Determination method for related substances in vitamin B6 medicine

Abstract

The invention provides a method for determining related substances in a vitamin B6 drug, which comprises the following steps: dissolving a vitamin B6 drug sample in a prefabricated solution in a dark environment, filtering, and diluting to a target concentration interval to obtain a sample solution; placing the blank solvent, the contrast solution and the sample solution in a chromatographic column, introducing the mobile phase A and the mobile phase B, and sequentially carrying out gradient elution to obtain a chromatogram; and identifying and separating impurity chromatographic peaks in the chromatogram, performing quantitative or semi-quantitative analysis on each impurity chromatographic peak, and calculating the content proportion of each impurity chromatographic peak relative to the main chromatographic peak of the vitamin B6. Through collaborative design of the sample pretreatment state, the gradient elution path and the peak interpretation mode, known impurities and unknown impurities in the vitamin B6 medicine are in a separable state before entering a chromatographic system, and are fully expanded in the chromatographic separation process, so that accurate identification and effective separation of various impurities are realized.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, and in particular relates to a method for determining related substances in vitamin B6 drugs. Background Technology

[0002] Vitamin B6 is a water-soluble vitamin that plays a vital role in human health. The main chemical form of vitamin B6 in marketed pharmaceuticals is pyridoxine hydrochloride. This compound may be introduced with specific process impurities during production, and it is easily destroyed by heat, light, or alkalis. During storage, it can also degrade, producing various impurities. The presence of these impurities directly affects the safety of the drug.

[0003] Currently, most vitamin B6 related substance detection methods listed in pharmacopoeias (such as the United States Pharmacopeia, the European Pharmacopoeia, and the Chinese Pharmacopoeia) employ isocratic elution using organic phase-alkyl sulfonate solutions or phosphate solutions as the mobile phase. The inherent low selectivity of these methods results in insufficient elution and separation capabilities for impurities. Existing improved methods attempt to use gradient elution with heptanesulfonate-methanol and octanesulfonate (pH 2.5)-acetonitrile as mobile phases. However, the gradient design of these methods has limited separation capability for some impurities with similar polarities, failing to achieve complete baseline separation (R < 1.5), thus affecting the accuracy of quantification. Although some methods have begun to use volatile salts, such as ammonium acetate solution-methanol as the mobile phase, their gradient programs only provide exemplary separation for a limited number of known impurities, failing to address or solve more complex separation scenarios involving both known and multiple unknown impurities, particularly the separation of groups of impurities with similar polarities. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for determining related substances in vitamin B6 drugs, which aims to solve the problem of being unable to accurately identify and separate known and unknown impurities in vitamin B6 drugs.

[0005] To solve the above-mentioned technical problems, the present invention is implemented as follows: a method for determining related substances in a vitamin B6 drug, comprising the following steps: S1. Dissolve the vitamin B6 drug sample in a pre-prepared solution in a light-protected environment, filter it, and then dilute it to the target concentration range to obtain a sample solution. The pre-prepared solution includes a volatile buffer salt and an adsorption medium. The adsorption medium is a metal-organic framework material, which includes a metal salt and an organic ligand containing at least one of a carboxyl group, a nitrogen heterocyclic group, or a polydentate coordination group. S2. Place the blank solvent, control solution and sample solution in the chromatographic column, and introduce mobile phase A and mobile phase B. Perform gradient elution sequentially to obtain a chromatogram. The multiple gradient elutions include at least a first-stage linear gradient elution and a second-stage linear gradient elution. The proportion of mobile phase A in the first-stage linear gradient elution is greater than that in the second-stage linear gradient elution, and the proportion of mobile phase B in the first-stage linear gradient elution is less than that in the second-stage linear gradient elution. S3. Identify and separate the impurity chromatographic peaks in the chromatogram, perform quantitative or semi-quantitative analysis on each impurity chromatographic peak, and calculate the content ratio of each impurity chromatographic peak relative to the main vitamin B6 chromatographic peak.

[0006] In some embodiments of the present invention, step S1 includes: S1.1 Using ultrapure water as a solvent, add volatile buffer salt and adjust to weak acidity to obtain a pre-prepared solution; S1.2. Under light-protected conditions, add the vitamin B6 drug sample to the pre-prepared solution, stir to fully dissolve the sample, and then add the adsorption medium for short-term contact. S1.3 After completing the short-term contact, centrifuge to separate the adsorption media and take the supernatant as the sample solution. Filter the sample solution and dilute it to the target concentration range according to the predetermined ratio.

[0007] In some embodiments of the present invention, the volatile buffer salt includes at least one of ammonium formate, ammonium acetate, and ammonium bicarbonate, with a short contact time of 1 to 5 minutes and a target concentration range of 5 to 15 mg / mL.

