Method for detecting nitrosamine impurities in starting material N-Boc-piperazine of tandospirone citrate

Abstract

The invention provides a method for detecting nitrosamine impurities in a starting material N-Boc-piperazine of tandospirone citrate, which comprises the following steps: carrying out linear gradient elution on a chromatographic column by taking octadecylsilane chemically bonded silica as a filler, a potassium hexafluorophosphate solution as a mobile phase A and acetonitrile as a mobile phase B; the pH value of the potassium hexafluorophosphate solution ranges from 4.3 to 5.5. The method is high in specificity, high in detection sensitivity and accuracy and good in linearity, and the nitrosamine impurity 1-nitrosopiperazine contained in N-Boc-piperazine can be accurately measured. According to the method, the technical barrier of detecting the nitrosamine impurities by using a gas chromatography-mass spectrometry or liquid chromatography-mass spectrometry method at present is overcome, the detection cost is reduced, and meanwhile, other impurities contained in the method do not influence the accurate detection of the nitrosamine impurities.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical analysis methods, and particularly relates to a method for detecting nitrosamine impurities in N-Boc-piperazine, a starting material of tandospirone citrate. Background Art

[0002] Tandospirone citrate has antianxiety and antidepressant effects. Its preparation method is similar to that of buspirone. The synthetic starting materials include N-derivatives of piperazine, substituted amides or anhydrides, and aliphatic chains containing four carbon atoms (An efficient synthesis of buspirone and its analogues, Jacek Cybulski), and N-Boc-piperazine is a common starting material.

[0003] In the field of pharmaceutical analysis, nitrosamine impurities belong to the "concern queue" substances mentioned in the ICH M7(R2) (Evaluation and Control of DNA-Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk) guideline.

[0004] As far as we know, nitrosamine impurities such as dimethylnitrosamine (NDMA) and N-nitrosodiethylamine (NDEA) may be generated through the nitrosation mechanism, that is, under certain conditions, amine compounds, especially secondary amines, react with sodium nitrite (NaNO2) or other nitrosating agents to produce nitrosamine impurities.

[0005] The possible sources of introduction of nitrosating agents include: nitrites, nitrite esters, nitrous acid, substances prepared from nitrites (such as sodium azide, etc.) and oxidation of amine compounds.

[0006] Therefore, N-Boc-piperazine is likely to generate the nitrosamine impurity 1-nitrosopiperazine through the following pathway:

[0007]

[0008] There are several main ways of introducing nitrosamines:

[0009] (I) Risks of nitrosamine impurities introduced by process

[0010] If materials that can introduce secondary amines and nitrosating agents (including starting materials, solvents, reagents, catalysts, intermediates, etc.) are used in the same process step, there is a high risk of introducing nitrosamine impurities. Even if materials that can introduce secondary amines and nitrosating agents are used in different process steps, nitrosamine impurities may still be generated.

[0011] 2. Risks Introduced by Pollution

[0012] The use of materials contaminated with nitrosamine impurities (starting materials, intermediates, solvents, reagents, catalysts, etc.) during the production of raw materials may bring the risk of nitrosamine impurities; the use of recycled materials also has the risk of introducing nitrosamine impurities; when producing different varieties on the same production line, cross-contamination may also be a potential cause of the introduction of nitrosamine impurities.

[0013] 3. Risks of degradation

[0014] Some drugs themselves will degrade to produce nitrosamine impurities, such as ranitidine, which will produce nitrosamine impurities at high temperatures.

[0015] 1-Nitrosopiperazine is mutagenic. EMA has announced that the acceptable daily intake (AI) of this impurity is 400 ng / day, and the limit of nitrosamine impurities in the starting material N-Boc-piperazine is strictly controlled by the limit of this impurity in the preparation (tandospirone citrate tablets).

[0016] According to the calculation formula:

[0017]

[0018] Where: AI is the acceptable daily intake of 400ng;

[0019] The maximum daily dose of tandospirone citrate tablets is 60 mg;

[0020] The limit of the impurity 1-nitrosopiperazine was calculated to be 6.7 ppm, and the limit was strictly controlled to 6 ppm.

[0021] Currently, the detection methods for nitrosamine impurities include column chromatography and gas phase detection methods, and no disclosure of liquid phase detection methods with high detection efficiency and convenient use has been retrieved. Since 1-nitrosopiperazine has weak retention and low limit, it is urgent to develop a liquid phase method that can accurately and effectively detect this impurity.

[0022] In addition, N-Boc-piperazine may contain the following impurities:

[0023]

[0024]

[0025] The presence of the above impurities may affect the accurate quantification of the nitrosamine impurities under study and increase the difficulty of the analysis. Summary of the invention

[0026] In order to fill the technical gap in detecting the impurity, the present invention provides a method for detecting nitrosamine impurities in N-Boc-piperazine, a starting material of tandospirone citrate, wherein the structural formula of the nitrosamine impurity is:

[0027]

[0028] In order to ensure the safety and quality of drugs and achieve effective risk control, the present invention achieves the effect of strictly controlling nitrosamine impurities. The technical solution of the present invention is as follows:

[0029] Detection method of nitrosamine impurities in N-Boc-piperazine, the starting material of tandospirone citrate,

[0030] The chromatographic column uses octadecylsilane bonded silica gel as a filler, potassium hexafluorophosphate solution as a mobile phase A, and acetonitrile as a mobile phase B, and linear gradient elution is performed; the pH value of the potassium hexafluorophosphate solution ranges from 4.3 to 5.5.

