Method for detecting content of polyethylene glycol powder

Abstract

The invention discloses a method for detecting the content of polyethylene glycol powder, which adopts a high performance liquid chromatography, and the chromatographic conditions are as follows: a differential refraction detector is adopted, an aqueous phase gel chromatographic column Waters Ultrahydrogel DP column is used as a chromatographic column, a mobile phase is a sodium sulfate solution, and the problem of interference of auxiliary materials in the polyethylene glycol powder on the main component polyethylene glycol can be solved. According to the method, the mobile phase components in the chromatographic conditions are few in variety and low in salt concentration, the influence on the service life of the chromatographic column is reduced, meanwhile, the mobile phase is convenient to prepare, the research and development cost is reduced, and the detection efficiency of the polyethylene glycol content is improved.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical quality control technology, and specifically relates to a method for testing the content of polyethylene glycol powder. Background Technology

[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.

[0003] Polyethylene glycol (PEG) is polymerized from ethylene glycol or ethylene oxide under alkaline catalyst catalysis, with a molecular weight between 200 and 35,000. PEG has excellent lubricity, moisturizing properties, dispersibility, and biocompatibility, and is widely used in pharmaceuticals and daily chemical products.

[0004] In the pharmaceutical field, polyethylene glycol 3350 can be used as a laxative to treat constipation, as a base for ointments and suppositories to adjust consistency and drug release rate, and as a coating material for tablets to improve appearance and moisture resistance.

[0005] Polyethylene glycol 3350 powder is a first-line treatment for constipation recommended by authoritative guidelines both domestically and internationally. This product is administered orally and requires the addition of flavoring agents such as citric acid, sodium citrate, sucralose, and flavorings. Therefore, the content of the main ingredient, polyethylene glycol 3350, needs to be monitored and controlled.

[0006] Currently, most pharmacopoeias of various countries use acid-base titration to determine the molecular weight of polyethylene glycol compounds, calculating the average molecular weight by measuring the number of hydroxyl groups at the ends of the molecular chains. This method is cumbersome, uses reagents that are highly toxic to laboratory personnel and the environment, and is influenced by numerous factors that can affect the experimental results.

[0007] High-performance gel permeation chromatography (GPC) can effectively separate PEG compounds using the size exclusion principle, and can perform statistical analysis of chromatographic behavior to determine key information such as molecular weight distribution and content. Existing technology provides a method for determining the content of polyethylene glycol 4000, specifically for compound polyethylene glycol electrolyte powders. This method employs high-performance liquid chromatography (HPLC) with a differential refractive index detector, using an aqueous gel chromatography column (PL aquagel-OH column). The mobile phase consists of a salt solution containing sodium chloride, potassium chloride, sodium bicarbonate, sodium sulfate, and sodium saccharin, diluted with water, and isocratic elution is used. However, the complex salt composition of this mobile phase affects the column's lifespan. Furthermore, when this method was applied to the detection of the main component in polyethylene glycol 3350 powder, excipients were found to interfere with the determination of the main component content. Existing technology also provides a method for determining the molecular weight and distribution of polyethylene glycol 3350 using a 0.1 mol / L sodium nitrate aqueous solution as the mobile phase. However, in liquid chromatography (LC) detection, the recommended buffer salt concentration in the mobile phase is generally no higher than 0.1 mol / L. This is because when a high-salt mobile phase is replaced with a low-salt or organic phase, salts may precipitate due to changes in solubility, clogging the sieve plate of the column head and the pores between the stationary phase particles. This leads to increased column pressure, decreased separation efficiency, and affects subsequent detection results. Residual salts may alter the polarity of the mobile phase, causing retention time deviations and decreased column efficiency. Residual salts can also corrode the sieve plate structure, causing problems such as column head depression. After using a high-salt mobile phase, the column must be rinsed with a high proportion of water to remove residual salts before storage. Higher salt concentrations shorten the column's lifespan.

[0008] Therefore, the research and development of polyethylene glycol powder still urgently needs a method that can accurately detect the polyethylene glycol content while reducing chromatographic column wear. Summary of the Invention

[0009] To address the shortcomings of existing technologies, the purpose of this invention is to provide a method for testing the content of polyethylene glycol powder.

[0010] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for testing the content of polyethylene glycol powder, which employs high-performance liquid chromatography (HPLC). The chromatographic conditions are as follows: a differential refractive index detector is used, a Waters Ultrahydrogel DP column is used as the chromatographic column, and sodium sulfate solution is used as the mobile phase. This method can solve the problem of interference between excipients and the main component polyethylene glycol in polyethylene glycol powder.

[0011] Preferably, the mobile phase is a sodium sulfate solution with a concentration of 0.005~0.05 mol / L.

[0012] Preferably, the chromatographic column is a Waters Ultrahydrogel DP 7.8mm×30cm.

[0013] Preferably, the column temperature is 30~40℃, more preferably 35℃.

[0014] Preferably, the mobile phase flow rate is 0.7~0.9 ml / min, more preferably 0.8 ml / min.

[0015] Preferably, the detection instrument is a Waters Arc high-performance liquid chromatograph.

[0016] Preferably, the steps include: S1, Preparation of sample solution: Preparation of citrate solution: Prepare the prescribed amount of citrate solution; Reference solution: Place polyethylene glycol reference standard in a container and add citrate solution to prepare a solution with a concentration of 1 ± 0.1 mg / ml; Test solution: Accurately weigh the sample and add water to prepare a solution with a concentration of 1 ± 0.1 mg / ml.

