Method for determining stability of vitamin B2 in compound zinc-iron-calcium oral solution

Abstract

The invention discloses a method for determining the stability of vitamin B2 in a compound zinc-iron-calcium oral solution. The method comprises the following steps: (1) determining the content of vitamin B2 in the compound zinc-iron-calcium oral solution by using a liquid chromatography; and (2) determining the content of a specific degradation product of vitamin B2 in the compound zinc-iron-calcium oral solution by using an improved liquid chromatography, adding the compound zinc-iron-calcium oral solution into a liquid chromatograph for gradient elution, and during gradient elution, eluting according to a two-isocratic and three-gradient alternate elution mode. And (3) determining the impurity proportion by utilizing the content of the degradation product of the vitamin B2, which can be obtained in the step (2). According to the method disclosed by the invention, experimental conditions are groped and optimized, so that the vitamin B2 degradation impurities lumiflavin and lumitin in the compound zinc-iron-calcium oral solution are effectively separated, the peak shape is good, and the vitamin B2 degradation impurities which cannot be detected by an existing method can be detected.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and in particular relates to a method for measuring and improving the stability of vitamin B2 in a compound zinc-iron-calcium oral solution. Background Art

[0002] Compound zinc iron calcium oral solution is a compound preparation suitable for patients with zinc, iron, calcium and vitamin B 2 Diseases caused by deficiency. Including calcium deficiency such as osteoporosis, tetany, bone dysplasia, rickets, etc.; zinc deficiency such as growth retardation, malnutrition, anorexia, etc.; iron deficiency anemia caused by chronic blood loss, malnutrition, pregnancy, childhood development, etc. due to various reasons; angular cheilitis, glossitis, keratitis, conjunctivitis, corneal ulcer, retinitis, neuritis, herpetic eye, scrotitis, seborrheic dermatitis, etc. The active ingredients in this product are calcium gluconate, zinc gluconate, ferrous gluconate and vitamin B 2 After consulting domestic and foreign pharmacopoeias, calcium gluconate, zinc gluconate, and ferrous gluconate were not subject to relevant substance control; however, EP and BP had no control over vitamin B12. 2 Strict control of relevant substances is carried out.

[0003] There are currently two manufacturers in China. One of them has a registration standard that controls the related substances of compound zinc iron calcium oral solution. The study found that according to the quality standard, the negative sample has more chromatographic peaks. The experiment found that it mainly comes from ferrous gluconate and sucrose. Different excipient sources and other factors have an impact on the chromatographic peaks of negative samples. This method is not operational, and the samples of the two manufacturers have no vitamin B under this chromatographic condition. 2 The degradation impurity photoflavin interferes with the detection of photopigment.

[0004] Vitamin B 2 Unstable to light, research has found that vitamin B in compound zinc iron calcium oral solution 2 The content can be reduced by 7% during the validity period. In order to ensure the safety and effectiveness of drugs, it is urgent to provide a method for determining the content of vitamin B in compound zinc iron calcium oral solution. 2 Considering that the compound zinc iron calcium oral solution is a compound preparation, it is necessary to accurately determine the vitamin B12 content in the solution when other components interfere. 2 The content of degradation impurities has always been a problem, and its stability is unknown. Summary of the invention

[0005] In view of the shortcomings of the prior art, the inventors of the present invention conducted numerous testing experiments and finally obtained a method for determining the content of vitamin B12 in compound zinc, iron and calcium oral solution. 2 The stability method can be used for vitamin B in compound zinc iron calcium oral solution2 The detection of degradation impurity content is accurate and reliable, highly specific, time-saving and labor-saving, easy to operate, and can eliminate the interference of auxiliary materials and detect impurity components that cannot be accurately detected by existing detection methods.

[0006] Principle

[0007] The inventors studied vitamin B 2 When testing the qualified rate and stability, it was found that the testing process was greatly disturbed and it was difficult to obtain accurate test data. After further in-depth research, the inventors found that the difficulty of testing was mainly concentrated in vitamin B 2 The degradation ratio test must first determine whether it can accurately reflect the 2 In the study, the inventors noticed that leucophorin, photopigment and vitamin B 2 However, the detection of these two substances is not easy, mainly due to two difficulties: (1) there is a large chromatographic peak at the relative retention time of 0.95 of photoflavin, which is easily misjudged as photoflavin; (2) during the detection process, sodium benzoate is difficult to separate from photoflavin, causing great interference.