[0031] Preferably, the pH value of the potassium hexafluorophosphate solution is 4.8.

[0032] Preferably, the concentration of the potassium hexafluorophosphate solution is in the range of 0.015 mol / L to 0.025 mol / L, preferably 0.02 mol / L.

[0033] Preferably, the gradient elution program is:

[0034] Within 0 to 6 minutes, the volume ratio of mobile phase A and mobile phase B remained unchanged at 95-100:5-0; within 6 to 6.1 minutes, the volume ratio of mobile phase A and mobile phase B linearly changed to 10:90; within 6.1 to 16 minutes, the volume ratio of mobile phase A and mobile phase B remained unchanged at 10:90; within 16 to 17 minutes, the volume ratio of mobile phase A and mobile phase B linearly changed from 10:90 to 95-100:5-0; within 17 to 27 minutes, the volume ratio of mobile phase A and mobile phase B remained unchanged at 95-100:5-0.

[0035] Preferably, the detection wavelength is 235±2 nm.

[0036] The column temperature of the chromatographic column is 30°C to 45°C, preferably 35°C.

[0037] The flow rate of the mobile phase is in the range of 0.7 ml / min to 1.3 ml / min, preferably 1 ml / min.

[0038] Preferably, the chromatographic column adopts a YMC-Triart C18, 4.6 mm×150 mm, 3 μm or a chromatographic column with equivalent performance.

[0039] Furthermore, the detection method also includes sample preparation:

[0040] Test solution: Dissolve the product in solvent and quantitatively dilute to make a solution containing approximately 15 mg of N-Boc-piperazine per 1 ml.

[0041] Reference solution: Take an appropriate amount of nitrosamine impurity reference substance, weigh accurately, dissolve in solvent and quantitatively dilute to make a solution containing 0.09 μg per 1 ml.

[0042] Furthermore, the solvent is a 1% potassium phosphate tripotassium aqueous solution with a pH value ranging from 11.5 to 12.5, and the preparation method is to weigh 1g of potassium phosphate tripotassium, add 100ml of water to dissolve, and shake well to obtain the solution.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] 1. The starting material N-Boc-piperazine of tandospirone citrate contains a secondary amine structure, and there may be a risk of the presence of related nitrosamine impurities. In order to ensure the safety and quality control of the drug and achieve effective risk control, nitrosamine impurities must be controlled. Based on this, the present invention analyzes nitrosamine impurities in the starting material N-Boc-piperazine, develops a detection method and formulates a control strategy.

[0045] 2. In view of the problem that the 1-nitrosopiperazine impurity is weakly retained and is basically not retained by conventional mobile phases, the present invention uses a chaotropic potassium hexafluorophosphate solution as mobile phase A and acetonitrile as mobile phase B to avoid the problem of long system equilibrium time and difficult method repeatability when using ion pair reagents. In addition, the problem of inconsistent retention time of impurities in the reference solution and the retention time of impurities in the spiked test sample is solved by adjusting the solvent to a specific pH value.

[0046] 3. The method of the present invention has strong specificity, high detection sensitivity and accuracy, good linearity, and can accurately determine the nitrosamine impurity 1-nitrosopiperazine contained in N-Boc-piperazine. It not only overcomes the technical barriers of currently commonly used gas chromatography-mass spectrometry or liquid chromatography-mass spectrometry methods for detecting nitrosamine impurities, but also reduces the detection cost. At the same time, other impurities contained in the method do not affect the accurate detection of nitrosamine impurities. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0048] Figure 1 It is a specificity test spectrum, in which the spectrum from bottom to top is blank solvent, interference solution, spiked test solution, test solution and 1-nitrosopiperazine reference solution.

[0049] Figure 2 It is the result diagram under other solvent and chromatographic column conditions in Comparative Example 2, wherein the spectrum from bottom to top is the spiked test solution and the 1-nitrosopiperazine reference solution.

[0050] Figure 3 This is the result diagram under other chromatographic conditions in Comparative Example 3, wherein the diagram from bottom to top includes solvent, interfering solution, spiked test solution and 1-nitrosopiperazine reference solution.

[0051] Figure 4 This is the chromatogram when the mobile phase A in Comparative Example 4 is pH 4.2.

[0052] Figure 5 This is the chromatogram when the mobile phase A in Comparative Example 5 is pH 5.6.

