Method for determining acetate and malate of compound electrolyte injection (II) by liquid chromatography
The detection of acetate and malate in compound electrolyte injection (II) was solved by liquid chromatography, and the problems of low accuracy and poor reproducibility in the prior art were solved, and efficient and accurate quality control detection was achieved.
Patent Information
- Application Number
- CN202411995033.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-31
- Publication Date
- 2025-04-15
AI Technical Summary
The existing enzyme-catalyzed ultraviolet spectrophotometry is used to determine the problem of low accuracy and poor reproducibility of acetate and malate in compound electrolyte injection (II).
Liquid chromatography was used to detect acetate and malate by high-performance liquid chromatography, and sulfonic acid polystyrene and divinylbenzene interpolymer cation exchange resin H type was used as the filler, and appropriate detection wavelength, flow rate, retention time and column temperature were set, and the appropriate amount of reference solution and test solution were combined for detection.
High accuracy and good reproducibility detection of acetate and malate in compound electrolyte injection (II) is achieved, meeting the quality control needs of the preparation.
Smart Images
Figure BDA0005224382870000021 
Figure BDA0005224382870000031 
Figure BDA0005224382870000032
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of drug detection, and specifically relates to a method for determining acetate and malate of compound electrolyte injection (II) by liquid chromatography. Background Art
[0002] Compound electrolyte injection (II) is a water and electrolyte supplement and alkalizer, and also has physiological functions. Malic acid can directly participate in human metabolism, promote aerobic basal metabolism, reduce blood ammonia concentration, reduce the damage of anticancer drugs to normal cells, and has an anti-fatigue effect. It can also treat anemia and is suitable for liver dysfunction and liver failure. It is helpful to human health and can also prevent edema, high blood pressure and fat accumulation.
[0003] Compound electrolyte injection (II) is used to treat isotonic dehydration accompanied by or expected to occur acidosis, and to replenish the loss of extracellular fluid. This product is a compound preparation, and its main components are (each 1000ml injection contains): sodium chloride 6.799g, potassium chloride 0.2984g, calcium chloride (CaCl2·2H2O) 0.3675g, magnesium chloride (MgCl2·6H2O) 0.2033g, sodium acetate (C2H3NaO2·3H2O) 3.266g, L-malic acid 0.671g, sodium hydroxide 0.200g, and water for injection.
[0004] At present, the quality standard of compound electrolyte injection (II) is not included in the pharmacopoeia of various countries, and only its import drug registration standard (JX20110067) is found. In this standard, acetate and malate are both determined by enzyme-catalyzed ultraviolet spectrophotometry, which is easily interfered by various factors during the detection process, and has low accuracy and poor reproducibility. Summary of the invention
[0005] The object of the present invention is to provide a method for determining acetate and malate in compound electrolyte injection (II) by liquid chromatography, thereby achieving quality control of the preparation.
[0006] The method for determining acetate and malate in compound electrolyte injection (II) by liquid chromatography is to detect acetate and malate by high performance liquid chromatography, sulfonic acid polystyrene and divinylbenzene copolymer cation exchange resin H type is used as filler, detection wavelength is 200-210nm, flow rate is 0.5-1.0ml / min, retention time of acetate is 16min, retention time of malate is 12min, column temperature is 50-90℃, injection volume is 5-50μl, and mobile phase is 0.005mol / L sulfuric acid solution. Preferably, the flow rate is 0.6ml per minute; column temperature is 80℃; detection wavelength is 210nm; injection volume is 20μl.
[0007] The specific steps of the method are:
[0008] (1) Reference solution: Sodium acetate and L-malic acid were taken as reference substances, respectively, dissolved in mobile phase and quantitatively diluted to prepare a mixed solution containing 2 mg of sodium acetate and 0.67 mg of L-malic acid per 1 ml. Two portions were prepared in parallel.
[0009] (2) Compound electrolyte injection was used as the test solution. 20 μl of the test solution and the reference solution were accurately measured and injected into the liquid chromatograph respectively. The chromatogram was recorded and the peak area was calculated by the external standard method. The calculation formula is as follows:
[0010]
[0011] Where:
[0012] A s , A i : are the peak areas of sodium acetate and L-malic acid in the reference solution and the test solution, respectively;
[0013] W s : is the sample weight of sodium acetate and L-malic acid in mg;
[0014] V s : dilution volume of sodium acetate and L-malic acid in the reference solution, ml;
[0015] P s : is the content of sodium acetate and L-malic acid reference substances, %;
[0016] M: The molecular weights of sodium acetate and L-malic acid are 82.03 and 134.09.
