Method for detecting related substances in cycloserine

Abstract

The invention relates to the technical field of drug detection, and particularly discloses a method for detecting related substances in cycloserine. The specific derivatization reagent is adopted, so that the impurities and the benzaldehyde derivative are subjected to derivatization reaction to generate amide, and the ultraviolet absorption response intensity of the impurities is improved; according to the method, a ZORBAX SB-Phenyl chromatographic column is adopted to carry out HPLC (High Performance Liquid Chromatography) analysis on a cycloserine sample, so that rapid and accurate detection on impurities such as D-serine, D-serine methyl ester hydrochloride, 3-chloro-D-alanine methyl ester hydrochloride and L-cycloserine in the cycloserine at the same time is realized. The detection method is high in specificity, low in detection limit and quantitation limit, good in linear relation, high in recovery rate, good in repeatability, high in stability, good in durability, easy and rapid to operate, low in detection cost and accurate and reliable in detection result, can be used for quality control and comprehensive evaluation of cycloserine, and provides reliable guarantee for improving and controlling the quality of cycloserine.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of drug detection, and particularly relates to a detection method of related substances in cycloserine. BACKGROUND

[0002] Cycloserine (Oxamycin) is also known as dextrorotatory-4-amino-3-tetrahydroisoxazolone, and its molecular formula is C3H6N2O2. The structural formula is shown as formula I. Cycloserine is an antibiotic drug, which has inhibitory effect on gram-positive bacteria (such as tubercle bacillus) and gram-negative bacteria (such as rickettsia). Although the inhibitory effect of cycloserine on tubercle bacillus is weaker than that of streptomycin, cycloserine is not prone to drug resistance. Therefore, cycloserine is mainly used for treating infections caused by drug-resistant tubercle bacillus in clinical practice. Related impurities are generated in the preparation process of cycloserine, such as D-serine (the structural formula is shown as formula II), D-serine methyl ester hydrochloride (the structural formula is shown as formula III), 3-chloro-D-alanine methyl ester hydrochloride (the structural formula is shown as formula IV), and L-cycloserine (the structural formula is shown as formula V). These impurities will affect the detection of the content of cycloserine, and the impurities need to be controlled to avoid affecting the content of cycloserine.

[0003]

[0004] Formula I

[0005] Formula II

[0006] Formula III

[0007] Formula IV

[0008] Formula V At present, there is no report on the detection method of related substances (D-serine, D-serine methyl ester hydrochloride, 3-chloro-D-alanine methyl ester hydrochloride, and L-cycloserine) in cycloserine. In order to realize accurate detection of the above related substances, multiple detections are required, and the operation process is complicated, time-consuming and laborious. Therefore, there is a need in the art to provide a detection method capable of quickly and accurately detecting related substances in cycloserine. SUMMARY

[0009] In order to solve the above problems, the application provides a detection method of related substances in cycloserine, which can quickly and accurately detect the impurities D-serine, D-serine methyl ester hydrochloride, 3-chloro-D-alanine methyl ester hydrochloride and L-cycloserine in cycloserine by using high performance liquid chromatography (HPLC).

[0010] To solve the above technical problems, the technical scheme provided by the application is: The application provides a detection method of related substances in cycloserine, which comprises the following steps: Preparation of the test sample solution: cycloserine samples are dissolved in water, and a derivatization reagent is added for reaction to obtain the test sample solution; the derivatization reagent comprises N-acetyl-L-cysteine, benzaldehyde derivative and borate buffer; Preparation of the control sample solution: D-serine control sample, D-serine methyl ester hydrochloride control sample, 3-chloro-D-alanine methyl ester hydrochloride control sample and L-cycloserine control sample are dissolved in water to obtain the control sample solution; The test sample solution is detected by using HPLC, and the chromatographic conditions of the HPLC comprise: A ZORBAX SB-Phenyl chromatographic column is used, mobile phase A comprises (95-100):(1-3) volume ratio of disodium hydrogen phosphate solution and acetonitrile, and mobile phase B comprises (35-45):(55-65) volume ratio of disodium hydrogen phosphate solution and acetonitrile, and gradient elution is performed.