[0017] S2, Determination Method: After the chromatographic system has been balanced, inject the reference solution and the test solution according to the chromatographic conditions described, record the chromatograms, and calculate the content of the main component of the product using the external standard method.

[0018] Preferably, the polyethylene glycol powder includes polyethylene glycol 3350 powder and polyethylene glycol 4000 powder. More preferably, it is polyethylene glycol 3350 powder.

[0019] Preferably, the excipients included in the polyethylene glycol powder are citric acid and sodium citrate.

[0020] The beneficial effects achieved by one or more embodiments of the present invention described above are as follows: 1. This invention provides a method for detecting the polyethylene glycol content in polyethylene glycol powder, which solves the problem of interference of excipient components in polyethylene glycol powder on the detection of the main component content, and is conducive to the effective and accurate detection of polyethylene glycol; 2. In the detection method provided by the present invention, the mobile phase has fewer components and lower salt concentration in the chromatographic conditions, which reduces the wear and tear of expensive gel permeation chromatography columns, helps to extend the service life of the chromatography columns, and reduces research and development costs; at the same time, the mobile phase is easy to prepare, which improves the efficiency of polyethylene glycol content detection. 3. Methodological validation has demonstrated that this method is simple and feasible, with strong specificity, good precision, recovery rate, and robustness, and is suitable for content testing of polyethylene glycol 3350 and other molecular weight polyethylene glycol powders. Attached Figure Description

[0021] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0022] Figure 1 This is a comparison diagram of the method validation specificity test in Example 3; from bottom to top, they are: diluent water, diluent citrate solution, blank excipient solution, reference standard positioning solution, and test solution.

[0023] Figure 2 This is a comparison diagram of the mobile phase in Example 4, which was 0.005 mol / L sodium sulfate solution; from bottom to top, the components are water (diluent), citrate solution (diluent), blank excipient, reference standard positioning solution, and test solution.

[0024] Figure 3 This is a comparison diagram of the mobile phase in Example 4, which was 0.01 mol / L sodium sulfate solution; from bottom to top, the components are water (diluent), citrate solution (diluent), blank excipient, reference standard positioning solution, and test solution.

[0025] Figure 4 This is a comparison diagram of the mobile phase in Example 4, which was 0.1 mol / L sodium sulfate solution; from bottom to top, the components are water (diluent), citrate solution (diluent), blank excipient, reference standard positioning solution, and test solution.

[0026] Figure 5 The diagram shows the comparison of the mobile phase in Comparative Example 1, which was 0.0001 mol / L sodium sulfate solution. From bottom to top, the components are: water (diluent), citrate solution (diluent), blank excipient, reference standard positioning solution, and test solution.

[0027] Figure 6 The diagram shows the comparison of the mobile phase in Comparative Example 2, which was 0.001 mol / L sodium sulfate solution. From bottom to top, the components are: water (diluent), citrate solution (diluent), blank excipient, reference standard positioning solution, and test solution.

[0028] Figure 7 The diagram shows the comparison of the mobile phase in Comparative Example 3, which was 0.05 mol / L sodium nitrate solution. From bottom to top, the components are: water (diluent), citrate solution (diluent), blank excipient, reference standard positioning solution, and test solution.

[0029] Figure 8 The diagram shows the comparison of the mobile phase in Comparative Example 3, which was 0.05 mol / L sodium bicarbonate solution. From bottom to top, the components are: water (diluent), citrate solution (diluent), blank excipient, reference standard positioning solution, and test solution.

[0030] Figure 9 The diagram shows the comparison of the mobile phase in Comparative Example 3, which was 0.05 mol / L ammonium acetate solution. From bottom to top, the components are: water (diluent), citrate solution (diluent), blank excipient, reference standard positioning solution, and test solution.

[0031] Figure 10 The diagram shows the comparison of the mobile phase in Comparative Example 3, which was 0.05 mol / L ammonium dihydrogen phosphate solution. From bottom to top, the components are: water (diluent), citrate solution (diluent), blank excipient, reference standard positioning solution, and test solution.

[0032] Figure 11 Comparative Example 4 shows a comparison chart of the existing methods used for testing the content of polyethylene glycol 3350; from bottom to top, the components are water (diluent), citrate solution (diluent), blank excipient, reference standard positioning solution, and test solution. Detailed Implementation

[0033] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0034] This invention provides a method for testing the content of polyethylene glycol powder, which employs high-performance liquid chromatography (HPLC). The chromatographic conditions are as follows: a differential refractive index detector is used, a Waters Ultrahydrogel DP column is used as the chromatographic column, and sodium sulfate solution is used as the mobile phase. This method can solve the problem of interference between excipients and the main component polyethylene glycol in polyethylene glycol powder.

[0035] In some embodiments of the present invention, the mobile phase is a sodium sulfate solution with a concentration of 0.005~0.05 mol / L.

[0036] In some embodiments of the present invention, the chromatographic column is a Waters Ultrahydrogel DP 7.8mm×30cm.

[0037] In some embodiments of the present invention, the column temperature of the chromatographic column is 30~40℃.

[0038] In some embodiments of the present invention, the mobile phase flow rate is 0.7~0.9 ml / min.

