A method for determining the content of dimer impurities in sitagliptin phosphate

Abstract

The present invention provides a method for determining the content of dimer impurities in sitagliptin phosphate, belongs to the technical field of drug analysis, and performs chromatographic analysis on a control solution, a test solution, and a test solution spiked with a sample. The control solution is a dimer impurity reference solution, the test sample is a sitagliptin phosphate test sample, and the test solution spiked with a sample is a mixed solution of the sitagliptin phosphate test sample and dimer impurities. The present invention establishes a method for determining the content of dimer impurities in sitagliptin phosphate by HPLC, and quantitatively detects dimer impurities that may exist in sitagliptin phosphate. The present invention has strong specificity, high sensitivity, good repeatability, and good durability, and is of great significance for the quality control of dimer impurities in sitagliptin phosphate, and provides a basis for the establishment of the quality standard of sitagliptin phosphate.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a method for determining the content of dimer impurities in sitagliptin phosphate. Background Art

[0002] Sitagliptin phosphate is a DPP-4 inhibitor, which is used in combination with other therapeutic drugs to control blood sugar in diabetes. It is a safe and effective oral drug for the treatment of type 2 diabetes with broad market prospects.

[0003] The chemical name of sitagliptin phosphate is 7-[(3R)-3-amino-1-oxo-4-(2,4,5-trifluorophenyl)butyl]-5,6,7,8-tetrahydro-3-(trifluoromethyl)-1,2,4-triazolone[4,3-α]pyrazine phosphate (1:1) monohydrate, and the molecular formula is C 16 H 15 F6N5O·H3PO4·H2O, the chemical structure is as follows:

[0004]

[0005] During the synthesis of the API of sitagliptin phosphate, the reaction byproduct dimer impurity is produced by the condensation reaction between sitagliptin phosphate and the intermediate II during the enzyme-catalyzed amination process. The dimer impurity is not included in the pharmacopoeias of various countries. According to the actual production data, the dimer impurity is controlled to be no more than 0.10%. Since the dimer impurity has low polarity and weak ultraviolet response, the repeatability and sensitivity are poor during detection, and the durability of the detection instrument is poor.

[0006] Therefore, it is very important to provide a stable, reliable, sensitive and durable liquid chromatography detection method for determining the dimer impurity content in sitagliptin phosphate. Summary of the invention

[0007] The purpose of the present invention is to provide a method for determining the content of dimer impurities in sitagliptin phosphate, so as to solve the technical problems of poor repeatability, low sensitivity and poor durability of detection instruments in the detection process of the prior art.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a method for determining the content of dimer impurities in sitagliptin phosphate, comprising the following steps:

[0010] (1) mixing a dimer impurity reference substance and a diluent to obtain a reference solution;

[0011] Mixing the sitagliptin phosphate test sample and the diluent to obtain a test sample solution;

[0012] The sitagliptin phosphate test sample, the dimer impurity and the diluent are mixed to obtain a test sample spiked solution;

[0013] (2) performing chromatographic detection on the control solution, the test solution, and the test spiked solution, and calculating the dimer impurity content in sitagliptin phosphate by peak area and concentration using an external standard method;

[0014] The mobile phase A of the chromatographic detection is a trifluoroacetic acid aqueous solution with a concentration of 0.01-0.1%, and the mobile phase B is acetonitrile, wherein the pH of the mobile phase A is 5-6, and the reagent for adjusting the pH value is triethylamine.

[0015] Further, the concentration of the control solution is 0.01 to 1.3 μg / mL;

[0016] The concentration of the test solution is 0.5-1.5 mg / mL;

[0017] The concentration of the sitagliptin phosphate test sample in the test sample spiked solution is 0.5-1.5 mg / mL, and the concentration of the dimer impurity is 0.01-1.3 μg / mL.

[0018] Furthermore, in step (1), the diluent is a mixed solution of acetonitrile and water in a volume ratio of 1:1.