[0008] In order to solve the above two major problems, the inventors tried various elution methods and finally found that when the pH value of the first mobile phase was adjusted to 3.5 with triethylamine and combined with a two-isocratic and three-gradient alternating elution method, the interference of impurities on the detection process can be greatly reduced, thereby improving the accuracy of detection.

[0009] Specifically, the present invention provides a method for determining the content of vitamin B12 in a compound zinc, iron and calcium oral solution. 2 A method for determining stability, the method comprising:

[0010] (1) Obtaining Vitamin B 2 The initial content of vitamin B12 in compound zinc iron calcium oral solution was determined by liquid chromatography. 2 Content;

[0011] (2) Determination of vitamin B in compound zinc, iron and calcium oral solution by improved liquid chromatography 2 Specific degradation products

[0012] The content of the substance, wherein the improved liquid chromatography method comprises:

[0013] (2.1) Chromatographic columns with octadecylsilane bonded silica gel as filler;

[0014] (2.2) Take the compound zinc iron calcium oral solution and filter it with a 0.45 μm microporous filter membrane, and take the filtrate as the sample to be tested.

[0015] Solution;

[0016] (2.3) 20 μl of the test solution and reference solution were injected into the liquid chromatograph respectively;

[0017] (2.4) Using 1‰ phosphoric acid solution adjusted to pH 3.5 with triethylamine as the first mobile phase A and acetonitrile as the second mobile phase B, adding them into a liquid chromatograph for gradient elution. When performing gradient elution, elution is performed in a two-isocratic and three-gradient alternating elution mode. This step comprises:

[0018] First isocratic elution: The gradient of mobile phase and elution time was as follows: 0-5 min, mobile phase A-mobile phase B (90:10) isocratic elution;

[0019] First gradient elution: 5-20 minutes, the proportion of mobile phase A gradually decreased from 90% to 80%, and the proportion of mobile phase B gradually increased from 10% to 20%;

[0020] Second isocratic elution: 20-25 min, mobile phase A-mobile phase B (80:20) isocratic elution;

[0021] Second gradient elution: 25-35 minutes, the proportion of mobile phase A gradually decreased from 80% to 50%, and the proportion of mobile phase B gradually increased from 20% to 50%;

[0022] The third isocratic elution: 35-45 minutes, mobile phase A-mobile phase B (50:50) isocratic elution;

[0023] (2.5) Vitamin B 2 The photodegradation solution is mixed with the test sample solution to prepare a system suitability solution;

[0024] (2.6) Using a liquid chromatograph, perform chromatographic detection on the test solution and the reference solution respectively;

[0025] (3) Using the vitamin B obtained in step 2 2 The content of degradation products and vitamin B 2 The content of the degradation product is used as the basis for judging the stability.

[0026] In a preferred implementation, when gradient elution is performed, mobile phase A is a 1‰ phosphoric acid solution.

[0027] In another preferred implementation, during gradient elution, the pH value of the phosphoric acid solution is adjusted to 3.5 with triethylamine. In another preferred implementation, vitamin B 2 The photodegradation solution was mixed with the test sample solution to prepare the system suitability solution.

[0028] In another preferred implementation, during liquid chromatography detection, the detection wavelength is selected to be 260 nm.

[0029] In another preferred implementation, during liquid chromatography detection, the flow rate is 1.0 ml / min and the column temperature is 40° C. On the other hand, the present invention provides a method for determining the oral dissolution of compound zinc, iron and calcium by the method described in claim 1.

[0030] Vitamin B in liquid 2 A method for degrading impurities, characterized in that the method comprises:

[0031] Determination of Vitamin B in Compound Zinc-Iron-Calcium Oral Solution by Improved Liquid Chromatography 2 Specific degradation products

[0032] The content of the substance, wherein the improved liquid chromatography method comprises:

[0033] (2.1) Chromatographic columns with octadecylsilane bonded silica gel as filler;

[0034] (2.2) Take the compound zinc iron calcium oral solution and filter it with a 0.45 μm microporous membrane. Take the filtrate as

[0035] Solution of the product to be tested;

[0036] (2.3) 20 μl of the test solution and reference solution were injected into the liquid chromatograph respectively;

[0037] (2.4) Use 1‰ phosphoric acid solution adjusted to pH 3.5 with triethylamine as the first mobile phase A and acetonitrile as the second mobile phase B, add them into the liquid chromatograph for gradient elution. When performing gradient elution, follow the second step.

