Method for detecting related substances of mannitol sorbitol injection

Abstract

The invention relates to the technical field of analytical chemistry, and particularly discloses a method for detecting related substances of a mannitol sorbitol injection. According to the method disclosed by the invention, maltitol, maltitol and D-iduronol in the mannitol sorbitol injection are detected by adopting a high performance liquid chromatography. According to the method, the strong cation calcium type exchange column with the sulfonated cross-linked styrene-divinyl benzene copolymer as the filler is adopted to quantitatively detect the related substances containing maltitol, maltitol and D-iduronol, and the related substances of the mannitol sorbitol injection can be accurately detected.

Description

Technical Field

[0001] The present invention relates to the technical field of analytical chemistry, and specifically discloses a method for detecting related substances in mannitol and sorbitol injection. Background Art

[0002] Mannitol and sorbitol injection, as a dehydrating agent, is widely used in the treatment of cerebral edema, diuresis, and reduction of intraocular pressure caused by various reasons. In the case of dealing with drug overdose or poisoning, this drug can promote the excretion of harmful substances and prevent nephrotoxicity. However, related substances generally exist in drugs, and these substances mainly come from starting materials, intermediates, polymers, side reaction products in the production process, and degradation products during storage. The content of related substances is a direct indicator to measure the purity of drugs, and their existence may affect the purity and quality of drugs, and further affect the safety and effectiveness of drugs. Therefore, it is crucial to establish an effective analytical method to control the content of these impurities.

[0003] The related substances of mannitol and sorbitol injection are generally considered to include maltitol and D-iditol, but the current detection methods are easily interfered by other impurities, resulting in poor separation of mannitol and sorbitol related substances, thus reducing the accuracy of detection. Therefore, providing a detection method with high accuracy is still the top priority of current research. Summary of the Invention

[0004] Aiming at the problem that cannot be accurately measured in the prior art, the present invention provides a method for detecting related substances in mannitol and sorbitol injection.

[0005] In order to solve the above technical problems, the technical solution provided by the present invention is:

[0006] A method for detecting related substances in mannitol and sorbitol injection, wherein the related substances include maltitol, maltitol and D-iditol, and high performance liquid chromatography is used for detection. The chromatographic conditions are as follows:

[0007] (1) Preparation of test solution and reference solution:

[0008] Take the test sample of mannitol and sorbitol injection and prepare a test solution with a solution;

[0009] Take the maltitol reference substance and prepare a maltitol reference solution with a solvent;

[0010] Take the maltitol reference substance and prepare a maltitol reference solvent with a solvent;

[0011] Take the D-iditol reference substance and prepare a D-iditol reference solvent with a solvent;

[0012] (2) The test solution and the reference solution are detected by high performance liquid chromatography, and the detection conditions of the high performance liquid chromatography are as follows:

[0013] Chromatographic column: a strong cation calcium type exchange column filled with sulfonated cross-linked styrene divinylbenzene copolymer;

[0014] Differential detector;

[0015] Mobile phase: water;

[0016] Isocratic elution.

[0017] Compared with the prior art, the method for detecting related substances of mannitol and sorbitol injection provided by the present invention uses a strong cation calcium type exchange column filled with sulfonated cross-linked styrene divinylbenzene copolymer to quantitatively detect related substances including maltitol, maltitol and D-iditol, and can accurately detect the related substances of mannitol and sorbitol injection. The method for detecting related substances of mannitol and sorbitol injection provided by the present invention is simple to operate, has good separation effect and low detection limit, provides an effective guarantee for improving and better controlling the quality of mannitol and sorbitol injection, is beneficial to ensuring the consistency and stability of the product quality of mannitol and sorbitol injection, and thus is beneficial to improving the clinical safety and effectiveness of mannitol and sorbitol injection.

[0018] Preferably, the detection temperature of the differential detector is 54 - 56 °C.

[0019] More preferably, the detection temperature of the differential detector is 55 °C.

[0020] The preferred detection temperature can improve the baseline stability of mannitol and sorbitol injection and avoid the occurrence of baseline drift problems due to temperature effects.

[0021] Preferably, the length of the strong cation calcium type exchange column is 200 mm - 400 mm, the inner diameter is 5.0 mm - 10.0 mm, and the filler diameter is 1.0 μm - 5.0 μm.

[0022] Preferably, the strong cation calcium type exchange column is a calcium type exchange column of Phenomenex.

[0023] The preferred chromatographic column can make the peak shape of mannitol and sorbitol injection, the resolution between mannitol and sorbitol and impurities, and the detection sensitivity all excellent, so as to be beneficial to the effective separation of mannitol and sorbitol from impurities, and the results are accurate, reliable and have good repeatability.