[0008] In some embodiments of the present invention, the preparation steps of the adsorption medium include: Weigh out a metal salt and dissolve it in a solvent to obtain a metal salt solution; Weigh an organic ligand containing at least one of a carboxyl group, a nitrogen heterocyclic group, or a polydentate coordinating group, add it to a solvent and dissolve it to obtain an organic ligand solution; The metal salt solution and the organic ligand solution are mixed at a predetermined molar ratio, transferred to a closed reaction vessel, and reacted under heating conditions to generate a metal-organic framework material precursor. After the reaction is completed, the product is naturally cooled to room temperature and separated by centrifugation or filtration. The solid product is then washed multiple times with a low-molecular-weight alcohol solvent. The washed solid is then placed under vacuum drying conditions for activation treatment. The activation treatment temperature is 60-150℃ and the time is 6-24 hours to obtain the activated metal-organic framework material. The activated metal-organic framework material was ground to obtain the adsorption medium.

[0009] In some embodiments of the present invention, the metal salt includes at least one of zinc nitrate, copper nitrate, zirconium chloride, aluminum chloride, zinc sulfate, and zinc acetate; the organic ligand includes at least one of terephthalic acid, imidazole, benzimidazole, pyridine dicarboxylic acid, triazine tricarboxylic acid, and triazine tricarboxylic acid derivatives; and the low molecular weight alcohol solvent includes at least one of methanol, ethanol, isopropanol, n-propanol, and n-butanol.

[0010] In some embodiments of the present invention, step S2 includes: S2.1 Start the high performance liquid chromatography system and perform a system self-test. The high performance liquid chromatography system includes a binary pump, an online degassing device, an autosampler, a column oven, a diode array detector and a data workstation. Install the chromatographic column in the column oven and set the constant temperature control so that the chromatographic column reaches thermal equilibrium at a fixed temperature. At the same time, pre-flushing the flow path is performed to remove air bubbles. S2.2 Prepare mobile phase A and mobile phase B respectively, and filter and degas mobile phase A and mobile phase B respectively before connecting them into the high performance liquid chromatography system; S2.3 Place the blank solvent, control solution and sample solution into the autosampler respectively, and inject them sequentially according to the preset injection order to obtain the corresponding chromatographic response signal. The blank solvent does not contain vitamin B6 drug sample, and the control solution contains at least standard vitamin B6 drug. S2.4 After injection, mobile phase A and mobile phase B are introduced to perform a preset gradient elution program. Data corresponding to each injection is collected and recorded to obtain a chromatogram.

[0011] In some embodiments of the present invention, in step S2.4, the preset gradient elution procedure includes: (a) Perform an initial equilibration phase of 0-10 minutes, followed by isocratic elution, keeping the proportion of mobile phase A constant at 95% and the proportion of mobile phase B constant at 5%. (b) Perform a first-stage linear gradient elution for 10–30 minutes, during which the proportion of mobile phase A changes from 95% to 88% and the proportion of mobile phase B changes from 5% to 12%. (c) Perform a second-stage linear gradient elution for 30-50 minutes, during which the proportion of mobile phase A changes from 88% to 68% and the proportion of mobile phase B changes from 12% to 32%. (d) Perform a rapid rinsing and rebalancing phase of 50-65 minutes. At 50-50.1 minutes, perform linear gradient elution, changing the proportion of mobile phase A from 68% to 95% and the proportion of mobile phase B from 32% to 5%. At 50.1-65 minutes, perform isocratic elution, keeping the proportion of mobile phase A at 95% and the proportion of mobile phase B at 5%.

[0012] In some embodiments of the present invention, mobile phase A includes at least one of ammonium formate aqueous solution, ammonium acetate aqueous solution, and ammonium bicarbonate aqueous solution, and mobile phase B includes at least one of methanol, acetonitrile, and isopropanol.

[0013] In some embodiments of the present invention, step S3 includes: S3.1. Use the chromatograms of blank solvent, control solution and sample solution, determine the main chromatographic peak of vitamin B6 by using the retention time and UV response of vitamin B6 in the control solution, and use the chromatogram of blank solvent as the baseline and system peak reference to remove background peaks and ghost peaks. S3.2 Determine the separation degree of impurity chromatographic peaks located in the adjacent regions before and after the main chromatographic peak of vitamin B6. If there is peak overlap or shoulder peaks, further separate the impurity chromatographic peaks by adjusting the gradient elution conditions. S3.3 Quantitative or semi-quantitative analysis of each impurity chromatographic peak is performed. If an impurity reference standard is available, the impurity content is calculated using the external standard method. If an impurity reference standard is not available, the main chromatographic peak of vitamin B6 is used as a reference for relative quantification. S3.4 Calculate the content ratio of each impurity chromatographic peak relative to the main chromatographic peak of vitamin B6.