[0038] The elution is performed by three-gradient alternating elution mode, and the step comprises:

[0039] First isocratic elution: The gradient of mobile phase and elution time was as follows: 0-5 min, mobile phase A-mobile phase B (90:10) isocratic elution;

[0040] First gradient elution: 5-20 minutes, the proportion of mobile phase A gradually decreased from 90% to 80%, and the proportion of mobile phase B gradually increased from 10% to 20%;

[0041] Second isocratic elution: 20-25 min, mobile phase A-mobile phase B (80:20) isocratic elution;

[0042] Second gradient elution: 25-35 minutes, the proportion of mobile phase A gradually decreased from 80% to 50%, and the proportion of mobile phase B gradually increased from 20% to 50%;

[0043] The third isocratic elution: 35-45 minutes, mobile phase A-mobile phase B (50:50) isocratic elution;

[0044] (2.5) Vitamin B 2 The photodegradation solution is mixed with the test sample solution to prepare a system suitability solution;

[0045] (2.6) Use liquid chromatography to perform chromatographic detection on the test solution and the reference solution respectively.

[0046] Beneficial Effects

[0047] The present invention has made a lot of optimizations on liquid reverse phase chromatography technology, so that vitamin B in compound zinc iron calcium oral solution can be accurately determined. 2 Degrade impurities, make up for the shortcomings of existing methods, and effectively detect vitamin B 2 The main impurities of photoflavin and photopigment are degraded to ensure the safety of medication. 2 It has the advantages of accuracy, reliability, short cycle and simple operation in degrading impurities. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The typical liquid chromatogram of the test solution of the related substances when the compound zinc iron calcium oral solution of manufacturer A is tested using the method of manufacturer B;

[0049] Figure 2 The typical liquid chromatogram of the test solution of the related substances when the compound zinc, iron and calcium oral solution of manufacturer B is tested using the method of manufacturer B;

[0050] Figure 3 The typical liquid chromatogram of the test solution of the related substances when the negative sample of the compound zinc iron calcium oral solution with mixed excipients from manufacturers A and B is tested using the method of manufacturer B;

[0051] Figure 4 The following is the liquid chromatogram of the test solution of the related substances when the compound zinc, iron and calcium oral solution of manufacturer A was tested after being destroyed by light;

[0052] Figure 5 The following is the liquid chromatogram of the test solution of the related substances when luciferin and luciferin were added to the compound zinc-iron-calcium oral solution of manufacturer A for testing;

[0053] Figure 6 This is the liquid chromatogram of the test solution of the relevant substances when the compound zinc, iron and calcium oral solution of manufacturer A was tested;

[0054] Figure 7 This is the liquid chromatogram of the test solution of the related substances when the compound zinc, iron and calcium oral solution of manufacturer B was tested;

[0055] Figure 8 As: UV spectrum of flavin reference solution;

[0056] Fig. 9 As: UV spectrum of photopigment reference solution;

[0057] Fig.10 It is: the liquid chromatogram of the test solution of the related substance when the negative sample of manufacturer A is tested;

[0058] Fig.11 This is the liquid chromatogram of the test solution of the related substance when the negative sample of manufacturer B is tested;

[0059] Fig.12 This is the chromatogram obtained by the method in the comparative example. DETAILED DESCRIPTION

[0060] The present invention will be described in detail below in conjunction with the accompanying drawings and embodiments thereof, but the protection scope of the present invention is not limited to the scope of the embodiments described herein.

[0061] 1. Raw material preparation

[0062] 1.1. Preparation of test solution

[0063] Take this product and filter it through a 0.45μm microporous membrane, and take the filtrate.

[0064] 1.2. Preparation of reference solution

[0065] Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the mark with water, and shake well.

[0066] 1.3. Preparation of system suitability solution

[0067] System suitability solution (1) Take vitamin B 2 Place 30 mg of the reference substance in an open container, add 1 ml of 0.5 mol / L sodium hydroxide solution to dissolve it, expose it to light for 4 hours, add 0.5 ml of glacial acetic acid, transfer it to a 100 ml volumetric flask, dilute it to the scale with water, shake it well, filter it, and take the filtrate.

