow rate is 0.4 mL / min, and after 2.3 min, flow rate is 0.2 mL / min.

[0045] The present study found that, through a specific elution procedure, the linear range of the standard curve of empagliflozin was 3.00-600 ng / mL, and the fitting degree R 2 The linear range of the metformin standard curve was 20.0-4000 ng / mL, and the fitting degree R 2 All are greater than 0.98. Too high a flow rate may result in insufficient separation of compounds, while too low a flow rate may prolong the analysis time and increase the risk of baseline drift. The solubility, distribution coefficient and elution rate of the compound can be improved by a specific mobile phase combination and a specific gradient change rate.

[0046] The parameters of the tandem mass spectrometer include: collision gas (CAD): 5-10 psi; curtain gas (CUR): 18-25 psi; ion source gas 1 (GS1): 40-60 psi; ion source gas 2 (GS2): 50-70 psi.

[0047] Preferably, the parameters of the tandem mass spectrometer include: collision gas (CAD): 7 psi; curtain gas (CUR): 20 psi; ion source gas 1 (GS1): 50 psi; ion source gas 2 (GS2): 60 psi.

[0048] The parameters of the tandem mass spectrometer also include: ion source spray voltage (IS): 4000-6000V; ion source temperature (TEM): 500-600°C.

[0049] Preferably, the parameters of the tandem mass spectrometer also include: ion source spray voltage (IS): 5000V; ion source temperature (TEM): 550°C.

[0050] Beneficial Effects

[0051] 1. The protein precipitation method is used to pre-prepare samples, and standard curve samples, quality control samples, pre-balanced samples, double blank samples, pre-balanced double blank samples, and SST (system suitability) samples are set at the same time, which effectively improves the system suitability and can process multiple samples at the same time, thereby improving the experimental efficiency.

[0052] 2. The gradient of the standard curve samples of empagliflozin and metformin is 8-12;

[0053] The concentration range of the curve samples is 3-4000ng / mL, which can take into account the detection accuracy, sensitivity and repeatability.

[0054] 3. By setting up quality control samples and selecting specific gradients, the intra-batch and inter-batch precision and accuracy can be improved.

[0055] 4. Through a specific elution procedure, the linear range of the standard curve of empagliflozin is 3.00-600 ng / mL, and the fitting degree R 2 The linear range of the metformin standard curve was 20.0-4000 ng / mL, and the fitting degree R 2 Both are greater than 0.98.

[0056] 5. The single-needle running time of this application test is 2.3 minutes, which effectively improves the detection efficiency, reduces the amount of reagents used and reduces the use time of the instrument.

[0057] 6. This application can detect both empagliflozin and metformin at the same time, and has passed a full set of methodological validation. All inspection items meet the acceptance criteria and can be used for clinical biological sample testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 This is the standard curve of empagliflozin in Example 1.

[0059] Figure 2 This is the metformin standard curve of Example 1. DETAILED DESCRIPTION

[0060] Example 1

[0061] A method for simultaneously determining empagliflozin and metformin in human plasma comprises the following steps:

[0062] S1, pre-preparation of samples using protein precipitation method;

[0063] S2, subjecting the pre-prepared samples to liquid chromatography-tandem mass spectrometry detection;

[0064] S3, chromatogram collection and chromatographic peak integration of analyte and internal standard were completed by AB SciexAnalyst software (version 1.7.2). The standard curve was obtained by regression using Watson LIMS system (version 7.6.1). The chromatographic response ratio of analyte to internal standard was used as the ordinate, and the weighted (W = 1 / x2) least squares method was used to perform linear regression with the concentration of analyte in plasma (x) and the response ratio (y). The obtained regression equation (y = ax + b) was the standard curve ( Figure 1 , Figure 2 ), determine the content of empagliflozin and metformin in the test object, wherein the regression equations are y=0.00602x-0.000770 and y=0.00152x+0.00695, respectively, and the drug concentration of the test sample is calculated by the standard curve equation obtained by fitting.