[0014] The method for determining related substances in vitamin B6 drugs in this invention has the following advantages compared with the prior art: By introducing a pre-prepared solution containing volatile buffer salts and adsorption media during sample pretreatment, vitamin B6 and its known and unknown impurities are in a separable state before entering the chromatographic system, significantly reducing the interference of excipients and inorganic salts on detection. Combined with a multi-stage gradient elution method, the elution ranges of different impurities are widened, avoiding peak masking and peak overlap. Furthermore, by using the main chromatographic peak as a unified reference for quantitative or semi-quantitative interpretation of each impurity, unknown impurities can be identified and evaluated even without reference standards, thereby achieving accurate separation and reliable determination of related substances in vitamin B6. Attached Figure Description

[0015] Figure 1 This is a chromatogram of the spiked solution used in elution procedure 1 of Example 4 of this invention; Figure 2 This is a chromatogram of the spiked solution used in elution procedure 2 of Example 4 of this invention; Figure 3 This is a chromatogram of the spiked solution used in elution procedure 3 of Example 4 of this invention; Figure 4 This is a chromatogram of the spiked solution used in elution procedure 4 of Example 4 of this invention; Figure 5This is the chromatogram of the spiked solution when using the Kromasil 100-5-C18 column in Example 5 of this invention; Figure 6 This is the chromatogram of the spiked solution when the column temperature is 25°C as in Example 6 of this invention; Figure 7 This is the chromatogram of the spiked solution when the column temperature is 35°C as in Example 6 of this invention; Figure 8 This is the chromatogram of the spiked solution when the column temperature is 40°C as in Example 6 of this invention; Figure 9 This is the chromatogram of the spiked solution when the flow rate is 0.8 ml / min as in Example 7 of this invention; Figure 10 This is the chromatogram of the spiked solution when the flow rate is 1.2 ml / min as in Example 7 of this invention; Figure 11 This is the chromatogram of the blank solvent in Example 10 of the present invention; Figure 12 This is the chromatogram of the control solution (raw material) in Example 10 of the present invention; Figure 13 This is a chromatogram of the raw material test solution in Example 10 of the present invention; Figure 14 This is the chromatogram of the control solution (preparation) in Example 10 of the present invention; Figure 15 This is a chromatogram of the test solution of the formulation in Example 10 of the present invention. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0017]

Example 1

[0018] In step S1.1, ultrapure water is used as the solvent, ammonium formate is added and adjusted to a weakly acidic state to prepare a pre-prepared solution, wherein the amount of ammonium formate added is 0.63 g / L. In step S1.2, a vitamin B6 drug sample is weighed under light-protected conditions and added to the pre-prepared solution. The sample is stirred to fully dissolve, and the sample dissolution concentration is set as an initial mother liquor of 20 mg / mL. After dissolution, an adsorption medium is added for short-term contact, wherein the amount of adsorption medium added is 5 mg / mL (based on the sample volume), and the short-term contact time is 3 min. In step S1.3, after the short-term contact is completed, centrifugation is performed to separate the layers. The centrifugation conditions are 8000 rpm and 5 min. The supernatant is taken as the sample solution after separating the adsorption medium. The sample solution is filtered through a 0.22 μm filter membrane and then diluted to the target concentration range according to a predetermined ratio. The concentration of the diluted sample solution is 10 mg / mL.

[0019]

Example 2

[0020]

Example 3

[0021] In S1.1, ultrapure water was used as the solvent, ammonium bicarbonate was added and adjusted to a weakly acidic state to obtain a pre-prepared solution, with an ammonium bicarbonate addition amount of 0.79 g / L; in S1.2, after dissolving to obtain a 20 mg / mL mother liquor, an adsorption medium was added, with an adsorption medium addition amount of 5 mg / mL and a short contact time of 5 min; in S1.3, the centrifugation conditions were 8000 rpm for 5 min, filtration was performed using a 0.22 μm filter membrane, and the concentration of the diluted sample solution was 10 mg / mL.

[0022] Comparative Example 1 Compared with Example 1, Comparative Example 1 only omits the step of "adding adsorption medium for short-term contact" in S1.2, that is, no metal-organic framework material is added after the sample is dissolved in the ammonium formate pre-prepared solution. The remaining steps S1.1 and S1.3 are the same as in Example 1.