[0011] Compared with the prior art, the detection method of related substances in cycloserine provided by the application uses specific derivatization reagents, so that the impurities and benzaldehyde derivative generate amides through derivatization reaction, thereby increasing the ultraviolet absorption response intensity of the impurities; then the cycloserine samples are analyzed by using the ZORBAX SB-Phenyl chromatographic column for HPLC analysis, so that the four related substances D-serine, D-serine methyl ester hydrochloride, 3-chloro-D-alanine methyl ester hydrochloride and L-cycloserine existing in the cycloserine and the contents thereof can be quickly and accurately detected at the same time. By limiting the chromatographic column and mobile phase of HPLC and the like, the application can realize the separation of cycloserine and impurities, each impurity peak does not interfere with the main peak, and each chromatographic peak can be completely separated, so as to improve the peak response intensity of the test sample solution, thereby improving the detection accuracy. The detection method has strong specificity, low detection limit and quantitative limit, good linear relationship, high recovery rate, good repeatability, strong stability, good durability, simple and fast operation, low detection cost, accurate and reliable detection results, and can be used for quality control and comprehensive evaluation of cycloserine, thereby providing reliable guarantee for improving and controlling the quality of cycloserine.

[0012] Preferably, the pH of the borate buffer is 9.2-9.8.

[0013] Preferably, the method for preparing the borate buffer solution includes the following steps: Boric acid, a strong base, and water are mixed, and the pH of the mixture is adjusted to obtain a borate buffer solution.

[0014] More preferably, the strong base includes sodium hydroxide.

[0015] More preferably, the mass-to-volume ratio of boric acid, strong alkali and water is (6~6.4)g:(2.6~3)g:1000mL.

[0016] For example, derivatizing reagents also include water.

[0017] Preferably, the benzaldehyde derivative includes o-phthalaldehyde.

[0018] Preferably, the mass-to-volume ratio of N-acetyl-L-cysteine, benzaldehyde derivative, water, and borate buffer in the derivatization reagent is (0.18~0.22)g:(0.15~0.19)g:50mL:(140~160)mL.

[0019] Preferably, the mass-to-volume ratio of cycloserine sample, derivatization reagent and water in the test solution is (4.5~5.5) mg: 10 mL: (13~15) mL.

[0020] Preferably, the reaction temperature is 10℃~40℃ and the reaction time is 40min~50min.

[0021] Preferably, the concentration of D-serine in the reference solution is 0.2 μg / mL to 2 μg / mL.

[0022] Preferably, the concentration of D-serine methyl ester hydrochloride in the reference solution is 0.2 μg / mL to 2 μg / mL.

[0023] Preferably, the concentration of 3-chloro-D-alanine methyl ester hydrochloride in the reference solution is 0.2 μg / mL to 2 μg / mL.

[0024] Preferably, the concentration of L-cycloserine in the reference solution is 0.2 μg / mL to 2 μg / mL.

[0025] More preferably, the concentrations of D-serine, D-serine methyl ester hydrochloride, 3-chloro-D-alanine methyl ester hydrochloride, and L-cycloserine in the reference solution are 0.3 μg / mL to 0.8 μg / mL.

[0026] Preferably, the ZORBAX SB-Phenyl chromatographic column has a length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm.

[0027] Preferably, the concentration of the disodium hydrogen phosphate solution is 0.018 mol / L to 0.022 mol / L.

[0028] Preferably, the pH of the disodium hydrogen phosphate solution is 6.8 to 7.2, and more preferably 7 to 7.2.

[0029] For example, a method for preparing a disodium hydrogen phosphate solution includes the following steps: Disodium hydrogen phosphate is dissolved in water, and the pH of the system is adjusted with phosphoric acid to obtain a disodium hydrogen phosphate solution.

[0030] Preferably, the mobile phase A comprises a disodium hydrogen phosphate solution and acetonitrile in a volume ratio of (96~100):2.

[0031] Preferably, the mobile phase B comprises a disodium hydrogen phosphate solution and acetonitrile in a volume ratio of (38~42):(58~62).

[0032] Preferably, the gradient elution procedure is as follows: 0 min ~ 15 min, 98% ~ 100% → 90% mobile phase A, 2% ~ 0% → 10% mobile phase B; 15 min to 35 min, 90% → 0 mobile phase A, 10% → 100% mobile phase B; 35 min ~ 37 min, 0 → 100% mobile phase A, 100% → 0 mobile phase B; 37 min to 45 min, 100% mobile phase A.

[0033] Preferably, the chromatographic conditions of the high performance liquid chromatography method further include: a detection wavelength of 330 nm to 340 nm.