[0065] The step S1 is specifically as follows:

[0066] a. Prepare a 2.2 mL 96-well polypropylene plate and mix 100 μL of each of the standard curve samples (except the upper limit of quantitation), quality control samples, pre-equilibrated samples, double blank samples, pre-equilibrated double blank samples, SST samples and test samples with 25 μL of internal standard sample (1000 μL) X-d6 / 2000 Y-d4 ng / mL, where X-d6 is metformin-d6, and Y-d4 is empagliflozin-d4) mixed;

[0067] b, mixing the double blank sample and the pre-equilibrated double blank sample with 50 wt % methanol aqueous solution respectively;

[0068] c, 100 μL of the standard curve sample with the upper limit of quantification was mixed with 50 wt % methanol aqueous solution;

[0069] d. Add 300 μL of acetonitrile to all the prepared samples, vortex mix at 25°C for 5 minutes, centrifuge the sample plate at 4°C, 4000 rpm for 5 minutes, transfer 100 μL of supernatant to another clean 96-well polypropylene plate at 25°C, add 100 μL of ultrapure water to all the samples, vortex mix at room temperature for 3 minutes and immediately place them in the autosampler.

[0070] The standard curve samples are respectively empagliflozin standard curve samples and metformin standard curve samples; the gradients of the empagliflozin standard curve samples and metformin standard curve samples are both 8; the gradient of the empagliflozin standard curve sample is: 3.00ng / mL, 6.00ng / mL, 15.0ng / mL, 60.0ng / mL, 120ng / mL, 240ng / mL, 480ng / mL and 600ng / mL, wherein 3.00ng / mL is the lower limit of quantification (LLOQ), and 600 ng / mL is the upper limit of quantification (ULOQ); the gradient of metformin standard curve samples is: 20.0 ng / mL, 40.0 ng / mL, 100 ng / mL, 400 ng / mL, 800 ng / mL, 1600 ng / mL, 3200 ng / mL and 4000 ng / mL; among them, 20.0 ng / mL is the lower limit of quantification (LLOQ), and 4000 ng / mL is the upper limit of quantification (ULOQ).

[0071] The quality control samples are empagliflozin quality control samples and metformin quality control samples; the gradients of the empagliflozin quality control samples and metformin quality control samples are 4, among which the gradient of the empagliflozin quality control sample is: lower limit of quantification (LLOQQC, 3.00ng / mL), low concentration (LQC, 9.00ng / mL), medium concentration (GMQC, 42.0ng / mL, MQC, 180ng / mL), and high concentration (HQC, 450ng / mL); the gradient of the metformin quality control sample is: lower limit of quantification (LLOQ QC, 20.0ng / mL), low concentration (LQC, 60.0ng / mL), medium concentration (GMQC, 280ng / mL, MQC, 1200ng / mL), and high concentration (HQC, 3000ng / mL).

[0072] The internal standard sample includes a solution prepared by metformin-d6 and empagliflozin-d4.

[0073] The standard curve samples and quality control samples were first diluted to 1.00 mg / mL using 50 wt % methanol aqueous solution, and the internal standard sample was diluted to 1.00 mg / mL using DMSO.

[0074] The standard curve samples and quality control samples were diluted to 16000 / 2400 ng / mL (metformin / empagliflozin), and then diluted to 1600 / 240 ng / mL (metformin / empagliflozin) using K2EDTA (dipotassium ethylenediaminetetraacetic acid) human plasma, and finally diluted to the target concentration using K2EDTA human plasma.

[0075] The internal standard sample was then diluted to the target concentration using DMSO.

[0076] The pre-equilibrium sample, double blank sample, double blank sample treated with pre-equilibrium, and SST sample are all K2EDTA human plasma.