[0068] System suitability solution (2) Take an equal volume of system suitability solution (1) and this product and mix them well.

[0069] 2.1. Obtaining Vitamin B 2 The initial content of vitamin B2 can be determined by the pharmacopoeia or standard method.

[0070] 2.2. Impurity detection conditions

[0071] Filler: Octadecylsilane bonded silica gel as filler (Ultimate XB, 4.6×250mm, 5μm or equivalent performance column);

[0072] Mobile phase B: acetonitrile;

[0073] Mobile phase A: Take 1‰ phosphoric acid solution and adjust the pH to 3.5 with triethylamine

[0074] Research found that BP vitamin B 2 Under the chromatographic conditions of drug substance related substances, some chromatographic columns can separate luminoflavin from adjacent impurity peaks, while the separation of luminoflavin and excipient sodium benzoate is poor. Because the pKa of sodium benzoate is 4.2, the pH value of the mobile phase should be adjusted when optimizing the chromatographic conditions. The Ultimate XB-C18 column was used, and the gradient elution was the same as BP. The mobile phase A is shown in the table below, and the mobile phase B is acetonitrile. The separation of luminoflavin and luminoflavin in the A sample test solution with appropriate amounts of luminoflavin and luminoflavin reference substances was investigated. The results are shown in Table 1.

[0075] Table 1 Mobile phase optimization results

[0076]

[0077] The inventors found during the detection process that there was a large chromatographic peak at the relative retention time of 0.95 of leucoflavin, which was easily misjudged as leucoflavin, thereby increasing the detection error. Therefore, the inventors continuously adjusted the composition and pH value of the mobile phase until the separation degree between the chromatographic peak and leucoflavin could be significantly improved.

[0078] In addition, the inventors found during the testing process that sodium benzoate and photopigment could not be separated under BP chromatography conditions in the multi-chromatographic column, so the inventors further optimized the elution procedure, elution conditions, mobile phase components and pH value until the separation degree of sodium benzoate and photopigment reached a satisfactory index. 2 The retention time of the degradation impurities was almost unaffected, but sodium benzoate was significantly affected. Under the chromatographic conditions of mobile phase A of 1:1000 v / v phosphoric acid solution, 2‰ phosphoric acid solution or 2‰ phosphoric acid solution, the separation degree of photochrome and sodium benzoate was less than 1.5; when the mobile phase A was 1‰ phosphoric acid solution with a pH value of 4.5, the retention time of sodium benzoate peak shifted forward, the peak shape became wider, and it overlapped with photoflavin; when the mobile phase A was 1‰ phosphoric acid solution with a pH value of 4.0, 3.5, and 3.0, photoflavin and photochrome were well separated, and the peak shape and separation were best when the phosphoric acid solution had a pH value of 3.5, so the pH value of mobile phase A was determined to be 3.5.

[0079] System suitability test: Because the detection of photoflavin and photopigment is easily interfered, this product is combined with vitamin B 2 The photodestruction solutions were mixed in equal amounts to form a system suitability solution, and the separation degree between photoflavin and the photopigment and the adjacent chromatographic peaks was required to be greater than 1.5 to ensure that photoflavin and the photopigment could be effectively separated.

[0080] Selection of detection wavelength: Vitamin B 2 The detection wavelength of the related substance BP is 267nm, the detection wavelength of ChP is 444nm, and the detection wavelength of vitamin B 2 , has maximum absorption at wavelengths of 267nm, 375nm and 444nm, leucoflavin has maximum absorption at wavelengths of 222nm and 265nm, and photopigment has maximum absorption at wavelengths of 219nm and 260nm. Considering that the products to be tested are compound preparations and vitamin B 2 The solution is unstable to light, and this variety of vitamin B 2 The relevant substances focus on the photodegradation impurities of flavin and photopigment. The results of subsequent sample detection and sample stability test showed that flavin was not detected, and the detection amount of photopigment was 0.29%-2.82%; the destruction experiment showed that flavin was a degradation product under alkaline conditions, while the preparation was acidic, and the increase of flavin under light irradiation of the preparation was not obvious, but the increase of photopigment was significant; the change of wavelength has a greater impact on the absorption value of photopigment. The absorption value of flavin at 260nm is about 86% of the absorption value at 265nm, while the absorption value of photopigment at 265nm is only about half of the absorption value at 260nm. Therefore, the maximum absorption wavelength of photopigment 260nm is selected as the detection wavelength.

