HPLC (High Performance Liquid Chromatography) detection method for related impurities in vitamin B6 injection

Abstract

The invention relates to the technical field of analytical chemistry, and particularly discloses an HPLC (High Performance Liquid Chromatography) detection method for related impurities in a vitamin B6 injection. The invention provides an HPLC detection method for related impurities in vitamin B6 injection. The HPLC detection method comprises the following steps: carrying out separation detection on a to-be-detected sample by adopting a chromatographic column taking octadecylsilane chemically bonded silica as a filler and a mobile phase gradient elution method; the mobile phase A is 0.04% sodium pentane sulfonate solution-methanol with the volume ratio of 95: 5, and the pH value of the 0.04% sodium pentane sulfonate solution is adjusted to 3.0 by adopting phosphoric acid; the mobile phase B is methanol. The HPLC detection method provided by the invention can detect 13 impurities in the vitamin B6 injection at the same time, the detection specificity is high, the sensitivity is high, the separation degree is good, and the quality of the vitamin B6 injection can be effectively controlled when the detection method is applied to production of the vitamin B6 injection.

Description

Technical Field

[0001] The present application relates to the technical field of analytical chemistry, and in particular to a HPLC detection method for relevant impurities in vitamin B6 injection. Background Art

[0002] Vitamin B6 is an important water-soluble vitamin with a wide range of applications in the medical field. Vitamin B6 injection is widely used to treat vitamin B6 deficiency, vomiting during pregnancy, and certain metabolic diseases due to its high efficiency and rapid absorption. However, vitamin B6 injection may produce a variety of impurities during the production and storage process, which not only affect the quality and stability of the drug, but may also cause adverse reactions to patients. Therefore, the development of an efficient and accurate detection method to ensure the safety and effectiveness of vitamin B6 injection has important clinical significance and technical value.

[0003] The impurity detection methods of existing vitamin B6 injection mainly include gas chromatography (GC), ultraviolet visible spectroscopy (UV-Vis) and high performance liquid chromatography (HPLC); among them, the HPLC method has become one of the most commonly used methods because of its high resolution and good quantitative ability. Although the existing HPLC detection method has achieved certain results, there are still some shortcomings in the actual production application of vitamin B6 injection. Especially when vitamin B6 injection contains multiple impurities (such as impurity A, impurity B, impurity C, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, impurity M, impurity N, impurity O and impurity P) at the same time, the existing detection method is often difficult to effectively separate all target impurities, resulting in some impurities failing to be separated or having problems such as merging peaks, thereby affecting the reliability and accuracy of the overall detection.

[0004] Therefore, in order to ensure the safety and effectiveness of vitamin B6 injection, it is crucial to provide a detection method for related impurities in vitamin B6 injection with strong specificity, high sensitivity and good separation. Summary of the invention

[0005] In order to quickly and accurately detect relevant impurities (impurity A, impurity B, impurity C, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, impurity M, impurity N, impurity O and impurity P) in vitamin B6 injection, the present application provides a HPLC detection method for relevant impurities in vitamin B6 injection.

[0006] The HPLC detection method of relevant impurities in vitamin B6 injection provided in the present application adopts the following technical scheme: A HPLC detection method of relevant impurities in vitamin B6 injection, comprising the following steps: using a chromatographic column filled with octadecylsilane bonded silica gel and a mobile phase gradient elution method to separate and detect the sample to be tested; the mobile phase gradient elution program is: 0-5min, mobile phase A was 100%, mobile phase B was 0%; 5-10 min, mobile phase A was gradually reduced to 95%, and mobile phase B was gradually increased to 5%; 10-22 min, mobile phase A was gradually reduced to 92%, and mobile phase B was gradually increased to 8%; From 22 to 60 min, mobile phase A was gradually reduced to 65%, and mobile phase B was gradually increased to 35%; 60-70 min, mobile phase A was gradually reduced to 40%, and mobile phase B was gradually increased to 60%; 70-80min, mobile phase A: 40%, mobile phase B: 60%; 80-81 min, mobile phase A was gradually increased to 100%, and mobile phase B was gradually decreased to 0%; 81-90 min, mobile phase A was 100%, mobile phase B was 0%; Wherein, the mobile phase A is 0.04% sodium pentane sulfonate solution-methanol in a volume ratio of 95:5, and the pH of the 0.04% sodium pentane sulfonate solution is adjusted to 3.0 using phosphoric acid; the mobile phase B is methanol; The relevant impurities include: impurity A, impurity B, impurity C, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, impurity M, impurity N, impurity O and impurity P.

