Method for simultaneously determining related substances of glycine and methionine

Abstract

The invention relates to a method for simultaneously determining related substances of glycine and methionine. The method comprises the following steps: step 1, preparing a blank auxiliary material solution; step 2, preparing a test solution; step 3, preparing a reference solution; step 4, preparing a system applicable solution; and step 5, detecting. Compared with a traditional method, the method has the advantages that the related substances in the glycine and the methionine can be simultaneously determined, the sample pretreatment is simple, the compound retention time is stable, the instrument configuration requirement is low, the analysis cost is reduced, the sensitivity is high, the impurity separation degree is good, and the related substances in the glycine and the methionine can be effectively separated.

Description

Technical Field

[0001] The invention belongs to the technical field of drug detection, and in particular relates to a method for simultaneously determining glycine and methionine-related substances. Background Art

[0002] At present, the 2020 edition of the Chinese Pharmacopoeia only contains the raw material standards for a single glycine or methionine, and does not contain the corresponding related substance detection methods. The USP and EP Pharmacopoeia also only contain the raw material standards for a single glycine or methionine. Although the related substance detection methods are included, the method for simultaneously detecting these two substances is not included. In addition, a method for simultaneously detecting related substances of glycine and methionine is also disclosed, but it uses a derivatization method, and its mobile phase and sample preparation process are complicated, and a fluorescence detector is used.

[0003] Disadvantages of traditional technology: 1. The traditional method is to detect a single component and fail to detect both glycine and methionine at the same time. 2. The post-column derivatization method needs to be equipped with a derivatizer and a fluorescence detector in addition to the conventional high performance liquid chromatograph, and the preparation of the derivatization solution uses a variety of reagents, making the preparation process complicated.

[0004] Cause: The traditional method uses a strong acid ion exchange resin as a filler (SHIM-PACKAMINO-NA, 6×100mm); sodium citrate solution (take 8.4g of citric acid monohydrate and 11.8g of trisodium citrate dihydrate, dissolve in water and dilute to 1000ml) as the mobile phase; the column temperature is 60°C. The chromatographic column is expensive and the column temperature is high, which can easily damage the chromatographic column. In addition, the detection method is a post-column derivatization method, the derivatization temperature is 60°C, and the derivatization solution (prepare a buffer solution containing 384mmol / L sodium carbonate, 219mmol / L boric acid and 108mmol / L potassium sulfate. Take 0.8g of o-phthalaldehyde, add 20ml of methanol to dissolve, add 900ml of the above buffer solution, mix well, add 1.0g of N-acetyl-L-cysteine, stir to dissolve, and dilute to 1000ml with buffer solution to obtain), and the detector is a fluorescence detector. It is necessary to weigh multiple buffer salts, perform multiple mixing steps and other tedious operations, and the derivatization solution preparation process is complicated. It is also necessary to equip a derivatizer and a fluorescence detector, which are relatively expensive. In the process of using this method, it was found that this method has a weak detection ability for some known impurities and cannot effectively separate multiple known impurities. Summary of the invention

[0005] The object of the present invention is to provide a method for simultaneously determining glycine and methionine related substances.

[0006] To achieve the above-mentioned object and other related objects, the technical solution provided by the present invention is: a method for simultaneously determining glycine and methionine-related substances, comprising the following steps:

[0007] Step 1: Blank excipient solution

[0008] Take blank auxiliary material, place it in a container, add mobile phase to dissolve and dilute, shake well, and filter to obtain;

[0009] Step 2: Preparation of test solution

[0010] Take the sample to be tested, place it in a container, add the mobile phase to dissolve and dilute it, shake it well, and filter it;

[0011] Step 3: Reference solution

[0012] Weigh glycine reference substance and methionine reference substance, place them in the same container, add mobile phase to dissolve and dilute, and shake well;

[0013] Step 4: System Suitability Solution

[0014] Take methionine impurity reference substance and glycine impurity reference substance, place them in a volumetric bottle respectively, dilute them to the mark with a diluent, shake well, and use them as methionine impurity reference substance impurity stock solution and glycine impurity reference substance stock solution; take methionine reference substance and glycine reference substance, place them in a container respectively, add 2 ml of methionine impurity A stock solution and 1 ml of other impurity stock solutions, and dilute them with a diluent to make a solution containing 2.5 mg of glycine and methionine, 12.5 μg of methionine impurity A, and 5 μg of other known impurities per ml;

[0015] Step 5: Detection

[0016] High performance liquid chromatography combined with ultraviolet detector was used, with octadecylsilane bonded silica gel as filler; the mobile phase was 20 mM potassium dihydrogen phosphate solution-acetonitrile = 35:65; the column temperature was 50°C; the isocratic elution was 20 minutes; the flow rate was 0.5 ml per minute; the detection wavelength was 200 nm; and the injection volume was 40 μl.

