Detection method of dibenzoyl thiamine disulfide and related substances thereof
The qualitative and quantitative analysis of diphenylthiamine and its related substances by high performance liquid chromatography solves the problem of the lack of detection methods in the existing technology, and achieves detection effect with high sensitivity and low detection limit, ensuring product quality and safety.
Patent Information
- Application Number
- CN202511868197.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-11
- Publication Date
- 2026-03-06
AI Technical Summary
The existing technology lacks effective detection methods for benzoyl disulfide and related substances, especially for monitoring 4-dimethylaminopyridine, synthesis reaction catalyst, starting material benzoyl disulfide and synthesis byproduct phenylthioamine, which affects product quality and safety.
High-performance liquid chromatography (HPLC) was used to construct a standard curve by recording chromatograms and peak areas, and combined with detection at different wavelengths to achieve qualitative and quantitative analysis of diphenylthiamine and its related substances.
It achieves high sensitivity and low detection limit for the detection of diphenylthiamine and related substances, ensuring product quality control, effectively monitoring the presence of related substances, and guaranteeing product safety.
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Figure CN121613012A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of analytical detection, and particularly relates to a method for detecting thiodibenzoyl disulfide and related substances. Background Technology
[0002] Benzoyl disulfide, also known as Bisbentiamine, has the molecular formula C2. 38 H 42 N8O6S2, with a molecular weight of 770.92, is a white crystalline powder or crystal. It is odorless, has a slightly bitter taste, and a melting point of 141-147℃. It is readily soluble in chloroform, slightly soluble in methanol and ethanol, and practically insoluble in benzene and ether. It is extremely soluble in aqueous solutions of inorganic salts and organic acids, and practically insoluble in water. The chemical structural formula of the diphenylthiamine is as follows: .
[0003] As a vitamin B1 derivative, diphenylthiamine has advantages over other derivatives, such as non-hygroscopicity, good stability, and resistance to decomposition by thiamine-degrading enzymes. Therefore, it is widely used in rice, flour, and dairy product processing and is an important nutritional supplement. However, to ensure its quality and safety, strict control must be exercised over substances that may be introduced during the production process. Currently, some enterprise standards do not cover the detection and control of such substances. For example, the synthesis catalyst 4-dimethylaminopyridine, the starting materials thiamine disulfide and benzoic acid, and the synthesis byproduct diphenylthiamine are all substances that need to be monitored. Therefore, establishing and improving methods for the detection and monitoring of these substances is crucial to ensuring the safety and efficacy of diphenylthiamine products. Summary of the Invention
[0004] To address the shortcomings of existing technologies, the present invention aims to provide a method for detecting diphenylthiamine disulfide and its related substances. This invention provides a high-performance liquid chromatography (HPLC) method for separating and analyzing related substances in diphenylthiamine (whether used in pharmaceutical production or as a food additive). This method exhibits good specificity, high sensitivity, low detection limit, and is simple to operate and highly efficient in separation, making it suitable for quality control of this compound.
[0005] The objective of this invention is achieved through the following technical solution: A method for detecting thiamine dibenzoyl disulfide and related substances includes the following steps: (1) Prepare reference solutions of benzoyl disulfide at different concentrations, and record the corresponding chromatograms and peak areas using high performance liquid chromatography; plot the standard curve of benzoyl disulfide with concentration as the abscissa and peak area as the ordinate. Similarly, standard curves for the relevant substances were plotted; the relevant substances were 4-dimethylaminopyridine (DMAP), thioamine disulfide, benzoic acid, and thioamine monophenylamine. (2) Inject the test sample solution into the high performance liquid chromatograph and record the chromatograms at wavelengths of 233 nm and 280 nm. By comparing the retention times of each peak in the test sample solution with the reference solution of benzoyl disulfide and related substances, the main peak and each impurity peak are identified.
[0006] Preferably, the concentration of the benzoyl disulfide thioamine reference solution in step (1) is 0.101~5.030 μg / mL.