[0007] In this application, in order to realize the simultaneous detection of impurities A, B, C, F, G, H, I, J, K, M, N, O and P in vitamin B6 injection, the inventors improved the method based on the Chinese Pharmacopoeia (ChP2020), and through a large number of experimental explorations, the mobile phase and mobile phase elution program in the detection method of related impurities in vitamin B6 injection were re-determined, and the above 13 kinds of impurities in vitamin B6 injection were detected quickly and accurately, and the detection was specific, sensitive, and the separation between the substances in the spectrum was good. Therefore, the HPLC detection method of related impurities in the vitamin B6 injection provided in the present application is used in the production of vitamin B6 injection, which can effectively control the quality of vitamin B6 injection.

[0008] In this application, the chemical names and structural formulas of relevant impurities in vitamin B6 injection are shown in Table 1 below: Table 1 Chemical names and structural formulas of relevant impurities in this application Optionally, during the separation and detection process, the detection wavelength is 291 nm.

[0009] Optionally, during the separation and detection process, the flow rate is 0.8-1.2 mL / min.

[0010] Optionally, during the separation and detection process, the column temperature is 25-35°C.

[0011] Optionally, during the separation and detection process, the injection volume is 10-15 μL.

[0012] Optionally, the sample to be tested needs to be diluted with mobile phase A to a test solution with a concentration of 1 mg / mL.

[0013] Optionally, the sample to be tested contains one or more of vitamin B6, impurity A, impurity B, impurity C, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, impurity M, impurity N, impurity O and impurity P.

[0014] Optionally, the chromatographic column with octadecylsilane bonded silica gel as filler is a GL Inertsil ODS-3 chromatographic column with specifications of 4.6 mm×250 mm and 5 μm.

[0015] In summary, this application has the following beneficial effects: 1. The present application provides an HPLC detection method for relevant impurities in vitamin B6 injection. The detection method uses phosphoric acid to adjust the pH of a 0.04% sodium pentane sulfonate solution to 3.0, and adopts a 0.04% sodium pentane sulfonate solution-methanol with a volume ratio of 95:5 as mobile phase A, methanol as mobile phase B, and a specific mobile phase gradient elution program, so that impurities A, impurity B, impurity C, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, impurity M, impurity N, impurity O and impurity P in vitamin B6 injection can be quickly and accurately detected simultaneously, and the detection has strong specificity, high sensitivity and good separation.

[0016] 2. The peaks of the detection spectrum obtained by the HPLC detection method for relevant impurities in vitamin B6 injection provided in this application have good symmetry, appropriate peak height, and a separation degree between the main component peak and the adjacent peak greater than 1.5. The peak positions of each substance are far apart, and the separation effect is obvious.

[0017] 3. The HPLC detection method for relevant impurities in vitamin B6 injection provided in this application solves the problems existing in the related technology such as some impurities not being eluted and the existence of impurity merged peaks. By using the above detection method to detect relevant impurities in vitamin B6 injection, the quality of vitamin B6 injection can be effectively controlled.

[0018] 4. Vitamin B6, impurity A, impurity B, impurity C, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, impurity M, impurity N, impurity O and impurity P were detected using the HPLC detection method for relevant impurities in the vitamin B6 injection provided in the present application. The quantitative limits and detection limits of each substance obtained are as follows: the detection limit of impurity K is 0.03 μg / mL, and the quantitative limit is 0.10 μg / mL; the detection limit of impurity G is 0.03 μg / mL, and the quantitative limit is 0.11 μg / mL; the detection limit of impurity I is 0.03 μg / mL, and the quantitative limit is 0.09 μg / mL; the detection limit of impurity H is 0.03 μg / mL, and the quantitative limit is 0.10 μg / mL; the detection limit of impurity P is 0.05 μg / mL, and the quantitative limit is 0.17 μg / mL; the detection limit of the main component vitamin B6 is The detection limit of impurity A is 0.03μg / mL, and the limit of quantification is 0.09μg / mL; the detection limit of impurity B is 0.03μg / mL, and the limit of quantification is 0.09μg / mL; the detection limit of impurity F is 0.03μg / mL, and the limit of quantification is 0.10μg / mL; the detection limit of impurity M is 0.01μg / mL, and the limit of quantification is 0.05μg / mL; the detection limit of impurity O is 0.01μg / mL, and the limit of quantification is 0.05μg / mL; the detection limit of impurity N is 0.02μg / mL, and the limit of quantification is 0.07μg / mL; the detection limit of impurity C is 0.03μg / mL, and the limit of quantification is 0.09μg / mL; the detection limit of impurity J is 0.02μg / mL, and the limit of quantification is 0.08μg / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a chromatogram obtained by detecting the mixed solution using the HPLC detection method provided in Example 1; Figure 2 is a chromatogram obtained by testing the system suitability solution using the HPLC detection method provided in Example 1; Figure 3 This is a chromatogram obtained by detecting the mixed solution using the HPLC detection method provided in Comparative Example 1; Figure 4 This is a chromatogram obtained by detecting the mixed solution using the HPLC detection method provided in Comparative Example 2; Figure 5This is the chromatogram obtained by detecting the mixed solution using the HPLC detection method provided in Comparative Example 6. DETAILED DESCRIPTION

