Analysis method for determining concentrations of sitagliptin and metformin in human plasma

Abstract

The invention discloses an analysis method for determining the concentration of sitagliptin and metformin in human plasma. The analysis method comprises the following steps: S1, preparing a matrix, a standard working solution, an internal standard working solution and a quality control working solution in advance; s2, setting parameters of liquid chromatography and mass spectrometry; and S3, detecting and analyzing data. According to the method, the human plasma is extracted by adopting a protein precipitation method, and the K2EDTA retarder is added into the plasma, so that background impurity interference is small during detection. Sitagliptin and metformin labeled by stable isotopes are adopted as internal standards, and can be used as effective internal standards of objects to be detected for accurate quantitative analysis. In the analysis and detection process, a dark brown glass bottle is adopted for storage, and ethanol is adopted as a solvent, so that the stability of the sample can be improved, the property is not changed in the detection process, and the detection accuracy deviation is controlled to be less than 15%. According to the method, sitagliptin and metformin can be detected at the same time, the detection speed is high, and the detection efficiency is high.

Description

Technical Field

[0001] The present invention relates to the field of G01N, to a method and technical field for testing or analyzing materials by measuring the chemical or physical properties of materials, and in particular to an analytical method for measuring the concentrations of sitagliptin and metformin in human plasma. Background Art

[0002] Sitagliptin is a highly effective and selective hypoglycemic drug, and metformin is an oral hypoglycemic drug. Sitagliptin and metformin are used in combination with each other and in conjunction with dietary control to achieve the treatment of type II hyperglycemia. In the actual treatment process, the methods for detecting sitagliptin and metformin vary greatly, and the methods for detecting the content of sitagliptin and metformin in plasma are not highly specific, easily affected by the environment, and unstable during the detection process, resulting in decreased detection accuracy and large detection deviation. Therefore, it is crucial to develop a detection and analysis method that can efficiently detect the concentrations of sitagliptin and metformin in plasma.

[0003] Chinese invention patent CN202111538156.1 discloses a method for determining the concentration of sitagliptin in plasma by liquid chromatography-mass spectrometry, using sitagliptin-d4 as an internal standard, adding a precipitant for protein precipitation, taking the supernatant and adding a diluent, pre-treating, separating by a chromatographic column, and detecting by a mass spectrometer. The detection speed is fast, the accuracy is high, and the sensitivity is excellent, but it cannot achieve the simultaneous detection of sitagliptin and metformin. Chinese invention patent CN202210069300.X discloses a method for detecting sitagliptin and metformin in biological samples, which uses LC-MS / MS method for detection, and can achieve high specificity, sensitivity, and rapid determination of the content of sitagliptin and metformin in tablets, but it has a large interference with drug detection in plasma and reduces accuracy. Summary of the invention

[0004] In order to develop a detection and analysis method that can efficiently detect the concentrations of sitagliptin and metformin in plasma, the first aspect of the present invention provides an analytical method for determining the concentrations of sitagliptin and metformin in human plasma, comprising the following steps:

[0005] S1 pre-prepared matrix, standard working solution, internal standard working solution, quality control working solution;

[0006] S2 sets the parameters of liquid chromatography and mass spectrometry;

[0007] S3 detects and analyzes data.

[0008] As a preferred embodiment, the standard working solution includes a sitagliptin standard working solution and a metformin standard working solution.

[0009] As a preferred embodiment, the internal standard working solution includes sitagliptin-d4 internal standard working solution and metformin-d6 internal standard working solution.

[0010] As a preferred embodiment, the concentration of the sitagliptin standard working solution is 5-1000 ng / mL, and the concentration of the metformin standard working solution is 20-4000 ng / mL.

[0011] As a preferred embodiment, the concentration of the sitagliptin standard working solution is 5ng / mL, 10ng / mL, 25ng / mL, 100ng / mL, 200ng / mL, 400ng / mL, 800ng / mL, 1000ng / mL, and the concentration of the metformin standard working solution is 20ng / mL, 40ng / mL, 100ng / mL, 400ng / mL, 800ng / mL, 1600ng / mL, 3200ng / mL, 4000ng / mL.

[0012] This analytical method uses stable isotope-labeled sitagliptin and metformin (sitagliptin-d4 and metformin-d6) as internal standards for the following reasons: sitagliptin-d4 and metformin-d6 have similar physicochemical properties and chromatographic behaviors to the analytes and can be used as effective internal standards for accurate quantitative analysis of the analytes.

[0013] Ultra-high performance liquid chromatography tandem mass spectrometry has the advantages of high sensitivity and good selectivity. Therefore, ultra-high performance liquid chromatography tandem mass spectrometry was used to determine K 2 Concentrations of sitagliptin and metformin in EDTA human plasma samples.

