Detection method of R-3-aminopiperidine dihydrochloride

Abstract

The invention belongs to the technical field of drug analysis and detection, and particularly relates to a detection method of R-3-aminopiperidine dihydrochloride. According to the method, 0.07-0.2 vol% formic acid aqueous solution and acetonitrile are taken as mobile phases, gradient elution is carried out according to a specific ratio, and qualitative and quantitative analysis can be carried out on R-3-aminopiperidine dihydrochloride by combining mass spectrometric detection; according to the present invention, the method has characteristics of rapid detection and rapid analysis, the target peak pattern is good, the peak can appear after about 0.6-0.9 min, the whole detection time is within 5 min, rapid detection and analysis can be achieved, methodological investigation results prove that the method has advantages of high precision, good repeatability, high accuracy and high sensitivity, and the measured R-3-aminopiperidine dihydrochloride still presents the good linear relationship in the extremely low concentration range of 1.018-203.6 ng / mL, the method is favorable for monitoring the R-3-aminopiperidine dihydrochloride in the production of the ridipeptidyl peptidase-4 inhibitor.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug analysis and detection, and more specifically, relates to a detection method for R-3-aminopiperidine dihydrochloride. Background Art

[0002] (R)-3-aminopiperidine dihydrochloride is a chiral compound and an important intermediate or starting material for the synthesis of chemical and pharmaceutical drugs. It has important applications in the chemical and pharmaceutical industries and is an important intermediate for new fine chemicals, low-toxic pesticides, high-value-added medicines and chemical additives. It is mainly used in the pharmaceutical industry to synthesize dipeptidyl peptidase-4 (DPP-IV) inhibitors, such as trelagliptin, alogliptin, linagliptin and other diabetes drugs. For example, the (R)-3-aminopiperidine dihydrochloride of the present invention is used as a reaction raw material for the synthesis of linagliptin, and its synthesis route is as follows:

[0003]

[0004] Among them, the residue of (R)-3-aminopiperidine dihydrochloride will also react with intermediates, solvents, etc., resulting in an increase in the impurity content in the final product, thereby affecting the purity and quality of the final product of DPP-IV inhibitor drugs. At the same time, according to the requirements of ICH guideline Q3A, (R)-3-aminopiperidine dihydrochloride needs to be studied with a limit of 0.1%. Therefore, it is necessary to establish a simple, accurate and rapid method for detecting (R)-3-aminopiperidine dihydrochloride to detect the final product to ensure the quality of the drug.

[0005] According to the structure, dipeptidyl peptidase inhibitors such as linagliptin have strong conjugated groups such as benzoheterocycles and strong ultraviolet absorption, while R-3-aminopiperidine dihydrochloride has no conjugated groups and weak ultraviolet absorption, and the polarity difference between the two is large, so it is difficult to detect using the conventional HPLC-UV method; if the HPLC-UV method is used, (R)-3-aminopiperidine dihydrochloride needs to be derivatized to obtain the conjugated group, but the derivatization method has high requirements for the derivatization matrix, and the experiment is cumbersome, and the recovery rate is difficult to meet the requirements; at the same time, the boiling point of R-3-aminopiperidine dihydrochloride is 241.9°C, which is difficult to vaporize due to its high boiling point, and it cannot enter the chromatographic column, and it is easy to decompose and deteriorate in the chromatographic column. At the least, no results can be measured, and at the worst, the chromatographic column is contaminated or even blocked; and conventional purity or content determination uses a gas chromatography column, but when it is used as an impurity determination, the limit is low, and the method specificity and sensitivity are required to be higher, which is difficult to meet the requirements when using gas chromatography. Chinese patent application CN15656389A discloses a method for determining the content of R-3-aminopiperidine dihydrochloride in alogliptin benzoate by LC-MS. The method adopts LC-MS coupling technology to directly analyze the content of R-3-aminopiperidine dihydrochloride, and the chromatographic and mass spectrometric information of the sample can be obtained without complicated separation and purification steps for R-3-aminopiperidine dihydrochloride, which effectively improves the detection effect. However, the R-3-aminopiperidine dihydrochloride disclosed in the method shows a good linear relationship in the range of 0.9979 to 4.5360 μg / mL, and still requires a large injection volume (≥30 μL) and a long elution time (>20min). When the content of R-3-aminopiperidine dihydrochloride is lower (<0.5 μg / mL), the above method cannot meet the detection requirements. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the prior art that it is impossible to accurately and quickly detect extremely low content (<0.5 μg / mL) of R-3-aminopiperidine dihydrochloride based on a low-dosage injection volume, and to provide a detection method for R-3-aminopiperidine dihydrochloride.

