Method for measuring content of lactic acid and / or acetic acid in cefuroxime sodium by HPLC (High Performance Liquid Chromatography) method

Abstract

The invention provides a method for determining the content of lactic acid and / or acetic acid in cefuroxime sodium by an HPLC (High Performance Liquid Chromatography) method. High performance liquid chromatography is adopted, and chromatographic conditions are as follows: a chromatographic column takes octadecyl silane bonded silica gel resistant to a 100% water phase as a filler; a phosphoric acid aqueous solution is used as a mobile phase A, and methanol or acetonitrile is used as a mobile phase B; and gradient elution. According to the method, the content of acetic acid and the content of lactic acid can be measured at the same time, the separation and detection efficiency is improved, and it is ensured that the quality of cefuroxime sodium is controllable.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of analytical chemistry, and particularly relates to a method for determining the content of lactic acid and / or acetic acid in cefuroxime sodium by HPLC. BACKGROUND

[0002] Cefuroxime sodium is the sodium salt of cefuroxime, and belongs to the second generation of cephalosporin antibiotics. It has a broad antibacterial spectrum, and is effective against most gram-negative bacteria, including Haemophilus influenzae, Streptococcus pneumoniae, Meningococcus, Escherichia coli, Klebsiella, Proteus mirabilis, Enterobacter, Citrobacter, Salmonella, Shigella and some indole-positive denitrifying bacteria. The antibacterial spectrum of gram-positive bacteria is similar to that of cefadroxil, mainly including Staphylococcus and Streptococcus. Cefuroxime sodium is relatively stable to β-lactamase, and its stability is not inferior to that of the third generation of cephalosporins. It is stable to almost all standard enzymes and β-lactamase extracted from clinically isolated negative bacilli, and many β-lactamase-producing bacteria, including gram-positive and gram-negative bacteria, are sensitive to it. Cefuroxime sodium is stable to negative bacillus enzyme, and has an effect on negative bacilli comparable to that of the third generation of cephalosporins. Moreover, it is stable to staphylococcus enzyme, and has a stronger effect on staphylococcus than the third generation of cephalosporins. These advantages make it the first choice for treating gram-negative bacterial or gram-negative and gram-positive bacterial mixed infections. In addition, cefuroxime sodium also has good pharmacokinetic properties, such as low protein binding rate, high free blood concentration, wide in vivo distribution, good tissue permeability, high bioavailability after intramuscular injection, and low nephrotoxicity.

[0003] In the existing synthesis process of cefuroxime sodium, sodium acetate and sodium lactate are often used in the final salting step (such as CN101812076A), resulting in the presence of acetic acid and lactic acid residues in the product. However, there are few reports on the determination of acid residues, especially the residual amount of acetic acid and lactic acid, in cephalosporin products. Excessive residues of acetic acid and lactic acid can cause damage to equipment and recipients.

[0004]

[0005] Since acetic acid and lactic acid have strong polarity, they are almost not retained in conventional reverse phase chromatography, and have relatively weak ultraviolet absorption. The product has a low limit, and a higher test sample concentration is required, which puts a heavy burden on the chromatographic column and chromatographic instrument, resulting in short service life of the chromatographic column and low repeatability.

[0006] On the other hand, lactic acid can be mostly decomposed in the stomach and is non-toxic, but it has sensitization effect on local damaged tissues. Since the recipient (such as humans and other mammals) does not have an enzyme system to metabolize D-lactic acid, D-lactic acid cannot be metabolized after entering the recipient, and excessive intake can cause an increase in blood uric acid level, causing metabolic disorders. Acetic acid is an impurity that needs to be controlled in the residual solvent determination method in the fourth appendix of the 2020 edition of the Chinese Pharmacopoeia (0861).

[0007] In order to further ensure the quality of the medicine, ensure the safety of the medicine, control the impurities within the safety limit, realize the controllable quality of the medicine, a method for effectively determining the content of lactic acid and / or acetic acid in cefuroxime sodium is needed to effectively control the quality of cefuroxime sodium. SUMMARY

[0008] In view of the deficiencies in the prior art, the present application provides a method for determining the content of lactic acid and / or acetic acid in cefuroxime sodium by HPLC.