[0039] In some embodiments of the present invention, the detection instrument is a Waters Arc high-performance liquid chromatograph.

[0040] In some embodiments of the present invention, the following steps are included: S1, Preparation of sample solution: Preparation of citrate solution: Prepare the prescribed amount of citrate solution; Reference solution: Place polyethylene glycol reference standard in a container and add citrate solution to prepare a solution with a concentration of 1 ± 0.1 mg / ml; Test solution: Accurately weigh the sample and add water to prepare a solution with a concentration of 1 ± 0.1 mg / ml.

[0041] S2, Determination Method: After the chromatographic system has reached equilibrium, inject the reference solution and the test solution under the described chromatographic conditions, record the chromatograms, and calculate the content of the main component of the product using the external standard method; the calculation formula is: ; In the formula: A i : Peak area of ​​the main peak in the test solution; A r The average peak area of ​​the main peak in the reference solution; m r The sample weight of polyethylene glycol 3350 reference standard in the reference solution is (mg). V i : The volume of the test solution diluted, in ml; V r : Dilution volume of the reference solution, ml; B r Content of polyethylene glycol 3350 reference standard.

[0042] In some embodiments of the present invention, the main component of the polyethylene glycol powder is polyethylene glycol 3350.

[0043] In some embodiments of the present invention, the excipients included in the polyethylene glycol powder include citric acid and sodium citrate.

[0044] In this embodiment, the formulation of the polyethylene glycol powder is as follows:

[0045] The present invention will be further described below with reference to the embodiments.

[0046] Example 1: Method for testing the content of polyethylene glycol 3350 1.1 Chromatographic conditions Instrument: Waters Arc high performance liquid chromatograph; Detector: differential refractive index detector, model 2414; Test method: ChP2025 General Chapter 4 0512 High Performance Liquid Chromatography; Column: Waters Ultrahydrogel DP 7.8mm×30cm (or other equivalent column); Mobile phase: 0.05 mol / L sodium sulfate solution (pH 6.3); Preparation of mobile phase: Dissolve 7.1 g of sodium sulfate in 1000 ml of water, shake well, and the solution is ready.

[0047] Flow rate: 0.8 ml / min; Column temperature: 35℃; Injection volume: 20 μl; Detector temperature: 35℃; Run time: 20 minutes (approximately 2.5 times the main peak retention time).

[0048] 1.2 Solution Preparation Preparation of citrate solution: Weigh 15 mg of citric acid and 24 mg of sodium citrate, add 1000 ml of water to dissolve, shake well, and the solution is ready.

[0049] Reference solution: Accurately weigh approximately 20 mg of polyethylene glycol 3350 reference standard, place it in a 20 ml volumetric flask, add citrate solution and shake to dissolve, then dilute to the mark with citrate solution and shake well. Prepare two parallel solutions, namely reference solution 1 and reference solution 2.

[0050] Test solution: Accurately weigh an appropriate amount of this product (approximately equivalent to 500 mg of polyethylene glycol 3350), place it in a 500 ml volumetric flask, add water and shake to dissolve, then dilute with water to the mark and shake well. Prepare two parallel solutions, namely test solution 1 and test solution 2.

[0051] 1.3 Determination Method After the system has reached equilibrium, inject the sample according to the chromatographic conditions described in section 1.1. Inject reference solution 1 five times consecutively, with the RSD of the main peak area ≤ 2.0%. In the first chromatogram, the tailing factor of the main peak should be between 0.8 and 2.0. Inject reference solution 2 twice consecutively, and inject each of the test solutions twice. The recovery rate of reference solution 2 should be between 98.0% and 102.0%. Inject test solution 1 twice consecutively, and then inject test solution 2 twice consecutively. At the end, as a follow-up control, inject reference solution 1 once more, with the RSD of the main peak area ≤ 2.0%. Record the chromatograms separately and calculate the content of the main component of the product using the external standard method.

[0052] 1.4 System Applicability Requirements The reference solution should be injected five times consecutively. The RSD of the main peak area should be ≤2.0%, and the tailing factor of the main peak should be between 0.8 and 2.0.

[0053] 1.5 Calculation Formula ; In the formula: A i: Peak area of ​​the main peak in the test solution; A r The average peak area of ​​the main peak in the reference solution (5 consecutive injections); m r The sample weight of polyethylene glycol 3350 reference standard in the reference solution is (mg). V i : The volume of the test solution diluted, in ml; V r : Dilution volume of the reference solution, ml; B r Content of polyethylene glycol 3350 reference standard; Labeled quantity: 96.03%.

[0054] 1.6 Acceptable Standards Based on the peak area calculated using the external standard method, the content of polyethylene glycol 3350 in this product should be 90.0% to 110.0% of the labeled amount.

[0055] 1.7 The results of the content inspection of the three batches of samples are shown in the table below:

[0056] Example 2: Comparison of the effects of diluents 2.1 The diluent is water. Blank excipients: Weigh 0.36g of sucralose, 2.52g of citric acid, 4.07g of sodium citrate, and 0.08g of sweet orange microcapsule powder flavoring according to the prescription ratio, and mix thoroughly to obtain the blank excipients.

[0057] Reference solution: Weigh approximately 20 mg of polyethylene glycol 3350 reference standard accurately, place it in a 20 ml volumetric flask, add water and shake to dissolve, then dilute with water to the mark and shake well to obtain the solution.