[0007] The object of the present invention is to provide the application of the method.

[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0009] The present invention protects a method for detecting R-3-aminopiperidine dihydrochloride in linagliptin, which uses liquid chromatography-mass spectrometry to detect the test sample;

[0010] The liquid chromatography conditions are: using octadecylsilane bonded silica gel as a chromatographic column as a filler, using 0.07-0.2 vol% formic acid aqueous solution as mobile phase A, acetonitrile as mobile phase B, and detecting under the following gradient elution conditions:

[0011]

[0012] The mass spectrometry conditions are: using an electrospray ion source and a multiple ion reaction positive ion detection mode.

[0013] After a lot of preliminary research, the inventor creatively discovered that according to the characteristics of the drug, the overall influence of the mobile phase, gradient elution conditions, mass spectrometry conditions and other detection parameters was continuously studied by using liquid chromatography-mass spectrometry, and a method for quickly and accurately detecting R-3-aminopiperidine dihydrochloride as an impurity in the drug was summarized. The method can be used to qualitatively and quantitatively analyze R-3-aminopiperidine dihydrochloride based on a low injection volume; and the peak shape of the target peak is good, and the peak can be produced in about 0.6 to 0.9 minutes. The entire detection time is within 5 minutes, and the method can be quickly detected and analyzed. The methodological investigation proves that the method of the present invention has high precision, good repeatability, high accuracy and high sensitivity. The measured R-3-aminopiperidine dihydrochloride still shows a good linear relationship in an extremely low concentration range of 1.018 to 203.6 ng / mL, which is beneficial to the monitoring of R-3-aminopiperidine dihydrochloride in the production of dipeptidyl peptidase-4 inhibitors to ensure the quality of the final product.

[0014] Preferably, the gradient conditions are as follows:

[0015]

[0016] Furthermore, the electrospray ion source is an AJS-ESI source.

[0017] Furthermore, the multiple ion reaction positive ion detection mode is an MRM (multiple reaction monitoring) mode.

[0018] Furthermore, the selection of quantitative and qualitative ions in the mass spectrometry detection is as follows:

[0019]

[0020] Furthermore, the drying gas temperature of the electrospray ion source is 250-350°C; the drying gas flow rate is 4-6 L / min; the sheath gas temperature is 200-300°C; the sheath gas flow rate is 9-12 L / min; the nebulizer pressure is 40-50 psi; the nozzle voltage is 400-600 V; and the capillary voltage range is 3000-4000 V.

[0021] Preferably, the chromatographic column is Agilent ZORBAX Eclipse plus C 18 .

[0022] Preferably, the specifications of the chromatographic column are 3.0×50 mm and 1.8 μm.

[0023] Preferably, the flow rate of the mobile phase is 0.3-0.5 mL / min.

[0024] Preferably, the column temperature is 25-30°C.

[0025] Preferably, the injection volume of the detection is 2 to 5 μL.

[0026] Preferably, the test sample is prepared into a test sample solution using a mixed solution of acetonitrile, water and formic acid for detection.

[0027] Preferably, the volume ratio of acetonitrile, water and formic acid is 20-40:60-80:0.07-0.2.

[0028] More preferably, the volume ratio of acetonitrile, water and formic acid is 30:70:0.07-0.2.

[0029] Furthermore, the detection limit of the R-3-aminopiperidine dihydrochloride is 5.0 ppm.

[0030] Furthermore, the limit of quantification of the R-3-aminopiperidine dihydrochloride is 10.0 ppm.

[0031] The present invention also protects the application of the detection method in monitoring R-3-aminopiperidine dihydrochloride in the preparation of dipeptidyl peptidase-4 inhibitor.

[0032] Preferably, the dipeptidyl peptidase-4 inhibitor includes trelagliptin, alogliptin or linagliptin.