[0017] Qualified products contain 22.8-25.2mmol / L of acetate and 4.75-5.25mmol / L of malate.
[0018] The method for determining acetate and malate in compound electrolyte injection (II) by liquid chromatography has strong specificity and high accuracy. DETAILED DESCRIPTION
[0019] 1. Exclusivity
[0020] (1) Blank excipient solution: The solution that does not contain acetate and malate in the preparation formula is the blank excipient solution.
[0021] (2) Test solution: Take this product, which is the test solution;
[0022] (3) Reference solution: Take appropriate amounts of sodium acetate and L-malic acid reference substances, weigh them accurately, add them to the mobile phase to dissolve them, and quantitatively dilute them to make a mixed solution containing approximately 2 mg of sodium acetate (anhydrous) and 0.67 mg of L-malic acid per 1 ml. Prepare 2 portions in parallel.
[0023] Accurately measure 20 μl of blank solvent, blank auxiliary material solution, test solution and reference solution, inject into liquid chromatography, and record the chromatogram. From the chromatogram, it can be seen that the blank solvent and blank auxiliary material do not interfere with the detection of acetate and malate, indicating that this method has good specificity.
[0024] 2. System precision
[0025] Take reference solution 1# (5 injections in a row) and reference solution 2# (2 injections in a row) under the specificity item and record the chromatogram. The results are shown in Table 1.
[0026] Table 1 System precision test results
[0027]
[0028] The test results show that: the theoretical plate number in the chromatogram of the reference solution is not less than 2000 according to L-malic acid and sodium acetate, the reference solution 1# was injected 5 times continuously, the RSD of the peak area of 5 injections was ≤2.0%, the RSD of the retention time was ≤1.0%, and the recovery rate of the response factor of the two reference solutions was between 98.0% and 102.0%, indicating that the system has good precision and can meet the test requirements.
[0029] 3. Linearity and range
[0030] Take appropriate amount of L-malic acid reference substance and sodium acetate reference substance, weigh them accurately, dilute them into solutions with limit concentration of 20% to 200%, accurately measure 20 μl of each of the above solutions, inject them into liquid chromatograph, and record the chromatogram. In the concentration range of 20% to 200%, linear regression analysis is performed with concentration as the horizontal axis (X) and peak area as the vertical axis (Y). The results are shown in Tables 2 and 3.
[0031] Table 2 Acetate linearity results
[0032]
[0033]
[0034] Table 3 Malate linearity results
[0035]
[0036] The test results show that the linear equation between the concentration and the peak area of acetate in the range of 4.704μg / ml to 47.037μg / ml is y=67.2871x+5.8220, r=1.0000; the linear equation between the concentration and the peak area of malate in the range of 0.938μg / ml to 9.375μg / ml is y=210.9669x+3.2105, r=1.0000. This shows that the method has a good linear relationship.
[0037] 4. Accuracy
[0038] Reference solution: Take appropriate amount of sodium acetate and L-malic acid reference substances respectively, weigh accurately, add mobile phase to dissolve and quantitatively dilute to make a mixed solution containing approximately 2 mg of sodium acetate (calculated as anhydrous substance) and 0.67 mg of L-malic acid per 1 ml, prepare 2 portions in parallel.
[0039] Spiked test solution: Prepare spiked test solution equivalent to 50%, 100%, and 150% concentration levels in triplicate.
[0040] Accurately measure 20 μl of each of the above solutions, inject them into the liquid chromatograph, and record the chromatogram. The results are shown in Table
[0041] Table 4 Accuracy results
[0042]
[0043] The test results show that the average recovery rates of acetate and malate (n=9) should be between 98.0% and 102.0%; the RSD of the recovery rates are all ≤2.0%, indicating that the method has good accuracy.
[0044] 5. Repeatability
[0045] Take this product to obtain the test solution, and prepare 6 portions in parallel.
[0046] Take appropriate amounts of sodium acetate and L-malic acid reference substances respectively, weigh accurately, add mobile phase to dissolve and quantitatively dilute to make a mixed solution containing approximately 2 mg of sodium acetate (anhydrous) and 0.67 mg of L-malic acid per 1 ml. Prepare 2 portions in parallel as reference substance solutions.