[0058] 80% recovery sample solution: Accurately weigh 0.4 g of polyethylene glycol 3350 and 20 mg of blank excipient, place them in a 500 ml volumetric flask, add water and shake to dissolve, then dilute with water to the mark and shake well. Prepare 3 portions using the same method.

[0059] 100% recovery sample solution: Accurately weigh 0.5 g of polyethylene glycol 3350 and 20 mg of blank excipient, place them in a 500 ml volumetric flask, add water and shake to dissolve, then dilute with water to the mark and shake well. Prepare 3 portions using the same method.

[0060] 120% recovery rate sample solution: Accurately weigh 0.6 g of polyethylene glycol 3350 and 20 mg of blank excipient, place them in a 500 ml volumetric flask, add water and shake to dissolve, then dilute with water to the mark and shake well. Prepare 3 portions using the same method.

[0061] Take the above solution, inject it into the liquid chromatograph, and record the chromatogram. Calculate the recovery rate of polyethylene glycol 3350 using the external standard method. The results are shown in the table below.

[0062] Table 1. Results of recovery test when water was used as the diluent for the reference solution.

[0063] As shown in Table 1, the individual recoveries ranged from 102.5% to 103.5%, with a mean of 103.1% and an RSD of 0.4%, which does not meet the acceptable standard (98.0%–102.0%). The recoveries were all too high. The reason for this might be that the test solution contained citric acid, sodium citrate, sucralose, and flavorings, and the matrix of the reference solution was inconsistent with that of the test solution when water was used as the diluent. To avoid the influence of the diluent on the polyethylene glycol 3350 content test, and to ensure that the matrix of the reference solution and the test solution were as consistent as possible, the diluent for the reference solution was changed to the prescribed amount of citrate solution for the experiment, since the amount of sucralose and sweet orange microcapsule powder flavoring added in the formulation was relatively small.

[0064] 2.2 The diluent is the prescribed amount of citrate solution. Diluent: Citrate solution (Weigh 15mg of citric acid and 24mg of sodium citrate, add 1000ml of water to dissolve, shake well, and you will get the solution).

[0065] Reference solution: Weigh approximately 20 mg of polyethylene glycol 3350 reference standard accurately, place it in a 20 ml volumetric flask, add citrate solution and shake to dissolve, then dilute to the mark with citrate solution and shake well to obtain the solution.

[0066] 80% recovery sample solution: Accurately weigh 0.4 g of polyethylene glycol 3350 and 20 mg of blank excipient, place them in a 500 ml volumetric flask, add water and shake to dissolve, then dilute with water to the mark and shake well. Prepare 3 portions using the same method.

[0067] 100% recovery sample solution: Accurately weigh 0.5 g of polyethylene glycol 3350 and 20 mg of blank excipient, place them in a 500 ml volumetric flask, add water and shake to dissolve, then dilute with water to the mark and shake well. Prepare 3 portions using the same method.

[0068] 120% recovery rate sample solution: Accurately weigh 0.6 g of polyethylene glycol 3350 and 20 mg of blank excipient, place them in a 500 ml volumetric flask, add water and shake to dissolve, then dilute with water to the mark and shake well. Prepare 3 portions using the same method.

[0069] Take the above solution, inject it into the liquid chromatograph, and record the chromatogram. Calculate the recovery rate of polyethylene glycol 3350 using the external standard method. The results are shown in the table below.

[0070] Table 2. Recovery test results when the diluent for the reference solution is the prescribed amount of citrate solution.

[0071] As shown in Table 2, the individual recovery rates were all between 99.6% and 101.0%, with a mean of 100.3% and an RSD of 0.5%, which met the acceptable standard (98.0% to 102.0%). Therefore, the diluent for the reference solution was changed to the prescribed amount of citrate solution.

[0072] Example 3: Methodological Validation of Polyethylene Glycol 3350 Disperse Content 3.1 System Applicability Diluent: Citrate solution (Weigh 15mg of citric acid and 24mg of sodium citrate, add 1000ml of water to dissolve, shake well, and you will get the solution).

[0073] Reference solution: Weigh approximately 20 mg of polyethylene glycol 3350 accurately, place it in a 20 ml volumetric flask, add diluent and shake to dissolve, then dilute to the mark with diluent and shake well to obtain the solution.

[0074] Take the above solution and inject it five times consecutively, recording the chromatograms; the results are shown in the table below.

[0075] Table 3. Results of Methodological Validation System Applicability

[0076] As shown in Table 3, the peak area RSD of the main peak was 0.5% after five consecutive injections of the reference solution, and the tailing factor of the main peak was 1.36, indicating that the system has good applicability.

[0077] 3.2 Specificity Diluent: Citrate solution (Weigh 15mg of citric acid and 24mg of sodium citrate, add 1000ml of water to dissolve, shake well, and you will get the solution).

[0078] Blank excipient solution: Weigh approximately 20 mg of the blank excipient of this product accurately, place it in a 500 ml volumetric flask, add water and shake to dissolve, then dilute with water to the mark and shake well to obtain the solution.

[0079] Reference standard positioning solution: Weigh approximately 20 mg of polyethylene glycol 3350 accurately, place it in a 20 ml volumetric flask, add diluent and shake to dissolve, then dilute to the mark with diluent and shake well to obtain the solution.

[0080] Test solution: Weigh approximately 0.52 g of this product accurately, place it in a 500 ml volumetric flask, add water and shake to dissolve, then dilute with water to the mark and shake well to obtain the test solution.