[0024] Preferably, the flow rate of the high performance liquid chromatography is 0.3 mL / min - 0.7 mL / min.

[0025] Preferably, the flow rate of the high performance liquid chromatography is 0.5 mL / min.

[0026] Preferably, the column temperature of the high performance liquid chromatography is 70°C - 90°C.

[0027] Preferably, the column temperature of the high performance liquid chromatography is 80°C.

[0028] Preferably, the injection volume of the high performance liquid chromatography is 15 μL - 25 μL.

[0029] Preferably, the injection volume of the high performance liquid chromatography is 20 μL.

[0030] Preferably, the solvent is water.

[0031] The preferred detection conditions can achieve a higher resolution between mannitol sorbitol injection and impurities, thereby improving the accuracy of detection, shortening the peak emergence time of each component in mannitol sorbitol injection, and improving the detection efficiency.

[0032] Preferably, based on the total mass of mannitol and sorbitol, the mass concentration of the test solution is 4 mg / mL - 6 mg / mL.

[0033] More preferably, based on the total mass of mannitol and sorbitol, the mass concentration of the test solution is 5 mg / mL.

[0034] Preferably, the mass concentration of the reference solution is 0.5 mg / mL - 1.5 mg / mL.

[0035] More preferably, the mass concentration of the reference solution is 1 mg / mL.

[0036] In summary, the method for detecting related substances of mannitol sorbitol injection provided by the present invention uses a strong cation calcium type exchange column filled with sulfonated cross-linked styrene divinylbenzene copolymer to quantitatively detect related substances including maltitol, maltitol and D-iditol, and can accurately detect the related substances of mannitol sorbitol injection. Description of the Drawings

[0037] Figure 1 It is the chromatogram of the maltitol reference solution in Example 1;

[0038] Figure 2 It is the chromatogram of the maltitol reference solution in Example 1;

[0039] Figure 3 It is the chromatogram of the D-iditol reference solution in Example 1;

[0040] Figure 4Chromatogram of the system suitability solution in Example 1. Detailed implementation manners

[0041] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only one embodiment of the present invention, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0042] Example 1

[0043] This example provides a method for detecting related substances in mannitol and sorbitol injection, which specifically includes the following content:

[0044] (1) Preparation of test solution and reference solution:

[0045] Take mannitol and sorbitol injection, and prepare a test solution with a mannitol concentration of 5 mg / mL and a sorbitol concentration of 5 mg / mL with water.

[0046] Take maltitol reference substance and prepare a maltitol reference solution with a concentration of 1 mg / mL with water.

[0047] Take maltosyl sorbitol reference substance and prepare a maltitol reference solution with a concentration of 1 mg / mL with water.

[0048] Take D-iditol reference substance and prepare a maltitol reference solution with a concentration of 1 mg / mL with water.

[0049] (2) Use high performance liquid chromatography to detect the test solution and the reference solution, and record the chromatogram. The specific detection conditions of high performance liquid chromatography include:

[0050] Chromatographic column: a calcium type exchange column of Phenomenex filled with sulfonated cross-linked styrene divinylbenzene copolymer;

[0051] Chromatographic column specification: 7.8 mm × 300 mm, 5.0 μm;

[0052] Mobile phase: water;

[0053] Column temperature: 80 °C;

[0054] Flow rate: 0.5 mL / min;

[0055] Injection volume: 20 μL.

[0056] Differential refractive index detector, detection temperature: 55 °C.

[0057] 1. Specificity investigation

[0058] Take mannitol reference substance and sorbitol reference substance, dissolve them in water, and prepare a system suitability solution containing 5 mg of mannitol and 5 mg of sorbitol per 1 mL.

[0059] Take 20 μL of each reference solution and the test solution, and perform high-performance liquid chromatography detection respectively according to the above conditions, record the chromatogram. The chromatograms of each reference solution and the test solution are as Figures 1 - 4 shown, and the results are shown in Table 1. Among them, the chromatogram of the maltitol reference solution is shown in the appendix Figure 1 ; the chromatogram of the maltitol alcohol reference solution is shown in the appendix Figure 2 ; the chromatogram of the D-iditol reference solution is shown in the appendix Figure 3 ; the chromatogram of the system suitability solution is shown in the appendix Figure 4 .