[0007] Preferably, the operating conditions of the high performance liquid chromatograph in step (1) are as follows: the packing material is octadecylsilane-bonded silica gel, the mobile phase is a mixture of 10 mmol / L potassium dihydrogen phosphate aqueous solution and methanol at a volume ratio of 35:65, the flow rate is 1.0 mL / min, the detection wavelength is 233 nm, the column temperature is 30 °C, and the injection volume is 10 μL.
[0008] Preferably, the octadecylsilane-bonded silica gel is Welch XB-C18.
[0009] Preferably, the potassium dihydrogen phosphate aqueous solution is adjusted to pH=5.5 with phosphoric acid.
[0010] Preferably, in step (1), the standard curves of thiamine disulfide, benzoic acid and thiamine monosulfide are all plotted by linear regression of their peak areas against their concentrations at a detection wavelength of 233 nm.
[0011] Preferably, in step (1), the standard curve of 4-dimethylaminopyridine is plotted by linear regression of its peak area against the concentration at a detection wavelength of 280 nm.
[0012] Preferably, in step (1), the linear concentration range of the standard curve of benzoic acid is 0.102~5.100 μg / mL.
[0013] Preferably, in step (1), the linear concentration range of the standard curve of thiamine disulfide is 0.097~4.825 μg / mL.
[0014] Preferably, in step (1), the linear concentration range of the standard curve of 4-dimethylaminopyridine is 10.28~514 ng / mL.
[0015] Compared with the prior art, the beneficial effects of the present invention include: (1) This invention can qualitatively identify related substances of diphenylthioamine, such as 4-dimethylaminopyridine (DMAP), dithioamine, and benzoic acid. It can also quantitatively detect 4-dimethylaminopyridine (DMAP) at 280 nm using an external standard method. Furthermore, it can quantitatively detect dithioamine and benzoic acid at 233 nm using principal component analysis with a correction factor.
[0016] (2) The limits of quantification of 4-dimethylaminopyridine of the present invention were 0.103 ng and the limit of detection were 0.0308 ng; the limits of quantification of dithiothioamine were 0.48 ng and the limit of detection were 0.14 ng; and the limits of quantification of benzoic acid were 0.26 ng and the limit of detection were 0.0765 ng.
[0017] (3) When the wavelength is 233 nm, the linear regression equation for diphenylthiamine in the concentration range of 0.101~5.030 μg / mL is A=36.952C+0.4975, the correlation coefficient r is 1.0000, and the linear relationship is good; the linear regression equation for benzoic acid in the concentration range of 0.102~5.100 μg / mL is A=38.664C+0.0924, the correlation coefficient is 1.0000, and the linear relationship is good, with a correction factor of 1.0 for diphenylthiamine; the linear regression equation for thiamine disulfide in the concentration range of 0.097~4.825 μg / mL is A=26.522C-0.1897, the correlation coefficient is 0.9998, and the linear relationship is good, with a correction factor of 1.4 for diphenylthiamine.
[0018] When the wavelength is 280 nm, the linear regression equation for DMAP in the concentration range of 10.28~514 ng / mL is A=0.0897C+0.5501, with a correlation coefficient of 0.9998, indicating a good linear relationship. Attached Figure Description
[0019] Figure 1 This is the linear response curve of diphenylthiamine within the measured concentration range.
[0020] Figure 2 The linear response curve of thioamine disulfide within the measured concentration range is shown.
[0021] Figure 3 This is the linear response curve of benzoic acid within the measured concentration range.
[0022] Figure 4 Linear response curves for DMAP were measured within a concentration range.
[0023] Figure 5 The liquid chromatogram is for the system suitability solution.
[0024] Figure 6 The liquid chromatogram of the solution of test sample 1 (batch 2 - long-term 9 months, 25℃) is shown.
[0025] Figure 7 The liquid chromatogram of the solution of test sample 2 (batch 6 - accelerated 3 months, 40°C) is shown.
[0026] Figure 8 To obtain the liquid chromatogram, take 4 mL of DMAP stock solution, dilute to 20 mL with mobile phase, and then detect the obtained liquid chromatogram at a wavelength of 280 nm. Detailed Implementation
[0027] 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.