[0009] To achieve the above-mentioned purpose, the technical scheme of the present application is:

[0010] A method for determining the content of lactic acid and / or acetic acid in cefuroxime sodium by HPLC, using high performance liquid chromatography, the chromatographic conditions include:

[0011] The chromatographic column is filled with octadecylsilane bonded silica gel resistant to 100% water phase;

[0012] Phosphoric acid aqueous solution is used as mobile phase A, and methanol or acetonitrile is used as mobile phase B;

[0013] Gradient elution: at 0 minutes, the volume fraction of mobile phase A is 95%-99%, and the volume fraction of mobile phase B is 1%-5%; at 5 minutes, the volume fraction of mobile phase A is 95%-99%, and the volume fraction of mobile phase B is 1%-5%; at 10 minutes, the volume fraction of mobile phase A is 45-55%, and the volume fraction of mobile phase B is 45%-55%; at 15 minutes, the volume fraction of mobile phase A is 45-55%, and the volume fraction of mobile phase B is 45%-55%; at 17 minutes, the volume fraction of mobile phase A is 95%-99%, and the volume fraction of mobile phase B is 1%-5%; at 25 minutes, the volume fraction of mobile phase A is 95%-99%, and the volume fraction of mobile phase B is 1%-5%.

[0014] According to the present application, the chromatographic column is selected from one of Agilent ZORBAX SB-Aq, Waters WelchUltimateLP-C18 or Phenomenex Titank C18, preferably Agilent ZORBAX SB-Aq 4.6*250 5-Micron.

[0015] According to the present application, the volume concentration of the phosphoric acid aqueous solution is 0.05%-0.2%, preferably 0.1%. The preparation method is to mix phosphoric acid and water to obtain, wherein the volume percentage of phosphoric acid in the volume of solvent water is 0.05%-0.2%.

[0016] According to the application, preferably, the mobile phase B is acetonitrile.

[0017] According to the application, preferably, the gradient elution is as follows: at 0 minute, the volume ratio of the mobile phase A is 99% and the volume ratio of the mobile phase B is 1%; at 5 minute, the volume ratio of the mobile phase A is 99% and the volume ratio of the mobile phase B is 1%; at 10 minute, the volume ratio of the mobile phase A is 50% and the volume ratio of the mobile phase B is 50%; at 15 minute, the volume ratio of the mobile phase A is 50% and the volume ratio of the mobile phase B is 50%; at 17 minute, the volume ratio of the mobile phase A is 99% and the volume ratio of the mobile phase B is 1%; at 25 minute, the volume ratio of the mobile phase A is 99% and the volume ratio of the mobile phase B is 1%.

[0018] According to the application, preferably, the chromatographic conditions further include that the column temperature of the chromatographic column is 20-40℃, preferably 30℃; the flow rate of the mobile phase is 0.9ml / min-1.1ml / min, preferably 1.0ml / min; the detection wavelength of the ultraviolet detector is 210nm; and the injection volume is 10μl.

[0019] According to the application, preferably, the sample preparation solvent is water.

[0020] According to the application, preferably, the impurity peak order is lactic acid, acetic acid in turn, and the separation degree of adjacent compound peaks is all greater than 1.5.

[0021] According to the application, in one preferred embodiment, the HPLC method for determining the content of lactic acid and / or acetic acid in cefuroxime sodium is as follows:

[0022] Solvent: water

[0023] Blank solution: water

[0024] Impurity mixed stock solution: lactic acid (LAC) and acetic acid (HAC) are weighed respectively, dissolved in solvent and constant volume, and diluted to prepare a solution containing 50μg of lactic acid and acetic acid per 1ml;

[0025] Reference solution: 1ml of the impurity mixed stock solution is placed in a 10ml volumetric flask, dissolved in solvent and constant volume, and diluted to prepare a solution containing 5μg of lactic acid and acetic acid per 1ml;

[0026] Test sample solution: cefuroxime sodium is weighed accurately, dissolved in solvent and constant volume, and diluted to prepare a solution containing about 1mg per 1ml;

[0027] Test sample solution: cefuroxime sodium is weighed accurately, dissolved in solvent and constant volume, and diluted to prepare a solution containing about 1mg per 1ml;

[0028] Chromatographic conditions: the chromatographic column is Agilent ZORBAX SB-Aq 4.6*250 5-Micron; the mobile phase A is 0.1% phosphoric acid aqueous solution; the mobile phase B is acetonitrile; the flow rate of the mobile phase is 1.0 ml / min; the ultraviolet detector detection wavelength is 210 nm; the column temperature is 30 DEG C; the injection volume is 10 mu l;