[0033] The invention has the following beneficial effects: the invention provides a method for detecting R-3-aminopiperidine dihydrochloride in linagliptin, wherein the method uses 0.07-0.2 vol% formic acid aqueous solution and acetonitrile as mobile phases, performs gradient elution in a specific ratio, and combines mass spectrometry detection to perform qualitative and quantitative analysis on R-3-aminopiperidine dihydrochloride; and the peak shape of the target peak is good, the peak can be produced in about 0.6-0.9 minutes, the entire detection time is within 5 minutes, and the method can be quickly detected and analyzed. Methodological investigation proves that the method of the invention has high precision, good repeatability, high accuracy and high sensitivity, and the measured R-3-aminopiperidine dihydrochloride still shows a good linear relationship in an extremely low concentration range of 1.018-203.6 ng / mL, which is beneficial to the monitoring of R-3-aminopiperidine dihydrochloride in the production of dipeptidyl peptidase-4 inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1This is a chromatogram of the test solution prepared using linagliptin in batch 20210421X in Example 1.

[0035] Figure 2 This is a chromatogram of the test solution prepared using linagliptin in batch 20210517X in Example 2.

[0036] Figure 3 This is a chromatogram of the test solution prepared using linagliptin in batch 20210823X in Example 3.

[0037] Figure 4 It is the chromatogram (quantitative ion pair) of the reference solution in Example 4.

[0038] Figure 5 The chromatogram (quantitative ion pair) of the test solution of Example 4.

[0039] Figure 6 This is a statistical diagram of the linear relationship of R-3-aminopiperidine dihydrochloride in Example 6 within a certain concentration range. DETAILED DESCRIPTION

[0040] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0041] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0042] The preparation methods of the reagents, instruments or samples to be tested used in the present invention are as follows:

[0043] R-3-aminopiperidine dihydrochloride was obtained from Emeishan Hongsheng Pharmaceutical Co., Ltd., batch number 2M018200901, purity 100%;

[0044] Linagliptin comes from Zhuzhou Qianjin Pharmaceutical Co., Ltd., with batch numbers 20210421X, 20210517X, and 20210823X.

[0045] Example 1 A method for detecting the content of R-3-aminopiperidine in linagliptin

[0046] 1. Preparation of test and reference substances

[0047] Sample preparation solvent (also known as blank reagent): 30% vol acetonitrile (containing 0.1% vol formic acid)

[0048] Test solution: Take about 10 mg of linagliptin raw material, weigh accurately, place in a 100 ml volumetric flask, add solvent to dissolve, dilute to the scale, and shake well.

[0049] Reference substance solution: take about 17 mg of (R)-3-aminopiperidine dihydrochloride (equivalent to about 10 mg of (R)-3-aminopiperidine), accurately weigh, place in a 100 ml volumetric flask, add solvent to dissolve, dilute to scale, shake well to obtain reference substance stock solution, accurately measure 1 ml of the obtained reference substance stock solution, place in a 20 ml volumetric flask, add solvent to dilute to scale, shake well to obtain reference substance dilution solution, accurately measure 2 ml of the obtained reference substance dilution solution, place in a 100 ml volumetric flask, add solvent to dilute to make a solution containing about 0.1 μg of (R)-3-aminopiperidine per 1 ml.

[0050] 2. HPLC conditions

[0051] Chromatographic mass spectrometry conditions: octadecylsilane bonded silica gel was used as filler (Agilent ZORBAX Eclipseplus C18, 3.0×50mm, 1.8μm); 0.1vol% formic acid aqueous solution was used as mobile phase A, acetonitrile was used as mobile phase B, elution was performed as shown in the table below, column temperature was 30°C, flow rate was 0.3ml per minute, injection volume was 2μl. The elution procedure and mass spectrometry related parameters are shown in Table 1 below.

[0052] Table 1 Gradient elution conditions

[0053]

[0054]

[0055] 3. Mass spectrometry conditions

[0056] An electrospray ion source was used, the ion source was AJS-ESI source, positive mode detection, MRM mode, and the selection of quantitative and qualitative ions is shown in Table 2;

[0057] Ion source parameters: drying gas temperature 300°C; drying gas flow rate 5 L / min; sheath gas temperature 250°C; sheath gas flow rate 11 L / min; nebulizer pressure 45 psi; nozzle voltage 500 V; capillary voltage 3500 V. Time segment setting: 0-1.5 min cut into the mass spectrometer, 1.5 min later cut into the waste liquid.