[0034] Preferably, the chromatographic conditions of the high performance liquid chromatography method further include: a column temperature of 25℃~35℃.

[0035] Preferably, the chromatographic conditions of the high performance liquid chromatography method further include a flow rate of 0.9 mL / min to 1.1 mL / min.

[0036] Preferably, the chromatographic conditions of the high performance liquid chromatography method further include: an injection volume of 18 μL to 22 μL.

[0037] The present invention has the following beneficial effects: Compared to other chromatographic columns, the ZORBAX SB-Phenyl chromatographic column of this invention has better stability, better separation effect on cyclic serine and related substances, and can be used under highly alkaline conditions.

[0038] This invention uses a mixture of disodium hydrogen phosphate solution and acetonitrile as the mobile phase, which has good stability at room temperature, is not prone to generating bubbles and salting out, and can achieve better separation of cycloserine and related substances, resulting in higher detection sensitivity.

[0039] In the derivatization reagent, phthalaldehyde can react with amino acids to generate derivatives with ultraviolet absorption, thereby improving the detection sensitivity in liquid chromatography; N-acetyl-L-cysteine ​​is a reducing agent that can synergistically react with phthalaldehyde and also help separate the corresponding isomers (the main component D-cycloserine and the impurity L-cycloserine); the borate buffer provides an alkaline environment, which can improve the stability of the derivatives.

[0040] The preferred chromatographic conditions of this invention can further improve the separation between the detection peaks of cyclic serine and related substances in the chromatogram. Attached Figure Description

[0041] Fig. 1 This is the HPLC chromatogram of the spiked test solution in Example 1 of the present invention; the chromatographic peaks in the chromatogram from left to right are D-serine, L-cyclic serine, cyclic serine, 3-chloro-D-alanine methyl ester hydrochloride and D-serine methyl ester hydrochloride. Fig. 2 This is the HPLC chromatogram of the test solution in Example 1 of the present invention; the chromatographic peaks in the chromatogram from left to right are D-serine, L-cycloserine, cycloserine and 3-chloro-D-alanine methyl ester hydrochloride; Fig. 3 This is the HPLC chromatogram of the derivatizing reagent in Example 1 of the present invention; Fig. 4 This is the HPLC chromatogram of the test solution in Example 2 of the present invention; Fig. 5 This is the HPLC chromatogram of the spiked test solution in Comparative Example 1 of the present invention; the chromatographic peaks in the chromatogram from left to right are D-serine, L-cyclic serine, cyclic serine, 3-chloro-D-alanine methyl ester hydrochloride and D-serine methyl ester hydrochloride. Fig. 6 This is the HPLC chromatogram of the test solution in Comparative Example 2 of the present invention; Fig. 7 This is the HPLC chromatogram of the test solution in Comparative Example 3 of the present invention. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0043] The preparation method of cyclic serine in this invention is described in Formulas 1 to 3, and includes the following steps: S1. Take 50 kg (475.77 mol) of D-serine and add it to 120 kg of methanol. Control the temperature at 0℃~10℃, add 82 kg (689.31 mol) of thionyl chloride, and after the addition, raise the temperature to 35℃~40℃ and react for 12h~14h. Then lower the temperature to -5℃~0℃, add 204 kg of ethyl acetate, and keep it at the temperature for crystallization for 2h~3h. Then filter and dry the resulting filter cake to obtain 66.68 kg (428.60 mol) of intermediate 1, with a yield of 90.08%.

[0044] S2. Add 50 kg (321.38 mol) of intermediate 1 to 79 kg of acetonitrile and 270 kg of dichloromethane, heat to 35℃~40℃, add 50 kg (420.31 mol) of thionyl chloride, keep the reaction at this temperature for 10 h~12 h, then cool to 0℃~10℃ and keep the temperature at this temperature for 1 h~1.5 h to allow crystallization. Then filter and dry the resulting filter cake to obtain 47.86 kg (275.02 mol) of intermediate 2, with a yield of 85.58%.