[0081] Flow rate: 1.0ml / min;

[0082] Column temperature: 40°C;

[0083] Considering that this product is a compound preparation, the chromatographic peaks reach more than 20 under the proposed chromatographic conditions. Taking into account the actual situation of the negative sample of this product, the attribution of the impurity peaks and the stability test, the vitamin B content of this variety is investigated by the content of leucoflavin and leucochrome. 2 Related substances. Studies have found that vitamin B 2 Under acidic conditions, it will not degrade to form leucophorin, so controlling the leucophorin can control the vitamin B in the compound zinc, iron and calcium oral solution. 2 degradation.

[0084] If there is a photochromatographic peak in the chromatogram of the test solution, its peak area shall not be greater than 1.5 times (1.5%) the main peak area of ​​the control solution.

[0085] 3. Measurement process

[0086] (1) Select a chromatographic column with octadecylsilane bonded silica gel as the filler;

[0087] (2) filtering the compound zinc-iron-calcium oral solution to prepare a test solution;

[0088] (3) 20 μl of the test solution and reference solution were injected into the liquid chromatograph respectively;

[0089] (4) using phosphate buffer as the first mobile phase A and acetonitrile as the second mobile phase B, adding the mixture into a liquid chromatograph for gradient elution, using a two-isocratic and three-gradient elution method;

[0090] (5) Use a liquid chromatograph to perform chromatographic detection on the test solution and the reference solution respectively.

[0091] 4. Method Validation

[0092] As described in the background art, the safety of drug use cannot be guaranteed according to the enterprise registration standards.

[0093] During the research process, the research team of the present invention repeatedly explored and optimized the experimental conditions to find the most suitable chromatographic method: ① pH value of mobile phase A; ② detection wavelength; ③ system suitability test. By optimizing these factors, the ability to separate leucoflavin, leucochrome and adjacent chromatographic peaks is optimized, and vitamin B in this product can be effectively detected. 2 The main impurities of photodegradation.

[0094] During the research process, the research team of the present invention repeatedly explored and optimized the experimental conditions and results to find the most suitable experimental method.

[0095] It can be seen from the above test results that only when the Ph value is adjusted to 3.0-4.0 and the phosphoric acid solution is 1‰, the separation of adjacent peaks of flavin and photopigment can reach the highest separation, while when the phosphoric acid solution is 0.5‰ and 2‰, either peak overlap occurs or the separation does not reach the standard requirement of 1.5.

[0096] 4.1 Destruction test

[0097] 4.1.1 Vitamin B 2 API Destruction Test

[0098] Vitamin B 2 It is relatively stable under acid, oxidation and high temperature conditions, but is easily degraded under alkaline and strong light conditions to produce photoflavin and photopigment, both of which can be effectively separated from their adjacent impurity peaks under this chromatographic condition. The results are shown in Table 2.

[0099] Table 2 Vitamin B 2 Raw material destruction test results

[0100]

[0101] 4.1.2 Destruction experiment of compound zinc, iron and calcium oral solution

[0102] The destruction experiment showed that the test sample was relatively stable under the above acid, alkali and high temperature conditions; the main peak was significantly degraded under oxidative conditions; the photopigment increased significantly under light, about 5 times that of the undestroyed one. The photoflavin produced by degradation and the photopigment photoflavin can be effectively separated from its adjacent impurity peaks under this chromatographic condition. The results are shown in Table 3.

[0103] Table 3 Destruction test results of compound zinc iron calcium oral solution

[0104]

[0105]

[0106] 4.2 Repeatability study

[0107] Take an appropriate amount of this product and determine the impurity content of this product according to the test method for related substances. Repeat the determination 6 times to examine the precision of the method. The results are shown in Table 4 below.

[0108] Table 4 Repeatability results of compound zinc iron calcium oral solution test solution

[0109]

[0110] The experimental results show that after repeated measurements for 6 times, the measurement deviation of the photopigment content was less than 1%, and the method has good precision.