[0058] Table 2 Selection of quantitative and qualitative ions

[0059]

[0060] The calculation formula of (R)-3-aminopiperidine impurity is as follows:

[0061]

[0062] In the formula, S 杂质 : The quantitative impurity peak area in the test solution;

[0063] S 对 : The quantitative impurity peak area in the reference solution, which is the average value of the results of repeated injections of the same sample;

[0064] M 对 : The amount of reference substance, mg;

[0065] M 供 : The amount of the test sample, mg;

[0066] ω 对 : Content of reference substance, %, ω 对 is 100%;

[0067] 0.5818: Conversion factor of (R)-3-aminopiperidine (100.10) relative to (R)-3-aminopiperidine dihydrochloride (172.05);

[0068] The dilution factor of the above test products is 100;

[0069] The dilution factor of the above-mentioned reference substance is 100,000.

[0070] 4. Test and results

[0071] Accurately measure the test solution and the reference solution. The reference solution is the same sample and is injected into the liquid chromatograph 5 times. The test solution is the same sample and is injected into the liquid chromatograph 2 times. Record the spectral data and refer to the above calculation formula to detect the content of (R)-3-aminopiperidine impurity in linagliptin in batch 20210421X.

[0072] Example 2 A method for detecting the content of R-3-aminopiperidine in linagliptin

[0073] The detection method of Example 2 is different from that of Example 1 in that the column temperature in the HPLC condition is 25°C; the ion source parameters in the mass spectrometry condition are as follows: drying gas temperature 250°C; drying gas flow rate 4 L / min; sheath gas temperature 200°C; sheath gas flow rate 9 L / min; nebulizer pressure 40 psi; nozzle voltage 400 V; capillary voltage 3000 V; the selection of quantitative and qualitative ions is shown in Table 3; for batch 20210517X The prepared test solution was measured.

[0074] Other steps and parameters refer to Example 1.

[0075] Table 3 Selection of quantitative and qualitative ions

[0076]

[0077] Example 3 A method for detecting the content of R-3-aminopiperidine in linagliptin

[0078] The detection method of Example 3 is different from that of Example 1 in that the flow rate in the HPLC condition is 0.5 mL / min; the ion source parameters in the mass spectrometry condition are as follows: drying gas temperature 350°C; drying gas flow rate 6 L / min; sheath gas temperature 300°C; sheath gas flow rate 12 L / min; nebulizer pressure 50 psi; nozzle voltage 600 V; capillary voltage 4000 V; the selection of quantitative and qualitative ions is shown in Table 4; for batch 20210823X The prepared test solution was measured.

[0079] Other steps and parameters refer to Example 1.

[0080] Table 4 Selection of quantitative and qualitative ions

[0081]

[0082] Test results of Examples 1 to 3

[0083] Table 5 Determination results of the content of (R)-3-aminopiperidine impurity in different batches of linagliptin

[0084]

[0085] Note: 1. All calculations use S quantitative, and S qualitative is used to assist in determining false positive results; 2. To avoid false positive results, the ratio of the peak area of ​​the qualitative and quantitative ions in the test sample should be within the range of ±25% of the ratio of the peak area of ​​the qualitative and quantitative ions in the reference sample, that is, the value of S qualitative / S quantitative is between 0.19 and 0.31.

[0086] As can be seen from the table, the content of (R)-3-aminopiperidine impurity in different batches of linagliptin is within 0.1%, and the value of S qualitative / S quantitative is within 0.19-0.31, indicating that there is no false positive result, indicating that this method can meet the quality control requirements of drugs.

[0087] Using different batches The chromatogram of the prepared test solution is as follows Figures 1 to 3 As shown, no other chromatographic peaks appear in the chromatogram, indicating that the blank solvent does not interfere with the detection and the detection meets the requirements.

[0088] Embodiment 4 Specificity Investigation

[0089] According to the method of Example 1, blank solvent, test solution and reference solution were sampled and tested. The test results are shown in Table 6. Figures 4-5 shown.