[0019] Furthermore, the conditions for the chromatographic detection are: flow rate of 0.9-1.1 mL / min, column temperature of 28-32° C., injection volume of 18-22 μL, detection wavelength of 260-270 nm, and detection time of 18-22 min.

[0020] Furthermore, in step (2), the chromatographic column used for chromatographic detection is an octadecylsilane bonded silica gel column with a column length of 150 to 300 mm, an inner diameter of 4 to 5 mm, a filler particle size of 3 to 5 μm, and the model of the chromatographic column is Waters XBridgeC18.

[0021] Furthermore, in step (2), the procedure of gradient elution in chromatographic detection is:

[0022] At 0-8 min, it is 95-5% mobile phase A, 5-95% mobile phase B;

[0023] From 8 to 10 minutes, excluding 8 minutes, it is 5% mobile phase A and 95% mobile phase B;

[0024] At 10-12 min, excluding 10 min, 5-95% mobile phase A and 95-5% mobile phase B;

[0025] During 12 to 20 minutes (excluding 12 minutes), the mobile phase A was 95% and the mobile phase B was 5%.

[0026] Beneficial effects of the present invention:

[0027] (1) The determination method of the present invention utilizes high performance liquid chromatography and a gradient elution program to elute the dimeric impurities with low polarity within a short analysis time, thereby shortening the sample analysis and detection time and saving time and reagent costs.

[0028] (2) The determination method of the present invention improves the detection sensitivity of dimer impurities, and the method has strong specificity, good repeatability and good durability.

[0029] (3) The determination method of the present invention adopts ordinary liquid chromatography, uses conventional chromatographic columns and reagents, has mild chromatographic conditions and is easy to operate, providing a basis for the establishment of the quality standard of sitagliptin phosphate. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The chromatogram is the blank solution detected in Example 1;

[0031] Figure 2 is a chromatogram detected by the control solution in Example 1;

[0032] Figure 3 The chromatogram is a test sample solution with batch number SS11-P-2110001 in Example 1. DETAILED DESCRIPTION

[0033] The present invention provides a method for determining the content of dimer impurities in sitagliptin phosphate, comprising the following steps:

[0034] (1) mixing a dimer impurity reference substance and a diluent to obtain a reference solution;

[0035] Mixing the sitagliptin phosphate test sample and the diluent to obtain a test sample solution;

[0036] The sitagliptin phosphate test sample, the dimer impurity and the diluent are mixed to obtain a test sample spiked solution;

[0037] (2) The control solution, the test solution and the test spiked solution were subjected to chromatographic detection, and the dimer impurity content in sitagliptin phosphate was calculated by the peak area and concentration using the external standard method.

[0038] In the present invention, a blank solution is also included when performing chromatographic detection. The blank solution is a diluent, and the diluent is a mixed solution of acetonitrile and water in a volume ratio of 1:1.

[0039] In the present invention, the mobile phase A of the chromatographic detection is a trifluoroacetic acid aqueous solution with a concentration of 0.01 to 0.1%, preferably 0.03 to 0.07%, and more preferably 0.05%, and the mobile phase B is acetonitrile, wherein the pH of the mobile phase A is 5 to 6, preferably 5.2 to 5.8, and more preferably 5.4 to 5.6, and the reagent for adjusting the pH value is preferably triethylamine.

[0040] In the present invention, the concentration of the control solution is 0.01 to 1.3 μg / mL, preferably 0.1 to 1.2 μg / mL, and more preferably 0.5 to 1 μg / mL;

[0041] The concentration of the test solution is 0.5-1.5 mg / mL, preferably 0.8-1.2 mg / mL, and more preferably 1 mg / mL;

[0042] The concentration of the sitagliptin phosphate test sample in the test sample spiked solution is 0.5-1.5 mg / mL, preferably 0.8-1.2 mg / mL, and more preferably 1 mg / mL; the concentration of the dimer impurity is 0.01-1.3 μg / mL, preferably 0.1-1.2 μg / mL, and more preferably 0.5-1 μg / mL.