[0023] Comparative Example 2 Compared with Example 1, Comparative Example 2 only changed the "short contact time" in S1.2 from 3 min to 15 min. The adsorption medium was still the metal-organic framework material prepared in Example 1, and the amount added was still 5 mg / mL. The rest of S1.1 and S1.3 were the same as in Example 1.

[0024] Experimental procedure: Sample turbidity testing procedure: After preparing the diluted sample solution according to the steps of the example / comparative example, take 10 mL of the sample solution and add it to a clean colorimetric bottle. Use a turbidimeter to measure the turbidity at room temperature and record the turbidity value (NTU). Measure each sample 3 times and take the average value.

[0025] Sample conductivity test procedure: Take 20 mL of sample solution into a beaker, use a conductivity meter to measure and record the conductivity (mS / cm) at room temperature. Before the test, calibrate the conductivity electrode with a standard solution, repeat the test 3 times and take the average value.

[0026] Vitamin B6 recovery test procedure (non-chromatographic): The diluted sample solution is used as the test object. The absorbance at the characteristic absorption wavelength of vitamin B6 is measured by ultraviolet spectrophotometer, and the concentration is converted by the calibration curve prepared with vitamin B6 standard. The recovery rate is calculated as measured concentration / theoretical concentration × 100%, where the theoretical concentration is calculated by the sample weight and dilution factor.

[0027] The experimental results are shown in Table 1: Table 1: This invention introduces a pre-prepared solution containing volatile buffer salts and an adsorption medium during the sample pretreatment stage. This effectively reduces the interference of excipients, inorganic salts, and non-target components on the detection of vitamin B6 drug samples during dissolution, resulting in a sample solution with high clarity and a stable ionic environment. Experimental results show that, compared with the control group without the adsorption medium, the sample solution prepared by this invention has significantly reduced turbidity and conductivity, while maintaining a vitamin B6 recovery rate of over 98%, avoiding undesirable loss of the main component. Through this pretreatment method, the sample solution is in a state of low matrix interference and good retention of the main component before entering the chromatographic system, providing a stable and reliable sample basis for the accurate separation and identification of related substances.

[0028] Unless otherwise specified, all materials and reagents used in the examples are commercially available.

[0029] Instrument: Shimadzu LC-2050C3D high performance liquid chromatograph; Chromatographic columns: Xselect HSST3 (250×4.6mm, 5μm) and Kromasil 100-5-C18 (250×4.6mm, 5μm).

[0030] The reference standard is used to prepare the reference solution. The source of the reference standard is: Vitamin B6 reference standard: China National Institutes for Food and Drug Control, content 100.0%; Imp-1 reference standard: CATO, purity 99.1%; Imp-2 reference standard: CATO, purity 96.6%; Imp-3 reference standard: self-made, purity 95.3%; Imp-4 reference standard: STD, purity 99.6%; Imp-5 reference standard: Shandong Wodsen Biotechnology Co., Ltd., content: 97.5%; Vitamin B6 drug sample: Dafeng Haijiano Pharmaceutical Co., Ltd.; Vitamin B6 sample solution: selected from the sample solution in Example 1.

[0031] Known impurities of vitamin B6 include, but are not limited to, those with structures listed in Table 2. Table 2: [Example 4] Selection of mobile phase gradient (1) Chromatographic conditions Chromatographic column: Xselect HSST3 column, dimensions: 4.6mm × 250mm × 5μm; Column temperature: 30℃; Flow rate: 1 ml / min; Injection volume: 10 μl; Detector: PDA, detection wavelength 291nm; Mobile phase: 20 mmol / L ammonium formate aqueous solution (pH 3.0) was used as mobile phase A, and methanol was used as mobile phase B. Elution was performed according to elution programs 1 to 4 respectively.

[0032] Elution program 1: 0-60 minutes, 85% mobile phase A.

[0033] Elution program 2: 0-15 minutes, 90% mobile phase A; 15-30 minutes, 90% → 85% mobile phase A; 30-45 minutes, 85% → 68% mobile phase A; 45-45.1 minutes, 68% → 90% mobile phase A; 45.1-60 minutes, 90% mobile phase A.

[0034] Elution program 3: 0-15 minutes, 90% mobile phase A; 15-30 minutes, 90% → 80% mobile phase A; 30-45 minutes, 80% → 68% mobile phase A; 45-45.1 minutes, 68% → 90% mobile phase A; 45.1-60 minutes, 90% mobile phase A.

[0035] Elution program 4: 0-10 minutes, 95% mobile phase A; 10-30 minutes, 95% → 88% mobile phase A; 30-50 minutes, 88% → 68% mobile phase A; 50-50.1 minutes, 68% → 95% mobile phase A; 50.1-65 minutes, 95% mobile phase A.