[0020] The present application provides a HPLC detection method for relevant impurities in vitamin B6 injection, comprising the following steps: (1) Prepare sample solution: (2) injecting the sample solution into a liquid chromatograph, and using a GL Inertsil ODS-3 chromatographic column (specifications: 4.6 mm×250 mm, 5 μm) and a mobile phase gradient elution method for separation and detection; during the separation and detection process, the column temperature is 25-35° C., the detection wavelength is 291 nm, the mobile phase flow rate is 0.8-1.2 mL / min, and the injection volume is 10-15 μL; The mobile phase gradient elution program is: 0-5min, mobile phase A was 100%, mobile phase B was 0%; 5-10 min, mobile phase A was gradually reduced to 95%, and mobile phase B was gradually increased to 5%; 10-22 min, mobile phase A was gradually reduced to 92%, and mobile phase B was gradually increased to 8%; From 22 to 60 min, mobile phase A was gradually reduced to 65%, and mobile phase B was gradually increased to 35%; 60-70 min, mobile phase A was gradually reduced to 40%, and mobile phase B was gradually increased to 60%; 70-80min, mobile phase A: 40%, mobile phase B: 60%; 80-81 min, mobile phase A was gradually increased to 100%, and mobile phase B was gradually decreased to 0%; 81-90 min, mobile phase A was 100%, mobile phase B was 0%; The mobile phase A is 0.04% sodium pentane sulfonate solution-methanol in a volume ratio of 95:5, and the pH of the 0.04% sodium pentane sulfonate solution is adjusted to 3.0 using phosphoric acid; the mobile phase B is methanol.

[0021] The sources of the raw materials used in this application are shown in Table 2 below, and the remaining raw materials, reagents, solvents, etc. can be obtained commercially.

[0022] Table 2 Impurities and sample sources in this application The present application is further described in detail below in conjunction with embodiments and performance testing experiments.

[0023] Example 1 Example 1 provides an HPLC detection method for relevant impurities in vitamin B6 injection, comprising the following steps: (1) Solution preparation: prepare sample solution; (2) Injecting the sample solution into a liquid chromatograph, and using a GL Inertsil ODS-3 chromatographic column (specifications: 4.6 mm×250 mm, 5 μm) and a mobile phase gradient elution method for separation and detection; during the separation and detection process, the column temperature was 30° C., the detection wavelength was 291 nm, the mobile phase flow rate was 1.0 mL / min, and the injection volume was 10 μL; The mobile phase gradient elution program is: 0-5min, mobile phase A was 100%, mobile phase B was 0%; 5-10 min, mobile phase A was gradually reduced to 95%, and mobile phase B was gradually increased to 5%; 10-22 min, mobile phase A was gradually reduced to 92%, and mobile phase B was gradually increased to 8%; From 22 to 60 min, mobile phase A was gradually reduced to 65%, and mobile phase B was gradually increased to 35%; 60-70 min, mobile phase A was gradually reduced to 40%, and mobile phase B was gradually increased to 60%; 70-80min, mobile phase A: 40%, mobile phase B: 60%; 80-81 min, mobile phase A was gradually increased to 100%, and mobile phase B was gradually decreased to 0%; 81-90 min, mobile phase A was 100%, mobile phase B was 0%; The mobile phase A is 0.04% sodium pentane sulfonate solution-methanol in a volume ratio of 95:5, and the pH of the 0.04% sodium pentane sulfonate solution is adjusted to 3.0 using phosphoric acid; the mobile phase B is methanol.

[0024] Exclusive inspection The specificity of the HPLC detection method for relevant impurities in the vitamin B6 injection provided in this application was investigated, and the specific process is as follows: (1) Prepare sample solution: Blank solution / diluent: mobile phase A.

[0025] Blank excipient solution: Measure 1 mL of blank excipient, place in a 100 mL volumetric flask, dilute to the mark with diluent, and shake well.

[0026] Test solution: Accurately measure 1 mL of vitamin B6 injection, place it in a 100 mL volumetric flask, dilute to the scale with diluent, and shake well.

[0027] Sensitivity solution: Accurately measure 1 mL of the test solution, place it in a 100 mL volumetric flask, dilute it to the scale with diluent, shake well, accurately measure 1 mL, place it in a 50 mL volumetric flask, dilute it to the scale with diluent, shake well.