[0077] The liquid chromatography-tandem mass spectrometry detection parameter settings are shown in Table 1:

[0078] Table 1

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[0080]

[0081]

[0082] Performance Testing Methods and Data

[0083] The method validation items and results are shown in Table 2:

[0084] Table 2

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[0088] In summary, the analytical method of this application for determining the concentrations of empagliflozin and metformin in K2EDTA human plasma by liquid chromatography tandem mass spectrometry complies with the relevant requirements of the 2020 edition of the Pharmacopoeia "Guidelines for Validation of Quantitative Analysis Methods for Biological Samples" and can be used for the analysis of plasma samples in clinical trials.

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Claims

1. A method for simultaneously determining empagliflozin and metformin in human plasma, characterized in that: The following steps are involved: S1, pre-preparation of samples using protein precipitation method; S2, subjecting the pre-prepared samples to liquid chromatography-tandem mass spectrometry detection; S3, fitting to obtain a linear regression equation, y=ax+b, to determine the contents of empagliflozin and metformin in the analyte, wherein y is the peak area ratio of the analyte and its internal standard, and x is the theoretical concentration of the standard curve sample of the analyte.

2. The method for simultaneously determining empagliflozin and metformin in human plasma according to claim 1, characterized in that: The step S1 specifically includes: a. Mix the standard curve samples except the upper limit of quantitation, quality control samples, pre-equilibrium samples, double blank samples, pre-equilibrium treated double blank samples, SST samples and test samples with the internal standard sample respectively; b, respectively mixing the double blank sample and the pre-equilibrated double blank sample with the methanol solution; c, mix the standard curve sample of the upper limit of quantitation with the methanol solution; d. Add acetonitrile to all samples and mix well.

3. The method for simultaneously determining empagliflozin and metformin in human plasma according to claim 2, characterized in that: The standard curve samples include empagliflozin standard curve samples and metformin standard curve samples; the gradients of the empagliflozin standard curve samples and metformin standard curve samples are 8-12; and the concentration range of the standard curve samples is 3-4000 ng / mL.

4. The method for simultaneously determining empagliflozin and metformin in human plasma according to claim 1 or 3, characterized in that: The parameters of the chromatographic column of the liquid chromatography are: inner diameter 1-3 mm, length 50-150 mm, particle size 1-5 μm, pore size 5. The method for simultaneously determining empagliflozin and metformin in human plasma according to claim 4, characterized in that: The column temperature of the liquid chromatography is 35-45°C, and the temperature of the automatic sample injector is 5-12°C.

6. The method for simultaneously determining empagliflozin and metformin in human plasma according to claim 5, characterized in that: The mobile phase of the liquid chromatography comprises a mobile phase A and a mobile phase B, wherein the mobile phase A comprises an aqueous solution containing 0.01-0.5 wt % acetic acid, and the mobile phase B comprises acetonitrile or an acetonitrile aqueous solution.

7. The method for simultaneously determining empagliflozin and metformin in human plasma according to claim 6, characterized in that: The liquid chromatography adopts gradient elution, and the procedure of the gradient elution includes: 0-1.2min, the volume ratio of mobile phase B to mobile phase is 3-10wt%; 1.2-1.8min, the volume ratio of mobile phase B to mobile phase is 80-95wt%; after 1.8-2min, the volume ratio of mobile phase B to mobile phase is 3-10wt%.

8. The method for simultaneously determining empagliflozin and metformin in human plasma according to claim 7, characterized in that: The gradient elution procedure also includes: 0-0.8min, flow rate is 0.1-0.3mL / min, 0.8-2.3min, flow rate is 0.2-0.6mL / min, and after 2.3min, the flow rate is 0.1-0.3mL / min.

9. The method for simultaneously determining empagliflozin and metformin in human plasma according to claim 8, characterized in that: The parameters of the tandem mass spectrometer include: CAD: 5-10 psi; CUR: 18-25 psi; GS1: 40-60 psi; GS2: 50-70 psi.

10. The method for simultaneously determining empagliflozin and metformin in human plasma according to claim 9, characterized in that: The parameters of the tandem mass spectrometry also include: IS: 4000-6000V; TEM: 500-600°C.

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