[0017] The preferred technical solution is: the mobile phase is a mixture of potassium dihydrogen phosphate solution and acetonitrile.

[0018] The preferred technical solution is: the methionine impurity reference substance is at least one of a methionine impurity A reference substance and a methionine impurity C reference substance.

[0019] The preferred technical solution is: the glycine impurity reference substance is at least one of a glycine impurity A reference substance, a glycine impurity B reference substance, a glycine impurity C reference substance, a glycine impurity I reference substance and a glycine impurity H reference substance.

[0020] The preferred technical solution is: the diluent is counted as the mobile phase; the diluent is a mixed solution consisting of 20mM potassium dihydrogen phosphate solution and acetonitrile in a volume ratio of 35:65.

[0021] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0022] Compared with the traditional method, the method of the present invention can simultaneously determine the related substances in glycine and methionine, and its sample pretreatment is simple, the compound retention time is stable, the instrument configuration requirement is low, the analysis cost is reduced, the sensitivity is high, the impurity separation is good, and the related substances in glycine and methionine can be effectively separated. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a comparative example detection spectrum.

[0024] Figure 2 This is the detection spectrum of Example 1 of the present invention. DETAILED DESCRIPTION

[0025] The following is a description of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0026] See also Figure 1-2 . It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings in this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the limiting conditions for the implementation of the present invention, so they have no substantial technical significance. Any modification of the structure, change in the proportional relationship, or adjustment of the size should still fall within the scope of the technical content disclosed by the present invention without affecting the effects and purposes that can be achieved by the present invention. At the same time, the terms such as "upper", "lower", "left", "right", "middle" and "one" quoted in this specification are only for the convenience of description, and are not used to limit the scope of the implementation of the present invention. Changes or adjustments in their relative relationships should also be regarded as the scope of the implementation of the present invention without substantially changing the technical content.

[0027] The instruments and equipment used in the embodiments of the present invention are as follows:

[0028] Ultimate 3000 high performance liquid chromatograph (ultraviolet detector), PB-10 acidometer. High performance liquid chromatography combined with ultraviolet detector, octadecylsilane bonded silica gel as filler (Merck SeQuant ZIC-HILIC, 5μm, 4.6×150mm or chromatographic column with equivalent performance); mobile phase is 20mM potassium dihydrogen phosphate solution-acetonitrile (35:65); column temperature is 50℃; isocratic elution is 20 minutes; flow rate is 0.5ml per minute; detection wavelength is 200nm; injection volume is 40μl.

[0029] The materials specifically used in the embodiments of the present invention are as follows:

[0030] Glycine reference substance: Source: China National Institute for Food and Drug Control; Content: 100%.

[0031] Methionine reference substance: Source: China National Institute for Food and Drug Control; Content: 100%.

[0032] Methionine impurity A reference substance: Source: SINCO; Content: 95.29%.

[0033] Methionine impurity C reference substance: Source: SINCO; Content: 97.44%.

[0034] Glycine impurity A reference substance: Source: SINCO; Content: 96.32%.

[0035] Glycine impurity B reference substance: source: SINCO; content: 95.80%).

[0036] Glycine impurity C reference substance: Source: SINCO; Content: 90.29%.

[0037] Glycine impurity H reference substance: source: SINCO; content: 98.10%).

[0038] Glycine impurity I reference substance: Source: SINCO; Content: 99.59%.

[0039] Comparative Example:

[0040] The sample preparation is as follows: take 20 tablets of this product, accurately weigh, grind, accurately weigh an appropriate amount, place in a 100ml volumetric flask, add water to dissolve and dilute to the scale, shake well, centrifuge, and take the supernatant. Filter, accurately measure 1ml of the filtrate, place in a 100ml volumetric flask, dilute to the scale with water, shake well, as the test solution, and then prepare the derivative solution (prepare a buffer solution containing 384mmol / L sodium carbonate, 219mmol / L boric acid and 108mmol / L potassium sulfate. Take 0.8g of o-phthalaldehyde, add 20ml of methanol to dissolve, add 900ml of the above buffer solution, mix well, add 1.0g of N-acetyl-L-cysteine, stir to dissolve, and dilute to 1000ml with the buffer solution to obtain), the test solution and the derivative solution enter the derivatizer at the same time, react in the derivatizer, and then enter the fluorescence detector for detection and analysis, and finally obtain the test results.