[0029] Gradient elution: at 0 min, the volume ratio of mobile phase A is 99%, and the volume ratio of mobile phase B is 1%; at 5 min, the volume ratio of mobile phase A is 99%, and the volume ratio of mobile phase B is 1%; at 10 min, the volume ratio of mobile phase A is 50%, and the volume ratio of mobile phase B is 50%; at 15 min, the volume ratio of mobile phase A is 50%, and the volume ratio of mobile phase B is 50%; at 17 min, the volume ratio of mobile phase A is 99%, and the volume ratio of mobile phase B is 1%; at 25 min, the volume ratio of mobile phase A is 99%, and the volume ratio of mobile phase B is 1%;

[0030] Determination method: accurately taking the blank solution, the test sample solution, the control sample solution and the test sample standard solution, respectively injecting into the liquid chromatograph, and recording the chromatogram; the content of lactic acid and / or acetic acid is calculated by the external standard method.

[0031] The technical features and beneficial effects of the present application are as follows:

[0032] The HPLC method provided by the present application can simultaneously determine the content of acetic acid and lactic acid in cefuroxime sodium, and has the advantages of simple method, low cost, strong specificity and applicability, high detection sensitivity, good linear relationship, high precision and accuracy, good durability of the detection method, and high detection efficiency, which has important significance for realizing the quality control of cefuroxime sodium.

[0033] In the method of the present application, increasing the proportion of organic phase B can significantly shorten the retention of the target substance, shorten the detection time, save the cost, and need to be strictly controlled.

[0034] The detection method of the present application as a whole, the specific type of chromatographic column, combined with specific mobile phase and elution conditions, etc., jointly act to realize the excellent effect of the present application; any change of condition will affect the detection effect. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 : High performance liquid chromatogram of blank solution in example 1;

[0036] Figure 2 : High performance liquid chromatogram of control sample solution in example 1;

[0037] Figure 3 : High performance liquid chromatogram of test sample solution in example 1;

[0038] Figure 4 HPLC chromatogram of test sample solution of Example 1;

[0039] Figure 5 Linear relationship graph of lactic acid in test example;

[0040] Figure 6 Linear relationship graph of acetic acid in test example. DETAILED DESCRIPTION

[0041] The preferred embodiments of the present application will be described in detail below, and the test methods of the specific conditions not mentioned in the preferred embodiments are generally carried out according to the conventional conditions. The described embodiments are only a part of the embodiments of the present application, which can better illustrate the content of the present application, but are not all the embodiments. Based on the embodiments in the present application, the person skilled in the art can make non-essential improvements and adjustments to the embodiments, which all belong to the scope of protection of the present application.

[0042] Example 1:

[0043] A method for determining the content of lactic acid and / or acetic acid in cefuroxime sodium by HPLC, which adopts high performance liquid chromatography,

[0044] High performance liquid chromatograph: Agilent 1260 high performance liquid chromatography system and workstation;

[0045] Solvent: water

[0046] Blank solution: water

[0047] Impurity mixed stock solution: Take appropriate amount of lactic acid (LAC) and acetic acid (HAC), respectively, dissolve and dilute to prepare a solution containing about 50 μg of lactic acid and acetic acid per 1 ml.

[0048] Control solution: Take 1 ml of impurity mixed stock solution and add solvent to a 10 ml volumetric flask, dissolve and dilute to prepare a solution containing about 5 μg of lactic acid and acetic acid per 1 ml.

[0049] Test sample solution: Take appropriate amount of cefuroxime sodium, accurately weigh and add solvent to dissolve and dilute to prepare a solution containing about 1 mg per 1 ml.

[0050] Test sample solution: Take appropriate amount of cefuroxime sodium, accurately weigh and add solvent to dissolve and dilute to prepare a solution containing about 1 mg per 1 ml.

[0051] Chromatographic conditions: the chromatographic column was Agilent ZORBAX SB-Aq 4.6*250 5-Micron; mobile phase A: 0.1% phosphoric acid aqueous solution; mobile phase B: acetonitrile; flow rate of mobile phase was 1.0 ml / min; UV detector detection wavelength was 210 nm; column temperature was 30°C; injection volume was 10 μl.

[0052] Gradient elution:

[0053]

[0054] Determination method: accurately measure the blank solution, test solution, control solution and test standard solution, respectively inject into the liquid chromatograph, and record the chromatogram. The content of lactic acid and acetic acid is calculated by external standard method.