[0111] 4.3 Durability test

[0112] Take 20 μl of the test solution and the control solution respectively and inject them into the liquid chromatograph. Change the chromatographic conditions such as the chromatographic column brand, column temperature and flow rate, and record the chromatogram. The results are shown in Table 5.

[0113] Table 5 Durability test results of related substances (system suitability test and sample measurement results)

[0114]

[0115]

[0116] The results showed that the method had good durability and the components to be measured could be effectively separated from adjacent peaks.

[0117] 4.4 Stability test of preparations

[0118] In order to investigate the stability of this product and establish reasonable limits for related substances, the influencing factors were investigated, including blank excipients, light, and samples placed at 40℃ and 60℃ for 30 days. See Table 6 for details.

[0119] Table 6: Related substances affecting factors of preparations

[0120]

[0121] The test results show that when the compound zinc-iron-calcium oral solution is placed under light at 40°C and 60°C for 30 days, only the photopigment in the above chromatographic peaks increases significantly.

[0122] 4. Production process optimization

[0123] Company A used the method of the present invention to investigate the stability of the following compound zinc-iron-calcium oral solution under light, 40°C and 60°C for 30 days: optimize the production process, adjust the vitamin B 2 The dissolving temperature was set and light was avoided during the production process; the prescription process was examined, vitamin C was added and nitrogen was filled for protection; the packaging materials were examined, and polyvinyl chloride, low-density polyethylene composite hard sheet pressed bottles and glass bottles were used for packaging.

[0124] The results showed that vitamin B 2 It is unstable under light and long-term storage. The content of degraded impurity photopigment increases significantly, but no increase is seen in photoflavin.

[0125] (1) Using improved process measures, vitamin B 2 There is no obvious degradation, which is better than the products produced by the original production process. However, after 30 days of testing of influencing factors, there is basically no significant difference from the products produced by the process before the improvement.

[0126] (2) The samples were tested for 30 days under three conditions: "adding vitamin C without nitrogen filling, adding vitamin C with nitrogen filling, and not adding vitamin C without nitrogen filling". The results showed no significant difference, indicating that oxygen in the production and storage process is not the cause of vitamin B 2 The main factor of degradation.

[0127] (3) For products packaged in glass bottles, except for the more obvious degradation of photopigments under strong light conditions (photopigment degradation is basically the same at 40°C and 60°C), there is no significant difference in other impurities between products packaged in glass bottles and those packaged in plastic bottles. Specific embodiment 1

[0129] A total of 10 batches of drugs from two commercially available compound zinc-iron-calcium oral solution manufacturers were investigated using the method of the present invention. The results are shown in Table 7.

[0130] The relevant substances were protected from light and determined according to the above improved liquid chromatography method.

[0131] Take the test solution and filter it through a 0.45 μm microporous filter membrane, and take the filtrate.

[0132] Control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the mark with water, and shake well.

[0133] System suitability solution (1) Take vitamin B 2 Place 30 mg of the reference substance in an open container, add 1 ml of 0.5 mol / L sodium hydroxide solution to dissolve it, expose it to light for 4 hours, add 0.5 ml of glacial acetic acid, transfer it to a 100 ml volumetric flask, dilute it to the scale with water, shake it well, filter it, and take the filtrate.

[0134] System suitability solution (2) Take an equal volume of system suitability solution (1) and this product and mix well.

[0135] Chromatographic conditions: Use octadecylsilane bonded silica gel as filler (Ultimate XB, 4.6×250mm, 5μm or chromatographic column with equivalent performance); take 1‰ phosphoric acid solution and adjust the pH to 3.5 with triethylamine as mobile phase A, acetonitrile as mobile phase B, and perform gradient elution according to the following table; column temperature is 40℃; detection wavelength is 260nm. Injection volume is 20μl.

[0136]

[0137] System suitability requirements System suitability solution (1) The order of peaks is vitamin B 2 , leucoflavin and leucochrome. System suitability solution (2) The resolution between leucoflavin, leucochrome and adjacent chromatographic peaks should be greater than 1.5.

[0138] Determination method: Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0139] If there is a photochromatographic peak in the chromatogram of the limit test solution, its peak area shall not be greater than 1.5 times (1.5%) the main peak area of ​​the control solution.