[0028] Vitamin B6 reference solution: Take about 10 mg of vitamin B6 reference, accurately weigh, place in a 100 mL volumetric flask, add diluent to dissolve and dilute to the scale, shake well, accurately measure 1 mL, place in a 100 mL volumetric flask, dilute to the scale with diluent, and shake well.

[0029] Impurity mixed stock solution I: Take about 5 mg each of impurity A, impurity B, impurity C, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, impurity M, impurity N, impurity O and impurity P reference substances, accurately weigh, place in a 25 mL volumetric flask, add methanol to dissolve and dilute to the scale with diluent, and shake well.

[0030] Impurity mixed stock solution II: Accurately measure 2.5 mL of impurity mixed stock solution I, place it in a 50 mL volumetric flask, dilute to the scale with diluent, and shake well.

[0031] Test sample spiked solution: Accurately measure 1 mL of vitamin B6 injection and place it in a 100 mL volumetric flask. Accurately add 1 mL of impurity mixed stock solution I, dilute to the scale with diluent, and shake well.

[0032] Impurity A stock solution: weigh about 1 mg of impurity A reference substance, place in a 10 mL volumetric flask, add methanol to dissolve and dilute to scale, shake well. Prepare impurity B, impurity C, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, impurity M, impurity N, impurity O and impurity P stock solutions in the same way.

[0033] Impurity A localization solution: Take 100 μl of impurity A stock solution and 900 μl of diluent and mix well. Prepare impurity B, impurity C, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, impurity M, impurity N, impurity O and impurity P localization solutions in the same way.

[0034] Vitamin B6 localization solution: that is, vitamin B6 reference solution.

[0035] Mixed solution: Take about 10 mg of vitamin B6 raw material, weigh accurately, place in a 10 mL volumetric flask, accurately add 1 mL of impurity mixed stock solution II, add diluent to dissolve and dilute to the scale, and shake well.

[0036] System suitability solution: Take about 10 mg of vitamin B6 reference substance, weigh accurately, place in a 10 mL volumetric flask, add 200 μl of impurity C, impurity M and impurity N stock solutions respectively, add diluent to dissolve and dilute to the scale, and shake well.

[0037] (2) Accurately measure the blank solution, blank excipient solution, system suitability solution, sensitivity solution, vitamin B6 reference solution, test solution, test spiked solution and each positioning solution, inject them into a liquid chromatograph, perform detection according to the method of Example 1, and record the chromatogram. The detection results are shown in Tables 3 and 4 below. The chromatogram of the mixed solution is shown in Figure 1 The chromatogram of the system suitability solution is shown in Figure 2 shown.

[0038] Table 3 Specificity investigation results 1 Table 4 Specificity investigation results 2 According to the test results of Table 3 and Table 4, under the liquid chromatography test conditions provided in this application, the blank solution and the blank auxiliary material solution do not interfere with the detection of vitamin B6 and related impurities. In the chromatogram of the mixed solution, the peak order is impurity K (RRT 0.26), impurity G (RRT 0.61), impurity I (RRT 0.65), impurity H peak 1 (RRT 0.82), impurity P (RRT 0.91), main component, impurity A (RRT 1.27), impurity B (RRT 1.32), impurity H peak 2 (RRT 1.50), impurity F (RRT 1.61), impurity M (RRT 1.85), impurity O (RRT 1.96), impurity N (RRT 1.99), impurity C (RRT 2.39), impurity J (RRT 3.19), and the separation between each impurity peak and between the main component and the adjacent peak is greater than 1.5 (the minimum value is 1.60), which meets the requirements. In the system suitability solution chromatogram, the order of peaks is main component, impurity M, impurity N, impurity C, the theoretical plate number of the main component peak is 26424, the separation between each peak is greater than 1.5 (the minimum value is 8.03), and in the sensitivity solution chromatogram, the main component peak height signal-to-noise ratio is 23.1, which meets the requirements. Therefore, it is shown that the HPLC detection method for related impurities in vitamin B6 injection provided by this application has good specificity.