[0041] ① Preparation of mobile phase: Take 8.4011g of citric acid monohydrate and 11.8017g of trisodium citrate dihydrate, dissolve in water and dilute to 1000ml. Mix well and filter.

[0042] ② Preparation of derivative solution: Take 109.921g of sodium carbonate, 13.627g of boric acid, and 18.821g of potassium sulfate, dissolve them in water and dilute to 1000ml to obtain a buffer solution. Take 0.8045g of o-phthalaldehyde, add 20ml of methanol to dissolve it, add 900ml of the above buffer solution, mix well, add 1.0010g of N-acetyl-L-cysteine, stir to dissolve it, dilute to 1000ml with the buffer solution, and filter to obtain the product.

[0043] ③ Glycine impurity A / B / C / D / E / F / G / H / I solution: Accurately weigh about 1 mg of each impurity and prepare a solution containing 10 μg of each impurity per 1 ml.

[0044] ④ Preparation of mixed solutions of various impurities: Weigh 200 mg of glycine raw material, place in a 20 ml volumetric flask, add solvent to dissolve and dilute to scale, shake well to obtain. Take 1 ml of the above solution and place in a 10 ml volumetric flask, add dissolve to dilute to scale, shake well to obtain. Take 0.1 ml of solution and 1 ml of each impurity solution of glycine, place in the same 10 ml volumetric flask, add solvent to dilute to scale, shake well to obtain.

[0045] ⑤ Determination results and spectra such as Figure 1 and as shown in the table below.

[0046]

[0047] From the above results, it can be seen that the traditional method can only detect impurities B / G / H / I, and the theoretical plate number of impurity G is low, and impurities B / H are not separated. This method has weak impurity detection ability and cannot effectively separate impurities.

[0048] Example 1: A method for simultaneously determining glycine and methionine-related substances

[0049] ① Preparation of mobile phase: weigh 5.4438 g of potassium dihydrogen phosphate, add appropriate amount of water to dissolve and dilute to 2000 ml, stir evenly, measure 1750 ml, mix with 3250 ml of acetonitrile, filter and sonicate to obtain.

[0050] ②Methionine impurity A / C, glycine impurity A / B / C / H / I solutions: Accurately weigh 5 mg of each impurity to prepare a solution containing 500 μg of each impurity per 1 ml.

[0051] ③ Preparation of mixed solutions of various impurities: Accurately weigh 25 mg of glycine raw material and 25 mg of methionine raw material, place them in the same 10 ml volumetric flask, add appropriate amount of diluent to dissolve, then add 0.25 ml of the above methionine impurity A solution, 0.1 ml of other glycine and methionine impurity solutions, make up to the scale, shake well, and the diluent is the mobile phase.

[0052] ④ The results of the test are as follows Figure 2 As shown in the following table:

[0053]

[0054]

[0055] It can be seen from the above results that the method in this embodiment is significantly better than the method in the comparative example in terms of its ability to detect impurities and to separate various impurities.

[0056] In order to overcome the problems of high instrument configuration requirements, complex sample pretreatment, weak impurity detection capability and low impurity separation when detecting related substances in glycine and methionine by traditional methods, the present invention provides a detection method for simultaneously determining related substances in glycine and methionine.

[0057] 1. The problem of high instrument configuration requirements has been solved: the instruments used in the traditional method are high performance liquid chromatograph, derivatization instrument and fluorescence detector, while the present invention only uses high performance liquid chromatograph and ultraviolet detector to complete the detection of relevant substances. It is not only cheaper than the traditional method in terms of cost, but also more convenient to use than the traditional method, which greatly improves the operability of personnel.

[0058] 2. The problem of complicated sample pretreatment in traditional methods is solved. The present invention takes 2 pieces of the sample to be tested, puts them in a 20mL volumetric bottle, adds mobile phase to dissolve and dilute to the scale, shakes well, filters, takes the filtrate as the test solution, and then directly enters the ultraviolet detector to obtain the analysis result. Compared with the traditional method, the sample pretreatment method of the present invention is not only faster and simpler to operate, but also greatly improves the repeatability of the results.

[0059] 3. Solved the problems of weak impurity detection ability and low impurity separation degree of traditional methods: the detection object of the related substance method is impurities in the final analysis, so the pros and cons of a related substance method can be judged from the impurity detection ability and the separation degree of each impurity.