[0060] Table 1 Results of system suitability test

[0061] Name Retention time / min Mannitol 13.206 Maltitol (peak 1) 14.869 Maltitol 16.871 Maltitol (peak 2) 17.467 Sorbitol 29.186 D-iditol 33.105

[0062] Comparing the appendix Figures 1 - 4 and Table 1, it can be seen that the separation effects of maltitol, maltitol alcohol, D-iditol, mannitol and sorbitol are good, and the specificity of this method is good.

[0063] 2. Linear range

[0064] Take the reference solutions and quantitatively dilute them with water to prepare a series of solutions with different concentrations as linear solutions. Perform high-performance liquid chromatography detection respectively according to the above conditions, record the peak areas. Take the peak areas as the ordinate and the concentrations of the reference solutions (μg / mL) as the abscissa, and make a linear regression curve. The results are shown in Tables 2-4.

[0065] Table 2 Results of linear test for D-iditol

[0066]

[0067] It can be seen from Table 2 that within the range of 0.0526 mg / mL - 1.0518 mg / mL for D-iditol, the relationship between the concentration and the peak area is good. The linear equation is y = 319415x - 2131.8, and R is 0.9999.

[0068] Table 3 Results of linear test for maltitol

[0069]

[0070] It can be seen from Table 3 that within the range of 0.0502 mg / mL - 1.0048 mg / mL for maltitol, the relationship between the concentration and the peak area is good. The linear equation is y = 333349x - 1479, and R is 0.9999.

[0071] Table 4 Results of the linearity test of maltitol

[0072]

[0073] As can be seen from Table 4, in the range of 0.0490 mg / mL - 0.9794 mg / mL, the relationship between the concentration of maltitol and the peak area is good. The linear equation is y = 329745x - 3167.7, and R is 0.9999.

[0074] 3. Detection of the limit of detection and limit of quantification

[0075] Take the control solutions and quantitatively dilute them with water to prepare a series of solutions with different concentrations as linear solutions. Perform high-performance liquid chromatography detection under the above conditions respectively. When the signal-to-noise ratio is 10:1, it is the limit of quantification; when the signal-to-noise ratio is 3:1, it is the limit of detection. The detection results are shown in Tables 5 - 6.

[0076] Table 5 Detection results of the limit of detection and limit of quantification

[0077]

[0078] Table 6 Repeated detection results of the limit of quantification

[0079] Peak area D-iditol Maltitol Maltitol Quantification limit 1 1465 1782 2700 Quantification limit 2 1557 1792 2635 Quantification limit 3 1521 1795 2710 Quantification limit 4 1377 1858 2420 Quantification limit 5 1529 1767 2591 Quantification limit 6 1543 1716 2646 Mean value 1498.7 1785.0 2617.0 RSD% 4.50 2.58 4.05

[0080] As can be seen from Tables 5 and 6, the limit of detection of D-iditol is 0.0026 mg / mL, which is equivalent to 0.002% of the sample concentration; the limit of quantification is 0.0053 mg / mL, which is equivalent to 0.004% of the sample concentration; the limit of detection of maltitol is 0.0025 mg / mL, which is equivalent to 0.002% of the sample concentration; the limit of quantification is 0.0050 mg / mL, which is equivalent to 0.003% of the sample concentration; the limit of detection of maltitol is 0.0025 mg / mL, which is equivalent to 0.002% of the sample concentration; the limit of quantification is 0.0098 mg / mL, which is equivalent to 0.007% of the sample concentration. It can be seen that the sensitivity of the impurities in the detection method of the mannitol and sorbitol injection provided by the present invention meets the detection requirements.

[0081] It can also be seen from Tables 5 and 6 that after repeated determination 6 times, the RSD% of the peak area of the limit of quantification of D-iditol is 4.50%, the RSD% of the peak area of the limit of quantification of maltitol is 2.58%, and the RSD% of the peak area of the limit of quantification of maltitol is 4.05%, all of which are less than 10%. It can be seen that the detection method of the related substances of the mannitol and sorbitol injection provided by the present invention meets the requirements of quantitative determination.

[0082] 4. Detection of solution stability

[0083] After placing each control solution for 0 h, 9 h, 15 h, and 67 h respectively, test and record the peak areas according to the above high performance liquid chromatography conditions. The results are shown in Table 7.

[0084] Table 7 Detection Results of Solution Stability

[0085] Peak area 0 h 9h 15h 67h Mean value RSD% D-iditol 1867613.2 1875476.1 1877645 1916691.4 1884356.4 1.17 Maltitol 1839752.7 1844665.9 1849111.7 1875459.6 1852247.5 0.86 Maltitol 1 852217.7 854949.5 855714.7 865684.2 857141.5 0.69 Maltitol 2 895188.7 880724.7 893278.5 895523 891178.7 0.79

[0086] As can be seen from Table 7, after standing at room temperature for 67 h, the peak areas of the mother liquors of each impurity showed no obvious change, indicating that the control solution was stable within at least 67 h when placed at room temperature.