[0055] Figure 1 The high performance liquid chromatogram of the blank solution is as follows: Figure 2 The high performance liquid chromatogram of the control solution is as follows: Figure 3 The high performance liquid chromatogram of the test solution is as follows: Figure 4 The high performance liquid chromatogram of the test standard solution is as follows. By calculation, the content of lactic acid in the test solution is 3.97 μg / ml, and acetic acid is not detected. The content of lactic acid in the test standard solution is 9.560 μg / ml, and the content of acetic acid is 4.963 μg / ml.

[0056] Test example:

[0057] Chromatographic conditions: the chromatographic column was Agilent ZORBAX SB-Aq 4.6*250 5-Micron; mobile phase A: 0.1% phosphoric acid aqueous solution; mobile phase B: acetonitrile; flow rate of mobile phase was 1.0 ml / min; UV detector detection wavelength was 210 nm; column temperature was 30°C; injection volume was 10 μl.

[0058] Gradient elution:

[0059]

[0060] 1. Linear test

[0061] Solvent: water;

[0062] Linear stock solution: take lactic acid (LAC) and acetic acid (HAC) appropriately, dissolve and constant volume with solvent, and dilute to prepare a solution containing about 50 μg of lactic acid and acetic acid per 1 ml.

[0063] Linear solution 1: take 0.5 mL of linear stock solution, add solvent to constant volume in a 25 mL volumetric flask, and shake well to obtain.

[0064] Linear solution 2: take 1 mL of linear stock solution 1, place in a 25 mL volumetric flask, dilute to volume with solvent and shake to mix.

[0065] Linear solution 3: take 2.5 mL of linear stock solution 1, place in a 25 mL volumetric flask, dilute to volume with solvent and shake to mix.

[0066] Linear solution 4: take 4 mL of linear stock solution 1, place in a 25 mL volumetric flask, dilute to volume with solvent and shake to mix.

[0067] Linear solution 5: take 5 mL of linear stock solution 1, place in a 25 mL volumetric flask, dilute to volume with solvent and shake to mix.

[0068] Take the above test solutions in turn into the high performance liquid chromatograph, and the test results are as shown in Table 1. The linear relationship diagram of lactic acid is as shown in Figure 5 The linear relationship diagram of acetic acid is as shown in Figure 6 .

[0069] Table 1 Linear results of lactic acid (LAC)

[0070]

[0071] Table 2 Linear results of acetic acid (HAC)

[0072]

[0073] From Table 1-Table 2 and Figure 5 and 6 It can be seen that the concentration of lactic acid (LAC) and acetic acid (HAC) is in the range of 1.0 μg / ml-10.0 μg / ml, and the concentration and peak area have a good linear relationship.

[0074] 2. Detection limit, limit of quantification test

[0075] Limit of quantification solution: accurately take linear solution 1 as the limit of quantification solution.

[0076] Detection limit solution: accurately take 3 ml of the limit of quantification solution into a 10 ml volumetric flask, dilute to the mark with solvent and shake to mix.

[0077] Take the above test solutions in turn into the high performance liquid chromatograph, and the test results are as shown in Table 1. The linear relationship diagram of lactic acid is as shown in

[0078] Table 3 Limit of quantification results

[0079]

[0080] Table 4 Detection limit results

[0081]

[0082] From table 3-table 4, the detection limit concentration of lactic acid (LAC) is 0.3ug / ml, and the quantitative limit concentration is 1.0ug / ml.

[0083] 3. Accuracy test

[0084] Control solution: take linear solution 3 as the control solution.

[0085] Test solution: accurately weigh 25.55mg of cefuroxime sodium, put it into a 25mL volumetric flask, dilute and dissolve with solvent, and then add solvent to constant volume.

[0086] Low concentration accuracy solution: weigh about 25mg of cefuroxime sodium, put it into a 25mL volumetric flask, add 0.5ml of linear stock solution, dissolve and dilute with solvent to constant volume, shake well, and prepare 3 parallel samples.

[0087] Medium concentration accuracy solution: weigh about 25mg of cefuroxime sodium, put it into a 50mL volumetric flask, add 2.5ml of linear stock solution, dissolve and dilute with solvent to constant volume, shake well, and prepare 3 parallel samples.

[0088] High concentration accuracy solution: weigh about 25mg of cefuroxime sodium, put it into a 50mL volumetric flask, add 5ml of linear stock solution, dissolve and dilute with solvent to constant volume, shake well, and prepare 3 parallel samples.