[0036] (2) Reference stock solution: Take appropriate amounts of Imp-1, Imp-2, Imp-3, Imp-4 and Imp-5 reference standards respectively, dissolve and dilute with water to prepare a solution containing about 2.5 mg of Imp-1, Imp-2, Imp-3, Imp-4 and Imp-5 per ml.

[0037] (3) Alkali-degraded solution: Take an appropriate amount of vitamin B6 sample solution from Example 1, adjust the pH value to 5.0 with 1 mol / L sodium hydroxide solution, place the solution at 60℃ for 30 days, and then take it out.

[0038] (4) Spiked solution: Accurately measure 5 ml of vitamin B6 base destruction solution, accurately add 0.5 ml of reference stock solution, place in a 50 ml volumetric flask, dilute with water to the mark, and shake well.

[0039] Experimental procedure: The spiked solution was injected using each elution program, and the chromatograms were recorded. The results are shown in Table 3. When elution program 4 was selected, the peak shapes of each impurity were good (tailing factor between 0.8 and 1.2), and the resolution between the main component and impurities, as well as between known and unknown impurities, was greater than 1.5. This indicates that the method can effectively separate 13 known and unknown impurities of vitamin B6. Therefore, elution program 4 was determined to be the appropriate elution condition.

[0040] Table 3: Results of mobile phase selection [Example 5] Selection of Chromatographic Column Mobile phase: 20 mmol / L ammonium formate aqueous solution (pH 3.0) was used as mobile phase A, and methanol was used as mobile phase B. Elution was performed according to the gradient in Table 4. Table 4: Chromatographic columns: Xselect HSST3 column, size: 4.6mm×250mm×5μm; Kromasil 100-5-C18 column, size: 4.6mm×250mm×5μm; other conditions are the same as the chromatographic conditions determined in Example 4.

[0041] Experimental procedure: The spiked solutions were detected using the chromatographic columns described above, and the chromatograms were recorded. The results are shown in Table 5.

[0042] Table 5. Results of column selection: [Example 6] Selection of column temperature Mobile phase: 20 mmol / L ammonium formate aqueous solution (pH 3.0) was used as mobile phase A, and methanol was used as mobile phase B. Gradient elution was performed according to Table 6 below. Table 6: Chromatographic column: Xselect HSST3 column was selected, with dimensions of 4.6 mm × 250 mm × 5 μm; Column temperatures: 25℃, 30℃, 35℃, 40℃; Other conditions are the same as the chromatographic conditions determined in Example 4; Experimental procedure: Different column temperatures were set to detect the spiked solution, and the chromatograms were recorded. The results are shown in Table 7.

[0043] Table 7 Column Temperature Selection Results [Example 7] Selection of Flow Rate Mobile phase: 20 mmol / L ammonium formate aqueous solution (pH 3.0) was used as mobile phase A, and methanol was used as mobile phase B. Gradient elution was performed according to Table 8.

[0044] Table 8: Chromatographic column: Kromasil 100-5-C18 column was selected, with dimensions of 4.6 mm × 250 mm × 5 μm; Flow rates: 0.8 ml / min, 1.0 ml / min, 1.2 ml / min; Other conditions are the same as the chromatographic conditions determined in Example 4; Experimental procedure: Different column temperatures were set to detect the spiked solution, and the chromatograms were recorded. The results are shown in Table 9.

[0045] Table 9 Flow velocity selection results:

Example 8

[0046] Table 10 Results of the linear experiment: Table 11 Calculation results of correction factors: [Example 9] Accuracy Test The recoveries of Imp-1, Imp-2, Imp-3, Imp-4 and Imp-5 were determined under the chromatographic conditions determined in Example 4 with impurity spike levels of 50%, 100% and 200%, respectively. The results are shown in Table 12.

[0047] Table 12 Accuracy test results: [Example 10] Determination of Vitamin B6 in a Sample Blank solvent: water; Raw material test solution: Take an appropriate amount of vitamin B6 raw material, accurately weigh it, dissolve it in water and quantitatively dilute it to prepare a solution containing approximately 1 mg / ml of vitamin B6 per ml; Preparation test solution: Accurately measure an appropriate amount of vitamin B6 sample solution from Example 4, dissolve it in water and quantitatively dilute it to prepare a solution containing approximately 1 mg / ml of vitamin B6 per ml; Control solution: Accurately measure 0.2 ml of the above test solution and place it in a 200 ml volumetric flask. Dilute with water to the mark and shake well.

[0048] Experimental Procedure: Accurately measure 20 μl each of blank solvent, test solution, and control solution, inject them into the liquid chromatograph, and determine the chromatographic conditions as determined in Example 4. Record the chromatograms, and calculate the amount of related substances in the test sample using the self-comparison method with correction factors. Results are shown in Table 13, and the chromatograms are shown in […]. Figures 11-15 .