[0014] As a preferred embodiment, the sitagliptin quality control working solution includes a minimum quality control concentration (LLOQQC) of 5 ng / mL, a low quality control concentration (LQC) of 15 ng / mL, a medium quality control concentration (GMQC) of 70 ng / mL, a medium quality control concentration (MQC) of 300 ng / mL, and a high quality control concentration (HQC) of 750 ng / mL.

[0015] As a preferred embodiment, the metformin quality control working solution includes a minimum quality control concentration (LLOQQC) of 20 ng / mL, a low quality control concentration (LQC) of 60 ng / mL, a medium quality control concentration (GMQC) of 280 ng / mL, a medium quality control concentration (MQC) of 1200 ng / mL, and a high quality control concentration (HQC) of 3000 ng / mL.

[0016] As a preferred embodiment, the mobile phase of the liquid chromatography comprises mobile phase A and mobile phase B, the mobile phase A is water containing 0.1-1 wt % formic acid, and the mobile phase B is acetonitrile.

[0017] As a preferred embodiment, the mobile phase elution program of the liquid chromatography is as follows: time 0 min, flow rate 0.3-0.8 mL / min, mobile phase B 30-40 vol%; time 0.3-0.5 min, flow rate 0.3-0.8 mL / min, mobile phase B 85-95 vol%; time 1-1.5 min, flow rate 0.3-0.5 mL / min, mobile phase B 85-95 vol%; time 1-1.5 min, flow rate 0.3-0.5 mL / min, mobile phase B 30-40 vol%; time 1-2 min, flow rate 0.3-0.5 mL / min, mobile phase B 30-40 vol%.

[0018] As a preferred embodiment, the mobile phase elution program of the liquid chromatography is as follows: time 0 min, flow rate 0.5 mL / min, mobile phase B 35 vol%; time 0.4 min, flow rate 0.4 mL / min, mobile phase B 90 vol%; time 1.2 min, flow rate 0.4 mL / min, mobile phase B 90 vol%; time 1.21 min, flow rate 0.4 mL / min, mobile phase B 35 vol%; time 1.5 min, flow rate 0.4 mL / min, mobile phase B 35 vol%.

[0019] As a preferred embodiment, the particle size of the filler in the chromatographic column in the liquid chromatography is The size of the chromatographic column is 50 mm×4.6 mm.

[0020] As a preferred embodiment, the column temperature of the chromatographic column in the liquid chromatography is 35-55°C, and the injection temperature of the automatic sampler in the liquid chromatography is 5-10°C.

[0021] As a preferred embodiment, the retention time of metformin in the liquid chromatography is 0.83 min, the retention time of sitagliptin is 1.026 min, the retention time of metformin-d6 is 0.827 min, and the retention time of sitagliptin-d4 is 1.026 min.

[0022] As a preferred embodiment, the mass spectrometry condition parameters are as shown in Table 1:

[0023] Table 1

[0024]

[0025]

[0026] In brown glass bottles, it has experimental stability under room temperature and white light. The implementation stability includes room temperature and white light stability, freeze-thaw cycle stability at -20℃ and -80℃, and long-term stability at -20℃ and -80℃. The sample is stored in brown glass bottles that can be protected from light and ethanol is used as a solvent to improve the stability of the sample. There will be no change in properties during the test, and the accuracy deviation of the test is controlled to be less than 15%.

[0027] As a preferred embodiment, the matrix includes a blank matrix, a high-fat matrix and a hemolytic matrix; the blank matrix is ​​human plasma, and the human plasma contains a retarder K 2 EDTA.

[0028] As a preferred embodiment, the high-fat matrix is ​​1 mg of low-density lipoprotein and 3 mg of triglycerides added to 1 mL of blank matrix.

[0029] As a preferred embodiment, the preparation method of the hemolytic matrix is: freezing human whole blood at -80°C for 30 minutes, vortexing for 1 minute after thawing, and mixing with a blank matrix at a volume ratio of 1:49 to obtain the hemolytic matrix.

[0030] The second aspect of the present invention provides an application of an analytical method for determining the concentrations of sitagliptin and metformin in human plasma, which is applied to the detection of human plasma.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) The analytical method of the present invention for determining the concentration of sitagliptin and metformin in human plasma is to extract human plasma using a protein precipitation method, and add K 2 EDTA retarder, less interference from background impurities during detection.

[0033] (2) The analytical method of the present invention for determining the concentration of sitagliptin and metformin in human plasma uses stable isotope-labeled sitagliptin and metformin as internal standards, which can be used as effective internal standards for the analytes for accurate quantitative analysis.