[0053] Figure 6 This is the chromatogram in Comparative Example 8. DETAILED DESCRIPTION

[0054] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0055] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0056] Example 1, Detection of nitrosamine impurities in N-Boc-piperazine, the starting material of tandospirone citrate, by high performance liquid chromatography

[0057] Chromatographic conditions: Use octadecylsilane bonded silica gel as filler (YMC-Triart C18, 4.6mm×150mm, 3μm or chromatographic columns with equivalent performance are recommended); use 0.02mol / L potassium hexafluorophosphate solution as mobile phase A, and adjust the pH value to 4.8 with phosphoric acid; use acetonitrile as mobile phase B, and perform linear gradient elution according to the table below; flow rate is 1.0ml per minute; detection wavelength is 235nm; column temperature is 35℃; injection volume is 20μl.

[0058]

[0059] Solvent: 1% tripotassium phosphate solution.

[0060] Test solution: Take about 300 mg of the product (N-Boc-piperazine), accurately weigh it, put it in a 20 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well.

[0061] Reference solution: Take an appropriate amount of 1-nitrosopiperazine reference substance, weigh accurately, dissolve in solvent and quantitatively dilute to make a solution containing approximately 0.09 μg per 1 ml.

[0062] Determination method:

[0063] Accurately measure the test solution and reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0064] Limit: If there are impurity peaks in the chromatogram of the test solution, the impurity 1-nitrosopiperazine shall not exceed 0.0006% (6ppm) when calculated by peak area using the external standard method.

[0065] Calculation formula:

[0066]

[0067] Where: A 样 is the peak area of ​​1-nitrosopiperazine in the chromatogram of the test solution;

[0068] A 对 is the peak area of ​​1-nitrosopiperazine in the chromatogram of the reference solution;

[0069] W 对 is the sample weight of 1-nitrosopiperazine reference substance, mg;

[0070] W 样 is the sample weight of the test product, mg;

[0071] P is the content of the reference substance;

[0072] V 对 is the dilution volume of the reference solution, ml;

[0073] V 样 is the dilution volume of the test solution, ml.

[0074] Based on this method, the nitrosamine impurities that may exist in the sample can be effectively detected and quantified.

[0075] Example 2, method verification test

[0076] 2.1 System suitability and specificity test

[0077] The chromatographic conditions in Example 1 were used.

[0078] Solvent: 1% tripotassium phosphate solution.

[0079] 1-Nitrosopiperazine reference substance stock solution ①: Take about 3.6 mg of 1-nitrosopiperazine reference substance, accurately weigh it, put it in a 200 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well.

[0080] 1-Nitrosopiperazine reference stock solution ②: Accurately measure 1 ml of 1-nitrosopiperazine reference stock solution ①, place in a 50 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0081] Reference solution: Accurately measure 5 ml of 1-nitrosopiperazine reference stock solution ②, place in a 20 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0082] Test solution: Take about 300 mg of the product, weigh accurately, place in a 20 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well.

[0083] Spiked test solution: Take about 300 mg of this product, accurately weigh it, and place it in a 20 ml volumetric flask. Accurately measure 5 ml of 1-nitrosopiperazine reference stock solution ② and place it in the same 20 ml volumetric flask. Add solvent to dissolve and dilute to the scale, and shake well.

[0084] Impurity interference solution: Weigh approximately 0.75 mg each of impurity III-1 reference substance, impurity III-3 reference substance, impurity III-4 reference substance, impurity III-9 reference substance, impurity III-11 reference substance and impurity III-12 reference substance, place them in the same 5 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well.

[0085] This method provides a detection method for one of the nitrosamines, 1-nitrosopiperazine, and the other two nitrosamine impurities, including impurities III-11 and III-12, are detected by other methods.

[0086] Accurately measure the solvent and reference solution, and inject them into the liquid chromatograph respectively. The reference solution is injected 5 times continuously, and the chromatogram is recorded.

[0087] Verification results: The reference solution was injected 5 times in a row, and the RSD of the retention time of 1-nitrosopiperazine peak was 0.2%, less than 1%, and the RSD of the peak area was 0.2%, less than 5%. In the chromatogram of the first reference solution, the theoretical plate number of the 1-nitrosopiperazine peak was 15937, greater than 5000, the tailing factor was 1.1, less than 1.5, and the signal-to-noise ratio of the 1-nitrosopiperazine peak height was 404.4, greater than 100, indicating that the system has good applicability.

[0088] Accurately measure the test solution, spiked test solution and interference solution, inject them into the liquid chromatograph respectively, and record the chromatogram. Figure 1 shown.

[0089] Verification results: The blank solvent, the main component and other impurities in the test solution and other specific impurities studied in other methods (impurity iii-1, impurity iii-3, impurity iii-4, impurity iii-9, impurity iii-11 and impurity iii-12) do not interfere with the detection of 1-nitrosopiperazine (iii-10 in the figure), indicating that the method has good specificity.

[0090] 2.2 Linearity and range tests

[0091] Solvent: 1% potassium phosphate aqueous solution.

[0092] 1-Nitrosopiperazine reference substance stock solution: Use the 1-nitrosopiperazine reference substance stock solution ② prepared under “2.1 System suitability and specificity test”.