[0045] S3. Dissolve 26.50 kg (381.36 mol) of hydroxylamine hydrochloride in 50 kg of purified water, cool to -15℃ to -10℃, add 148.5 kg of liquid sodium hydroxide, and mix thoroughly; cool to -20℃ to -15℃, add an aqueous solution containing 50 kg of intermediate 2, and stir for 0.5 h to 1 h; heat to 20℃ to 25℃ and react for 4 h to 6 h, adjusting the pH to 9.7 to 11.0 with liquid sodium hydroxide during the reaction; heat to 40℃ to 45℃ and react for 0.5 h to 1 h, adjusting the pH to 9.7 to 11.0 with liquid sodium hydroxide during the reaction; after the reaction, cool to 20℃ to 25℃ and adjust the pH to 11.3 to 11.7 with liquid sodium hydroxide; cool to 0℃ to 10℃, add 1100 kg of anhydrous ethanol, and maintain the temperature for crystallization for 1 h to 2 h; then filter to obtain filter cake and mother liquor.

[0046] Take the mother liquor, add glacial acetic acid at 0℃~10℃ to adjust the pH to 6.0~6.5, keep warm and stir for 0.5h~1h; then filter to obtain filter cake.

[0047] The filter cakes were collected twice and dried to obtain 19.28 kg (188.85 mol) of crude D-cycloserine, with a yield of 65.73%.

[0048] S4. Take 50 kg (489.76 mol) of crude D-cyclic serine and add it to 91.50 kg of ammonia water and 55 kg of purified water, mix well; cool to -5℃~0℃, keep warm and stir for 0.5 h~1 h, then add 600 kg of mixed alcohol solution (containing 300 kg of anhydrous ethanol and 300 kg of isopropanol), control the temperature at -5℃~0℃ and stir for 0.5 h~1 h; then add 3 kg of activated carbon, control the temperature at -5℃~0℃ and stir for 0.5 h~1 h; after the reaction is complete, filter, collect the mother liquor, control the temperature at 2℃~7℃, add 108 kg of glacial acetic acid in isopropanol solution (containing 46.5 kg of isopropanol and 61.5 kg of glacial acetic acid), adjust the pH to 8~8.5, keep warm to crystallize for 0.5 h~1 h; then filter, dry the obtained filter cake to obtain 32.69 kg (320.21 mol) of D-cyclic serine product, yield 65.38%.

[0049]

[0050] Formula 1

[0051] Formula 2

[0052] Formula 3 This invention uses D-serine as the starting material. During the synthesis reaction, some starting materials do not fully participate in the reaction, and a very small portion may remain in the final product (i.e., cyclic serine). For example, the product obtained from Formula 1 may contain D-serine. D-serine methyl ester hydrochloride is intermediate 1, and 3-chloro-D-alanine methyl ester hydrochloride is intermediate 2. The synthesis of the final product involves multiple steps, and the conversion rate of intermediates in each step may be insufficient, resulting in some intermediates not being completely converted into the next step product. L-cyclic serine is an isomer of cyclic serine. During the reaction or purification process, the product may undergo isomerization, and isomers have highly similar physicochemical properties, making them difficult to completely remove from the final product.

[0053] For example, the product obtained by Formula 1 may contain D-serine, the product obtained by Formula 2 may contain D-serine and D-serine methyl ester hydrochloride, and the product obtained by Formula 3 may contain D-serine, D-serine methyl ester hydrochloride, 3-chloro-D-alanine methyl ester hydrochloride and L-cyclic serine.

[0054] To better illustrate the present invention, further examples are provided below.

[0055] Example 1 This embodiment provides a method for detecting impurities in cyclic serine, including the following steps: (1) Solution preparation.

[0056] Borate buffer: Accurately weigh 6.2g boric acid and 2.8g sodium hydroxide, transfer to a 1000mL volumetric flask, dilute with water to the mark, and check the pH value, which should be 9.5±0.1 to obtain borate buffer.

[0057] N-acetyl-L-cysteine ​​solution: Accurately weigh 0.4 g of N-acetyl-L-cysteine, transfer it to a 50 mL volumetric flask to dissolve, and dilute with water to the mark to obtain an N-acetyl-L-cysteine ​​solution.

[0058] Phthalate solution: Accurately weigh 0.34g of phthalaldehyde, transfer it to a 50mL volumetric flask and dissolve it. Dilute with water to the mark to obtain phthalaldehyde solution.

[0059] Derivatization reagent: Transfer 25 mL each of N-acetyl-L-cysteine ​​solution and phthalaldehyde solution into a 200 mL volumetric flask, and dilute to the mark with borate buffer to obtain the derivatization reagent.