[0049] Table 13 Results of the detection of related substances in vitamin B6 samples: like Figure 1 As shown, under the detection condition of 291 nm, the main chromatographic peak of vitamin B6 appeared at approximately 8.3 min, with a sharp peak shape and high response intensity, accounting for approximately 98% of the total area. Within different retention time intervals before and after the main peak, multiple impurity peaks such as Imp-1, Imp-2, Imp-3, and Imp-4 could still be clearly observed, while Imp-5 was not eluted.

[0050] like Figure 2 As shown, the main peak of vitamin B6 is located at approximately 13.4 min. A well-defined and highly distinct Imp-5 impurity peak appears in the middle to later retention period, around 46 min. Simultaneously, several low-content impurity peaks are distributed within the 20–35 min range. This chromatographic behavior indicates that Imp2 and Imp-3 cannot be effectively separated, and the elution process lacks selective splitting and control capabilities.

[0051] like Figure 3 As shown in the figure, the Imp-4 peak overlaps with the unknown impurity, indicating that the intensity increase of elution procedure 3 is insufficient to separate the similar components that respond together in this range.

[0052] like Figure 4 As shown, Imp-5 impurity formed a significant peak in the 47-48 min range, while the main peak of vitamin B6 and the early-emerging impurity peak remained well separated, without any distortion of the main peak or increased tailing. This result indicates that even with a relatively high content of a certain impurity, the method of this invention can still ensure that the detection of the main component is not interfered with, demonstrating the method's tolerance and stability to fluctuations in impurity content, and meeting the requirements for extreme sample conditions in the detection of related substances in pharmaceuticals.

[0053] like Figure 5 As shown, the retention time of the vitamin B6 main peak was 17.902 seconds, the tailing factor was 3.219, and baseline separation was not achieved between the Imp-4 peak and the unknown impurity peak. These results demonstrate that the present invention, through the synergistic design of sample pretreatment and gradient elution conditions, effectively avoids the problem of main peak tailing and impurity ingestion under high-response conditions, further verifying the synergistic effect between steps S1 and S2.

[0054] like Figure 6 As shown, a significant increase in the number of impurity peaks appears within a relatively wide retention time range of 25–45 min. Each impurity peak maintains a narrow peak shape and is well separated from the others. This chromatogram demonstrates that even when the number of impurities in the sample increases and the impurity spectrum becomes more complex, the method of this invention can still ensure that each impurity is fully developed without peak overlap or "blurring," showcasing the good adaptability of this method to complex impurity systems.

[0055] like Figure 7 As shown, the retention time difference between the vitamin B6 main peak and the Imp-5 impurity peak exceeds 30 minutes, with the impurity peaks unfolding sequentially within the intermediate region, demonstrating a clear elution order. This result visually demonstrates the effective stretching effect of segmented gradient elution on the elution window, thus strongly supporting the inventiveness of this invention in its gradient elution strategy.

[0056] like Figure 8 As shown, multiple unknown impurity peaks with an area percentage below 0.1% can be observed in the chromatogram. These peaks remain stable and clearly defined under different analytical conditions. This result demonstrates that the method of the present invention has good sensitivity and detection capability, and can reliably detect and relatively evaluate even extremely low levels of unknown impurities, which is of great significance for drug quality control.

[0057] like Figure 9 As shown, the area ratio of Imp-5 impurities increased significantly to approximately 1.5%, while the area of ​​the vitamin B6 main peak remained above 96%, and the separation between the main peak and the various impurity peaks was good. This result indicates that even with increased impurities, the method of this invention can still accurately distinguish between the main component and impurity peaks, ensuring the reliability of related substance interpretation. This demonstrates that the method is applicable not only to ideal samples but also to situations with significant impurity fluctuations in actual production.

[0058] Figure 10At a wavelength of 291 nm, the main peak of vitamin B6 was observed at approximately 14 min. The main peak was sharp and well-symmetrical, dominating the total area. Simultaneously, multiple impurity peaks, including Imp-1, Imp-2, Imp-3, and Imp-5 appearing in the longer retention time range, were clearly distinguishable within different retention time intervals before and after the main peak. These impurity peaks exhibited high separation between themselves and between themselves and the main peak. This figure demonstrates that, after sample pretreatment and gradient elution using the methods of this invention, vitamin B6 and its known and unknown related substances were fully separated without main peak masking or severe co-elution, showcasing the method's overall separation capability for complex impurity systems.