[0090] Table 6 Specificity results

[0091] name RT / min S Quantification S qualitative / S quantitative Blank solvent / / / Reference solution 0.655 1143877 0.24 Test solution 0.663 16894 0.26

[0092] Note: 1. All calculations use S quantitative, and S qualitative is used to assist in determining false positive results; 2. To avoid false positive results, the ratio of the peak area of ​​the qualitative and quantitative ions in the test sample should be within the range of ±25% of the ratio of the peak area of ​​the qualitative and quantitative ions in the reference sample, that is, the value of S qualitative / S quantitative is between 0.19 and 0.31.

[0093] Figures 4-5 The data results in Table 6 show that no chromatographic peaks were detected in the blank solvent, and in the reference solution ( Figure 4 ) and test solution ( Figure 5 ), no other chromatographic peaks appeared in the chromatogram, indicating that the blank solvent did not interfere with the detection; the value of Squalitative / Squantitative was between 0.19 and 0.31, indicating that there were no false positive results and the specificity met the requirements.

[0094] Example 5 Determination of detection limit and quantification limit

[0095] In order to analyze the detection ability of R-3-aminopiperidine dihydrochloride, the reference substance stock solution prepared in Example 1 was diluted step by step to investigate the limit of detection (LOD) and limit of quantification (LOQ) of impurities. The results are shown in Tables 7 and 8.

[0096] Table 7 Detection limit results

[0097] name RT / min S Quantification S qualitative / S quantitative S / N Detection limit / ppm R-3-aminopiperidine 0.663 6727 0.29 34.8 5.0

[0098] Table 8 Quantitation Limit Results

[0099] name RT / min S Quantification S qualitative / S quantitative S / N Limit of quantification / ppm R-3-aminopiperidine 0.663 14017 0.28 147.1 10.0

[0100] The results showed that the detection method had a low detection limit and quantification limit for R-3-aminopiperidine dihydrochloride, had good sensitivity, and met the detection requirements.

[0101] Example 6 Determination of linear equation

[0102] An appropriate amount of the reference substance stock solution prepared in Example 1 was accurately measured and quantitatively diluted with solvent to prepare a series of linear solutions containing about 1, 5, 20, 100, and 200 ng / mL R-3-aminopiperidine (1% to 200% limit concentration) per 1 mL. The linear solutions of each concentration were taken and tested according to the relevant method disclosed in Example 1. Linear regression was performed with concentration as the abscissa and peak area as the ordinate, and the regression equation and correlation coefficient were calculated. The results are shown in Tables 9 and Figure 6 .

[0103] Table 9 Linearity results

[0104]

[0105] The results showed that R-3-aminopiperidine dihydrochloride had a good linear relationship within the concentration range of 1.018 ng / mL to 203.6 ng / mL.

[0106] Example 7 Investigation of Accuracy

[0107] Take about 10 mg of linagliptin, accurately weigh 9 portions, place in a 100 mL volumetric flask, add an appropriate amount of solvent to dissolve, accurately measure 1 mL, 2 mL, and 3 mL of the reference substance dilution in Example 1, place in the same volumetric flask, add solvent to dilute to the scale, shake well, and prepare spiked test solutions equivalent to 50%, 100%, and 150% of the limit concentration, respectively, and prepare 3 portions in parallel for each concentration. Test according to the method conditions of Example 1, and the results are shown in Table 10.

[0108] Table 10 Accuracy results

[0109]

[0110]

[0111] The results showed that the recovery rate of R-3-aminopiperidine dihydrochloride was between 91.8% and 119.3%, and the RSD was 8.9% (n=9), indicating that the method had good accuracy.

[0112] Example 8 Repeatability Study

[0113] Spiked test solution: Take about 10 mg of linagliptin, accurately weigh 6 portions, put them in a 100 ml volumetric flask, add solvent to dissolve, accurately add 2 ml of reference substance diluent, and dilute to the scale with ethanol, and shake well.

[0114] The six spiked test solutions and the reference solution were tested according to the method of Example 1. The results are shown in Table 11.