[0060] Test solution: Take a cyclic serine sample, dissolve it in water to prepare a cyclic serine sample solution with a concentration of 1 mg / mL, transfer 5 mL of the cyclic serine sample solution to a 25 mL volumetric flask containing 10 mL of derivatization reagent, let it stand at room temperature for 45 min, and then dilute to volume with water to obtain the test solution.

[0061] D-Serine reference solution: Dissolve D-serine reference standard in water to obtain a D-serine reference solution with a concentration of 100 μg / mL.

[0062] D-Serine Methyl Ester Hydrochloride Reference Solution: Dissolve D-Serine Methyl Ester Hydrochloride Reference Standard in water to obtain a D-Serine Methyl Ester Hydrochloride Reference Solution with a concentration of 100 μg / mL.

[0063] 3-Chloro-D-alanine methyl ester hydrochloride reference solution: Dissolve 3-chloro-D-alanine methyl ester hydrochloride reference standard in water to obtain a 100 μg / mL 3-chloro-D-alanine methyl ester hydrochloride reference solution.

[0064] L-Cycloserine reference solution: Dissolve L-cycloserine reference standard in water to obtain an L-cycloserine reference solution with a concentration of 100 μg / mL.

[0065] Spiked test sample stock solution: Take a cycloserine sample, dissolve it in water to prepare a cycloserine sample solution with a concentration of 0.1 mg / mL, transfer 1 mL of cycloserine sample solution, 1.5 mL of D-serine reference solution, 1.5 mL of D-serine methyl ester hydrochloride reference solution, 1.5 mL of 3-chloro-D-alanine methyl ester hydrochloride reference solution and 1.5 mL of L-cycloserine reference solution into a 20 mL volumetric flask, add water to make up to volume, and obtain the spiked test sample stock solution.

[0066] Spiked test solution: Transfer 1 mL of the spiked test solution to a 25 mL volumetric flask containing 10 mL of derivatization reagent. Allow the solution to stand at room temperature for 45 min, then dilute to volume with water to obtain the spiked test solution. The concentration of cycloserine in the spiked test solution is 0.2 μg / mL, and the concentrations of D-serine reference standard, D-serine methyl ester hydrochloride reference standard, 3-chloro-D-alanine methyl ester hydrochloride reference standard, and L-cycloserine reference standard are all 0.3 μg / mL.

[0067] (2) The above derivatization reagents, test solutions, and spiked test solutions were detected by HPLC. Specific HPLC chromatographic conditions included: A ZORBAX SB-Phenyl column (250 mm × 4.6 mm, 5 μm) was used.

[0068] Weigh 2.84 g of anhydrous disodium hydrogen phosphate, dissolve it in 1000 mL of water, and adjust the pH to 7.0 with phosphoric acid to prepare a 0.02 mol / L disodium hydrogen phosphate solution with a pH of 7.0.

[0069] Mobile phase A: disodium hydrogen phosphate solution and acetonitrile in a volume ratio of 98:2; Mobile phase B: disodium hydrogen phosphate solution and acetonitrile in a volume ratio of 40:60.

[0070] The gradient elution procedure is shown in Table 1.

[0071] Table 1 Gradient elution degree

[0072] The flow rate was 1 mL / min, the detection wavelength was 335 nm, the column temperature was 30 °C, and the injection volume was 20 μL.

[0073] The HPLC chromatograms of the spiked test solution and the test solution are as follows: Figs. 1-3 As shown.

[0074] Methodological Validation (1) Exclusivity The separation degree of each component in the spiked test solution was detected, and the test results are shown in Table 2. Figs. 1-3As can be seen from Table 2, the method for detecting related substances in cyclic serine provided in this embodiment does not interfere with the detection of each component with the derivatization reagent (blank solvent), the separation degree between each component meets the requirements, and the method has good specificity.

[0075] Table 2 Retention times and resolutions of each component in the spiked test solution

[0076] (2) Limit of detection and limit of quantitation Accurately weigh appropriate amounts of D-serine reference standard, D-serine methyl ester hydrochloride reference standard, 3-chloro-D-alanine methyl ester hydrochloride reference standard, and L-cycloserine reference standard, dissolve in water to prepare mixed reference standard solutions of different concentrations, and detect them according to the chromatographic conditions in Example 1. Record the chromatograms. The limit of quantitation (LOQ) is defined as the peak height being 10 times the baseline noise, and the limit of detection (LOD) is defined as the peak height being 3 times the baseline noise. The test results for the LQ and LOD are shown in Table 3, and the repeatability test results for the LQ are shown in Table 4. As can be seen from Tables 3 and 4, the detection method provided by this invention has low LQ and LQ for detecting related substances in cycloserine, and exhibits high sensitivity for detecting related substances in cycloserine. The maximum relative standard deviation (RSD) of the peak area of ​​each impurity after six repeated determinations is 5.561%, indicating that the detection method provided by this invention has good repeatability for the LQ.