[0093] Linear solution: Accurately measure 1.0ml, 3.0ml, 5.0ml, 8.0ml and 12.0ml of the 1-nitrosopiperazine reference stock solution, place them in different 20ml volumetric flasks, dilute to the scale with solvent, shake well, and use them as 20%, 60%, 100%, 160%, 240% linear solutions (as the 2nd to 6th points of the linear test respectively).

[0094] Verification results: The concentration C of 1-nitrosopiperazine showed a good linear relationship with the peak area in the concentration range of 0.0027 μg / ml to 0.2126 μg / ml. The linear equation was A=77236.6337C-22.3329. The linear correlation coefficient r was 1.0000, which was greater than 0.99. The absolute value of the intercept was equivalent to 0.32% of the response value of 100%, which was less than 25%, indicating that the method had a good linear relationship.

[0095] 2.3 Limit of quantitation and limit of detection test

[0096] Solvent: 1% potassium phosphate aqueous solution.

[0097] Quantitation limit solution: Accurately measure 3 ml of the 20% linear solution under "2.2 Linearity and range test" and place it in a 20 ml volumetric flask. Dilute to the mark with solvent and shake well.

[0098] Detection limit solution: Accurately measure 3 ml of the quantitative limit solution, place it in a 10 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0099] The signal-to-noise ratio S / N≈10 of the 1-nitrosopiperazine peak height was used as the limit of quantification, and the signal-to-noise ratio S / N≈3 was used as the limit of detection. The lowest quantitative concentration (μg / ml) and the lowest detection concentration (μg / ml) were reported as percentages relative to the test sample concentration and relative to the limit concentration. Six solutions of the limit of quantification concentration were prepared and injected sequentially, and the chromatograms were recorded to examine the repeatability of the limit of quantification test.

[0100] Verification results: The minimum quantitative concentration (quantification limit) of 1-nitrosopiperazine is 0.0027μg / ml (0.0540ng); the quantitative limit concentration is equivalent to 0.00002% of the test sample concentration, which is less than 0.00006%; the quantitative limit concentration is equivalent to 3.0% of the limit concentration, which is less than 10%. The minimum detection concentration (detection limit) of 1-nitrosopiperazine is 0.0008μg / ml (0.0160ng); the detection limit concentration is equivalent to 0.00001% of the test sample concentration, which is less than 0.00003%; the detection limit concentration is equivalent to 0.9% of the limit concentration, which is less than 5%.

[0101] In the repeatability test of the limit of quantitation, the RSD of the peak area of ​​1-nitrosopiperazine was 5.5%, which was less than 10%, indicating that the repeatability results of the limit of quantitation were good.

[0102] 2.4 Solution stability test

[0103] Solvent: 1% potassium phosphate aqueous solution.

[0104] 1-Nitrosopiperazine reference substance stock solution: Use the 1-nitrosopiperazine reference substance stock solution ② prepared under “2.1 System suitability and specificity test”.

[0105] Reference solution: Accurately measure 5 ml of 1-nitrosopiperazine reference stock solution, place it in a 20 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0106] Test solution: Take about 300 mg of the product, weigh accurately, place in a 20 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well.

[0107] Spiked test solution: Take about 300 mg of this product, accurately weigh it, and place it in a 20 ml volumetric flask. Accurately measure 5 ml of 1-nitrosopiperazine reference stock solution and place it in the same 20 ml volumetric flask. Dilute to the scale with solvent and shake well.

[0108] The reference solution, test solution and spiked test solution were placed at room temperature, and samples were injected and analyzed at the end of 0 h, 3 h, 9 h and 15 h, and the chromatograms were recorded. The ratio of the peak area of ​​1-nitrosopiperazine at each time point in the chromatograms of the reference solution and the spiked test solution to the peak area at 0 hour was calculated to examine whether there were impurity peaks at the elution position of 1-nitrosopiperazine in the test solution at each time point.

[0109] Verification results: In the chromatograms of the reference solution and the spiked test solution, the ratio of the peak area of ​​1-nitrosopiperazine at each time point to the peak area at 0h was between 80% and 115%, and the test solution and the spiked test solution did not degrade to produce impurities that affect the detection of 1-nitrosopiperazine. This indicates that the reference solution, the test solution, and the spiked test solution have good stability at room temperature for at least 15h.

[0110] 2.5 Repeatability test

[0111] Solvent: 1% potassium phosphate aqueous solution.

[0112] 1-Nitrosopiperazine reference substance stock solution ①: Take about 3.6 mg of 1-nitrosopiperazine reference substance, accurately weigh it, put it in a 200 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well.

[0113] 1-Nitrosopiperazine reference stock solution ②: Accurately measure 2 ml of 1-nitrosopiperazine reference stock solution ①, place in a 100 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0114] Reference solution: Accurately measure 5 ml of 1-nitrosopiperazine reference stock solution ②, place in a 20 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0115] Spiked test solution: Take about 300 mg of this product, accurately weigh it, and place it in a 20 ml volumetric flask. Accurately measure 5 ml of 1-nitrosopiperazine reference stock solution ② and place it in the same 20 ml volumetric flask. Dilute to the mark with solvent and shake well. Prepare 6 portions in the same way.