[0081] Take the above solutions, inject them into the liquid chromatograph, and record the chromatograms; the results are shown in Table 4 and Figure 1 .

[0082] Table 4 Results of Method Validation Specificity Tests

[0083] As can be seen from the above test results and comparison charts, no peaks were detected in the diluent and blank excipient, and neither interfered with the content test of polyethylene glycol 3350 in this product; the retention time of the main peak of the test solution was consistent with the retention time of the main peak of the reference solution; indicating that this method has good specificity for the content test of this product.

[0084] 3.3 Limit of detection and limit of quantitation Diluent: Citrate solution (Weigh 15mg of citric acid and 24mg of sodium citrate, add 1000ml of water to dissolve, shake well, and you will get the solution).

[0085] Limit of Quantitation (LOQ) Stock Solution: Accurately weigh approximately 50 mg of polyethylene glycol 3350, place it in a 50 ml volumetric flask, add diluent to dissolve and dilute to the mark, and shake well to obtain the solution.

[0086] Limit of Detection Solution: Accurately measure 1 ml of the limit of quantitation stock solution, place it in a 100 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0087] Limit of Quantitation Solution: Accurately measure 1 ml of the limit of quantitation stock solution, place it in a 50 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0088] Inject the above-mentioned limit of detection solution once, and the limit of quantitation solution six times consecutively, and record the chromatograms; the results are shown in Tables 5 and 6.

[0089] Table 5. Results of the method validation test for the detection limit.

[0090] As shown in Table 5, the signal-to-noise ratio of the polyethylene glycol 3350 peak is 25.0, and the detection limit concentration is 10.1360 μg / ml, which is equivalent to 1% of the sample concentration; this meets the acceptable standard, indicating that the method has good sensitivity.

[0091] Table 6 Results of Limit of Quantitation Tests for Method Validation

[0092] As shown in Table 6, the signal-to-noise ratio of polyethylene glycol 3350 peak is 34.7~56.0, the peak area RSD is 10.7%, and the limit of quantitation concentration is 20.2720 μg / ml, which is equivalent to 2% of the sample concentration; these values ​​meet the acceptable standard, indicating that this method has good sensitivity for the quantitative detection of polyethylene glycol 3350.

[0093] 3.4 Linear Diluent: Citrate solution (Weigh 15mg of citric acid and 24mg of sodium citrate, add 1000ml of water to dissolve, shake well, and you will get the solution).

[0094] Linear solution (1): Weigh approximately 25 mg of polyethylene glycol 3350 accurately, place it in a 50 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and the solution is ready.

[0095] Linear solution (2): Weigh approximately 40 mg of polyethylene glycol 3350 accurately, place it in a 50 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and you will get the solution.

[0096] Linear solution (3): Weigh approximately 50 mg of polyethylene glycol 3350 accurately, place it in a 50 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and you will get the solution.

[0097] Linear solution (4): Weigh approximately 60 mg of polyethylene glycol 3350 accurately, place it in a 50 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and the solution is ready.

[0098] Linear solution (5): Weigh approximately 75 mg of polyethylene glycol 3350 accurately, place it in a 50 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and you will get the solution.

[0099] Take the above linear solutions, inject them into the liquid chromatograph, and record the chromatograms; the results are shown in Table 7.

[0100] Table 7 Results of Linear Experiments for Methodological Validation

[0101] As shown in Table 7, the correlation coefficient r of polyethylene glycol 3350 in the range of 0.5092 mg / ml to 1.5188 mg / ml (equivalent to 50% to 150% of the concentration of the test sample) is 0.9999, which is greater than 0.999, indicating that the concentration and peak area of ​​polyethylene glycol 3350 have a good linear relationship in this concentration range.

[0102] 3.5 accuracy Diluent: Citrate solution (Weigh 15mg of citric acid and 24mg of sodium citrate, add 1000ml of water to dissolve, shake well, and you will get the solution).

[0103] Reference solution: Weigh approximately 20 mg of polyethylene glycol 3350 reference standard accurately, place it in a 20 ml volumetric flask, add citrate solution and shake to dissolve, then dilute to the mark with citrate solution and shake well to obtain the solution.

[0104] 80% recovery sample solution: Accurately weigh 0.4 g of polyethylene glycol 3350 and 20 mg of blank excipient, place them in a 500 ml volumetric flask, add water and shake to dissolve, then dilute with water to the mark and shake well. Prepare 3 portions using the same method.

[0105] 100% recovery sample solution: Accurately weigh 0.5 g of polyethylene glycol 3350 and 20 mg of blank excipient, place them in a 500 ml volumetric flask, add water and shake to dissolve, then dilute with water to the mark and shake well. Prepare 3 portions using the same method.

[0106] 120% recovery rate sample solution: Accurately weigh 0.6 g of polyethylene glycol 3350 and 20 mg of blank excipient, place them in a 500 ml volumetric flask, add water and shake to dissolve, then dilute with water to the mark and shake well. Prepare 3 portions using the same method.

[0107] Take the above solutions, inject them into the liquid chromatograph, and record the chromatograms; calculate the recovery rate according to the external standard method; the results are shown in Table 8.