[0077] Table 3. Test results for limits of quantitation and limits of detection.

[0078] Table 4 Results of repeatability tests at the limit of quantitation (peak area)

[0079] (3) Linear relationship Accurately weigh appropriate amounts of D-serine reference standard, D-serine methyl ester hydrochloride reference standard, 3-chloro-D-alanine methyl ester hydrochloride reference standard, L-cycloserine reference standard, and cycloserine reference standard, and prepare sample solutions of six concentration levels respectively. Detect these sample solutions under the chromatographic conditions described in Example 1, and measure the peak area of ​​the chromatogram. Plot a standard working curve with the component concentration in the sample solution as the abscissa and the peak area as the ordinate, and establish a linear equation. The final experimental results are shown in Tables 5 to 9. As can be seen from the data in Tables 5 to 9, the detection method provided by this invention exhibits a good linear relationship between the concentration of cycloserine and related substances and the peak area measured by chromatography.

[0080] Table 5. Results of linear relationship experiments for D-serine.

[0081] Table 6. Experimental results of linear relationship of D-serine methyl ester hydrochloride.

[0082] Table 73. Linearity test results of chloro-D-alanine methyl ester hydrochloride

[0083] Table 8. Results of linear relationship experiment for L-cycloserine.

[0084] Table 9. Results of linear relationship test for cycloserine.

[0085] (4) Recovery rate Accurately weigh appropriate amounts of D-serine reference standard, D-serine methyl ester hydrochloride reference standard, 3-chloro-D-alanine methyl ester hydrochloride reference standard, and L-cycloserine reference standard, dissolve in water to prepare a mixed reference solution with a concentration of 7.5 μg / mL (i.e., the concentration of each reference standard is 7.5 μg / mL). Take a cycloserine sample, dissolve in water to prepare a cycloserine sample solution with a concentration of 1 mg / mL, transfer 5 mL of the cycloserine sample solution to a 25 mL volumetric flask containing 10 mL of derivatization reagent, prepare 9 parallel aliquots, and accurately add 0.5 mL, 1.0 mL, and 1.5 mL of the above mixed reference solution to each aliquot. After standing at room temperature for 45 min, dilute to volume with water to obtain the spiked test solution for recovery. Detect according to the detection method and chromatographic conditions in Example 1, and perform parallel detection of each spiked test solution in 3 parallel tests. The final recoveries of each related substance in the cycloserine sample are shown in Tables 10-13. As can be seen from Tables 10 to 13, the recovery rates of each impurity were between 91% and 113% under the three different addition amounts, with a maximum RSD of 8.48%, indicating that the detection method provided by this invention has good accuracy.

[0086] Table 10 Recovery rate of D-serine

[0087] Table 11 Recovery of D-serine methyl ester hydrochloride

[0088] Table 1. Recovery rates of 23-chloro-D-alanine methyl ester hydrochloride

[0089] Table 13 Recovery rate of L-cycloserine

[0090] (5) Repeatability Six groups of cyclic serine samples from the same batch were taken, and test solutions were prepared according to the method in Example 1. The solutions were then injected under the chromatographic conditions described in Example 1, and the contents of related substances in different groups of cyclic serine samples were detected. The results are shown in Table 14. Table 14 shows that the detection method provided by this invention yields essentially consistent results for repeated detection of related substances in the same batch of cyclic serine, demonstrating good repeatability.

[0091] Table 14 Repeatability test results for the content of each impurity

[0092] (6) Intermediate precision Six cyclic serine samples from the same batch were taken, forming two groups. Different analysts prepared the test solutions at different times according to the method described in Example 1, and the solutions were then analyzed using different instruments under the detection method and chromatographic conditions of Example 1. The results are shown in Table 15. Table 15 shows that the detection method provided by this invention yields essentially consistent results for repeated detection of related substances in the same batch of cyclic serine, demonstrating good precision.