[0089] Take the above test solutions in turn and inject them into the high performance liquid chromatograph, and the test results are as follows.

[0090] Table 5 lactic acid (LAC) accuracy results

[0091]

[0092]

[0093] Table 6 acetic acid (HAC) accuracy results

[0094]

[0095] From table 5-table 6, the accuracy of the method of the present application is good.

[0096] The method of the present application can simultaneously determine the content of acetic acid and lactic acid, improve the efficiency of separation and detection, and ensure the controllable quality of cefuroxime sodium. The detection method has a short running time (25min), simple mobile phase preparation (0.1% phosphoric acid solution), high sensitivity, and quantitative limit concentration: lactic acid 1ug / ml, acetic acid 1ug / ml.

Claims

1. A method for determining the content of lactic acid and / or acetic acid in cefuroxime sodium by HPLC, characterized in that, High performance liquid chromatography (HPLC) was used, and the chromatographic conditions included: The chromatographic column is packed with octadecylsilane-bonded silica gel that is resistant to 100% aqueous phase. Using an aqueous phosphoric acid solution as mobile phase A and methanol or acetonitrile as mobile phase B; Gradient elution: At 0 minutes, the volume percentage of mobile phase A is 95%-99%, and the volume percentage of mobile phase B is 1%-5%; at 5 minutes, the volume percentage of mobile phase A is 95%-99%, and the volume percentage of mobile phase B is 1%-5%; at 10 minutes, the volume percentage of mobile phase A is 45%-55%, and the volume percentage of mobile phase B is 45%-55%; at 15 minutes, the volume percentage of mobile phase A is 45%-55%, and the volume percentage of mobile phase B is 45%-55%; at 17 minutes, the volume percentage of mobile phase A is 95%-99%, and the volume percentage of mobile phase B is 1%-5%; at 25 minutes, the volume percentage of mobile phase A is 95%-99%, and the volume percentage of mobile phase B is 1%-5%.

2. The method for determining the content of lactic acid and / or acetic acid in cefuroxime sodium by HPLC according to claim 1, characterized in that, The chromatographic column is selected from one of Agilent ZORBAX SB-Aq, Waters Welch Ultimate LP-C18 or Phenomenex Titan C18, preferably Agilent ZORBAX SB-Aq 4.6*250 5-Micron.

3. The method for determining the content of lactic acid and / or acetic acid in cefuroxime sodium by HPLC according to claim 1, characterized in that, The volume concentration of the phosphoric acid aqueous solution is 0.05%-0.2%, preferably 0.1%; it is prepared by mixing phosphoric acid and water, wherein the volume percentage of phosphoric acid to the volume of the solvent water is 0.05%-0.2%.

4. The method for determining the content of lactic acid and / or acetic acid in cefuroxime sodium by HPLC according to claim 1, characterized in that, Mobile phase B is acetonitrile.

5. The method for determining the content of lactic acid and / or acetic acid in cefuroxime sodium by HPLC according to claim 1, characterized in that, Gradient elution: At 0 minutes, the volume percentage of mobile phase A is 99% and that of mobile phase B is 1%; at 5 minutes, the volume percentage of mobile phase A is 99% and that of mobile phase B is 1%; at 10 minutes, the volume percentage of mobile phase A is 50% and that of mobile phase B is 50%; at 15 minutes, the volume percentage of mobile phase A is 50% and that of mobile phase B is 50%; at 17 minutes, the volume percentage of mobile phase A is 99% and that of mobile phase B is 1%; at 25 minutes, the volume percentage of mobile phase A is 99% and that of mobile phase B is 1%.

6. The method for determining the content of lactic acid and / or acetic acid in cefuroxime sodium by HPLC according to claim 1, characterized in that, The chromatographic conditions also include: a column temperature of 20-40℃, preferably 30℃; a mobile phase flow rate of 0.9ml / min-1.1ml / min, preferably 1.0ml / min; a UV detector wavelength of 210nm; and an injection volume of 10μl.

7. The method for determining the content of lactic acid and / or acetic acid in cefuroxime sodium by HPLC according to claim 1, characterized in that, The solvent used to prepare the sample was water.

8. The method for determining the content of lactic acid and / or acetic acid in cefuroxime sodium by HPLC according to claim 1, characterized in that, The impurity peaks eluted in the order of lactic acid and acetic acid, and the resolution of adjacent compound peaks was greater than 1.5.

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