[0028] Example 1 Plotting the standard curve (1) Chromatographic conditions: A Welch XB-C18 4.6×250mm 5µm column was used; the flow rate was 1.0mL / min; the column temperature was 30℃; the detection wavelengths were 233nm and 280nm; the injection volume was 10µL; the mobile phase was a mixture of 10 mmol / L potassium dihydrogen phosphate aqueous solution and methanol at a volume ratio of 35:65, and the pH of the potassium dihydrogen phosphate buffer was adjusted to 5.5 with phosphoric acid.
[0029] (2) Solution preparation Reference stock solution: Accurately weigh 10.06 mg of diphenylthiamine reference standard, place it in a 20 mL volumetric flask, add mobile phase to dissolve and dilute to the mark, shake well, and use it as diphenylthiamine stock solution.
[0030] Weigh 10.28 mg of DMAP reference standard accurately and place it in a 100 mL volumetric flask. Dissolve and dilute to the mark with the mobile phase, shake well, and then accurately measure 1 mL and place it in a 200 mL volumetric flask as DMAP stock solution.
[0031] Accurately weigh 9.65 mg of thiamine disulfide reference standard, place it in a 20 mL volumetric flask, add mobile phase to dissolve and dilute to the mark, and shake well to prepare thiamine disulfide stock solution.
[0032] Accurately weigh 10.2 mg of benzoic acid reference standard, place it in a 20 mL volumetric flask, add mobile phase to dissolve and dilute to the mark, shake well, and use it as benzoic acid stock solution.
[0033] Linear stock solution: Take 1 mL each of the diphenylthiamine stock solution, thiamine disulfide stock solution and benzoic acid stock solution from step (1), place them in 100 mL volumetric flasks, dilute to the mark with mobile phase, and shake well to obtain the linear stock solution.
[0034] Linear solutions: Take 1 mL, 2 mL, 4 mL, 10 mL, and 20 mL of linear stock solutions of diphenyl thiamine, thiamine disulfide, and benzoic acid, respectively, and place each volume into a 20 mL volumetric flask. Dilute to the mark with mobile phase and shake well to obtain a series of linear solutions for each component. Detect at a wavelength of 233 nm.
[0035] Preparation of DMAP linear solution Take 1 mL, 2 mL, 4 mL, 10 mL, and 20 mL of DMAP stock solution and place them in 20 mL volumetric flasks respectively. Dilute to the mark with mobile phase, shake well, and you will get a series of linear DMAP solutions. Detect them at a wavelength of 280 nm.
[0036] (3) Measurement results For diphenylthiamine, thiamine disulfide, and benzoic acid, accurately measure 10 μL of each of the linear solutions (5 concentrations) of the above reference standards and inject them into the liquid chromatograph, recording the chromatograms. Including the limit of quantitation, perform linear regression with concentration C (μg / mL) on the x-axis and peak area A on the y-axis. The linear results are shown in [Figure number missing]. Figures 1-3 .
[0037] See Figures 1-3 We can see that: the linear regression equation for diphenylthiamine is A=36.952C+0.4975, with a correlation coefficient r of 1.0000; the linear regression equation for thiamine disulfide is A=26.522C-0.1897, with a correlation coefficient of 0.9998; and the linear regression equation for benzoic acid is A=38.664C+0.0924, with a correlation coefficient of 1.0000.
[0038] For DMAP, accurately measure 10 μL of each of the five linear solutions (concentrations) of the above reference standards and inject them into the liquid chromatograph, recording the chromatograms. Including the limit of quantitation, perform linear regression with concentration C (ng / mL) on the x-axis and peak area A on the y-axis. The linear results are shown in [Figure number missing]. Figure 4 .
[0039] See Figure 4 We can see that the linear regression equation for DMAP is A = 0.0897C + 0.5501, and the correlation coefficient is 0.9998.
[0040] Figure 8 To obtain the liquid chromatogram, take 4 mL of DMAP stock solution, dilute to 20 mL with mobile phase, and then detect the obtained liquid chromatogram at a wavelength of 280 nm.
[0041] Example 2 Detection limit and quantitation limit Chromatographic conditions: Same as in Example 1.