[0116] Accurately measure the reference solution and the spiked test solution, inject them into the liquid chromatograph, and record the chromatogram. Calculate the content of 1-nitrosopiperazine in the spiked test solution by the external standard method using the peak area of ​​1-nitrosopiperazine.

[0117] Verification results: In the 6 spiked test solutions, the RSD of the content of 1-nitrosopiperazine was 0.9%, which was less than 6%, indicating that the method had good repeatability.

[0118] 2.6 Intermediate precision test

[0119] Different analysts prepared 1 reference solution and 6 spiked test solution at different times according to the repeatability test method. The intermediate precision of the method was investigated by measuring the reference solution and the spiked test solution in the same way on different liquid chromatographs according to the repeatability test method. The reference solution and the spiked test solution were accurately measured and injected into the liquid chromatograph respectively, and the chromatogram was recorded. The content of 1-nitrosopiperazine was calculated by the peak area according to the external standard method.

[0120] Verification results: Using the same batch of samples, different analysts, on different dates, according to the operating procedures of the repeatability test, prepared 6 spiked test solutions and reference solutions in the same way, and used different liquid chromatographs for determination in the same way. In the chromatograms of the 6 spiked test solutions determined by staff 2, the RSD of the content of 1-nitrosopiperazine was 0, less than 6%. The 6 test results and the 6 results of the repeatability test, a total of 12 data, were jointly calculated. The RSD of the content of 1-nitrosopiperazine in the 12 spiked test solutions was 3.0%, less than 11%, indicating that the precision of the method is good.

[0121] 2.7 Accuracy test

[0122] Solvent: 1% potassium phosphate aqueous solution.

[0123] Test solution: Take about 300 mg of the product, weigh accurately, place in a 20 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well.

[0124] 1-Nitrosopiperazine reference substance stock solution: Use the 1-nitrosopiperazine reference substance stock solution ② prepared under “2.5 Repeatability test”.

[0125] Reference solution: Accurately measure 5 ml of 1-nitrosopiperazine reference stock solution, place it in a 20 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0126] 40% recovery solution: Take about 300 mg of this product, weigh accurately, and place in a 20 ml volumetric flask. Accurately measure 2 ml of 1-nitrosopiperazine reference stock solution, place in the same 20 ml volumetric flask, dilute to scale with solvent, and shake well. Prepare 3 copies in parallel.

[0127] 100% recovery solution: Take about 300 mg of this product, weigh accurately, and place in a 20 ml volumetric flask. Accurately measure 5 ml of 1-nitrosopiperazine reference stock solution, place in the same 20 ml volumetric flask, dilute to scale with solvent, and shake well. Prepare 3 copies in parallel.

[0128] 200% recovery solution: Take about 300 mg of this product, accurately weigh it, and place it in a 20 ml volumetric flask. Accurately measure 10 ml of 1-nitrosopiperazine reference stock solution, place it in the same 20 ml volumetric flask, dilute to the mark with solvent, and shake well. Prepare 3 copies in parallel.

[0129] Accurately measure the reference solution, test solution and recovery solutions of each concentration, inject them into the liquid chromatograph respectively, record the chromatogram, calculate the measured amount of 1-nitrosopiperazine by peak area according to the external standard method and calculate the recovery rate.

[0130] The test results are shown in Table 1.

[0131]

[0132] Table 1 Accuracy test results

[0133]

[0134] Verification results: In the concentration range of 40% to 200%, the recovery rate of 1-nitrosopiperazine was 100.46% to 104.25%, and the average recovery rate was 101.9%, which was between 80% and 115%, indicating that the accuracy of this method was high.

[0135] 2.8 Durability test

[0136] Reference solution: Use the reference solution prepared under “Repeatability test”.

[0137] Spiked test solution: Take the spiked test solution prepared under the "Repeatability test".

[0138] The durability test inspection items, chromatographic column specifications and models, and durability test results are shown in Tables 2 to 4.

[0139] Table 2 Summary of durability inspection items

[0140]

[0141]

[0142] Table 3 Chromatographic columns of different brands

[0143] Chromatographic columns 1 2 3 model YMC-Triart C18 GL C18 Thermo BDS C18 Specification 4.6mm*150mm, 3μm 4.6mm*150mm, 3μm 4.6mm*150mm, 3μm

[0144] Table 4 Effect of different conditions on the content determination in the spiked test solution

[0145]

[0146] Verification results: Under each condition, the detected amount of nitrosamine impurities in the chromatogram of the spiked test solution did not change, indicating that the method has good durability.

[0147] Comparative Example 1, other mobile phases

[0148] The chromatographic conditions were as follows: octadecylsilane bonded silica gel was used as the filler (using YMC Triart C18, 4.6 mm × 150 mm, 3 μm); 0.02 mol / L phosphate buffer (pH adjusted to 9.0 with 10% sodium hydroxide solution)-acetonitrile (90:10) was used as the mobile phase; the flow rate was 1.0 ml per minute; the detection wavelength was 240 nm; the column temperature was 35°C; and the injection volume was 10 μl.