[0115] Table 11 Repeatability results

[0116]

[0117] The results showed that the range of the content of R-3-aminopiperidine dihydrochloride was 0.01% (n=6), indicating that the method had good repeatability.

[0118] Example 9 Investigation of Solution Stability

[0119] Take the spiked test solution and reference solution in Example 1, place the solution at 4°C, inject 2 μL of each sample at 0h and 2h, inject into the liquid spectrometer, record the chromatogram, measure and calculate the change rate of the peak area at each time point compared with the peak area at 0h, and see Table 12 for the results.

[0120] Table 12 Solution stability test results (4°C temperature control)

[0121]

[0122] The results show that the peak area change rate of the reference solution and the spiked test solution is less than 5.0% when placed at 4°C for 2 hours, indicating that they are stable within 2 hours at 4°C. Since the analysis time of the method is 5 minutes, the solution stability of 2 hours is sufficient to complete the sample determination.

[0123] Through the above verification process and verification results, it is proved that the invention can accurately and effectively detect the content of R-3-aminopiperidine dihydrochloride. This method has strong specificity, high sensitivity, stability and reliability, and has important research significance for the quality research of linagliptin. This method has wide applicability and can also be used for quality monitoring of other dipeptidyl peptidase-4 (DPP-IV) inhibitors.

[0124] Comparative Example 1 A method for detecting the content of R-3-aminopiperidine dihydrochloride

[0125] Comparative Example 1 provides a method for detecting the content of R-3-aminopiperidine dihydrochloride, which is mainly different from Example 1 in that gas chromatography is used for detection. The specific method comprises the following steps:

[0126] It is determined according to the residual solvent determination method (Method 2, General Rules 0861, Part IV, 2 of the 2020 edition of the Chinese Pharmacopoeia).

[0127] Chromatographic conditions: a capillary column with 5% phenyl-95% methylpolysiloxane as the stationary phase was used as the chromatographic column; the initial column temperature was 60°C, which was increased to 200°C at a rate of 20°C per minute, and then increased to 250°C at a rate of 100°C per minute, and maintained for 8 min; the injection port temperature was 260°C; the detector (FID) temperature was 300°C; the column flow rate was 1.0 mL / min, the carrier gas was nitrogen, and the split ratio was 2:1; the injection volume was 0.5 μL

[0128] Reference substance solution: Take 17 mg of R-3-aminopiperidine dihydrochloride (approximately equivalent to 10 mg of (R)-3-aminopiperidine), accurately weigh, place in a 50 mL volumetric flask, add 0.4 mL of 1 mol / L sodium hydroxide solution, add appropriate amount of methanol, ultrasonically dissolve and dilute to the scale, shake well, as the reference substance stock solution, accurately measure 2 mL, place in a 20 mL volumetric flask, add 0.1 mL of 1 mol / L sodium hydroxide solution, and dilute to the scale with methanol.

[0129] Spiked test solution: Take about 0.3g of linagliptin, weigh accurately, place in a 10mL volumetric flask, add 0.2mL of 1mol / L sodium hydroxide solution, and then ultrasonicate with methanol to dissolve, accurately measure 1mL, 2mL, and 3mL of reference substance stock solution and place in the above volumetric flask, dilute to the mark with methanol, and use as 50%, 100%, and 150% spiked test solution. Prepare 2 samples for each concentration.

[0130] The reference solution and the spiked test solution were sampled and tested. The results are shown in Table 13.

[0131] Table 13 Recovery results of spiked test samples

[0132]

[0133] The results showed that the recovery rate was far beyond the range of 80% to 120%, which did not meet the requirements of the chemical drug impurity determination standards in the pharmacopoeia, indicating that this method was not suitable for the content determination of R-3-aminopiperidine dihydrochloride.

[0134] Comparative Example 2 A method for detecting the content of R-3-aminopiperidine dihydrochloride in gliptin

[0135] This method mainly refers to the literature (Qu Xilong, Li Tiejian, Liu Zhijun, Zhang Guimin. Rapid pre-column derivatization HPLC method for the detection of (R)-3-aminopiperidine in trelagliptin [J]. Analysis Laboratory, 2021, 40(03): 361-365. DOI: 10.13595 / j.cnki.issn1000-0720.2020.072601.), and the specific method is as follows:

[0136] Derivatization reagent: weigh 282 mg of o-phthalaldehyde into a 25 mL brown volumetric flask, add 5 mL of methanol to dissolve it, then add 305 μL of 3-mercaptopropionic acid, dilute to the scale with borate buffer solution (weigh about 25 g of boric acid, 8 g of NaOH, add 1000 mL of water to dissolve it, and adjust the pH to 10.5), and let it stand in the dark for 30 min.

