A nuclear magnetic quantitative detection method for xanthan gum

Abstract

The present invention relates to a nuclear magnetic quantitative detection method for xanthan gum, wherein a sample is treated with an inorganic alkali solution, so that the ester group in the xanthan gum reacts with the inorganic alkali for hydrolysis, and then the content of the xanthan gum in the sample is detected by a nuclear magnetic method. The effective significance of the present invention is that the viscosity of the xanthan gum after being dissolved in heavy water is reduced by hydrolyzing the ester group in the xanthan gum with the inorganic alkali solution, and the product of the hydrolysis reaction produces a quantitative absorption peak with high separation degree in nuclear magnetic resonance, and then the detection of low-concentration xanthan gum can be completed by using a nuclear magnetic quantitative method. The detection method is simple to operate, has a fast detection speed, a short analysis time, and a high accuracy of the detection result, and provides method support for the detection of auxiliary material components in original research drugs and the consistency evaluation of generic drugs.

Description

Technical Field

[0001] The invention belongs to the field of detection technology, and in particular relates to a nuclear magnetic quantitative detection method for xanthan gum. Background Art

[0002] Xanthan gum, also known as xanthan gum, is a widely used microbial extracellular polysaccharide produced by Xanthomonas campestris using carbohydrates (such as corn starch) as the main raw material through fermentation engineering. It has unique rheological properties, good water solubility, and good compatibility with a variety of salts. As a thickener, suspending agent, emulsifier, stabilizer, it is widely used in more than 20 industries such as food, petroleum, and medicine. It is currently the world's largest-scale microbial polysaccharide with the widest range of uses.

[0003] Xanthan gum is used in pharmaceutical preparations and plays an important role in drug sustained release, water retention and bacteria isolation in the affected area. Therefore, the research on the detection of xanthan gum content in pharmaceutical preparations is of great significance, such as:

[0004] (1) Conduct drug consistency evaluation on generic drugs that have been marketed, that is, require that the quality and efficacy of generic drugs are consistent with those of original drugs, so as to improve the quality of generic drugs in my country and the overall development level of the pharmaceutical industry, and ensure the safety and effectiveness of drug use by the public.

[0005] (2) The content of xanthan gum, an excipient in the original drug, is tested to provide a reference for the process development of generic drugs, thus saving process development time.

[0006] At present, the content determination method of xanthan gum, a pharmaceutical excipient included in the four volumes of the "Chinese Pharmacopoeia", is not recorded. The "National Food Safety Standard Food Additive Xanthan Gum" (Draft for Comments) published in 2023 records the content determination method of pure xanthan gum, which is the drying loss method. This is not effectively helpful for the detection of lower concentrations of xanthan gum in pharmaceutical preparations. Summary of the invention

[0007] The present invention provides a nuclear magnetic quantitative detection method for xanthan gum, develops an effective method for detecting low-content xanthan gum in pharmaceutical preparations, and performs methodological verification, thereby providing method support for the detection of auxiliary material components in original research drugs and the consistency evaluation of generic drugs.

[0008] The technical solution adopted by the present invention is: a nuclear magnetic quantitative detection method for xanthan gum, wherein a sample is treated with an inorganic alkali solution so that the ester group in the xanthan gum reacts with the inorganic alkali for hydrolysis, and then the xanthan gum content in the sample is detected by a nuclear magnetic method.

[0009] Preferably, the inorganic alkaline solution is a sodium hydroxide solution.

[0010] Preferably, the specific detection steps are as follows:

[0011] Prepare internal standard solution: take internal standard substance and dissolve it in water to prepare internal standard solution;

[0012] Prepare xanthan gum reference solution: take xanthan gum reference, add inorganic alkali solution and internal standard solution, dissolve to prepare xanthan gum reference solution;

[0013] Prepare the test solution: take the test sample, add the inorganic base solution and the internal standard solution, and dissolve to prepare the test solution;

[0014] The xanthan gum reference solution and the test solution were sampled and tested in nuclear magnetic resonance. The internal standard peak area was set to 100, the xanthan gum peak was marked, and the xanthan gum content was calculated by the peak area using the external standard method.