[0149] 1-Nitrosopiperazine reference substance stock solution: Take about 3.6 mg of nitrosamine impurity reference substance, place it in a 200 ml volumetric flask, add water to dissolve and dilute to the scale, shake well, accurately measure 1 ml, place it in a 50 ml volumetric flask, dilute to the scale with water, and shake well.

[0150] 1-Nitrosopiperazine reference solution: Accurately measure 5 ml of 1-nitrosopiperazine reference stock solution, place it in a 20 ml volumetric flask, dilute to the scale with water, and shake well.

[0151] Accurately measure the 1-nitrosopiperazine reference solution, inject it into the liquid chromatograph, and record the chromatogram.

[0152] Result: 1-Nitrosopiperazine was not retained.

[0153] Comparative Example 2, other solvents and other chromatographic columns

[0154] The chromatographic conditions were as follows: dioctadecylsilane bonded silica gel as filler (using ChromaNik SunArmor RP-AQUA, 4.6 mm×150 mm, 3 μm); 0.02 mol / L potassium hexafluorophosphate solution as mobile phase A, acetonitrile as mobile phase B, and linear gradient elution according to the table below; the flow rate was 1.0 ml per minute; the detection wavelength was 240 nm; the column temperature was 35°C; and the injection volume was 10 μl.

[0155]

[0156] 1-Nitrosopiperazine reference substance stock solution: Take about 3.6 mg of 1-nitrosopiperazine reference substance, place it in a 200 ml volumetric flask, add water to dissolve and dilute to the scale, shake well, accurately measure 1 ml, place it in a 50 ml volumetric flask, dilute to the scale with water, and shake well.

[0157] 1-Nitrosopiperazine reference solution: Accurately measure 5 ml of 1-nitrosopiperazine reference stock solution, place it in a 20 ml volumetric flask, dilute to the scale with water, and shake well.

[0158] Spiked test solution: Take about 300 mg of this product (N-Boc-piperazine), accurately weigh it, place it in a 20 ml volumetric flask, add appropriate amount of water to dissolve, accurately measure 5 ml of 1-nitrosopiperazine reference stock solution, place it in the same 20 ml volumetric flask, dilute to the scale with water, and shake well.

[0159] Accurately measure the 1-nitrosopiperazine reference solution and the spiked test solution, inject them into the liquid chromatograph respectively, and record the chromatogram. The results are shown in Figure 2 .

[0160] As a result, the retention time of 1-nitrosopiperazine in the chromatographic peak of 1-nitrosopiperazine reference solution (RT=2.806min) and the retention time of 1-nitrosopiperazine in the chromatographic peak of spiked test solution (RT=3.135min) differed by about 0.3min. This may be due to the different matrices in the spiked test solution and the reference solution, resulting in a difference in the pH value of the solution and leading to a difference in the retention time of 1-nitrosopiperazine.

[0161] The pH values ​​of 1-nitrosopiperazine reference solution and spiked test solution were measured respectively:

[0162] Results: The pH value of the 1-nitrosopiperazine reference solution was 6.1, and the pH value of the spiked test solution was 10.6.

[0163] Confirm that the addition of alkaline N-Boc-piperazine to the spiked sample changes the pH of the solution. Consider changing the solvent from water to an alkaline solvent (1% tripotassium phosphate).

[0164] Comparative Example 3, other chromatographic columns

[0165] The chromatographic conditions were as follows: dioctadecylsilane bonded silica gel as filler (using ChromaNik Sun Armor RP-AQUA, 4.6 mm×150 mm, 3 μm); 0.02 mol / L potassium hexafluorophosphate solution as mobile phase A, acetonitrile as mobile phase B, and linear gradient elution according to the table below; the flow rate was 1.0 ml per minute; the detection wavelength was 235 nm; the column temperature was 35°C; and the injection volume was 10 μl.

[0166]

[0167] Solvent: 1% tripotassium phosphate solution.

[0168] 1-Nitrosopiperazine reference substance stock solution: use the "1-nitrosopiperazine reference substance stock solution" prepared under Comparative Example 1.

[0169] 1-Nitrosopiperazine reference solution: Accurately measure 5 ml of 1-nitrosopiperazine reference stock solution, place it in a 20 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0170] Test solution: Take about 150 mg of the product, weigh accurately, place in a 10 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well.

[0171] Spiked test solution: Take about 150 mg of this product, accurately weigh it, place it in a 10 ml volumetric flask, add an appropriate amount of solvent to dissolve it, accurately measure 2.5 ml of 1-nitrosopiperazine reference stock solution, place it in the same 10 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0172] Interference solution stock solution ①: Take about 5 mg each of impurity iii-1 reference substance, impurity iii-3 reference substance, impurity iii-4 reference substance and impurity iii-9 reference substance, accurately weigh, place in the same 25 ml volumetric flask, add 5 ml of acetonitrile to dissolve, dilute with water to the scale, and shake well.