[0047] Accurately measure 20 μl of the test solution and the reference solution, inject them into the liquid chromatograph, and record the chromatogram. The results are shown in Table 5.
[0048] Table 5 Repeatability test results
[0049]
[0050] The test results show that when 6 samples were measured in parallel, the RSD of acetate and malate contents was less than 2.0%, indicating that the method has good repeatability.
[0051] 6. Intermediate precision
[0052] Another tester performed the same operation as in the repeatability test. The results are shown in Table 6.
[0053] Table 6 Intermediate precision test results
[0054]
[0055] The test results show that: the RSDs of acetate and malate contents in 12 samples were all less than 2.0% when the method was used for parallel determination, indicating that the intermediate precision of the method was good.
[0056] 7. Solution stability
[0057] Take this product to obtain the test sample solution.
[0058] Take appropriate amounts of sodium acetate and L-malic acid reference substances respectively, weigh accurately, add mobile phase to dissolve and quantitatively dilute to make a mixed solution containing approximately 2 mg of sodium acetate (anhydrous) and 0.67 mg of L-malic acid per 1 ml, as the reference substance solution.
[0059] Place at room temperature, and accurately measure 20 μl of the reference solution and the test solution at 0, 4, 12, 24, 36, and 48 hours, inject into the liquid chromatograph, and record the chromatogram. The results are shown in Tables 7 and 8.
[0060] Table 7 Reference solution stability solution results
[0061]
[0062] Table 8 Test solution stability results
[0063]
[0064] The test results show that: at room temperature, the reference solution and the test solution are stable after being placed for 48 hours.
[0065] 8. Durability
[0066] Take the solution prepared according to the prescribed amount (excluding sodium acetate and L-malic acid) as the blank excipient solution.
[0067] Take this product to obtain the test sample solution.
[0068] Take appropriate amounts of sodium acetate and L-malic acid reference substances respectively, weigh accurately, add mobile phase to dissolve and quantitatively dilute to make a mixed solution containing approximately 2 mg of sodium acetate (anhydrous) and 0.67 mg of L-malic acid per 1 ml, as the reference substance solution.
[0069] Under standard conditions, flow rate 0.6±0.1ml / min, column temperature 80±5℃, different serial number chromatographic columns, accurately measure 20μl of blank solvent (mobile phase), blank auxiliary material solution, reference solution (5 injections in a row) and test solution, and inject them into the chromatograph, record the chromatogram, and the results are shown in Table 9
[0070] Table 9 Durability results
[0071]
[0072] Conclusion: After fine-tuning the chromatographic conditions, there was no interference in the detection of blank solvent and blank auxiliary material solution. Compared with the standard conditions, the content ratio of acetate and malate under each condition should be between 98.0% and 102.0%, indicating that this method has good durability.
Claims
1. A method for determining acetate and malate in compound electrolyte injection (II) by liquid chromatography, characterized in that: The method is to use high performance liquid chromatography to detect acetate and malate, sulfonic acid polystyrene and divinylbenzene copolymer cation exchange resin H type as filler, detection wavelength of 200-210nm, flow rate of 0.5-1.0ml / min, retention time of acetate of 16min, retention time of malate of 12min, column temperature of 50-90℃, injection volume of 5-50μl, mobile phase of 0.005mol / L sulfuric acid solution.
2. The method according to claim 1, characterized in that The specific steps of the method are: (1) Reference solution: Sodium acetate and L-malic acid were taken as reference substances, respectively, dissolved in mobile phase and quantitatively diluted to prepare a mixed solution containing 2 mg of sodium acetate and 0.67 mg of L-malic acid per 1 ml. Two portions were prepared in parallel. (2) Compound electrolyte injection was used as the test solution. 20 μl of the test solution and the reference solution were accurately measured and injected into the liquid chromatograph respectively. The chromatogram was recorded and the peak area was calculated by the external standard method. The calculation formula is as follows: Where: A s , A i : are the peak areas of sodium acetate and L-malic acid in the reference solution and the test solution, respectively; W s : is the sample weight of sodium acetate and L-malic acid in mg; V s : dilution volume of sodium acetate and L-malic acid in the reference solution, ml; P s : is the content of sodium acetate and L-malic acid reference substances, %; M: The molecular weights of sodium acetate and L-malic acid are 82.03 and 134.09.