[0015] Preferably, the internal standard is maleic acid, and the concentration of the internal standard solution is 100 μg / mL.

[0016] Preferably, the prepared xanthan gum reference solution and test solution are placed for 1 hour, shaken well, and sampled and tested in a nuclear magnetic resonance.

[0017] Preferably, the detection conditions for sample injection detection in nuclear magnetic resonance are detection sequence zg, scanning number 16 times, and delay time 5 seconds.

[0018] Application of nuclear magnetic quantitative detection method of xanthan gum in the method of testing pharmaceutical excipients or evaluating drug consistency.

[0019] The advantages and positive effects of the present invention are as follows: the viscosity of the xanthan gum after being dissolved in heavy water is reduced by hydrolyzing the ester group in the xanthan gum with an inorganic alkaline solution, and the product of the hydrolysis reaction produces a quantifiable absorption peak with high separation degree in nuclear magnetic resonance, and then the low-concentration xanthan gum can be detected by using a nuclear magnetic quantitative method. The detection method is simple to operate, has a fast detection speed, a short analysis time, and a high accuracy of the detection result, and provides method support for the detection of auxiliary material components in original research drugs and the consistency evaluation of generic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the NMR spectrum of the solution obtained by directly dissolving xanthan gum in heavy water;

[0021] Figure 2 is the nuclear magnetic spectrum of heavy water in Example 2 of the present invention;

[0022] Figure 3 is the NMR spectrum of the internal standard solution in Example 2 of the present invention;

[0023] Figure 4 is the NMR spectrum of the blank auxiliary material solution in Example 2 of the present invention;

[0024] Figure 5The NMR spectrum of the xanthan gum reference substance identification solution in Example 2 of the present invention;

[0025] Figure 6 The NMR spectrum of the sample identification solution in Example 2 of the present invention;

[0026] Figure 7 The NMR spectrum of the xanthan gum reference solution in Example 2 of the present invention;

[0027] Figure 8 is the NMR spectrum of the test solution in Example 2 of the present invention;

[0028] Fig. 9 This is a linear graph of xanthan gum peak area-concentration in Example 4 of the present invention. DETAILED DESCRIPTION

[0029] The embodiments of the present invention are described below with reference to the accompanying drawings.

[0030] The invention relates to a nuclear magnetic quantitative detection method for xanthan gum. The method reduces the viscosity of a xanthan gum solution by hydrolyzing the xanthan gum in an inorganic alkaline solution, improves the separation degree of absorption peaks generated in nuclear magnetic detection, and uses the absorption peak of the hydrolysis reaction product as a quantifiable peak to accurately determine the content of low-concentration xanthan gum in a test sample.

[0031] The inorganic alkaline solution can be sodium hydroxide solution, potassium hydroxide solution, etc., preferably sodium hydroxide solution.

[0032] The specific detection method provided by the embodiment of the present invention is as follows:

[0033] Prepare internal standard solution: take internal standard substance and dissolve it in water to prepare internal standard solution;

[0034] Prepare xanthan gum reference solution: take xanthan gum reference, add inorganic alkali solution and internal standard solution, dissolve to prepare xanthan gum reference solution;

[0035] Prepare the test solution: take the test sample, add the inorganic base solution and the internal standard solution, and dissolve to prepare the test solution;

[0036] The xanthan gum reference solution and the test solution were sampled and tested in nuclear magnetic resonance. The internal standard peak area was set to 100, the xanthan gum peak was marked, and the xanthan gum content was calculated by the peak area using the external standard method.

[0037] When xanthan gum is dissolved in heavy water, it is in a viscous gel state. When it is injected and scanned in a nuclear magnetic resonance, no quantitative absorption peak with high separation degree can be obtained (see Appendix Figure 1), while in this detection method, the inorganic alkali solution reacts with the methyl acetate in the xanthan gum structure to hydrolyze, which not only reduces the viscosity of the xanthan gum after dissolving in heavy water, but also the hydrolysis product, the methyl group in the acetic acid, has a highly separated and quantitative absorption peak at the position of 1.8ppm in the NMR scanning spectrum of the solution obtained after the hydrolysis reaction (see Appendix Figure 5 ), the xanthan gum content can be calculated accordingly.