[0140] Table 7 Determination of related substances in compound zinc iron calcium oral solution

[0141]

[0142] As can be seen from the above table, the method for determining related substances in the compound zinc iron calcium oral solution provided by the present invention can be used for the determination of vitamin B12 in the compound zinc iron calcium oral solution. 2 The stability test and degradation impurity detection not only make up for the omissions of existing detection technologies, but also are accurate and reliable, highly specific, time-saving and labor-saving, and easy to operate.

[0143] As shown in Table 8, through methodological research, the substance determination method of the present invention has good specificity, accuracy and precision, and is suitable for the determination of related substances in the compound zinc iron calcium oral solution.

[0144] Table 8 Summary of verification of substance determination methodology of the present invention

[0145]

[0146]

[0147] Comparative Example

[0148] Experimental comparison of BP and vitamin B listed in Part II of the 2015 edition of the Chinese Pharmacopoeia 2 Chromatographic conditions of substances related to the registered standards and the results are shown in Table 11.

[0149] Vitamin B in the Chinese Pharmacopoeia 2 The conditions used for the test are:

[0150]

[0151] Table 11 Determination results of different mobile phases

[0152]

[0153]

[0154] 1. From the table and Fig.12 It can be seen from the peaks in the chromatogram of the Chinese Pharmacopoeia (wavelength 267nm) that the method in the Chinese Pharmacopoeia, λ = 444nm: the chromatographic peaks of negative samples have less interference, However, the number and quality of impurities detected in the sample solution The wavelength was significantly reduced, and the photopigment in the sample could not be detected; referring to BP, only the wavelength was adjusted to 267nm, and there were many negative interferences, which could increase the detection of impurities. The peak width is wide and the peak shape is poor .

[0155] 2. According to the test method in the registration standard,

[0156] ① The relevant substances were tested according to this quality standard, and the test results were far greater than the limit of 2.0%. After communicating with Company B, the company proposed that sucrose degradation interfered with the detection of related substances. When the company tested this project, it did not deduct the chromatographic peaks before the retention time of 0.55 relative to the main peak according to the standard, but deducted all chromatographic peaks before the main peak as auxiliary materials. The company also did not conduct an attribution study on impurities. The applicant confirmed through experiments that deducting the chromatographic peaks before the retention time of 0.55 relative to the main peak according to this quality standard did not actually completely deduct the interference of the auxiliary material peaks. Under this chromatographic condition, the negative sample has more chromatographic peaks. Experiments have found that they are mainly derived from ferrous gluconate and sucrose. Factors such as different sources of auxiliary materials have an impact on the chromatographic peaks of negative samples. Therefore, our hospital believes that it is not operational and cannot control the relevant substances in this product.

[0157] ②Sodium benzoate in sample A interferes with the detection of photoflavin, and the number of impurities detected is small, with only 4 impurity peaks detected; other impurities in sample B also interfere with the detection of photoflavin.

[0158] Although the principles of the present invention are described in detail above in conjunction with the preferred embodiments of the present invention, those skilled in the art should understand that the above embodiments are merely explanations of the exemplary implementations of the present invention, and are not intended to limit the scope of the present invention. The details in the embodiments do not constitute limitations on the scope of the present invention, and any obvious changes such as equivalent transformations, simple replacements, etc. based on the technical solution of the present invention, without departing from the spirit and scope of the present invention, fall within the protection scope of the present invention.