[0060] Example 2: A method for simultaneously determining glycine and methionine-related substances

[0061] High performance liquid chromatography combined with ultraviolet detector was used, with octadecylsilane bonded silica gel as filler (Merck SeQuant ZIC-HILIC, 5μm, 4.6×150mm or chromatographic column with equivalent performance); the mobile phase was 20mM potassium dihydrogen phosphate solution-acetonitrile (35:65); the column temperature was 50°C; the isocratic elution was 20 minutes; the flow rate was 0.5ml per minute; the detection wavelength was 200nm; and the injection volume was 40μl.

[0062] The above method was used to test three batches of commercial samples of a certain product. The specific implementation plan is as follows:

[0063] 1. Instrument:

[0064] Ultimate 3000 high performance liquid chromatograph (UV detector), PB-10 acidity meter

[0065] 2. Drug testing:

[0066] Glycine reference substance (Source: China National Institute for Food and Drug Control; Content: 100%)

[0067] Methionine reference substance (Source: China National Institute for Food and Drug Control; Content: 100%)

[0068] Methionine impurity A reference substance (source: SINCO; content: 95.29%)

[0069] Methionine impurity C reference substance (Source: SINCO; Content: 97.44%)

[0070] Glycine impurity A reference substance (Source: SINCO; Content: 96.32%)

[0071] Glycine impurity B reference substance (Source: SINCO; Content: 95.80%)

[0072] Glycine impurity C reference substance (Source: SINCO; Content: 90.29%)

[0073] Glycine impurity H reference substance (Source: SINCO; Content: 98.10%)

[0074] Glycine impurity I reference substance (Source: SINCO; Content: 99.59%)

[0075] 3. Chromatographic conditions:

[0076] Column: Merck SeQuant ZIC-HILIC, 5 μm, 4.6 × 150 mm.

[0077] Mobile phase: 20 mM potassium dihydrogen phosphate solution-acetonitrile (35:65).

[0078] Column temperature: 50℃.

[0079] Isocratic elution: 20 min.

[0080] Flow rate: 0.5 ml per minute.

[0081] Detection wavelength: 200nm.

[0082] Injection volume: 40 μl.

[0083] 4. Sample preparation process:

[0084] (1) Blank excipient solution: Weigh 220 mg of blank excipient into a 20 ml volumetric flask, add mobile phase to dissolve and dilute to scale, shake well, and filter to obtain the solution.

[0085] (2) Preparation of test solution: Take 2 tablets of this product, place them in a 20 ml volumetric flask, add mobile phase to dissolve and dilute to the scale, shake well, filter, and obtain the solution.

[0086] (3) Reference solution: Accurately weigh 12.5 mg of glycine and 12.5 mg of methionine, place in the same 25 ml volumetric flask, add mobile phase to dissolve and dilute to scale, shake well. Accurately measure 1 ml of the above solution, place in a 100 ml volumetric flask, add mobile phase to dilute to scale, shake well, and the solution is ready.

[0087] (4) System suitability solution: Take 1.25 mg of methionine impurity A reference substance, 1 mg of methionine impurity C reference substance, 1 mg of glycine impurity A reference substance, 1 mg of glycine impurity B reference substance, 1 mg of glycine impurity C reference substance, 1 mg of glycine impurity I reference substance, and 1 mg of glycine impurity H reference substance, place them in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to prepare the impurity stock solutions. Take 50 mg of methionine reference substance and 50 mg of glycine reference substance, place them in a 20 ml volumetric flask, add 2 ml of methionine impurity A stock solution and 1 ml of each other impurity stock solution, and dilute with diluent to prepare a solution containing glycine, 2.5 mg of methionine, 12.5 μg of methionine impurity A, and 5 μg of each other known impurity per 1 ml.

[0088] 5. Measurement results

[0089] (1) System suitability results:

[0090]

[0091]

[0092] It can be seen from the results in the above table that the separation degree between the impurities of this method meets the requirements, and the separation degree between the main peak and adjacent impurities basically meets the requirements.

[0093] (2) Sample test results

[0094]

[0095] This method was used to test three batches of commercial samples of a certain product. The results showed that this method has accurate detection results and good reproducibility, and is suitable for the detection of glycine and methionine-related substances.

[0096] The present invention solves the problem of high instrument configuration requirements: the instruments used in the comparative example are a high performance liquid chromatograph, a derivatization instrument and a fluorescence detector, while the present invention only uses a high performance liquid chromatograph and an ultraviolet detector to complete the detection of relevant substances, which is not only cheaper than traditional methods in terms of cost, but also more convenient to use than traditional methods, greatly improving the operability of personnel.