[0038] The internal standard used in the embodiment of the present invention is maleic acid, and the concentration of the internal standard solution is 100 μg / mL; the inorganic alkali solution is a sodium hydroxide solution, and the present application does not limit its concentration and addition amount. When the xanthan gum content is detected according to the present detection method, it is determined that the added inorganic alkali solution can fully hydrolyze the ester group in the xanthan gum based on the sample information that has been mastered. Whether the inorganic alkali is just completely reacted or slightly excessive does not affect the detection of the xanthan gum content. After the xanthan gum reference solution and the test solution are prepared, they should be placed for 1 hour to allow the hydrolysis reaction to fully occur, and then shaken and tested in the nuclear magnetic resonance to improve the accuracy of the test results.

[0039] The detection conditions of the sample injection detection in the nuclear magnetic resonance of the embodiment of the present invention are detection sequence zg, scanning number 16 times, and delay time 5 seconds. The calculation formula of xanthan gum content in the test sample is as follows:

[0040] Xanthan gum content = A 样 *V 样 / F 响应 *m 样

[0041] Where: A 样 : xanthan gum peak area in the test solution;

[0042] V 样 : Preparation volume of test solution;

[0043] F 响应 : Xanthan gum response factor;

[0044] m 样 : The quality of the test product;

[0045] The xanthan gum response factor was calculated according to the following formula:

[0046] F 响应 =

[0047] Where: A: xanthan gum peak area in xanthan gum reference solution;

[0048] C: Xanthan gum concentration in xanthan gum reference solution (mg / mL).

[0049] The following is a group of embodiments as an example, and the scheme of the present invention is explained in conjunction with the accompanying drawings. Among them, the experimental methods without specific operating steps are all carried out in accordance with the corresponding product instructions. Unless otherwise specified, the instruments, reagents, and consumables used in the embodiments can all be purchased from commercial companies.

[0050] The instrument used in this group of examples is a nuclear magnetic resonance spectrometer, model BRUKER AVIII 400MHz. The test conditions of the nuclear magnetic quantitative analysis method are: sequence zg, NS=16, D1=5. The peak near 5.8ppm of maleic acid is selected as the quantitative peak, the peak area is set to 100, and the methyl peak at 1.8ppm of xanthan gum is integrated.

[0051] This group of examples uses tetracaine hydrochloride gel with known xanthan gum content as a test sample to verify the feasibility and accuracy of content detection. This group of examples is a confirmatory content test, so the xanthan gum from the following sources is used to prepare tetracaine hydrochloride gel (test sample) and also as a xanthan gum reference substance.

[0052] It is known that the tetracaine hydrochloride gel used contains 1.9% xanthan gum, and the rest is blank excipients; among which:

[0053] Xanthan gum: Batch number: F201C230201, purity 100%, source: Hubei Gedian Renfu Pharmaceutical Co., Ltd.;

[0054] Blank excipients include: tetracaine hydrochloride gel - batch number: 100456-201602, source: China Food and Drug Inspection Institute; potassium dihydrogen phosphate - batch number: 20221020, source: Tianjin Comeo Chemical Reagent Co., Ltd.; sodium methylparaben - batch number: 20231103, source: Tianjin Comeo Chemical Reagent Co., Ltd.; carbomer - batch number: 20230501, source: Gu'an Xixing Pharmaceutical Co., Ltd.; purified water, homemade.

[0055] The materials used in this group of embodiments also include:

[0056] Heavy water - Batch number: M3734, source: Cambridge Isotope Laboratories, Inc.

[0057] Maleic acid - Batch number: G1067945, purity: 99.66%, source: DREHRENSTORFER.

[0058] Sodium hydroxide solution - Batch number: 212017, concentration: 50wt%, source: Fisher chemical.