[0042] Solution preparation: (1) Accurately measure 1 mL of the diphenylthiamine stock solution, thiamine disulfide stock solution and benzoic acid stock solution in Example 1, place them in a 10 mL volumetric flask, add the mobile phase to dilute to the mark, and shake well; this is the limit of quantitation solution. (2) Accurately measure 3 mL of the solution from step (1), place it in a 10 mL volumetric flask, dilute with water to the mark, and shake well; use it as the detection limit solution; and perform detection at a wavelength of 233 nm; (3) Prepare the limit of quantitation solution and limit of detection solution of DMAP according to the steps (1) and (2), and perform detection at a wavelength of 280 nm.
[0043] Measurement results: Limit of quantitation: Accurately measure 10 μL of the limit of quantitation solution and inject it into the liquid chromatograph. The signal-to-noise ratio (S / N) is 10:1. According to the detection results, the limit of quantitation concentration of thiamine disulfide is 0.048 μg / mL; the limit of quantitation concentration of benzoic acid is 0.026 μg / mL; and the limit of quantitation concentration of DMAP is 10.3 ng / mL.
[0044] Detection limit: Accurately measure 10 μL of the detection limit solution and inject it into the liquid chromatograph. With a signal-to-noise ratio (S / N) of 3:1, the detection limit concentrations of thiamine disulfide, benzoic acid, and DMAP are 0.014 μg / mL, 0.00765 μg / mL, and 3.08 ng / mL, respectively.
[0045] Example 3 Durability of the method for determining diphenylthiamine-related substances - pH value assessment (1) Preparation of solution: blank solvent: mobile phase.
[0046] (2) Preparation of system suitability solution: Take appropriate amounts of diphenylthioamine, 4-dimethylaminopyridine, thiodisulfide and benzoic acid, accurately weigh them, add them to the mobile phase to dissolve and quantitatively dilute them to prepare a mixed solution containing 1 mg of diphenylthioamine, 2 µg of 4-dimethylaminopyridine, thiodisulfide and benzoic acid per 1 mL, which is used as the system suitability solution.
[0047] (3) Preparation of test solution: Take an appropriate amount of diphenylthiamine, dissolve and dilute it with the mobile phase to prepare a solution containing 1 mg per 1 mL, and use it as the test solution. Prepare 6 portions in the same way.
[0048] (5) High performance liquid chromatography conditions: Except for adjusting the pH of the potassium dihydrogen phosphate buffer in the mobile phase to 5.7, 5.6 and 5.5 respectively for investigation, the other conditions were the same as in Example 1.
[0049] Sample introduction and result analysis Accurately measure 10 μL each of the system suitability solution and the test solution, inject them into the liquid chromatograph, and record the chromatogram.
[0050] The liquid chromatogram of the system suitability solution is as follows: Figure 5 As shown, the pH of the potassium dihydrogen phosphate buffer solution is 5.5.
[0051] The retention times of substances in the system suitability solution are shown in Table 1. The retention times of substances in the test sample solution are shown in Table 2.
[0052] Table 1 Statistical results of system suitability solutions
[0053] Table 2 Statistical results of the test solution
[0054] Referring to Tables 1 and 2, we can conclude that in the chromatograms of the system suitability solution, the peak shape of benzoic acid is significantly improved when the mobile phase pH is 5.5, and the separation of other impurities from the main component meets the requirements. However, the peak shape and peak area of benzoic acid are poor when the pH increases, which should be noted.
[0055] Example 4 Determination of related substances in the active pharmaceutical ingredient diphenylthiamine (1) Solution preparation: blank solvent: mobile phase; (2) Preparation of reference solution: Take an appropriate amount of diphenylthiamine reference, accurately weigh it, add the mobile phase to dissolve and quantitatively dilute it to a solution containing 1 mg of diphenylthiamine per 1 mL.
[0056] (3) Preparation of test solution: Take an appropriate amount of the raw material of the workshop production that has been stored stably (equivalent to 25 mg of diphenyl thiamine), weigh it accurately, put it in a 25 mL volumetric flask, add the mobile phase to dissolve and dilute to the mark, and shake well.