[0034] (3) The analytical method of the present invention for determining the concentrations of sitagliptin and metformin in human plasma adopts a method of liquid chromatography-mass spectrometry and a smaller injection volume, and the accuracy and precision of sitagliptin and metformin detection are relatively high.

[0035] (4) The analytical method of the present invention for determining the concentration of sitagliptin and metformin in human plasma uses a brown glass bottle that can be protected from light during the analytical detection process, and uses ethanol as a solvent to improve the stability of the sample, so that no property changes will occur during the detection process, and the accuracy deviation of the detection is controlled to be less than 15%.

[0036] (5) The analytical method of the present invention for determining the concentrations of sitagliptin and metformin in human plasma can achieve simultaneous detection of sitagliptin and metformin with fast detection speed and high detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the product ion scanning spectrum of sitagliptin;

[0038] Figure 2 It is the product ion scanning spectrum of sitagliptin-d4;

[0039] Figure 3 This is the standard curve of sitagliptin;

[0040] Figure 4 This is the metformin daughter ion scanning spectrum;

[0041] Figure 5 It is the metformin-d6 daughter ion scanning spectrum;

[0042] Figure 6 This is the standard curve of metformin. DETAILED DESCRIPTION

[0043] Example 1

[0044] An analytical method for determining the concentration of sitagliptin and metformin in human plasma comprises the following steps:

[0045] S1 pre-prepared matrix, standard working solution, internal standard working solution, quality control working solution;

[0046] S2 sets the parameters of liquid chromatography and mass spectrometry;

[0047] S3 detects and analyzes data.

[0048] The standard working solution includes a sitagliptin standard working solution and a metformin standard working solution. The concentration of the sitagliptin standard working solution is 5ng / mL, 10ng / mL, 25ng / mL, 100ng / mL, 200ng / mL, 400ng / mL, 800ng / mL, 1000ng / mL, and the concentration of the metformin standard working solution is 20ng / mL, 40ng / mL, 100ng / mL, 400ng / mL, 800ng / mL, 1600ng / mL, 3200ng / mL, 4000ng / mL.

[0049] The internal standard working solution includes sitagliptin-d4 internal standard working solution and metformin-d6 internal standard working solution. The concentrations of the sitagliptin-d4 internal standard working solution and the metformin-d6 internal standard working solution are both 1 mg / mL.

[0050] The quality control working solution includes sitagliptin quality control working solution and metformin quality control working solution; the sitagliptin quality control working solution includes a minimum quality control concentration (LLOQ QC) of 5 ng / mL, a low quality control concentration (LQC) of 15 ng / mL, a medium quality control concentration (GMQC) of 70 ng / mL, a medium quality control concentration (MQC) of 300 ng / mL, and a high quality control concentration (HQC) of 750 ng / mL.

[0051] The metformin quality control working solution includes a minimum quality control concentration (LLOQ QC) of 20 ng / mL, a low quality control concentration (LQC) of 60 ng / mL, a medium quality control concentration (GMQC) of 280 ng / mL, a medium quality control concentration (MQC) of 1200 ng / mL, and a high quality control concentration (HQC) of 3000 ng / mL.

[0052] The sitagliptin and metformin were diluted with 50 wt % methanol aqueous solution to prepare standard curve sample working solution and quality control sample working solution, which were stored in amber glass bottles at -20°C.

[0053] The sitagliptin-d4 and metformin-d6 were diluted with 50 wt % methanol aqueous solution to prepare internal standard working solutions, which were stored in amber glass bottles at -20°C.

[0054] The standard working solution is as follows:

[0055]

[0056] The quality control working solution is as follows:

[0057]

[0058] The internal standard working solution is as follows:

[0059]

[0060] Note: X is metformin and Y is sitagliptin.

[0061] The matrix includes a blank matrix, a high-fat matrix and a hemolytic matrix; the blank matrix is ​​human plasma, and the human plasma contains a retarder K 2 EDTA. The human plasma was sourced from Zibo Traditional Chinese Medicine Hospital.

[0062] The high-fat matrix is ​​1 mg of low-density lipoprotein and 3 mg of triglyceride added to 1 mL of blank matrix.

[0063] The preparation method of the hemolytic matrix is ​​as follows: freezing human whole blood at -80°C for 30 minutes, vortexing for 1 minute after thawing, and mixing with a blank matrix at a volume ratio of 1:49. The human whole blood comes from Zibo Traditional Chinese Medicine Hospital.