[0059] Example 1: Detection of xanthan gum content in the test sample

[0060] The method for detecting the xanthan gum content in tetracaine hydrochloride gel (test sample) by nuclear magnetic quantitative method provided in this embodiment is a content detection experiment performed after the methodological verification of the test sample selected in this embodiment. The relevant experiments of the methodological verification are specifically described in Examples 2-9.

[0061] This embodiment specifically includes the following steps:

[0062] (1) Preparation of internal standard solution: accurately weigh 10 mg of maleic acid, dissolve it in an appropriate amount of heavy water, accurately transfer it to a 100 mL volumetric flask, and make up to volume with heavy water to prepare an internal standard solution with a concentration of 100 μg / mL;

[0063] (2) Preparation of xanthan gum reference solution: accurately weigh 1.8 mg of xanthan gum reference solution, accurately add 90 μL of blank excipient, 20 μL of sodium hydroxide solution and 2890 μL of internal standard solution, oscillate and mix to prepare a xanthan gum reference solution with a xanthan gum concentration of 0.6 mg / mL;

[0064] (3) Preparation of test solution: Take about 30 mg of the test sample, accurately weigh its mass, accurately add 20 μL of sodium hydroxide solution and 980 μL of internal standard solution, oscillate and mix to prepare the test solution;

[0065] (4) After the xanthan gum reference solution and the test solution were placed for 1 hour, they were shaken and tested in NMR.

[0066] (5) Calculation of xanthan gum content in the test sample: Calculate the xanthan gum content by peak area using the external standard method. The calculation formula is as follows:

[0067] Xanthan gum content = A 样 *V 样 / F 响应 *m 样

[0068] Where: A 样 : xanthan gum peak area in the test solution;

[0069] V 样 : Preparation volume of test solution;

[0070] F 响应 : Xanthan gum response factor;

[0071] m 样 : The quality of the test product;

[0072] The xanthan gum response factor was calculated according to the following formula:

[0073] F 响应 =

[0074] Where: A: xanthan gum peak area in xanthan gum reference solution;

[0075] C: Xanthan gum concentration in xanthan gum reference solution (mg / mL).

[0076] The test results of this batch of test samples: the xanthan gum content in the test samples is 1.87%.

[0077] Example 2: Specificity Experiment

[0078] Investigate whether the components in heavy water, internal standard solution and blank auxiliary materials interfere with the chromatographic peak of xanthan gum.

[0079] (1) Preparation of internal standard solution: accurately weigh 10 mg of maleic acid, dissolve it in an appropriate amount of heavy water, accurately transfer it to a 100 mL volumetric flask, and make up to volume with heavy water to prepare an internal standard solution with a concentration of 100 μg / mL;

[0080] (2) Prepare blank excipient solution: accurately measure 30 μL of blank excipient, accurately add 20 μL of sodium hydroxide solution and 950 μL of heavy water, mix well, and prepare blank excipient solution;

[0081] (3) Prepare xanthan gum reference substance identification solution: accurately weigh 1.5 mg of xanthan gum reference substance, accurately add 20 μL of sodium hydroxide solution and 980 μL of heavy water, mix well, and prepare xanthan gum reference substance identification solution;

[0082] (4) Prepare the test sample identification solution: accurately weigh 30 mg of the test sample, accurately add 20 μL of sodium hydroxide solution and 980 μL of heavy water, mix well, and prepare the test sample identification solution;

[0083] (5) Preparation of xanthan gum reference solution: accurately weigh 1.8 mg of xanthan gum reference solution, accurately add 90 μL of blank excipient, 20 μL of sodium hydroxide solution and 2890 μL of internal standard solution, oscillate and mix to prepare a xanthan gum reference solution with a xanthan gum concentration of 0.6 mg / mL;

[0084] (6) Preparation of test solution: Take about 30 mg of the test sample, accurately weigh its mass, accurately add 20 μL of sodium hydroxide solution and 980 μL of internal standard solution, oscillate and mix to prepare the test solution;

[0085] (7) After the solutions prepared in steps (3) to (6) were placed for 1 hour and then shaken, heavy water and the solutions prepared in steps (2) to (7) were sampled and tested in NMR. The test results were as shown in the attached table. Figure 2 ~ Attached Figure 8 shown.