[0059] Figure 11 The results for blank solvent injection at 291 nm show a stable baseline throughout the analysis time, with only slight systemic fluctuations at the end of the gradient change. No characteristic peaks corresponding to the retention times of vitamin B6 or its related substances were detected. This result indicates that the mobile phase system, gradient program, and detection conditions themselves do not introduce interfering peaks, providing a reliable baseline reference for the subsequent determination of impurity peaks in the sample and ensuring that the small peaks appearing in the chromatogram originate from the sample itself rather than from the system background.

[0060] Figure 12 A single chromatographic peak appeared only at approximately 16.65 min, corresponding to the main component of vitamin B6. This peak was symmetrical, had a high theoretical plate number, a tailing factor close to 1, and an area percentage of 100%, indicating that the control solution was uncontaminated and had a low concentration after dilution. This provided time and a basis for the accurate identification of the main peak in the subsequent sample solution.

[0061] Figure 13 The main peak of vitamin B6 was located at approximately 16.29 min, covering nearly 100% of the area, with only a tiny impurity peak of extremely low concentration appearing in the long retention time range. These results indicate that for samples with low impurity content, the method of this invention does not introduce additional impurity signals due to pretreatment or gradient elution, maintaining the integrity of the main component and detection stability, while still possessing the ability to detect trace impurities.

[0062] Figure 14 Similarly, a single vitamin B6 main peak appeared at approximately 16.5 min, with peak shape highly consistent with the aforementioned control solution. The theoretical plate number and tailing factor were both within a good range, and no obvious impurity peaks were detected. This figure further illustrates that the detection method has good reproducibility for the main vitamin B6 component under different batches or different preparation conditions, and the chromatographic system and method parameters are stable and reliable.

[0063] Figure 15The main peak of vitamin B6 was located at approximately 16.19 min, with an area ratio still above 99%. Meanwhile, several low-content impurity peaks, including Imp-3 and several unknown impurities, were distinguishable in the later retention time range. The resolution between these impurity peaks and the main peak was significantly greater than 1, and they were eluted sequentially in the long gradient section. This indicates that the method is not only suitable for high-purity samples but can also effectively develop and identify relevant substances in cases with more complex impurity spectra.

[0064] 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, and 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 determining related substances in a vitamin B6 drug, characterized in that the steps include... include: S1. Dissolve the vitamin B6 drug sample in a pre-prepared solution in a light-protected environment, filter it, and then dilute it to the target concentration range to obtain a sample solution. The pre-prepared solution includes a volatile buffer salt and an adsorption medium. The adsorption medium is a metal-organic framework material, which includes a metal salt and an organic ligand containing at least one of a carboxyl group, a nitrogen heterocyclic group, or a polydentate coordination group. S2. Place the blank solvent, control solution and sample solution in the chromatographic column, and introduce mobile phase A and mobile phase B. Perform gradient elution sequentially to obtain a chromatogram. The multiple gradient elutions include at least a first-stage linear gradient elution and a second-stage linear gradient elution. The proportion of mobile phase A in the first-stage linear gradient elution is greater than that in the second-stage linear gradient elution, and the proportion of mobile phase B in the first-stage linear gradient elution is less than that in the second-stage linear gradient elution. S3. Identify and separate the impurity chromatographic peaks in the chromatogram, perform quantitative or semi-quantitative analysis on each impurity chromatographic peak, and calculate the content ratio of each impurity chromatographic peak relative to the main vitamin B6 chromatographic peak.

2. The method for determining related substances in a vitamin B6 drug according to claim 1, characterized in that, Step S1 includes: S1.1 Using ultrapure water as a solvent, add volatile buffer salt and adjust to weak acidity to obtain a pre-prepared solution; S1.

2. Under light-protected conditions, add the vitamin B6 drug sample to the pre-prepared solution, stir to fully dissolve the sample, and then add the adsorption medium for short-term contact. S1.3 After completing the short-term contact, centrifuge to separate the adsorption media and take the supernatant as the sample solution. Filter the sample solution and dilute it to the target concentration range according to the predetermined ratio.

3. A method for determining related substances in a vitamin B6 drug according to claim 1 or 2, characterized in that, Volatile buffer salts include at least one of ammonium formate, ammonium acetate, and ammonium bicarbonate, with a short contact time of 1 to 5 minutes and a target concentration range of 5 to 15 mg / mL.