[0039] The quantitative limit and the detection limit use the signal-to-noise ratio as an evaluation index to examine the detection sensitivity of the HPLC detection method for relevant impurities in the vitamin B6 injection provided in this application. It is required to use the corresponding concentration when the signal-to-noise ratio is ≥10 as the quantitative limit, and the corresponding concentration when the signal-to-noise ratio is ≥3 as the detection limit. The details are as follows: Take vitamin B6 reference solution, impurity A, impurity B, impurity C, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, impurity M, impurity N, impurity O and impurity P stock solution, mix and dilute in appropriate amounts; prepare quantitative limit solution and detection limit solution, inject the quantitative limit solution and detection limit solution into a liquid chromatograph, and then detect according to the liquid chromatography detection conditions of Example 1. The detection limit and quantitative limit test results are shown in Table 5 below; Table 5 Detection limit and quantification limit test results According to the test results in Table 5, the detection limit of impurity K is 0.03μg / mL, and the limit of quantification is 0.10μg / mL; the detection limit of impurity G is 0.03μg / mL, and the limit of quantification is 0.11μg / mL; the detection limit of impurity I is 0.03μg / mL, and the limit of quantification is 0.09μg / mL; the detection limit of impurity H is 0.03μg / mL, and the limit of quantification is 0.10μg / mL; the detection limit of impurity P is 0.05μg / mL, and the limit of quantification is 0.17μg / mL; the detection limit of the main component vitamin B6 is 0.02μg / mL, and the limit of quantification is 0.06μg / mL; the detection limit of impurity A is 0.03μg / mL, and the limit of quantification is 0 .09μg / mL; the detection limit of impurity B is 0.03μg / mL, and the limit of quantification is 0.09μg / mL; the detection limit of impurity F is 0.03μg / mL, and the limit of quantification is 0.10μg / mL; the detection limit of impurity M is 0.01μg / mL, and the limit of quantification is 0.05μg / mL; the detection limit of impurity O is 0.01μg / mL, and the limit of quantification is 0.05μg / mL; the detection limit of impurity N is 0.02μg / mL, and the limit of quantification is 0.07μg / mL; the detection limit of impurity C is 0.03μg / mL, and the limit of quantification is 0.09μg / mL; the detection limit of impurity J is 0.02μg / mL, and the limit of quantification is 0.08μg / mL. Therefore, the sensitivity of the HPLC detection method for related impurities in vitamin B6 injection provided in the present application is good.

[0040] Linear range The linearity of each impurity content in the range of LOQ to 1% was examined, requiring linear regression with concentration as the horizontal axis and peak area as the vertical axis, with a correlation coefficient of r≥0.998, and the Y-axis intercept should be within 25% of the 100% limit response value.

[0041] (1) Prepare the following solution: Linear stock solution 1: Mixed stock solution I.

[0042] Linear stock solution 2: Take about 10 mg of vitamin B6 reference substance, weigh accurately, place in a 50 mL volumetric flask, add diluent to dissolve and dilute to the scale, and shake well.

[0043] Linear stock solution 3: Accurately measure 1 mL of linear stock solution 1 and 1 mL of linear stock solution 2, place in a 20 mL volumetric flask, dilute to the mark with diluent, and shake well.

[0044] Accurately measure a certain volume of linear stock solution 3, prepare it according to Table 6 below, and dilute it with diluent to prepare each linear solution.

[0045] Table 6 Concentration of linear solution (2) Accurately measure 10 μL of each of the above linear solutions and inject them into a liquid chromatograph; then perform detection according to the liquid chromatography detection conditions of Example 1, record the chromatogram; and perform linear regression with concentration as the abscissa (X) and peak area as the ordinate (Y). The linear relationship of each impurity is shown in Table 7.

[0046] Table 7 Linear relationship of impurities According to the test results in Table 7, impurities A, B, C, F, G, H, I, J, K, M, N, O, P and the main component vitamin B6 have a good linear relationship within the range of approximately LOQ to 1%. Therefore, it is shown that the HPLC detection method for relevant impurities in the vitamin B6 injection provided by the present application has good linearity.

[0047] Accuracy To investigate the accuracy of HPLC detection method for relevant impurities in vitamin B6 injection.

[0048] Accuracy test was conducted when the impurity A, impurity B, impurity C, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, impurity M, impurity N, impurity O and impurity P were all about 0.04%, 0.20%, 0.30% (i.e. 20%, 100%, 150% limits), and the average recovery rate of each impurity content should be between 70% and 130%, RSD (n=9) ≤15%. If as shown in Table 8 below.

[0049] Table 8 Accuracy test results According to the test results in Table 8, the average recoveries of impurities A, B, C, F, G, H, I, J, K, M, N, O and P at contents of approximately 0.04%, 0.20% and 0.30% (approximately equivalent to limits of 20%, 100% and 150%) are all within the range of 70% to 130%, and the RSDs are all less than 15% (the maximum value is 8.94%); therefore, it is shown that the HPLC detection method for relevant impurities in vitamin B6 injection provided in the present application has good accuracy.

[0050] Comparative Example 1 Comparative Example 1 provides an HPLC detection method for relevant impurities in vitamin B6 injection.

[0051] The method standard used in Comparative Example 1 is BP2024 reproduction. A capped octadecyl bonded silica gel column (Thermo Hypurity C18, 4.6 mm×250 mm, 5 μm) and an isocratic elution method of the mobile phase were used for separation and detection; the flow rate during the separation and detection process was 1.0 mL / min; the column temperature was 25°C; the detection wavelength was 210 nm; and the injection volume was 5 μL.