[0108] Table 8 Results of Method Validation Accuracy Test

[0109] As shown in Table 8, the individual recovery rates were all between 100.1% and 101.2%, with a mean of 100.7% and an RSD of 0.4%, which meet the acceptable standard. This indicates that the method is accurate for testing the content of polyethylene glycol 3350 in this product.

[0110] 3.6 Precision Preparation of citrate solution: Weigh 15 mg of citric acid and 24 mg of sodium citrate, add 1000 ml of water to dissolve, shake well, and the solution is ready.

[0111] Reference solution: Weigh approximately 20 mg of polyethylene glycol 3350 reference standard accurately, place it in a 20 ml volumetric flask, add citrate solution and shake to dissolve, then dilute to the mark with citrate solution and shake well to obtain the solution.

[0112] Test solution: Accurately weigh approximately 520 mg of this product, place it in a 500 ml volumetric flask, add water and shake to dissolve, then dilute with water to the mark and shake well. Prepare 6 portions using the same method.

[0113] Take the above solutions, inject them into a liquid chromatograph, record the chromatograms, and calculate the relative standard deviation of the main component content in the 6 test solutions using the external standard method. This is the repeatability. In the same laboratory, at different times, different analysts performed the above repeatability test, and the relative standard deviation of the main component content in the 12 test solutions was calculated. This is the intermediate precision. The results of the intermediate precision are shown in Table 9.

[0114] Table 9. Results of Precision Tests for Method Validation

[0115] As shown in Table 9, the RSD of the polyethylene glycol 3350 content in the 6 sample solutions was 0.3%, indicating that the method has good repeatability for the determination of polyethylene glycol 3350 content in this product.

[0116] The RSD of the polyethylene glycol 3350 content results in the 12 sample solutions was 0.3%, indicating that the method has good precision for the determination of polyethylene glycol 3350 content in this product.

[0117] 3.7 Durability 3.7.1 Solution stability Preparation of citrate solution: Weigh 15 mg of citric acid and 24 mg of sodium citrate, add 1000 ml of water to dissolve, shake well, and the solution is ready.

[0118] Reference solution: Weigh approximately 20 mg of polyethylene glycol 3350 reference standard accurately, place it in a 20 ml volumetric flask, add citrate solution and shake to dissolve, then dilute to the mark with citrate solution and shake well to obtain the solution.

[0119] Test solution: Accurately weigh about 520 mg of this product, place it in a 500 ml volumetric flask, add water and shake to dissolve, then dilute with water to the mark and shake well to obtain the test solution.

[0120] The above-mentioned reference solution and test solution were placed at room temperature for 50 h, respectively. At appropriate time points, 20 μl of each solution was taken and injected into the liquid chromatograph, and the chromatograms were recorded to examine the solution stability. The stability results are shown in Table 10.

[0121] Table 10 Results of Solution Stability Tests for Method Validation

[0122] As shown in Table 10, after 50 hours at room temperature, the peak area ratios of both the reference solution and the test solution at each time point to 0 h were between 1.00 and 1.01. No impurity peaks interfering with the determination of the main component were generated in the test solution. This indicates that both the reference solution and the test solution exhibited good stability within 50 hours at room temperature.

[0123] 3.7.2 Chromatographic condition robustness Diluent: Citrate solution (Weigh 15mg of citric acid and 24mg of sodium citrate, add 1000ml of water to dissolve, shake well, and you will get the solution).

[0124] Blank excipient solution: Weigh approximately 20 mg of the blank excipient of this product accurately, place it in a 500 ml volumetric flask, add water and shake to dissolve, then dilute with water to the mark and shake well to obtain the solution.

[0125] Reference solution: Weigh approximately 20 mg of polyethylene glycol 3350 accurately, place it in a 20 ml volumetric flask, add diluent and shake to dissolve, then dilute to the mark with diluent and shake well to obtain the solution.

[0126] Test solution: Weigh approximately 0.52 g of this product accurately, place it in a 500 ml volumetric flask, add water and shake to dissolve, then dilute with water to the mark and shake well to obtain the test solution.

[0127] Fine-tuning the chromatographic conditions, under normal conditions, column temperature (30℃, 40℃), flow rate (0.7 ml / min, 0.9 ml / min), and with different batches of chromatographic columns, 20 μl of each of the above solutions were injected into the liquid chromatograph, and the chromatograms were recorded. The robustness test results are shown in Table 11.

[0128] Table 11 Results of Methodological Validation Robustness Tests

[0129] As shown in Table 11, after fine-tuning the chromatographic conditions, the RSD of the main peak area of ​​the reference solution after five consecutive injections was between 0.2% and 0.6%; the tailing factor of the main peak of the reference solution was between 1.32 and 1.38, which met the system suitability requirements.

[0130] The percentage ratio of the main component content measured in the test solution to the content result under normal conditions was between 99.5% and 100.8%, indicating that the method has good robustness for testing the content of polyethylene glycol 3350 in this product.