[0173] Impurity III-11 reference solution: Take about 1.5 mg of impurity III-11 reference, accurately weigh, place in a 10 ml volumetric flask, add water to dissolve and dilute to the scale, and shake well.

[0174] Impurity III-12 reference solution: Take about 1.5 mg of impurity III-12 reference, accurately weigh, place in a 10 ml volumetric flask, add water to dissolve and dilute to the scale, and shake well.

[0175] Interference solution: Accurately measure 2 ml each of the interference solution stock solution ①, impurity iii-11 reference solution and impurity iii-12 reference solution, place them in the same 25 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0176] Accurately measure the solvent, 1-nitrosopiperazine reference solution, spiked test solution and interference solution, inject them into the liquid chromatograph respectively, record the chromatogram, and the results are shown in Figure 3 .

[0177] As can be seen from Comparative Example 3, the retention time of the 1-nitrosopiperazine peak in the chromatogram of the 1-nitrosopiperazine reference solution is substantially consistent with the retention time of the 1-nitrosopiperazine peak in the chromatogram of the spiked test solution, indicating that the solvent is suitable. However, the unknown impurity peak in the interfering solution also peaks at the 1-nitrosopiperazine peak position, interfering with the detection of 1-nitrosopiperazine, and the peak of 1-nitrosopiperazine is relatively wide. It can be seen that the effect of the C28 chromatographic column is not as good as that of the C18 chromatographic column.

[0178] From the comparison results of Comparative Example 2 and Comparative Example 3, it can be seen that in Comparative Example 2, the retention time of the 1-nitrosopiperazine peak in the chromatographic peak of the 1-nitrosopiperazine reference solution (RT = 2.806min) and the retention time of the 1-nitrosopiperazine peak in the chromatographic peak of the spiked test solution (RT = 3.135min) differ by about 0.3min. The reasons are analyzed as follows:

[0179] According to the retention mechanism of the chromatographic column, the pH value of the mobile phase and the solvent will affect the retention of the analyte in the stationary phase. The mobile phase pH is about 5, the pH value of the nitrosamine impurity reference solution is about 6, and the N-Boc-piperazine in the spiked test sample is an alkaline compound, the solution is alkaline, and the pH value is 10.6. When the injector draws a small amount of sample into the chromatographic column, the pH value will change. The pH value of the nitrosamine impurity in the chromatographic column is lower than the pH value of the test solution, resulting in a difference in the pH value of the solution and a difference in the retention time of 1-nitrosopiperazine. It is confirmed that N-Boc-piperazine in the spiked test sample changes the pH value of the solution. Therefore, using an alkaline solvent (1% tripotassium phosphate) (pH is about 12) as a solvent can ensure that the retention time of nitrosamine impurities in the chromatogram of the reference solution and the chromatogram of the spiked test solution is consistent, thereby ensuring the accuracy of the test results.

[0180] Comparative Example 4,

[0181] 0.02 mol / L potassium hexafluorophosphate solution was used as mobile phase A, the pH value was 4.2, and other chromatographic conditions were the same as those in Example 1.

[0182] Solvent: 1% tripotassium phosphate solution.

[0183] Interference solution: Weigh about 0.8 mg each of impurity III-1 reference substance, impurity III-3 reference substance, impurity III-4 reference substance, impurity III-9 reference substance, impurity III-11 reference substance and impurity III-12 reference substance, place them in the same 5 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well.

[0184] 1-Nitrosopiperazine: Take an appropriate amount of 1-nitrosopiperazine reference substance, dissolve it in solvent and quantitatively dilute it to make a solution containing approximately 0.09 μg per 1 ml.

[0185] The test results are as follows Figure 4 As shown: Other known impurities in the interfering solution interfere with the detection of 1-nitrosopiperazine (RT = 2.7 min).

[0186] Comparative Example 5,

[0187] 0.02 mol / L potassium hexafluorophosphate solution was used as mobile phase A, the pH value was 5.6, and other chromatographic conditions were the same as those in Example 1.

[0188] Solvent: 1% tripotassium phosphate solution.

[0189] Interference solution: Weigh about 0.8 mg each of impurity III-1 reference substance, impurity III-3 reference substance, impurity III-4 reference substance, impurity III-9 reference substance, impurity III-11 reference substance and impurity III-12 reference substance, place them in the same 5 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well.

[0190] 1-Nitrosopiperazine: Take an appropriate amount of 1-nitrosopiperazine reference substance, dissolve it in solvent and quantitatively dilute it to make a solution containing approximately 0.09 μg per 1 ml.

[0191] The test results are as follows Figure 5 As shown: Other known impurities in the interfering solution interfere with the detection of 1-nitrosopiperazine (RT = 4.8 min).

[0192] Comparative Example 6,

[0193] The pH value range of the feasible solvent is 11.5-12.5. The effect of solvents with different pH values ​​on the retention time of 1-nitrosopiperazine is shown in Table 5.