[0052] Preparation of mobile phase: Weigh 2.72 g of potassium dihydrogen phosphate into 900 mL of water, adjust the pH to 3.0 with dilute phosphoric acid, and dilute to 1000 mL with water.

[0053] Comparative Example 2 Comparative Example 2 provides an HPLC detection method for relevant impurities in vitamin B6 injection.

[0054] The method standard adopted in Comparative Example 2 is ChP2020 reproduction, and the difference from Example 1 is: mobile phase and elution procedure.

[0055] Octadecylsilane bonded silica gel column (GL Inertsil ODS-3V, 4.6mm×250mm, 5μm) and isocratic elution method of mobile phase were used for separation and detection; flow rate during separation and detection was 1.0mL / min; column temperature was 30℃; detection wavelength was 291nm; injection volume was 10μL.

[0056] The mobile phase was 0.04% sodium pentane sulfonate solution-methanol in a volume ratio of 85:15; the pH value of the 0.04% sodium pentane sulfonate solution was adjusted to 3.0 with glacial acetic acid.

[0057] Comparative Example 3 Comparative Example 3 provides an HPLC detection method for relevant impurities in vitamin B6 injection.

[0058] The difference between Comparative Example 3 and Example 1 is that the pH value of the 0.04% sodium pentane sulfonate solution in mobile phase A is adjusted to 3.0 using glacial acetic acid.

[0059] Comparative Example 4 Comparative Example 4 provides an HPLC detection method for relevant impurities in vitamin B6 injection.

[0060] The difference between Comparative Example 4 and Example 1 is: the ratio of mobile phase A.

[0061] Mobile phase A was 0.04% sodium pentane sulfonate solution-methanol in a volume ratio of 85:15; the pH value of the 0.04% sodium pentane sulfonate solution was adjusted to 3.0 with phosphoric acid; and mobile phase B was methanol.

[0062] Comparative Example 5 Comparative Example 5 provides an HPLC detection method for relevant impurities in vitamin B6 injection.

[0063] The difference between Comparative Example 5 and Example 1 is: the mobile phase gradient elution procedure.

[0064] The mobile phase gradient elution program of Comparative Example 5 is: 0-5min, mobile phase A was 100%, mobile phase B was 0%; 5-10 min, mobile phase A was gradually reduced to 95%, and mobile phase B was gradually increased to 5%; 10-22 min, mobile phase A was gradually reduced to 92%, and mobile phase B was gradually increased to 8%; From 22 to 48 min, mobile phase A was gradually reduced to 65%, and mobile phase B was gradually increased to 35%; 48-49 min, mobile phase A was gradually increased to 100%, and mobile phase B was gradually decreased to 0%; 49-60min, mobile phase A was 100%, mobile phase B was 100%.

[0065] Mobile phase A was 0.04% sodium pentane sulfonate solution-methanol in a volume ratio of 95:5; the pH value of the 0.04% sodium pentane sulfonate solution was adjusted to 3.0 with phosphoric acid; and mobile phase B was methanol.

[0066] Comparative Example 6 Comparative Example 6 provides an HPLC detection method for relevant impurities in vitamin B6 injection.

[0067] The difference between Comparative Example 6 and Example 1 is: the mobile phase gradient elution procedure.

[0068] The mobile phase gradient elution program used in Comparative Example 6 is: 0-5min, mobile phase A was 100%, mobile phase B was 0%; 5-10 min, mobile phase A was gradually reduced to 95%, and mobile phase B was gradually increased to 5%; 10-22 min, mobile phase A was gradually reduced to 92%, and mobile phase B was gradually increased to 8%; From 22 to 48 min, mobile phase A was gradually reduced to 65%, and mobile phase B was gradually increased to 35%; 48-53 min, mobile phase A was gradually reduced to 40%, and mobile phase B was gradually increased to 60%; 53-54min, mobile phase A: 40%, mobile phase B: 60%; 54-65min, mobile phase A was gradually increased to 100%, and mobile phase B was gradually decreased to 0%; Mobile phase A was 0.04% sodium pentane sulfonate solution-methanol in a volume ratio of 95:5; the pH value of the 0.04% sodium pentane sulfonate solution was adjusted to 3.0 with phosphoric acid; and mobile phase B was methanol.