[0131] Example 4: Results of mobile phase content testing for other salt concentration solutions Based on the chromatographic conditions of Example 1, the concentration of the salt solution in the mobile phase was adjusted. The mobile phases used were 0.005 mol / L sodium sulfate solution (prepared by dissolving 0.71 g of sodium sulfate in 1000 ml of water and shaking well), 0.01 mol / L sodium sulfate solution (dissolving 1.42 g of sodium sulfate in 1000 ml of water and shaking well), and 0.1 mol / L sodium sulfate solution (dissolving 14.2 g of sodium sulfate in 1000 ml of water and shaking well). Other chromatographic conditions were the same as in Example 1. The preparation methods for the diluent, blank excipient, reference solution, and test solution were the same as in Example 1. Each of the above solutions was injected into the liquid chromatograph, and the chromatograms were recorded. The results are shown in Table 12 and... Figures 2-4 : Table 12 Results of mobile phase inspection for different salt solutions

[0132] The above experimental results and comparison charts show that when 0.005~0.1 mol / L sodium sulfate solution is used as the mobile phase, neither the diluent nor the blank excipient interferes with the detection of polyethylene glycol 3350 content in this product, and the inverted peak after the main peak does not interfere with the detection of polyethylene glycol 3350. This method is suitable for the detection of polyethylene glycol 3350 content in this product. However, because the concentration of 0.1 mol / L sodium sulfate solution is relatively high, it is easy to cause column wear, and this concentration of mobile phase is not suitable for sample content detection.

[0133] Comparative Example 1: The content of polyethylene glycol 3350 was examined using chromatographic methods with different mobile phases. Based on the chromatographic conditions of Example 1, the concentration of the salt solution in the mobile phase was reduced. A 0.0001 mol / L sodium sulfate solution was used as the mobile phase. The mobile phase was prepared by dissolving 14.2 mg of sodium sulfate in 1000 ml of water and shaking well. Other chromatographic conditions were the same as in Example 1. The preparation methods for the diluent, blank excipient, reference solution, and test solution were the same as in Example 1. The above solutions were injected into the liquid chromatograph, and the chromatograms were recorded. The results are shown in Table 14 and... Figure 5 As shown: Table 14 Results for 0.0001 mol / L sodium sulfate solution as mobile phase

[0134] As can be seen from the above test results and comparison chart, both the diluent and the blank excipient interfere with the detection of polyethylene glycol 3350 content in this product under this method. Therefore, this method is not suitable for the detection of polyethylene glycol 3350 content in this product.

[0135] Comparative Example 2: The content of polyethylene glycol 3350 was examined using chromatographic methods with different mobile phases. Based on the chromatographic conditions of Example 1, the concentration of the salt solution in the mobile phase was reduced. A 0.001 mol / L sodium sulfate solution was used as the mobile phase. The mobile phase was prepared by dissolving 0.142 g of sodium sulfate in 1000 ml of water and shaking well. Other chromatographic conditions were the same as in Example 1. The preparation methods for the diluent, blank excipient, reference solution, and test solution were the same as in Example 1. Each of the above solutions was injected into the liquid chromatograph, and the chromatograms were recorded. The results are shown in Table 15 and... Figure 6 As shown: Table 15 Results for a mobile phase of 0.001 mol / L sodium sulfate solution

[0136] As can be seen from the above test results and comparison chart, the diluent and blank excipient do not interfere with the detection of polyethylene glycol 3350 content in this product under this method. However, the inverted peak after the main peak interferes with the detection of polyethylene glycol 3350. Therefore, this method is not suitable for the detection of polyethylene glycol 3350 content in this product.

[0137] Comparative Example 3: The content of polyethylene glycol 3350 was detected using chromatographic methods with different mobile phases. Based on the chromatographic conditions of Example 1, the composition of the salt solution in the mobile phase was changed, and the mobile phase was prepared using different salt compositions as described below: 3.1 The mobile phase used was a 0.05 mol / L sodium nitrate solution. The preparation method of the mobile phase was as follows: take 4.25 g of sodium nitrate, add 1000 ml of water to dissolve it, and shake well.

[0138] 3.2 The mobile phase used was a 0.05 mol / L sodium bicarbonate solution. The preparation method of the mobile phase was as follows: take 4.2 g of sodium bicarbonate, add 1000 ml of water to dissolve it, and shake well.

[0139] 3.3 The mobile phase used was 0.05 mol / L ammonium acetate solution. The preparation method of the mobile phase was as follows: take 3.85 g of ammonium acetate, add 1000 ml of water to dissolve it, and shake well.

[0140] 3.4 The mobile phase used was 0.05 mol / L ammonium dihydrogen phosphate solution. The preparation method of the mobile phase was as follows: take 5.75 g of ammonium dihydrogen phosphate, add 1000 ml of water to dissolve it, and shake well.

[0141] Other chromatographic conditions were the same as in Example 1. The preparation methods for diluent, blank excipient, reference solution, and test solution were the same as in Example 1. Each of the above solutions was injected into the liquid chromatograph, and the chromatograms were recorded. The results are shown in Table 16. Figures 7-10 As shown.

[0142] Table 16 Results of mobile phase inspection for different salt solutions

[0143] From Table 16 and Figures 7-10 The results show that when the mobile phase contains 0.05 mol / L sodium nitrate or 0.05 mol / L sodium bicarbonate, neither the diluent nor the blank excipient interferes with the detection of polyethylene glycol 3350 content in this product; however, the inverted peak after the main peak interferes with the detection of polyethylene glycol 3350. When the mobile phase contains 0.05 mol / L ammonium acetate, the normal chromatographic peak cannot be detected. When the mobile phase contains 0.05 mol / L ammonium dihydrogen phosphate, both the diluent and the blank excipient interfere with the detection of polyethylene glycol 3350 content in this product, and the inverted peak after the main peak interferes with the detection of polyethylene glycol 3350. The chromatographic conditions of the mobile phase with the above components are not suitable for the detection of polyethylene glycol 3350 content in this product.