[0087] 5. Repeatability Test

[0088] Take 5 mL of mannitol and sorbitol injection, dilute it with water to 10 mL to obtain the test solution.

[0089] Take 1 mL of the test solution and dilute it with water to 100 mL to obtain the control solution.

[0090] Prepare 6 parallel samples, test and record the chromatograms according to the above high performance liquid chromatography conditions. The detection results are shown in Table 8.

[0091] Table 8 Detection Results of Repeatability

[0092]

[0093] 6. Intermediate Precision Test

[0094] Take 5 mL of mannitol and sorbitol injection, dilute it with water to 10 mL to obtain the test solution.

[0095] Take 1 mL of the test solution and dilute it with water to 100 mL to obtain the control solution.

[0096] Prepare 6 parallel samples, test and record the chromatograms according to the above high performance liquid chromatography conditions, and compare with the detection results of repeatability. The detection results are shown in Table 9.

[0097] Table 9 Detection Results of Intermediate Precision

[0098]

[0099] The test results show that for 12 repeated determinations of the test solution, the RSD of the content determination results is 0.03%, indicating that the intermediate precision of the detection method provided by the present invention is good and meets the detection requirements.

[0100] 7. Accuracy Test

[0101] Take 300 mg of maltitol reference substance and prepare a maltitol reference substance mother liquor with a concentration of 15 mg / mL with water.

[0102] Take 300 mg of maltitol control substance, and prepare a mother solution of maltitol control substance with a concentration of 15 mg / mL using water.

[0103] Take 300 mg of D-iditol control substance, and prepare a mother solution of D-iditol control substance with a concentration of 15 mg / mL using water.

[0104] Take 1 mL of the D-iditol mother solution and 1 mL of each of the maltitol mother solutions, and add water to make up to 10 mL to obtain a mixed control solution.

[0105] Take 1 mL of the maltitol mother solution, and add water to make up to 10 mL to obtain a maltitol control solution.

[0106] Take 5 mL of mannitol and sorbitol injection, dilute it with water to 10 mL to obtain a blank test solution.

[0107] Take 5 mL of mannitol and sorbitol injection, 0.5 mL of the D-iditol mother solution and 0.5 mL of the maltitol mother solution, dilute it with water to 10 mL to obtain a recovery solution A.

[0108] Take 5 mL of mannitol and sorbitol injection, 1 mL of the D-iditol mother solution and 1 mL of the maltitol mother solution, dilute it with water to 10 mL to obtain a recovery solution B.

[0109] Take 5 mL of mannitol and sorbitol injection, 1.5 mL of the D-iditol mother solution and 1.5 mL of the maltitol mother solution, dilute it with water to 10 mL to obtain a recovery solution C.

[0110] Take 5 mL of mannitol and sorbitol injection, 0.5 mL of the D-iditol mother solution and 0.5 mL of the maltitol control substance solution, dilute it with water to 10 mL to obtain a recovery solution D.

[0111] Take 5 mL of mannitol and sorbitol injection, 1 mL of the D-iditol mother solution and 1 mL of the maltitol control substance solution, dilute it with water to 10 mL to obtain a recovery solution E.

[0112] Take 5 mL of mannitol and sorbitol injection, 1.5 mL of the D-iditol mother solution and 1.5 mL of the maltitol control substance solution, dilute it with water to 10 mL to obtain a recovery solution F.

[0113] Take 5 mL of mannitol and sorbitol injection, 0.5 mL of the maltitol mother solution and 0.5 mL of the maltitol control substance solution, dilute it with water to 10 mL to obtain a recovery solution G.

[0114] Take 5 mL of mannitol and sorbitol injection, 1 mL of the maltitol mother solution and 1 mL of the maltitol control substance solution, dilute it with water to 10 mL to obtain a recovery solution H.

[0115] Take 5 mL of mannitol sorbitol injection, 1.5 mL of maltitol mother liquor and 1.5 mL of maltitol mother liquor, dilute with water to 10 mL to obtain the recovery rate I solution;

[0116] The recovery rate A solution - the recovery rate I solution was prepared in parallel for 3 portions. Test and record the chromatogram according to the above high performance liquid chromatography conditions, and the test results are shown in Table 10 - Table 12.