[0093] Table 15 Intermediate precision test results for the content of each impurity

[0094] (7) Solution stability The test solution was prepared according to the method in Example 1. After being placed at room temperature for 0 h, 1.5 h, 3 h, 6 h, 9 h, 12 h, and 15 h, respectively, the stability of the solution was detected under the chromatographic conditions in Example 1. The results of the stability test are shown in Table 16. As can be seen from Table 16, when the test solution was placed at room temperature for 15 h and detected using the same method, D-serine methyl ester hydrochloride was not detected; D-serine was unstable within 1.5 h, with a cumulative RSD% of peak area of ​​20.695%, and after 15 h, the cumulative RSD% of peak area was 18.469%; within 15 h, the cumulative RSD% of peak area of ​​3-chloro-D-alanine methyl ester hydrochloride was 5.091%; L-cycloserine was unstable within 1.5 h, with a cumulative RSD% of peak area of ​​50.785%, and after 15 h, the cumulative RSD% of peak area was 56.596%. This indicates that the S-3-chloro-D-alanine methyl ester hydrochloride in the test solution is stable, while D-serine and L-cycloserine are unstable.

[0095] Table 16 Stability of the test solution (peak area)

[0096] (8) Durability Accurately weigh L-cycloserine reference standard, dissolve it in water to obtain an L-cycloserine reference standard solution with a concentration of 2.5 μg / mL.

[0097] Accurately weigh the cyclic serine sample, dissolve it in water to obtain a cyclic serine sample solution with a concentration of 1 mg / mL.

[0098] Transfer 3 mL of L-cycloserine reference solution and 5 mL of cycloserine sample solution into a 25 mL volumetric flask containing 10 mL of derivatization reagent (prepared in the same way as in Example 1). After standing for 45 min, dilute with water to the mark to obtain a system suitability solution, wherein the concentration of L-cycloserine is 0.3 μg / mL and the concentration of cycloserine is 200 μg / mL.

[0099] By varying only the column temperature, detection wavelength, flow rate, and mobile phase (initial mobile phase ratio and pH in the elution program) to control single-factor variables, detection was performed according to the detection method of Example 1. The results are shown in Table 17. Table 17 shows that, with each change in conditions, the resolution of cyclic serine and L-cyclic serine was generally greater than 1.5, meeting the requirements for impurity resolution in related substances studies. However, after changing the pH of the disodium hydrogen phosphate solution in the mobile phase to 6.8, the resolution of cyclic serine and L-cyclic serine was less than 1.5, indicating poor robustness. Under other conditions, the robustness of this example was good.

[0100] Table 17 System Suitability: Separation Degree of Cycloserine and L-Cycloserine in Solutions

[0101] Example 2 This embodiment provides a method for detecting impurities in cycloserine, similar to Example 1, except that: (1) 4.5 mL of the cycloserine sample solution is transferred to a 25 mL volumetric flask containing 10 mL of derivatization reagent in the prepared test solution. After standing at room temperature for 40 min, the solution is diluted with water to obtain the test solution. The conditions for the remaining solution preparation and HPLC detection are the same as in Example 1, and will not be repeated here.

[0102] The HPLC chromatogram of the test solution is as follows: Fig. 4 As shown.

[0103] Comparative Example 1 This comparative example provides a method for detecting related substances in cyclic serine, similar to Example 1, except that N-acetyl-L-cysteine ​​in the derivatization reagent is replaced with mercaptoethanol. Other solution preparation and HPLC detection conditions are the same as in Example 1 and will not be repeated.

[0104] The HPLC chromatogram of the spiked test solution is shown below.Fig. 5 As shown, the baseline exhibited abnormal fluctuations (baseline drift) after 20 minutes; the main peak (15.629 min) and the similar impurity peak (14.633 min) differed by only 1 minute, indicating insufficient separation; the impurity peaks at 14.633 min and 20.931 min had poor shapes; the main peak had a high phase response intensity, but some impurity peak signals were weak, resulting in low detection sensitivity.

[0105] Comparative Example 2 This comparative example provides a method for detecting related substances in cyclic serine, similar to Example 1, except that the chromatographic column is replaced with an Agilent ZORBAX SB-Phenyl column (4.6 mm × 250 mm, 5 μm). Other solution preparation and HPLC detection conditions are the same as in Example 1 and will not be repeated here.