Claims

1. A method for determining the stability of vitamin B2 in a compound zinc, iron and calcium oral solution, characterized in that: The method comprises: (1) obtaining the initial content of vitamin B2 or determining the content of vitamin B2 in the compound zinc iron calcium oral solution by liquid chromatography; (2) Determine the content of specific degradation products of vitamin B2 in the compound zinc iron calcium oral solution by using an improved liquid chromatography method, wherein the improved liquid chromatography method includes: (2.1) Chromatographic columns with octadecylsilane bonded silica gel as filler; (2.2) Filter the compound zinc iron calcium oral solution through a 0.45 μm microporous filter membrane and take the filtrate as the test solution; (2.3) 20 μl of the test solution and reference solution were injected into the liquid chromatograph respectively; (2.4) Using 1‰ phosphoric acid solution adjusted to pH 3.5 with triethylamine as the first mobile phase A and acetonitrile as the second mobile phase B, adding them into a liquid chromatograph for gradient elution. When performing gradient elution, elution is performed in a two-isocratic and three-gradient alternating elution mode. This step comprises: First isocratic elution: The gradient of mobile phase and elution time was as follows: 0-5 min, mobile phase A-mobile phase B (90:10) isocratic elution; First gradient elution: 5-20 minutes, the proportion of mobile phase A gradually decreased from 90% to 80%, and the proportion of mobile phase B gradually increased from 10% to 20%; Second isocratic elution: 20-25 min, mobile phase A-mobile phase B (80:20) isocratic elution; Second gradient elution: 25-35 minutes, the proportion of mobile phase A gradually decreased from 80% to 50%, and the proportion of mobile phase B gradually increased from 20% to 50%; The third isocratic elution: 35-45 minutes, mobile phase A-mobile phase B (50:50) isocratic elution; (2.5) Mix the vitamin B2 photodegradation solution with the test sample solution to prepare a system suitability solution; (2.6) Using a liquid chromatograph, perform chromatographic detection on the test solution and the reference solution respectively; (3) Using the content of the degradation product of vitamin B2 obtained in step 2 and the content of vitamin B2 to determine the degradation product, as a basis for determining the stability.

2. The method for determining vitamin B2 degradation impurities in compound zinc, iron and calcium oral solution according to claim 1, characterized in that: When performing gradient elution, mobile phase A is 1‰ phosphoric acid solution.

3. The method for determining vitamin B2 degradation impurities in compound zinc, iron and calcium oral solution according to claim 1, characterized in that: When gradient elution is performed, the pH value of the phosphoric acid solution is adjusted to 3.0-4.0 with triethylamine.

4. The method for determining vitamin B2 degradation impurities in compound zinc, iron and calcium oral solution according to claim 3, characterized in that: The pH value of the phosphoric acid solution was adjusted to 3.5 with triethylamine.

5. The method for determining vitamin B2 degradation impurities in compound zinc, iron and calcium oral solution according to claim 1, characterized in that: Mix the vitamin B2 photodegradation solution with the test sample solution to prepare a system suitability solution.

6. The method for determining vitamin B2 degradation impurities in compound zinc, iron and calcium oral solution according to claim 1, characterized in that: When liquid chromatography was used for detection, the detection wavelength was selected as 260 nm.

7. The method for determining vitamin B2 degradation impurities in compound zinc, iron and calcium oral solution according to claim 1, characterized in that: During liquid chromatography detection, the flow rate was 1.0 ml / min and the column temperature was 40°C.

8. A method for determining vitamin B2 degradation impurities in compound zinc, iron and calcium oral solution using the method described in claim 1, characterized in that: The method comprises: The content of specific degradation products of vitamin B2 in compound zinc-iron-calcium oral solution was determined by improved liquid chromatography, wherein the improved liquid chromatography method includes: (2.1) Chromatographic columns with octadecylsilane bonded silica gel as filler; (2.2) Filter the compound zinc iron calcium oral solution through a 0.45 μm microporous filter membrane and take the filtrate as the test solution; (2.3) 20 μl of the test solution and reference solution were injected into the liquid chromatograph respectively; (2.4) Using 1‰ phosphoric acid solution adjusted to pH 3.5 with triethylamine as the first mobile phase A and acetonitrile as the second mobile phase B, adding them into a liquid chromatograph for gradient elution. When performing gradient elution, elution is performed in a two-isocratic and three-gradient alternating elution mode. This step comprises: First isocratic elution: The gradient of mobile phase and elution time was as follows: 0-5 min, mobile phase A-mobile phase B (90:10) isocratic elution; First gradient elution: 5-20 minutes, the proportion of mobile phase A gradually decreased from 90% to 80%, and the proportion of mobile phase B gradually increased from 10% to 20%; Second isocratic elution: 20-25 min, mobile phase A-mobile phase B (80:20) isocratic elution; Second gradient elution: 25-35 minutes, the proportion of mobile phase A gradually decreased from 80% to 50%, and the proportion of mobile phase B gradually increased from 20% to 50%; The third isocratic elution: 35-45 minutes, mobile phase A-mobile phase B (50:50) isocratic elution; (2.5) Mix the vitamin B2 photodegradation solution with the test sample solution to prepare a system suitability solution; (2.6) Use liquid chromatography to perform chromatographic detection on the test solution and the reference solution respectively.

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