[0043] In the present invention, the conditions for the chromatographic detection are:

[0044] The flow rate is 0.9 to 1.1 mL / min, preferably 1 mL / min;

[0045] The column temperature is 28-32°C, preferably 29-31°C, and more preferably 30°C;

[0046] The injection volume is 18 to 22 μL, preferably 19 to 21 μL, and more preferably 20 μL;

[0047] The detection wavelength is 260-270 nm, preferably 264-268 nm, and more preferably 266 nm;

[0048] The detection time is 18 to 22 minutes, preferably 19 to 21 minutes, and more preferably 20 minutes.

[0049] In the present invention, in the step (2), the chromatographic detection uses an octadecylsilane bonded silica gel column with a column length of 150 to 300 mm, preferably 180 to 250 mm, and more preferably 250 mm; an inner diameter of 4 to 5 mm, preferably 4.2 to 4.8 mm, and more preferably 4.6 mm; a filler particle size of 3 to 5 μm, preferably 3.5 to 5 μm, and more preferably 5 μm; and the model of the chromatographic column is preferably Waters XBridge C18.

[0050] In the present invention, in step (2), the procedure of gradient elution in chromatographic detection is:

[0051] At 0-8 min, it is 95-5% mobile phase A, 5-95% mobile phase B;

[0052] From 8 to 10 minutes, excluding 8 minutes, it is 5% mobile phase A and 95% mobile phase B;

[0053] At 10-12 min, excluding 10 min, 5-95% mobile phase A and 95-5% mobile phase B;

[0054] During 12 to 20 minutes (excluding 12 minutes), the mobile phase A was 95% and the mobile phase B was 5%.

[0055] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0056] The structural formula of the dimer impurity used in the embodiment of the present invention is The manufacturer is Heyuan Broadspect Biotechnology Co., Ltd., the batch number is G44200629-01, and the content is 85.20%; the manufacturer of sitagliptin phosphate test sample is Jiangsu Weiqida Pharmaceutical Co., Ltd., the batch numbers are SS11-P-2105001, SS11-P-2105002, SS11-P-2110001, SS11-2110006 and SS11-2110012; the manufacturer of acetonitrile is Merck (Merck), the batch number is 20220324-18; the water uses Wahaha purified water; the manufacturer of trifluoroacetic acid is Aladdin Biochemical Technology Co., Ltd. (aladdin), the batch number is F2008095; the manufacturer of triethylamine is Sinopharm Group, the batch number is 20210304-05, and the content is ≥99.0%.

[0057] The electronic balance used in the embodiments of the present invention is manufactured by Mettler, and the models are XS205 and XPR10; the pH meter is manufactured by Mettler, and the model is S220; the high performance liquid chromatograph is manufactured by Waters Technology (Shanghai) Co., Ltd. (Waters), and the model is Waters e2695 / UV2489; the chromatographic column is manufactured by Waters, and the model is Waters XBridge C18; the workstation is manufactured by Agilent, and the model is Openlab CDS2.4.

[0058] Example 1

[0059] The diluent was used as a blank solution, and the diluent was a mixed solution of acetonitrile and water in a volume ratio of 1:1;

[0060] The dimer impurity and the diluent are mixed to obtain a control solution, wherein the concentration of the dimer impurity in the control solution is 1 μg / mL;

[0061] The test sample solutions of batch numbers SS11-P-2105001, SS11-P-2105002, SS11-P-2110001, SS11-2110006 and SS11-2110012 were mixed with the diluent to obtain test sample solutions, and the concentration of the test sample solutions was 1 mg / mL;

[0062] The dimer impurity, the test solution and the diluent are mixed to obtain a test spike solution, wherein the concentration of the dimer impurity in the test spike solution is 1 μg / mL, and the concentration of the test solution is 1 mg / mL;