[0106] The HPLC chromatogram of the test solution is shown below. Fig. 6 As shown, the separation between the impurity peaks and the main peak is poor, and the response value of the impurity peaks is significantly reduced.

[0107] Comparative Example 3 This comparative example provides a method for detecting related substances in cyclic serine, similar to Example 1, except that mobile phase A is replaced with disodium hydrogen phosphate (0.02 mol / L, pH=7.0), methanol, and acetonitrile in a volume ratio of 95:2.5:2.5. Other solution preparation and HPLC detection conditions are the same as in Example 1 and will not be repeated here.

[0108] The HPLC chromatogram of the test solution is shown below. Fig. 7 As shown, the baseline fluctuations became significantly larger, and a double peak appeared at 15.837 min, which affected the experimental results, and the separation between the main peak and the impurity peak was insufficient.

[0109] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting related substances in cyclic serine, characterized in that, Includes the following steps: Preparation of the test solution: The cycloserine sample was dissolved in water, and a derivatization reagent was added to react and obtain the test solution; the derivatization reagent included N-acetyl-L-cysteine, benzaldehyde derivative and borate buffer. Preparation of reference solutions: D-serine reference standard, D-serine methyl ester hydrochloride reference standard, 3-chloro-D-alanine methyl ester hydrochloride reference standard and L-cycloserine reference standard were dissolved in water to obtain reference solutions; The test solution was analyzed using high-performance liquid chromatography (HPLC), and the chromatographic conditions for HPLC included: A ZORBAX SB-Phenyl column was used. Mobile phase A consisted of disodium hydrogen phosphate solution and acetonitrile in a volume ratio of (95~100):(1~3), and mobile phase B consisted of disodium hydrogen phosphate solution and acetonitrile in a volume ratio of (35~45):(55~65), with gradient elution.

2. The method for detecting related substances in cyclic serine as described in claim 1, characterized in that, The pH of the borate buffer solution is 9.2~9.8; The benzaldehyde derivatives include o-phthalaldehyde.

3. The method for detecting related substances in cyclic serine as described in claim 1 or 2, characterized in that, The mass-to-volume ratio of N-acetyl-L-cysteine, benzaldehyde derivative, water, and borate buffer in the derivatization reagent is (0.18~0.22)g:(0.15~0.19)g:50mL:(140~160)mL.

4. The method for detecting related substances in cyclic serine as described in claim 1, characterized in that, The mass-to-volume ratio of cycloserine sample, derivatization reagent, and water in the test solution is (4.5~5.5) mg: 10 mL: (13~15) mL; The reaction temperature is 10℃~40℃, and the reaction time is 40min~50min.

5. The method for detecting related substances in cyclic serine as described in claim 1, characterized in that, The concentrations of D-serine, D-serine methyl ester hydrochloride, 3-chloro-D-alanine methyl ester hydrochloride, and L-cycloserine in the reference solution were 0.2 μg / mL to 2 μg / mL.

6. The method for detecting related substances in cyclic serine as described in claim 1, characterized in that, The ZORBAX SB-Phenyl chromatographic column has a length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm.

7. The method for detecting related substances in cyclic serine as described in claim 1, characterized in that, The concentration of the disodium hydrogen phosphate solution is 0.018 mol / L to 0.022 mol / L, and the pH is 6.8 to 7.

2.

8. The method for detecting related substances in cyclic serine as described in claim 1, characterized in that, The gradient elution procedure is as follows: 0 min ~ 15 min, 98% ~ 100% → 90% mobile phase A, 2% ~ 0% → 10% mobile phase B; 15 min to 35 min, 90% → 0 mobile phase A, 10% → 100% mobile phase B; 35 min ~ 37 min, 0 → 100% mobile phase A, 100% → 0 mobile phase B; 37 min to 45 min, 100% mobile phase A.

9. The method for detecting related substances in cyclic serine as described in claim 1, characterized in that, The chromatographic conditions for the high-performance liquid chromatography method also include: a detection wavelength of 330 nm to 340 nm and a column temperature of 25 °C to 35 °C.

10. The method for detecting related substances in cyclic serine as described in claim 1, characterized in that, The chromatographic conditions for the high-performance liquid chromatography method also include: a flow rate of 0.9 mL / min to 1.1 mL / min and an injection volume of 18 μL to 22 μL.

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