[0086] Compare with Figure 1 , Attachment Figure 5 and attached Figure 6 , attached Figure 5 The xanthan gum reference substance treated with sodium hydroxide solution showed a highly separated and quantitative absorption peak at around 1.8ppm. Figure 6 It shows that the sample treated with sodium hydroxide solution also has this absorption peak at around 1.8ppm; comprehensive comparative analysis of the attached Figure 2 -Attached Figure 8 Heavy water, internal standard solution and blank auxiliary materials treated with sodium hydroxide solution have no absorption peaks at this position and have no interference with the elution peak of xanthan gum.

[0087] Example 3: System Precision Experiment

[0088] Take the xanthan gum reference solution with a xanthan gum concentration of 0.6 mg / mL and inject it 6 times continuously, record the chromatogram, and calculate the RSD of the xanthan gum peak area. The RSD of the xanthan gum peak area for 6 consecutive injections is listed in Table 1. It can be seen from Table 1 that the RSD of the xanthan gum peak area is 1.2%, which is less than 5%. Therefore, the system precision of this method is good.

[0089] Table 1 System precision test results

[0090]

[0091] Example 4: Linearity and Range

[0092] Xanthan gum reference solutions with xanthan gum concentrations of 0.313 mg / mL, 0.476 mg / mL, 0.596 mg / mL, 0.721 mg / mL, 0.906 mg / mL and 1.218 mg / mL were prepared, and solutions of different concentrations were injected in sequence from low to high. Linear regression was performed with concentration as the abscissa and peak area as the ordinate. The obtained xanthan gum peak area-concentration linear graph is shown in the figure below: Fig. 9 The concentration and peak area results are shown in Table 2. The obtained linear equation of xanthan gum is y=111.8x+1.8848, and the correlation coefficient r=0.9995>0.990. It can be seen that xanthan gum has a good linear relationship in the concentration range of 0.313mg / mL~1.218mg / mL.

[0093] Table 2 Xanthan gum concentration and peak area results

[0094]

[0095] Example 5: Accuracy Experiment

[0096] Three different xanthan gum reference solutions of low, medium and high concentrations were prepared respectively, and three solutions of each concentration were prepared to investigate the accuracy of the method.

[0097] Prepare internal standard solution: accurately weigh 10 mg of maleic acid, add appropriate amount of heavy water to dissolve it, accurately transfer it to a 100 mL volumetric flask, add heavy water to make up to volume, and prepare an internal standard solution with a concentration of 100 μg / mL.

[0098] Prepare different accuracy solutions according to the proportions in Table 3. "Preparation volume" refers to the volume of different accuracy solutions, except for blank excipients and sodium hydroxide solution, the rest is supplemented with internal standard solution. Prepare three copies of each level of solution in parallel. Inject the above solutions for analysis.

[0099] Table 3 Accuracy solution preparation

[0100]

[0101] Acceptable criteria: The solution recovery rate for each level of accuracy should be in the range of 90% to 108%.

[0102] Results: The solution recovery rates at each level of accuracy were between 97.1% and 102.6%, see Table 4.

[0103] Table 4 Accuracy results

[0104]

[0105] Conclusion: This method has good accuracy.

[0106] Example 6: Repeatability Experiment

[0107] Two portions of xanthan gum reference solution and six portions of test solution were prepared to investigate the repeatability of the method.

[0108] Acceptable standard: The RSD of the content of 6 replicate solutions of the test sample should not be greater than 5%.

[0109] Results: The RSD of the content of the 6 repeatable solutions of the test sample was 2.2%, see Table 5.

[0110] Table 5 Repeatability results

[0111]

[0112] Conclusion: This method has good reproducibility.

[0113] Example 7: Intermediate precision experiment

[0114] To investigate the effect of random variation on precision, another analyst re-prepared the xanthan gum reference solution and test solution on different dates and investigated their recoveries.

[0115] Acceptable standard: The RSD of the content of 6 intermediate precision solutions of the test sample should not be greater than 5%.