[0194] Table 5 Effect of different pH ranges of solvent on retention time

[0195] pH of solvent Reference solution Spiked test solution Is it consistent? pH11.5 4.001min 4.098min Consistency pH12.5 4.109min 4.117min Consistency

[0196] The pH value of the solvent was adjusted to 11.4 with phosphoric acid, and the samples were prepared according to the chromatographic conditions and methods of Example 1. The retention time of nitrosamine impurities in the reference solution and the retention time of nitrosamine impurities in the spiked test solution differed by 0.2 min (the retention times of nitrosamine impurities in the reference solution and the spiked test solution were 3.802 min and 4.013 min, respectively). The test results showed that when the pH value of the solvent was 11.4, the retention time of nitrosamine impurities in the reference solution and the spiked test solution still deviated.

[0197] Comparative Example 7

[0198] The pH value of the solvent was adjusted to 12.6 with 10% potassium hydroxide solution. The retention time of nitrosamine impurities in the chromatogram of the reference solution and the retention time of nitrosamine impurities in the spiked test solution differed by 0.3min (the retention times of nitrosamine impurities in the reference solution and the spiked test solution were 4.451min and 4.176min, respectively). The test results showed that when the pH value of the solvent was 12.6, the retention time of nitrosamine impurities in the reference solution and the spiked test solution still deviated.

[0199] Comparative Example 8

[0200] Inspection method: High performance liquid chromatography method, general chapter 0512 of Part IV of the Chinese Pharmacopoeia 2020 edition.

[0201] Solvent: acetonitrile-water (1:1).

[0202] Reference solution: Take an appropriate amount of 1-nitrosopiperazine reference substance, weigh accurately, dissolve in solvent and quantitatively dilute to make a solution containing approximately 0.09 μg per 1 ml.

[0203] Chromatographic conditions: Use octadecylsilane bonded silica gel as filler (Waters C18, 4.6mm×150mm, 5μm or chromatographic column with equivalent performance is recommended); use phosphate buffer (take 1.08g of sodium 1-octane sulfonate, 1.21g of potassium dihydrogen phosphate, 0.83g of potassium hydrogen phosphate, add 1000ml of water to dissolve) as mobile phase A; use acetonitrile as mobile phase B, and perform linear gradient elution according to the table below; flow rate is 1.0ml per minute; detection wavelength is 235nm; column temperature is 35℃; injection volume is 20μl.

[0204]

[0205] Determination: Accurately measure the solvent and reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0206] The test results are as follows Figure 6 As shown: the reference substance did not produce a peak, indicating that this method cannot detect nitrosamine impurities.

[0207] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting nitrosamine impurities in N-Boc-piperazine, a starting material of tandospirone citrate, characterized in that: The structural formula of the nitrosamine impurity is: The chromatographic column uses octadecylsilane bonded silica gel as a filler, potassium hexafluorophosphate solution as a mobile phase A, and acetonitrile as a mobile phase B, and linear gradient elution is performed; the pH value of the potassium hexafluorophosphate solution ranges from 4.3 to 5.

5.

2. The method according to claim 1, characterized in that The pH value of the potassium hexafluorophosphate solution is 4.

8.

3. The method according to claim 1, characterized in that The concentration of the potassium hexafluorophosphate solution is in the range of 0.015 mol / L to 0.025 mol / L, preferably 0.02 mol / L.

4. The method according to claim 1, characterized in that The gradient elution program was: Within 0 to 6 minutes, the volume ratio of mobile phase A and mobile phase B remained unchanged at 95-100:5-0; within 6 to 6.1 minutes, the volume ratio of mobile phase A and mobile phase B linearly changed to 10:90; within 6.1 to 16 minutes, the volume ratio of mobile phase A and mobile phase B remained unchanged at 10:90; within 16 to 17 minutes, the volume ratio of mobile phase A and mobile phase B linearly changed from 10:90 to 95-100:5-0; within 17 to 27 minutes, the volume ratio of mobile phase A and mobile phase B remained unchanged at 95-100:5-0.

5. The method according to claim 1, characterized in that The detection wavelength is 235±2nm.

6. The method according to claim 1, characterized in that The column temperature of the chromatographic column is 30°C to 45°C, preferably 35°C.

7. The method according to claim 1, characterized in that The flow rate of the mobile phase is in the range of 0.7 ml / min to 1.3 ml / min, preferably 1 ml / min.

8. The method according to claim 1, characterized in that The chromatographic column used is YMC-Triart C18, 4.6 mm × 150 mm, 3 μm or a chromatographic column with equivalent performance.

9. The method according to claim 1, characterized in that: The detection method also includes sample preparation: Test solution: Take an appropriate amount of the product, weigh accurately, dissolve in solvent and quantitatively dilute to make a solution containing about 15 mg of N-Boc-piperazine per 1 ml; Reference solution: Take an appropriate amount of nitrosamine impurity reference substance, weigh accurately, dissolve in solvent and quantitatively dilute to make a solution containing 0.09 μg per 1 ml.

10. The method according to claim 9, characterized in that The solvent is a 1% tripotassium phosphate aqueous solution with a pH range of 11.5 to 12.5.

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