[0144] Comparative Example 4: Reproduction of the detection method in the prior art Chromatographic conditions: Chromatographic column: Waters Ultrahydrogel DP 7.8mm×30cm, the same packing material as the method in patent CN110672738, both being aqueous gel chromatography columns (different brands of chromatographic columns). A salt solution stock solution was prepared by adding water to 500 ml of sodium chloride, potassium chloride, sodium bicarbonate, sodium sulfate, and sodium saccharin, along with 0.175 g of sodium chloride, 0.090 g of potassium chloride, 0.200 g of sodium bicarbonate, 0.685 g of sodium sulfate, and 0.012 g of sodium saccharin. 40 ml of the stock solution was then added to 1000 ml of water to form the mobile phase. The flow rate was 0.8 ml / min; the column temperature was 35℃; the differential refractive index detector temperature was 35℃; the injection volume was 20 μL; and the elution method was isocratic elution.

[0145] Solution preparation: Diluent: Water or citrate solution (Weigh 15mg of citrate and 24mg of sodium citrate, add 1000ml of water to dissolve, shake well, and you have the solution).

[0146] Blank excipient solution: Weigh approximately 20 mg of the blank excipient of this product accurately, place it in a 500 ml volumetric flask, add water and shake to dissolve, then dilute with water to the mark and shake well to obtain the solution.

[0147] Reference standard positioning solution: Weigh approximately 20 mg of polyethylene glycol 3350 accurately, place it in a 20 ml volumetric flask, add citrate solution and shake to dissolve, then dilute to the mark with citrate solution and shake well to obtain the solution.

[0148] Test solution: Weigh approximately 0.52 g of this product accurately, place it in a 500 ml volumetric flask, add water and shake to dissolve, then dilute with water to the mark and shake well to obtain the test solution.

[0149] Take the above solutions, inject them into the liquid chromatograph, and record the chromatograms; the results are shown in Table 17 and... Figure 11 As shown.

[0150] Table 17 Results of Existing Techniques for Testing Polyethylene Glycol 3350 Content

[0151] From Table 17 and the test results and Figure 11 As can be seen from the comparison chart, the overall concentration of buffer salt was relatively high in this experiment, and the inverted peak disappeared under this experimental system. However, under these chromatographic conditions, both the diluent and the blank excipient eluted near the main peak, interfering with the detection of polyethylene glycol 3350 content in this product. Adjusting the ratio of mobile phase salt components did not improve the situation, indicating that this patented method is not suitable for the content detection of this product. At the same time, due to the high overall concentration of buffer salt, it has an adverse effect on the chromatographic column and is not suitable for the content detection of large batches of samples.

[0152] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for testing the content of polyethylene glycol powder, characterized in that, High performance liquid chromatography (HPLC) was used under the following chromatographic conditions: a differential refractive index detector was used, a Waters Ultrahydrogel DP column was used as the chromatographic column, and sodium sulfate solution was used as the mobile phase.

2. The method for testing the content of polyethylene glycol powder according to claim 1, characterized in that, The mobile phase is a sodium sulfate solution with a concentration of 0.005~0.05 mol / L.

3. The method for testing the content of polyethylene glycol powder according to claim 1, characterized in that, The chromatographic column was a Waters Ultrahydrogel DP 7.8mm × 30cm.

4. The method for testing the content of polyethylene glycol powder according to claim 1, characterized in that, The column temperature is 30~40℃.

5. The method for testing the content of polyethylene glycol powder according to claim 1, characterized in that, The mobile phase flow rate is 0.7~0.9 ml / min.

6. The method for testing the content of polyethylene glycol powder according to claim 1, characterized in that, The detection instrument was a WatersArc high-performance liquid chromatograph.

7. The method for testing the content of polyethylene glycol powder according to claim 1, characterized in that, Includes the following steps: S1, Preparation of sample solution: Preparation of citrate solution: Prepare the prescribed amount of citrate solution; Reference solution: Place polyethylene glycol reference standard in a container and add citrate solution to prepare a solution with a concentration of 1 ± 0.1 mg / ml; Test solution: Accurately weigh the sample and add water to prepare a solution with a concentration of 1 ± 0.1 mg / ml; S2, Determination Method: After the chromatographic system has reached equilibrium, inject the reference solution and the test solution under the described chromatographic conditions, record the chromatograms, and calculate the content of the main component of the product using the external standard method; the calculation formula is: ； In the formula: A i : Peak area of ​​the main peak in the test solution; A r The average peak area of ​​the main peak in the reference solution; m r The sample weight of polyethylene glycol 3350 reference standard in the reference solution is (mg). V i : The volume of the test solution diluted, in ml; A r : Dilution volume of the reference solution, ml; B r Content of polyethylene glycol 3350 reference standard.

8. The method for testing the content of polyethylene glycol powder according to claim 1, characterized in that, The polyethylene glycol powder includes polyethylene glycol 3350 powder and polyethylene glycol 4000 powder.

9. The method for testing the content of polyethylene glycol powder according to claim 1, characterized in that, The polyethylene glycol powder is polyethylene glycol 3350 powder.

10. The method for testing the content of polyethylene glycol powder according to claim 1, characterized in that, The polyethylene glycol powder contains excipients including citric acid and sodium citrate.

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