[0137] R-3-aminopiperidine reference substance mother solution: Take 21 mg of R-3-aminopiperidine dihydrochloride (approximately equivalent to 12.5 mg of (R)-3-aminopiperidine), place it in a 25 mL volumetric flask, add acetonitrile-water (50:50) to dissolve and dilute to the scale, accurately measure 0.2 mL of the reference substance stock solution, place it in a 10 mL volumetric flask, dissolve and dilute to the scale with acetonitrile-water (50:50) as the R-3-aminopiperidine reference substance mother solution.

[0138] R-3-aminopiperidine reference solution: Accurately measure 1.0 mL of reference stock solution and place it in a 10 mL volumetric flask. Add 1.0 mL of derivatization reagent and dilute to the mark with borate buffer solution. Shake for 30 seconds and let stand at room temperature for 1 minute.

[0139] Chromatographic column: YMC-Triart C 18 Column (150mm×4.6mm, 5μm); mobile phase A is 0.05mol / L sodium acetate (pH6.0), mobile phase B is methanol, gradient elution is performed according to the program in the following table, flow rate: 1.0mL / min; column temperature: 30℃; detection wavelength: 331nm; injection volume: 20μL, elution gradient see Table 14.

[0140] Table 14 Gradient elution conditions

[0141]

[0142] Take the reference solution and inject it for testing. At the same time, take the reference solution and place it at 2-8℃ for 1 hour before injecting it for testing. The results are shown in Table 15.

[0143] Table 15 Stability test results of reference solution

[0144] Time (h) 0 1 Peak area 45597 38966 Peak area change rate (%) / -14.5

[0145] The results showed that the solution stability of this method was extremely poor. When the reference solution was placed at 2-8°C for 1 hour, the main peak area degraded by 14.5%, indicating that this method was not easy to operate and was not suitable for sample analysis.

[0146] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A detection method for R-3-aminopiperidine dihydrochloride, characterized in that, Liquid chromatography-mass spectrometry was used to detect the test products; The liquid chromatography conditions are: using octadecylsilane bonded silica gel as a chromatographic column as a filler, using 0.07-0.2 vol% formic acid aqueous solution as mobile phase A, acetonitrile as mobile phase B, and detecting under the following gradient elution conditions: The mass spectrometry conditions are: using an electrospray ion source and a multiple ion reaction positive ion detection mode.

2. The detection method according to claim 1, characterized in that: The selection of quantitative and qualitative ions in the mass spectrometry conditions is as follows:

3. The detection method according to claim 1, characterized in that: The drying gas temperature of the electrospray ion source is 250-350°C; the drying gas flow rate is 4-6 L / min; the sheath gas temperature is 200-300°C; the sheath gas flow rate is 9-12 L / min; the nebulizer pressure is 40-50 psi; the nozzle voltage is 400-600 V; and the capillary voltage is 3000-4000 V.

4. The detection method according to claim 1, characterized in that: The chromatographic column is Agilent ZORBAX Eclipseplus C 18 .

5. The detection method according to claim 1, characterized in that: The flow rate of the mobile phase is 0.3-0.5 mL / min.

6. The detection method according to claim 1, characterized in that: The column temperature is 25-30°C.

7. The detection method according to claim 1, characterized in that: The injection volume of the detection is 2 to 5 μL.

8. The detection method according to claim 1, characterized in that: The test sample is prepared into a test sample solution using a mixed solution of acetonitrile, water and formic acid for detection.

9. The detection method according to claim 8, characterized in that: The volume ratio of acetonitrile, water and formic acid is 20-40:60-80:0.07-0.

2.

10. Use of the detection method according to any one of claims 1 to 9 in monitoring R-3-aminopiperidine dihydrochloride in the preparation of dipeptidyl peptidase-4 inhibitors.

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