[0116] Results: The RSD of the content of the 6 intermediate precision solutions of the test samples was 1.8%, and the RSD of the total 12 repeatability solutions was 2.2%, which met the requirements, see Table 6.

[0117] Table 6 Intermediate precision results

[0118]

[0119] Conclusion: This method has good intermediate precision.

[0120] Example 8: Stability test

[0121] Separately take xanthan gum reference solution and test solution (which has been placed for 1 hour after preparation to fully react with the inorganic alkali solution) and place them at room temperature. Samples were injected and analyzed at 0h, 1h, 2h, 4h, and 6h to examine their room temperature stability.

[0122] Results: The peak area ratio of the xanthan gum reference solution within 6 hours to that at 0 hours was in the range of 98.2%~102.0%, and the peak area ratio of the test solution within 6 hours to that at 0 hours was in the range of 99.8%~101.8%, both within the range of 95% to 105%, so the stability was good, see Table 7.

[0123] Table 7 Stability results

[0124]

[0125] Example 9: Durability Test

[0126] The test was conducted to examine the tolerance of the measurement results to a small change (D1±1, NS±2) in the spectral detection conditions without being affected.

[0127] The first xanthan gum reference solution and the first test solution prepared in the intermediate precision experiment were sampled and analyzed under different durability conditions, and the xanthan gum content was calculated by the external standard method using the peak area. The durability test results obtained by adjusting the scanning number D1±2 and the delay time NS±1 are shown in Table 8. From the data in Table 8, it can be seen that after adjusting the parameter D1, the RSD of the xanthan gum content in the test solution is 1.9%, and after adjusting the parameter NS, the RSD of the xanthan gum content in the test solution is 1.4%, both of which are less than 5%. Therefore, this method has good durability.

[0128] Table 8 Durability test results

[0129]

[0130] The embodiments of the present invention are described in detail above, but the contents described are only preferred embodiments of the present invention and cannot be considered to limit the scope of implementation of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A nuclear magnetic quantitative detection method for xanthan gum, characterized in that: The sample is treated with an inorganic alkali solution to allow the ester group in the xanthan gum to undergo a hydrolysis reaction with the inorganic alkali, and then the content of acetic acid generated by the hydrolysis reaction is detected by a nuclear magnetic resonance method, thereby calculating the content of the xanthan gum in the sample.

2. The nuclear magnetic quantitative detection method of xanthan gum according to claim 1, characterized in that, The inorganic alkali solution is a sodium hydroxide solution.

3. The nuclear magnetic quantitative detection method of xanthan gum according to claim 1 or 2, characterized in that, The specific detection steps are as follows: Prepare internal standard solution: take internal standard substance and dissolve it in water to prepare internal standard solution; Prepare xanthan gum reference solution: take xanthan gum reference, add inorganic alkali solution and internal standard solution, dissolve to prepare xanthan gum reference solution; Prepare the test solution: take the test sample, add the inorganic base solution and the internal standard solution, and dissolve to prepare the test solution; The xanthan gum reference solution and the test solution were sampled and tested in nuclear magnetic resonance. The internal standard peak area was set to 100, the xanthan gum peak was marked, and the xanthan gum content was calculated by the peak area using the external standard method.

4. The nuclear magnetic quantitative detection method of xanthan gum according to claim 3, characterized in that, The internal standard was maleic acid, and the concentration of the internal standard solution was 100 μg / mL.

5. The nuclear magnetic quantitative detection method of xanthan gum according to claim 3, characterized in that, After the prepared xanthan gum reference solution and test solution were placed for 1 hour, they were shaken and tested by NMR.

6. The nuclear magnetic quantitative detection method of xanthan gum according to claim 3, characterized in that, The detection conditions for sample injection detection in NMR were detection sequence zg, 16 scans, and a delay time of 5 seconds.

7. Application of the nuclear magnetic quantitative detection method of xanthan gum according to any one of claims 1 to 6 in a method for testing pharmaceutical excipients or a method for evaluating drug consistency.

Images