[0064] Transfer 100 μL of the blank matrix, standard working solution, internal standard working solution, and quality control working solution to another clean 96-well polypropylene plate at room temperature, add 100 μL of water to all samples, vortex mix at room temperature for 3 minutes and immediately place in the autosampler.

[0065] The parameters of liquid chromatography are shown in Table 2 below.

[0066] Table 2

[0067]

[0068]

[0069] The mass spectrometry parameters are shown in Table 1.

[0070] (1) Standard curve test:

[0071] The test results are shown in Tables 3 and 4.

[0072] Table 3

[0073]

[0074]

[0075] Table 4

[0076]

[0077]

[0078] (2) Accuracy test

[0079] The test results are shown in Tables 5 and 6.

[0080] Table 5

[0081]

[0082]

[0083]

[0084] Table 6

[0085]

[0086]

[0087]

[0088]

[0089] Note: #: Does not meet the acceptance criteria, but is still included in the calculation.

[0090] (3) Extraction recovery test

[0091] The test results are shown in Tables 7 and 8.

[0092] Table 7

[0093]

[0094]

[0095] Table 8

[0096]

[0097]

[0098] (4) Internal standard recovery rate

[0099] The test results are shown in Tables 9 and 10.

[0100] Table 9

[0101]

[0102] Table 10

[0103]

[0104]

[0105] (4) Matrix effect

[0106] The results of the matrix stability test are shown in Tables 11 and 12.

[0107] Table 11

[0108]

[0109] Table 12

[0110]

[0111]

[0112] Sitagliptin product ion scanning spectrum see Figure 1 ; Sitagliptin-d4 daughter ion scanning spectrum see Figure 2 ; The standard curve of sitagliptin is shown in Figure 3 ; Metformin daughter ion scanning spectrum see Figure 4 ; Metformin-d6 daughter ion scanning spectrum see Figure 5 ; The standard curve of metformin is shown in Figure 6 .

Claims

1. An analytical method for determining the concentration of sitagliptin and metformin in human plasma, characterized in that: The following steps are involved: S1 pre-prepared matrix, standard working solution, internal standard working solution, quality control working solution; S2 sets the parameters of liquid chromatography and mass spectrometry; S3 detects and analyzes data.

2. The analytical method for determining the concentration of sitagliptin and metformin in human plasma according to claim 1, characterized in that: The standard working solution includes a sitagliptin standard working solution and a metformin standard working solution.

3. The analytical method for determining the concentration of sitagliptin and metformin in human plasma according to claim 1, characterized in that: The internal standard working solution includes sitagliptin-d4 internal standard working solution and metformin-d6 internal standard working solution.

4. The analytical method for determining the concentration of sitagliptin and metformin in human plasma according to claim 2, characterized in that: The concentration of the sitagliptin standard working solution is 5-1000 ng / mL, and the concentration of the metformin standard working solution is 20-4000 ng / mL.

5. The analytical method for determining the concentration of sitagliptin and metformin in human plasma according to claim 1, 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 is water containing 0.1-1 wt % formic acid, and the mobile phase B is acetonitrile.

6. The analytical method for determining the concentration of sitagliptin and metformin in human plasma according to claim 1, characterized in that: The mobile phase elution program of the liquid chromatography is as follows: time 0 min, flow rate 0.3-0.8 mL / min, mobile phase B 30-40 vol%; time 0.3-0.5 min, flow rate 0.3-0.8 mL / min, mobile phase B 85-95 vol%; time 1-1.5 min, flow rate 0.3-0.5 mL / min, mobile phase B 85-95 vol%; time 1-1.5 min, flow rate 0.3-0.5 mL / min, mobile phase B 30-40 vol%; time 1-2 min, flow rate 0.3-0.5 mL / min, mobile phase B 30-40 vol%.

7. The analytical method for determining the concentration of sitagliptin and metformin in human plasma according to claim 1, characterized in that: The particle size of the filler in the chromatographic column of the liquid chromatography is The size of the chromatographic column is 50 mm×4.6 mm.

8. The analytical method for determining the concentration of sitagliptin and metformin in human plasma according to claim 1, characterized in that: The column temperature of the chromatographic column in the liquid chromatography is 35-55°C, and the injection temperature of the automatic sampler in the liquid chromatography is 5-10°C.

9. The analytical method for determining the concentration of sitagliptin and metformin in human plasma according to claim 1, characterized in that: The matrix includes a blank matrix, a high-fat matrix and a hemolytic matrix; the blank matrix is ​​human plasma, and the human plasma contains a retarder K2EDTA.

10. An application of the analytical method for determining the concentration of sitagliptin and metformin in human plasma according to any one of claims 1 to 9, characterized in that: Applied in the detection of human plasma.

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