[0063] The blank solution, control solution, test solution and test spiked solution were subjected to chromatographic detection, and the dimer impurity content in sitagliptin phosphate was calculated by peak area and concentration using the external standard method;

[0064] The conditions for chromatographic detection were as follows: chromatographic column: Waters XBridge C18, 4.6×250mm×5μm; column temperature: 30°C; sample chamber temperature: 4°C; wavelength: 266nm; flow rate: 1.0mL / min; injection volume: 20μL; running time: 20min; mobile phase A: 0.05% trifluoroacetic acid aqueous solution, adjusted to pH 5.5 with triethylamine; mobile phase B: acetonitrile;

[0065] The gradient elution program was:

[0066] At 0-8 min, it is 95-5% mobile phase A, 5-95% mobile phase B;

[0067] From 8 to 10 minutes, excluding 8 minutes, it is 5% mobile phase A and 95% mobile phase B;

[0068] At 10-12 min, excluding 10 min, 5-95% mobile phase A and 95-5% mobile phase B;

[0069] During 12 to 20 minutes (excluding 12 minutes), the mobile phase A was 95% and the mobile phase B was 5%.

[0070] The test solution in Example 1 was tested. According to the external standard method, the dimer impurity in the test solution should not exceed 0.1%. The results of the determination of the dimer impurity content in the sitagliptin phosphate test solution are shown in Table 1.

[0071] Table 1 Dimer impurity content determination results

[0072] Test sample batch number Dimer impurity content SS11-P-2105001 Not detected SS11-P-2105002 Not detected SS11-P-2110001 Not detected SS11-2110006 Not detected SS11-2110012 Not detected

[0073] Figure 1 The chromatogram is the blank solution detected in Example 1; Figure 2 is a chromatogram detected by the control solution in Example 1; Figure 3 The chromatogram is a test sample solution with batch number SS11-P-2110001 in Example 1. Figures 1 to 3 As shown in Table 1, dimer impurities were not detected in multiple batches of sitagliptin phosphate test samples, and the test results met the requirements.

[0074] The specificity test of the determination method of the present invention was carried out: the blank solution, the test sample spiked solution and the control solution were respectively tested according to the chromatographic conditions in Example 1, with one injection for each. The test results are shown in Table 2.

[0075] Table 2 Specificity test results

[0076]

[0077] As shown in Table 2, the blank solution has no interference with the detection of dimer impurities, and the absolute difference in the retention time of dimer impurities in the test spiked solution and the control solution meets the requirements, indicating that the determination method of the present invention has good specificity.

[0078] The system suitability test of the determination method of the present invention was carried out: blank solution and control solution were tested according to the chromatographic conditions in Example 1, the blank solution was injected into 1 needle, and the control solution was injected into 5 needles. The test results are shown in Table 3.

[0079] Table 3 System suitability test results

[0080] name 1 2 3 4 5 RSD(%) Dimer impurity peak area 10.74 10.73 10.75 10.75 10.76 0.11

[0081] As shown in Table 3, the RSD of the peak area of ​​dimer impurities in the reference solution for 5 consecutive times is less than 10%, indicating that the system applicability of the determination method of the present invention meets the requirements.

[0082] The detection limit and quantification limit of the determination method of the present invention were tested: the control solution was diluted to a certain concentration, the concentration when the peak signal-to-noise ratio was about 10:1 was taken as the quantification limit concentration, and 6 injections of the quantification limit solution were injected continuously; the concentration when the peak signal-to-noise ratio was about 3:1 was taken as the detection limit concentration, and 2 injections of the detection limit solution were injected continuously. The test results are shown in Table 4.

[0083] Table 4 Detection limit and quantification limit test results

[0084]

[0085] As can be seen from Table 4, the detection limit and quantification limit of the dimer impurity are extremely low, indicating that the detection sensitivity of the determination method of the present invention is high and can meet the determination requirements.