[0097] The present invention solves the problem of complex sample pretreatment in traditional methods: the sample is prepared as follows in the traditional method: take 20 tablets of the product, accurately weigh, grind, accurately weigh an appropriate amount, put it in a 100ml volumetric bottle, add water to dissolve and dilute to the scale, shake well, centrifuge, and take the supernatant. Filter, accurately measure 1 ml of the filtrate, put it in a 100 ml volumetric flask, dilute it with water to the scale, shake it well, and use it as the test solution. Then prepare a derivative solution (prepare a buffer solution containing 384 mmol / L sodium carbonate, 219 mmol / L boric acid and 108 mmol / L potassium sulfate. Take 0.8 g of o-phthalaldehyde, add 20 ml of methanol to dissolve it, add 900 ml of the above buffer solution, mix well, add 1.0 g of N-acetyl-L-cysteine, stir to dissolve it, and dilute it to 1000 ml with the buffer solution to obtain the product). The test solution and the derivative solution enter the derivatizer at the same time, react in the derivatizer, and then enter the fluorescence detector for detection and analysis, and finally obtain the test results. The sample preparation process of the present invention is as follows: take 2 tablets of this product, put them in a 20 ml volumetric flask, add the mobile phase to dissolve and make up to the scale, shake it well, filter it, take the filtrate as the test solution, and then directly enter the ultraviolet detector to obtain the analysis results. Compared with traditional methods, the sample pretreatment method of the present invention is not only faster and simpler to operate, but also greatly improves the repeatability of the results.

[0098] The present invention solves the problems of weak impurity detection capability and low impurity separation degree in traditional methods: the detection object of the related substance method is ultimately impurities, so the quality of a related substance method can be judged from aspects such as the impurity detection capability and the separation degree of each impurity.

[0099] Embodiment 3:

[0100] The above description is only used to explain the preferred embodiments of the present invention, and is not intended to limit the present invention in any form. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included in the scope of protection intended by the present invention.

Claims

1. A method for simultaneously determining glycine and methionine related substances, characterized in that: The following steps are involved: Step 1: Blank excipient solution Take blank auxiliary material, place it in a container, add mobile phase to dissolve and dilute, shake well, and filter to obtain; Step 2: Preparation of test solution Take the sample to be tested, place it in a container, add mobile phase to dissolve and dilute, shake well, and filter to obtain the sample; Step 3: Reference solution Weigh glycine reference substance and methionine reference substance, place them in the same container, add mobile phase to dissolve and dilute, and shake well; Step 4: System Suitability Solution Take methionine impurity reference substance and glycine impurity reference substance, place them in a volumetric bottle respectively, dilute them to the mark with a diluent, shake well, and use them as methionine impurity reference substance impurity stock solution and glycine impurity reference substance stock solution; take methionine reference substance and glycine reference substance, place them in a container respectively, add 2 ml of methionine impurity A stock solution and 1 ml of other impurity stock solutions, and dilute them with a diluent to make a solution containing 2.5 mg of glycine and methionine, 12.5 μg of methionine impurity A, and 5 μg of other known impurities per ml; Step 5: Detection High performance liquid chromatography combined with ultraviolet detector was used, with octadecylsilane bonded silica gel as filler; the mobile phase was 20 mM potassium dihydrogen phosphate solution-acetonitrile = 35:65; the column temperature was 50°C; the isocratic elution was 20 minutes; the flow rate was 0.5 ml per minute; the detection wavelength was 200 nm; and the injection volume was 40 μl.

2. The method for simultaneously determining related substances of glycine and methionine according to claim 1, characterized in that: The mobile phase was a mixture of potassium dihydrogen phosphate solution and acetonitrile.

3. The method for simultaneously determining related substances of glycine and methionine according to claim 1, characterized in that: The methionine impurity reference substance is at least one of a methionine impurity A reference substance and a methionine impurity C reference substance.

4. The method for simultaneously determining related substances of glycine and methionine according to claim 1, characterized in that: The glycine impurity reference substance is at least one of a glycine impurity A reference substance, a glycine impurity B reference substance, a glycine impurity C reference substance, a glycine impurity I reference substance, and a glycine impurity H reference substance.

5. The method for simultaneously determining related substances of glycine and methionine according to claim 1, characterized in that: The diluent is the mobile phase; the diluent is a mixture of 20 mM potassium dihydrogen phosphate solution and acetonitrile in a volume ratio of 35:65.

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