[0069] Test results The mixed solution was tested using the methods provided in Example 1 and Comparative Examples 1-6, and the minimum separation between the peaks in the spectrum was obtained. The results are shown in Table 9 below: Table 9 Minimum separation in the mixed solution detection spectra obtained by each detection method Minimum separation Example 1 1.60 Comparative Example 1 2.33 Comparative Example 2 1.19 Comparative Example 3 1.03 Comparative Example 4 1.31 Comparative Example 5 1.63 Comparative Example 6 2.04 (1) The chromatogram obtained by detecting the mixed solution using the detection method provided in Example 1 is as follows Figure 1 As shown, according to Figure 1 It can be seen that the minimum separation between the peaks is 1.60 (> 1.5), and the baseline in the spectrum is stable, the peak shape is symmetrical, there is no tailing or fronting phenomenon, and there are no other turtle peaks or impurity peaks. Therefore, it is shown that the detection method provided in Example 1 can achieve effective separation and detection of 13 impurities in vitamin B6 injection.

[0070] (2) The chromatogram obtained by detecting the mixed solution using the detection method provided in Comparative Example 1 is as follows Figure 3 As shown, the peak table is shown in Table 10 below; Table 10 Chromatogram peak table obtained by detecting the mixed solution using the detection method provided in Comparative Example 1 Detector A210nm Peak Retention time area high area% Tailing Factor Theoretical Plate Number (USP) Resolution (USP) 1 6.891 22028 2129 0.05 1.09 9540 - 2 8.303 12489 1267 0.03 1.06 15264 5.13 3 9.561 42337676 1602219 99.03 4.25 2221 2.33 4 15.405 118444 5572 0.28 1.17 11752 8.47 5 17.019 61838 3276 0.14 1.08 18195 3.01 6 18.225 71649 3660 0.17 1.08 19584 2.35 7 20.939 64260 2977 0.15 1.06 21139 4.95 8 25.615 30516 1119 0.07 1.08 19942 7.18 9 30.499 34571 1095 0.08 1.11 20801 6.22 total 42753471 1623313 100.00 according to Figure 3As can be seen from the test results in Table 10, the mixed solution was tested using the test conditions in the prior art BP2024. The results showed that multiple impurities such as impurity J in the mixed solution did not show peaks, and there was a situation where impurity peaks merged. Therefore, it is explained that the test conditions in the prior art BP2024 provided in Comparative Example 1 are difficult to achieve effective separation and detection of 13 impurities in vitamin B6 injection.

[0071] (3) The chromatogram obtained by detecting the mixed solution using the detection method provided in Comparative Example 2 is as follows Figure 4 As shown, the peak table is shown in Table 11 below; Table 11 Chromatogram peak table obtained by detecting the mixed solution using the detection method provided in Comparative Example 2 Detector A 291nm Peak Retention time area high area% Tailing Factor Theoretical Plate Number (USP) Resolution (USP) 1 3.817 9022 1120 0.04 1.42 5090 - 2 5.779 9273 1039 0.04 1.03 8730 8.50 3 6.107 5723 633 0.02 1.16 9355 1.31 4 6.683 14354 868 0.06 0.82 3413 1.62 5 7.407 3085 208 0.01 0.80 6454 1.75 6 8.555 25118847 1481393 99.28 0.65 5555 2.78 7 11.093 19334 1426 0.08 0.98 14121 6.10 8 11.588 23973 1671 0.09 1.10 13951 1.29 9 14.543 16469 875 0.07 1.06 13283 6.59 10 16.102 13834 669 0.05 0.96 12907 2.91 11 16.748 13065 689 0.05 1.12 16502 1.19 12 22.632 11367 349 0.04 1.04 10762 8.44 13 29.970 19096 501 0.08 1.12 12511 7.55 14 33.131 24635 572 0.10 1.14 13591 2.86 total 25302079 1492015 100.00 according to Figure 4 As can be seen from the test results in Table 11, the mixed solution was tested using the test conditions in the prior art ChP2020, and the results showed that only impurities C and impurity J did not show peaks in the mixed solution, and there was no impurity merging peak. Therefore, it is explained that the mobile phase and elution procedure in the prior art ChP2020 provided in Comparative Example 2 are difficult to achieve effective separation and detection of 13 impurities in vitamin B6 injection.

[0072] (4) According to the detection results in Table 9, the detection method provided in Comparative Example 3 detects the mixed solution, and the minimum separation degree between the peaks in the detection spectrum is only 1.03 (<1.5). This indicates that Comparative Example 3 uses mobile phase A prepared by adjusting 0.04% sodium pentane sulfonate solution with glacial acetic acid as eluent to elute the mixed solution, and the elution separation effect between impurities in the mixed solution is not good, and it is difficult to achieve effective detection of the impurity content in B6 injection.

[0073] (5) According to the detection results in Table 9, the detection method provided in Comparative Example 4 detects the mixed solution, and the minimum separation degree between the peaks in the detection spectrum is only 1.31 (<1.5). This indicates that Comparative Example 4 uses 0.04% sodium pentane sulfonate solution-methanol with a volume ratio of 85:15 as mobile phase A to elute the mixed solution, and the elution separation effect between impurities in the mixed solution is not good, and it is difficult to achieve effective detection of the impurity content in B6 injection.