[0086] The linearity and range of the determination method of the present invention were tested: linear solutions of different concentrations were prepared with reference substances and injected separately. The concentration range of dimer impurities in the linear solutions was 0.0171-1.2946 μg / mL. A standard curve was drawn with concentration as the abscissa and peak area as the ordinate. The test results of linearity and range are shown in Table 5.

[0087] Table 5 Linearity and range test results

[0088] name Linear equations Correlation coefficient scope Dimer impurity y=12.459x+0.1294 0.9991 0.0171~1.2946μg / mL

[0089] It can be seen from Table 5 that the dimer impurity has a good linear relationship using the determination method of the present invention.

[0090] The accuracy of the determination method of the present invention was tested: the test sample, dimer impurity and diluent were mixed to prepare recovery solutions, the concentrations of dimer impurities in the recovery solutions were 80%, 100% and 120%, respectively, and 3 portions of high, medium and low concentrations were prepared in parallel. The accuracy results are shown in Table 6.

[0091] Table 6 Accuracy test results

[0092]

[0093] As shown in Table 6, in the determination method of the present invention, the recovery rates of dimer impurities are all within the range of 80-120%, and the RSD of 9 recovery rates is less than 10%, indicating that the determination method of the present invention has good accuracy.

[0094] The determination method of the present invention was subjected to a precision test: Experimenter A prepared 1 portion of the control solution and 6 portions of the test sample spiked solution in parallel according to the preparation method of the control solution and the test sample spiked solution in Example 1, and tested them separately as the repeatability results. Experimenter B used different instruments and different chromatographic columns on different dates to prepare 1 portion of the control solution and 6 portions of the test sample spiked solution in parallel according to the preparation method of the control solution and the test sample spiked solution in Example 1, and tested them separately as the intermediate precision results, and calculated the RSD of the 12 results. The precision results are shown in Table 7.

[0095] Table 7 Precision test results

[0096]

[0097] As shown in Table 7, in the 12 results, the RSD of the dimer impurity content was less than 10%, indicating that the determination method of the present invention has good precision.

[0098] The solution stability test of the determination method of the present invention was carried out: a control solution and a test solution were prepared according to Example 1, and then placed at 4°C for several hours. The RSD of the dimer impurity peak area in the control solution and the RSD value of the dimer impurity content in the test solution at each time point were examined. The solution stability test results are shown in Table 8.

[0099] Table 8 Stability test results

[0100] Solution name Control solution Test solution Time Dimer impurity peak area Dimer impurity content (%) 0h 10.62 Not detected 6h 10.69 Not detected 10h 10.69 Not detected 18h 10.71 Not detected 22h 10.68 Not detected 29h 10.65 Not detected 36h 10.62 Not detected RSD(%) 0.34 N / A

[0101] As can be seen from Table 8, the control solution and the test solution were placed at 4°C for 36 hours, and the RSD of the peak area of ​​the dimer impurity in the control solution at each time point was less than 10%, and the dimer impurity was not detected in the test solution at each time point. The control solution and the test solution had good stability when placed at 4°C for 36 hours.

[0102] Example 2

[0103] Compared with Example 1, the only difference is that the flow rate of the chromatographic detection is 0.9 mL / min.

[0104] Example 3

[0105] Compared with Example 1, the only difference is that the flow rate of the chromatographic detection is 1.1 mL / min.

[0106] Example 4

[0107] Compared with Example 1, the only difference is that the injection volume for chromatographic detection is 18 μL.

[0108] Example 5

[0109] Compared with Example 1, the only difference is that the injection volume for chromatographic detection is 22 μL.

[0110] Example 6

[0111] Compared with Example 1, the only difference is that the column temperature of the chromatographic detection is 28°C.

[0112] Example 7

[0113] Compared with Example 1, the only difference is that the column temperature of the chromatographic detection is 32°C.