4. The method for determining related substances in a vitamin B6 drug according to claim 2, characterized in that, The preparation steps of the adsorption medium include: Weigh out a metal salt and dissolve it in a solvent to obtain a metal salt solution; Weigh an organic ligand containing at least one of a carboxyl group, a nitrogen heterocyclic group, or a polydentate coordinating group, add it to a solvent and dissolve it to obtain an organic ligand solution; The metal salt solution and the organic ligand solution are mixed at a predetermined molar ratio, transferred to a closed reaction vessel, and reacted under heating conditions to generate a metal-organic framework material precursor. After the reaction is completed, the product is naturally cooled to room temperature and separated by centrifugation or filtration. The solid product is then washed multiple times with a low-molecular-weight alcohol solvent. The washed solid is then placed under vacuum drying conditions for activation treatment. The activation treatment temperature is 60-150℃ and the time is 6-24 hours to obtain the activated metal-organic framework material. The activated metal-organic framework material was ground to obtain the adsorption medium.

5. The method for determining related substances in a vitamin B6 drug according to claim 4, characterized in that, The metal salt includes at least one of zinc nitrate, copper nitrate, zirconium chloride, aluminum chloride, zinc sulfate, and zinc acetate; the organic ligand includes at least one of terephthalic acid, imidazole, benzimidazole, pyridine dicarboxylic acid, triazine tricarboxylic acid, and triazine tricarboxylic acid derivatives; and the low molecular weight alcohol solvent includes at least one of methanol, ethanol, isopropanol, n-propanol, and n-butanol.

6. The method for determining related substances in a vitamin B6 drug according to claim 1, characterized in that, Step S2 includes: S2.1 Start the high performance liquid chromatography system and perform a system self-test. The high performance liquid chromatography system includes a binary pump, an online degassing device, an autosampler, a column oven, a diode array detector and a data workstation. Install the chromatographic column in the column oven and set the constant temperature control so that the chromatographic column reaches thermal equilibrium at a fixed temperature. At the same time, pre-flushing the flow path is performed to remove air bubbles. S2.2 Prepare mobile phase A and mobile phase B respectively, and filter and degas mobile phase A and mobile phase B respectively before connecting them into the high performance liquid chromatography system; S2.3 Place the blank solvent, control solution and sample solution into the autosampler respectively, and inject them sequentially according to the preset injection order to obtain the corresponding chromatographic response signal. The blank solvent does not contain vitamin B6 drug sample, and the control solution contains at least standard vitamin B6 drug. S2.4 After injection, mobile phase A and mobile phase B are introduced to perform a preset gradient elution program. Data corresponding to each injection is collected and recorded to obtain a chromatogram.

7. The method for determining related substances in a vitamin B6 drug according to claim 6, characterized in that, In step S2.4, the preset gradient elution procedure includes: (a) Perform an initial equilibration phase of 0-10 minutes, followed by isocratic elution, keeping the proportion of mobile phase A constant at 95% and the proportion of mobile phase B constant at 5%. (b) Perform a first-stage linear gradient elution for 10–30 minutes, during which the proportion of mobile phase A changes from 95% to 88% and the proportion of mobile phase B changes from 5% to 12%. (c) Perform a second-stage linear gradient elution for 30-50 minutes, during which the proportion of mobile phase A changes from 88% to 68% and the proportion of mobile phase B changes from 12% to 32%. (d) Perform a rapid rinsing and rebalancing phase of 50-65 minutes. At 50-50.1 minutes, perform linear gradient elution, changing the proportion of mobile phase A from 68% to 95% and the proportion of mobile phase B from 32% to 5%. At 50.1-65 minutes, perform isocratic elution, keeping the proportion of mobile phase A at 95% and the proportion of mobile phase B at 5%.

8. A method for determining related substances in a vitamin B6 drug according to claim 6 or 7, characterized in that, Mobile phase A includes at least one of ammonium formate aqueous solution, ammonium acetate aqueous solution, and ammonium bicarbonate aqueous solution, and mobile phase B includes at least one of methanol, acetonitrile, and isopropanol.

9. The method for determining related substances in a vitamin B6 drug according to claim 1, characterized in that, Step S3 includes: S3.

1. Use the chromatograms of blank solvent, control solution and sample solution, determine the main chromatographic peak of vitamin B6 by using the retention time and UV response of vitamin B6 in the control solution, and use the chromatogram of blank solvent as the baseline and system peak reference to remove background peaks and ghost peaks. S3.2 Determine the separation degree of impurity chromatographic peaks located in the adjacent regions before and after the main chromatographic peak of vitamin B6. If there is peak overlap or shoulder peaks, further separate the impurity chromatographic peaks by adjusting the gradient elution conditions. S3.3 Quantitative or semi-quantitative analysis of each impurity chromatographic peak is performed. If an impurity reference standard is available, the impurity content is calculated using the external standard method. If an impurity reference standard is not available, the main chromatographic peak of vitamin B6 is used as a reference for relative quantification. S3.4 Calculate the content ratio of each impurity chromatographic peak relative to the main chromatographic peak of vitamin B6.

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