[0074] (6) The mixed solution was tested by the method provided in Comparative Example 5. Impurity J did not show a peak within 60 min of operation. This indicates that the mobile phase elution program time provided in Comparative Example 5 is too short to effectively separate impurity J in B6 injection, and thus it is difficult to effectively detect the content of impurity J.

[0075] (7) The chromatogram obtained by detecting the mixed solution using the detection method provided in Comparative Example 6 is as follows Figure 5As shown, the peak table is shown in Table 12 below; Table 12 Chromatogram peak table obtained by detecting the mixed solution using the detection method provided in Comparative Example 6 PDA.Ch1.291nm Peak Retention time area high area% Tailing Factor Theoretical Plate Number (USP) Resolution (USP) 1 5.314 9077 995 0.04 1.26 7269 - 2 13.061 7481 644 0.03 1.08 27083 27.34 3 13.714 10239 843 0.04 1.24 28697 2.04 4 17.837 14075 1068 0.06 1.10 40022 12.12 5 21.817 24513174 1070635 99.54 2.30 18286 7.94 6 27.789 15791 1003 0.06 1.02 67695 11.14 7 28.792 19936 1334 0.08 1.02 80386 2.41 8 31.867 5580 458 0.02 1.00 140619 8.24 9 34.370 15732 1132 0.06 1.24 138595 7.06 10 44.642 16523 1337 0.07 1.09 296569 29.47 total 24627607 1079448 100.00 according to Figure 5 As can be seen from the test results in Table 12, the mixed solution was separated and tested by the mobile phase gradient elution program provided in Comparative Example 6. In the test spectrum, impurity J in the mixed solution did not show a peak, and there was no impurity merge peak. This indicates that the mobile phase elution program provided in Comparative Example 6 cannot separate impurity J, so it is difficult to achieve effective separation and detection of 13 impurities in vitamin B6 injection.

[0076] In summary, the HPLC detection method for relevant impurities in vitamin B6 injection provided in the present application can quickly and accurately detect 13 impurities in vitamin B6 injection at the same time by adjusting the mobile phase and the mobile phase gradient elution program, and the detection has strong specificity, high sensitivity and good separation.

[0077] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A HPLC detection method for related impurities in vitamin B6 injection, characterized in that: The following steps are involved: The chromatographic column using octadecylsilane bonded silica as filler and the mobile phase gradient elution method were used to separate and detect the samples to be tested; the mobile phase gradient elution procedure was: 0-5min, mobile phase A was 100%, mobile phase B was 0%; 5-10 min, mobile phase A was gradually reduced to 95%, and mobile phase B was gradually increased to 5%; 10-22 min, mobile phase A was gradually reduced to 92%, and mobile phase B was gradually increased to 8%; From 22 to 60 min, mobile phase A was gradually reduced to 65%, and mobile phase B was gradually increased to 35%; 60-70 min, mobile phase A was gradually reduced to 40%, and mobile phase B was gradually increased to 60%; 70-80 min, mobile phase A: 40%, mobile phase B: 60%; 80-81 min, mobile phase A was gradually increased to 100%, and mobile phase B was gradually decreased to 0%; 81-90 min, mobile phase A was 100%, mobile phase B was 0%; Wherein, the mobile phase A is 0.04% sodium pentane sulfonate solution-methanol in a volume ratio of 95:5, and the pH of the 0.04% sodium pentane sulfonate solution is adjusted to 3.0 using phosphoric acid; the mobile phase B is methanol; The relevant impurities include: impurity A, impurity B, impurity C, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, impurity M, impurity N, impurity O and impurity P.

2. The HPLC detection method according to claim 1, characterized in that During the separation and detection process, the detection wavelength is 291 nm.

3. The HPLC detection method according to claim 1, characterized in that During the separation and detection process, the flow rate is 0.8-1.2 mL / min.

4. The HPLC detection method according to claim 1, characterized in that During the separation and detection process, the column temperature is 25-35°C.

5. The HPLC detection method according to claim 1, characterized in that During the separation and detection process, the injection volume is 10-15 μL.

6. The HPLC detection method according to claim 1, characterized in that The sample to be tested needs to be diluted with mobile phase A to a test solution with a concentration of 1 mg / mL.

7. The HPLC detection method according to claim 1, characterized in that The sample to be tested contains one or more of vitamin B6, impurity A, impurity B, impurity C, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, impurity M, impurity N, impurity O and impurity P.

8. The detection method according to claim 1, characterized in that: The chromatographic column with octadecylsilane bonded silica gel as filler is a GL Inertsil ODS-3 chromatographic column with a specification of 4.6 mm×250 mm and 5 μm.

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