[0114] Example 8

[0115] Compared with Example 1, the only difference is that the pH value of the mobile phase A for chromatographic detection is 5.3.

[0116] Example 9

[0117] Compared with Example 1, the only difference is that the pH value of the mobile phase A for chromatographic detection is 5.7.

[0118] The durability test was carried out on Examples 1 to 9. The RSD of the dimer impurity peak area in the control solution was used to examine the RSD value of the dimer impurity content in the test sample spiked solution. The durability results are shown in Table 9.

[0119] Table 9 Durability test results

[0120]

[0121] As shown in Table 8, when the flow rate, injection volume, column temperature and mobile phase pH are changed, the RSD of the dimer impurity peak area in the control solution is less than 10%, and the RSD of the dimer impurity content is less than 10%, indicating that the determination method of the present invention has good durability.

[0122] As can be seen from the above embodiments, the present invention provides a method for determining the content of dimer impurities in sitagliptin phosphate, by performing chromatographic analysis on a control solution, a test solution and a test solution spiked with a sample. The control solution is a dimer impurity reference solution, the test sample is a sitagliptin phosphate test sample, and the test solution spiked with a sample is a mixed solution of the sitagliptin phosphate test sample and dimer impurities. The present invention establishes a method for determining the content of dimer impurities in sitagliptin phosphate by HPLC, and quantitatively detects dimer impurities that may exist in sitagliptin phosphate. The present invention has strong specificity, high sensitivity, good repeatability, and good durability, and is of great significance for the quality control of dimer impurities in sitagliptin phosphate, and provides a basis for the establishment of the quality standard of sitagliptin phosphate.

[0123] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for determining the content of dimer impurities in sitagliptin phosphate, characterized in that: The following steps are involved: (1) Mixing the dimer impurity reference substance and the diluent to obtain a reference solution; Mixing the sitagliptin phosphate test sample and the diluent to obtain a test sample solution; The sitagliptin phosphate test sample, the dimer impurity and the diluent are mixed to obtain a test sample spiked solution; (2) Perform chromatographic detection on the control solution, the test solution, and the test spiked solution, and calculate the dimer impurity content in sitagliptin phosphate by peak area and concentration using the external standard method; The mobile phase A of the chromatographic detection is a trifluoroacetic acid aqueous solution with a concentration of 0.01-0.1%, and the mobile phase B is acetonitrile, wherein the pH of the mobile phase A is 5-6, and the reagent for adjusting the pH value is triethylamine; The conditions of the chromatographic detection are: flow rate of 0.9-1.1 mL / min, column temperature of 28-32°C, injection volume of 18-22 µL, detection wavelength of 260-270 nm, and detection time of 18-22 min; In the step (2), the chromatographic detection uses an octadecylsilane bonded silica gel column with a column length of 250 mm, an inner diameter of 4.6 mm, and a filler particle size of 5 μm; In the step (2), the procedure of gradient elution in the chromatographic detection is: At 0-8 min, it was 95-5% mobile phase A and 5-95% mobile phase B; From 8 to 10 minutes, excluding 8 minutes, it is 5% mobile phase A and 95% mobile phase B; At 10-12 min, excluding 10 min, it is 5-95% mobile phase A and 95-5% mobile phase B; From 12 to 20 minutes, excluding 12 minutes, it is 95% mobile phase A and 5% mobile phase B; The structural formula of the dimer impurity is .

2. The measuring method according to claim 1, characterized in that The concentration of the control solution is 0.01~1.3µg / mL; The concentration of the test solution is 0.5~1.5mg / mL; The concentration of the sitagliptin phosphate test sample in the test sample spiked solution is 0.5-1.5 mg / mL, and the concentration of the dimer impurity is 0.01-1.3 µg / mL.

3. The measuring method according to claim 1 or 2, characterized in that In the step (2), the model of the chromatographic column is Waters XBridge C18.

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