[0057] Accurately measure 10 μL of each of the above solutions and inject them into the liquid chromatograph (test conditions are the same as in Example 1). Record the chromatograms and calculate the related substances of diphenyl thiamine in the test solution by peak area at wavelengths of 233 nm and 280 nm using the external standard method of principal component. The results are shown in Table 3. Table 3 Test results of various substances in the active pharmaceutical ingredient
[0058] According to the test results in Table 3, we can see that, following the above chromatographic conditions and sample preparation requirements, after 12 batches of testing, all batches of related substances met the standard limits. This indicates that the present invention can effectively detect related substances in pharmaceutical-grade diphenylthiamine, and can perform qualitative and quantitative analysis on all mentioned related substances, which can be used for routine quality monitoring of the product.
[0059] Figure 6 The liquid chromatogram of the solution of test sample 1 (batch 2 - long-term 9 months, 25℃) is shown.
[0060] Figure 7 The liquid chromatogram of the solution of test sample 2 (batch 6 - accelerated 3 months, 40°C) is shown.
[0061] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.
Claims
1. A method for detecting dibenzoyl thiamine disulfide and its related substances, characterized by, It comprises the following steps: (1) Prepare different concentrations of dibenzoyl dithiothiamine reference solution, use high performance liquid chromatograph to record corresponding chromatogram and corresponding peak area; take concentration as abscissa and peak area as ordinate to draw dibenzoyl dithiothiamine standard curve; In the same way, draw the standard curves of related substances respectively; the related substances are 4-dimethylamino pyridine, dithiothiamine, benzoic acid and monobenzylthiamine; (2) Inject the sample solution to be tested into high performance liquid chromatograph, record the chromatogram under 233 nm and 280 nm wavelength, and qualitatively analyze the main peak and each impurity peak by comparing the retention time of each peak in the sample solution to be tested, dibenzoyl dithiothiamine reference solution and related substance solution.
2. The method for detecting thiamine disulfide and related substances according to claim 1, characterized in that, The concentration of the dibenzoyl dithiothiamine reference solution in step (1) is 0.101-5.030 μg / mL.
3. The method of claim 1, wherein the dibenzoyl thiamine disulfide and related substances are detected by, The working conditions of the high performance liquid chromatograph in step (1) are as follows: the filler is octadecylsilane bonded silica gel, the mobile phase is 10 mmol / L potassium dihydrogen phosphate aqueous solution mixed with methanol at a volume ratio of 35:65, the flow rate is 1.0 mL / min, the detection wavelength is 233 nm, the column temperature is 30℃, and the injection amount is 10 μL.
4. The method for detecting dibenzoyl thiamine and its related substances according to claim 3, characterized in that, The octadecylsilane bonded silica gel is Welch XB-C18.
5. The method of claim 3, wherein the dibenzoyl dithiolamine and the related substances thereof are detected by HPLC. The potassium dihydrogen phosphate aqueous solution is adjusted to pH=5.5 with phosphoric acid.
6. The method of claim 1, wherein the dibenzoyl dithiothiamine and the related substances thereof are detected by HPLC. In step (1), the standard curves of dithiothiamine, benzoic acid and monobenzylthiamine are drawn by linear regression of their peak areas to concentrations under 233 nm detection wavelength.
7. The method of claim 1, wherein the dibenzoyl dithiothiamine and the related substances thereof are detected by HPLC. In step (1), the standard curve of 4-dimethylamino pyridine is drawn by linear regression of its peak area to concentration under 280 nm detection wavelength.
8. The method of claim 1, wherein the dibenzoyl dithiolamine and the related substances thereof are detected by HPLC. In step (1), the linear concentration range of the standard curve of benzoic acid is 0.102-5.100 μg / mL.
9. The method of claim 1, wherein the dibenzoyl dithiolamine and the related substances thereof are detected by HPLC. In step (1), the linear concentration range of the standard curve of dithiothiamine is 0.097-4.825 μg / mL.
10. The method of claim 1, wherein the dibenzoyl dithiolamine and the related substances thereof are detected by HPLC. In step (1), the linear concentration range of the standard curve of 4-dimethylamino pyridine is 10.28-514 ng / mL.