[0117] Table 10 Results of the recovery rate test of D-iditol (maltitol)

[0118]

[0119]

[0120] Table 11 Results of the recovery rate test of D-iditol (maltitol)

[0121]

[0122] Table 12 Results of the recovery rate test of maltitol (maltitol)

[0123]

[0124] It can be seen from Table 10 - Table 12 that the recovery rate of D-iditol is between 93.78% and 102.31%, the average value is 100.1%, and the RSD of 9 measurement results is 2.52%; the recovery rate of maltitol is between 94.01% and 99.58%, the average value is 98.6%, and the RSD of 9 measurement results is 1.76%; the recovery rate of maltitol is between 100.10% and 104.18%, the average value is 101.7%, and the RSD of 9 measurement results is 1.54%. Thus, it can be seen that the accuracy of the detection method for related substances of the mannitol sorbitol injection provided by the present invention is good.

[0125] 8. Durability detection

[0126] Take mannitol sorbitol injection and prepare a test solution with a mannitol concentration of 5 mg / mL and a sorbitol concentration of 5 mg / mL with water;

[0127] Take maltitol reference substance and prepare a maltitol reference solution with a concentration of 1 mg / mL with water;

[0128] Take maltitol reference substance and prepare a maltitol reference solution with a concentration of 1 mg / mL with water;

[0129] Take D-iditol reference substance and prepare a maltitol reference solution with a concentration of 1 mg / mL with water;

[0130] Fine-tune the above high performance liquid chromatography conditions, and perform high performance liquid chromatography detection by changing the type of chromatographic column, flow rate, column temperature and detector temperature respectively, and record the chromatograms. The detection results are shown in Table 13.

[0131] Table 13 Results of durability test

[0132]

[0133] As can be seen from Table 13, after the flow rate and column temperature are changed within a small range, the contents of each impurity and the total impurity results do not change significantly. After replacing the chromatographic columns from different manufacturers, the contents of each impurity and the total impurity results do not change significantly. Therefore, the detection method for mannitol and sorbitol injection provided by the present invention has good durability.

Claims

1. A method for detecting related substances in mannitol and sorbitol injection, characterized in that, The related substances include maltitol, maltitol and D-iditol, and are detected by high performance liquid chromatography. The chromatographic conditions are as follows: (1) Preparation of test solution and reference solution: Take the mannitol and sorbitol injection as the test sample and prepare a test solution with a solvent; Take the maltitol reference substance and prepare a maltitol reference solution with a solvent; Take the maltitol reference substance and prepare a maltitol reference solution with a solvent; Take the D-iditol reference substance and prepare a D-iditol reference solution with a solvent; (2) Detect the test solution and reference solution by high performance liquid chromatography. The detection conditions of the high performance liquid chromatography are as follows: Chromatographic column: a strong cation calcium type exchange column filled with sulfonated cross-linked styrene divinylbenzene copolymer; Differential refractive index detector; Mobile phase: water; Isocratic elution.

2. The detection method for related substances of mannitol and sorbitol injection according to claim 1, characterized in that, The detection temperature of the differential refractive index detector is 54-56 °C.

3. The method for detecting related substances in mannitol and sorbitol injection according to claim 1, characterized in that, The length of the strong cation calcium type exchange column is 200 mm - 400 mm, the inner diameter is 5.0 mm - 10.0 mm, and the packing diameter is 3.0 μm - 7.0 μm.

4. The method for detecting related substances in mannitol and sorbitol injection according to claim 3, wherein, The strong cation calcium type exchange column is a calcium type exchange column of Phenomenex.

5. The method for detecting related substances in mannitol and sorbitol injection according to claim 1, characterized in that, The flow rate of the high performance liquid chromatography is 0.3 mL / min - 0.7 mL / min.

6. The method for detecting related substances in mannitol sorbitol injection according to claim 5, characterized in that, The flow rate of the high performance liquid chromatography is 0.5 mL / min.

7. The method for detecting related substances in mannitol and sorbitol injection according to claim 1, wherein, The column temperature of the high performance liquid chromatography is 70 °C - 90 °C.

8. The detection method for related substances of mannitol sorbitol injection according to claim 7, characterized in that, The column temperature of the high performance liquid chromatography is 80 °C.

9. The detection method for related substances of mannitol and sorbitol injection according to claim 1, characterized in that, The injection volume of the high performance liquid chromatography is 15 μL - 25 μL.

10. The method for detecting related substances in mannitol and sorbitol injection according to claim 9, characterized in that, The injection volume of the high performance liquid chromatography is 20 μL.

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