Method for detecting 1, 4-naphthoquinone and 1, 2-naphthoquinone in cefaclor raw material

Abstract

The invention relates to the technical field of pharmaceutical analysis, in particular to a method for detecting 1, 4-naphthoquinone and 1, 2-naphthoquinone in cefaclor raw materials. According to the method, a 0.1% formic acid aqueous solution is used as a mobile phase A; methanol is used as a mobile phase B; gradient elution is carried out, and the elution flow rate is 1.0 ml per minute; the column temperature is 40 DEG C; the sample injection volume is 20 microliters; the detection wavelength is 250nm. The invention also provides specific solution preparation and detection steps. The high performance liquid chromatography detection method for 1, 4-naphthoquinone and 1, 2-naphthoquinone in the cefaclor raw material provided by the invention has good specificity, linear relationship, repeatability, intermediate precision, accuracy, stability and durability.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, specifically to a method for detecting 1,4-naphthoquinone and 1,2-naphthoquinone in cefaclor raw materials. Background Technology

[0002] Cefaclor is a second-generation oral cephalosporin derivative with good antibacterial activity against Gram-positive bacteria such as Staphylococcus aureus, Staphylococcus epidermidis, hemolytic and viridans streptococci, and Streptococcus pneumoniae. Its structural formula is shown in Formula I. The antibacterial mechanism of cefaclor is through inactivating transpeptidase, interfering with bacterial cell wall synthesis, and thus inhibiting peptidoglycan cross-linking.

[0003]

[0004] Two impurities, 1,4-naphthoquinone and 1,2-naphthoquinone, generated during the current synthesis process of cefaclor active pharmaceutical ingredient (API), belong to the quinone class of compounds. According to the ICH M7 guidelines, quinones are potential mutagenic impurities containing warning structures, with an acceptable daily intake (TTC) of 20 μg / day (for treatment duration > 1–12 months). Genotoxic impurities can cause damage to human genetic material even at extremely low concentrations, posing a significant threat to drug safety; therefore, their content in cefaclor API needs strict control. However, to date, no sensitive and accurate detection method has been reported for 1,4-naphthoquinone and 1,2-naphthoquinone genotoxic impurities in cefaclor. Therefore, developing a simple, accurate, and reliable detection method to control 1,4-naphthoquinone and 1,2-naphthoquinone in cefaclor API is of great significance. Summary of the Invention

[0005] To address the problem that existing technologies cannot effectively detect 1,4-naphthoquinone and 1,2-naphthoquinone in cefaclor, this invention provides an accurate and reliable detection method. It is achieved through the following technical solution:

[0006] A method for detecting 1,4-naphthoquinone and 1,2-naphthoquinone in cefaclor raw material, comprising the following steps: High performance liquid chromatography (HPLC) is used to determine 1,4-naphthoquinone and 1,2-naphthoquinone in cefaclor raw material.

[0007] Step (1): Preparation of test solution: Weigh 60 mg of cefaclor accurately, place it in a 10 ml volumetric flask, add 5 ml of 0.5% ammonia water to dissolve it, dilute to the mark with acetonitrile, and shake well;

[0008] Step (2): Preparation of reference solution: Accurately measure 1 ml of reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with solution, and shake well;

[0009] Step (3): Inject the reference solution and the test solution into the liquid chromatograph separately and record the chromatograms. The high-performance liquid chromatography conditions are as follows:

[0010] Chromatographic column: C18 column;

[0011] Mobile phase A: 0.1% formic acid aqueous solution;

[0012] Mobile phase B: Methanol;

[0013] Gradient elution, wherein the gradient elution conditions are as follows:

[0014] 0 min, mobile phase A accounts for 90%, mobile phase B accounts for 10%;

[0015] 15 min, mobile phase A accounts for 60% and mobile phase B accounts for 40%;

[0016] 25 min, mobile phase A accounts for 40%, mobile phase B accounts for 60%;

[0017] 28 min, mobile phase A accounted for 10%, and mobile phase B accounted for 90%;

[0018] 35 min, mobile phase A accounts for 10%, mobile phase B accounts for 90%;

[0019] 35.1 min, mobile phase A accounts for 90%, mobile phase B accounts for 10%;

[0020] For 45 minutes, mobile phase A accounted for 90% and mobile phase B accounted for 10%.

[0021] Preferably, the test solution in step (1) is a freshly prepared solution ready for use.

[0022] Preferably, the preparation step of the reference stock solution in step (2) is as follows: accurately weigh 1,4-naphthoquinone reference standard, add acetonitrile to dissolve and quantitatively dilute to prepare a solution containing approximately 300 ng per 1 ml.

[0023] Preferably, the diluting solution in step (2) is a 0.5% ammonia-acetonitrile (50:50) solution.

[0024] Preferably, the chromatographic column in step (3) is a GL Sciences Inertsustain C18.

[0025] More preferably, the chromatographic column has the following specifications: 250mm × 4.6mm, and the packing material diameter is 5μm or a column with equivalent performance.

[0026] Preferably, in step (3), the elution flow rate is 1.0 ml per minute; the column temperature is 40°C; the injection volume is 20 μl; and the detection wavelength is 250 nm.

[0027] Preferably, the 1,4-naphthoquinone is calculated by peak area using the external standard method, and the 1,2-naphthoquinone is calculated by peak area after correction of 1,4-naphthoquinone (multiplied by a correction factor of 0.3). The total amount of 1,4-naphthoquinone and 1,2-naphthoquinone shall not exceed 5 ug / ml.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] This invention employs high performance liquid chromatography (HPLC) to determine the content of 1,4-naphthoquinone and 1,2-naphthoquinone in cefaclor raw material. The experiment uses 0.5% ammonia-acetonitrile (50:50) as the dilution solution. 1,4-naphthoquinone is calculated by peak area using the external standard method, and 1,2-naphthoquinone is calculated by the peak area after correction for 1,4-naphthoquinone (multiplied by a correction factor of 0.3). The total amount of 1,4-naphthoquinone and 1,2-naphthoquinone must not exceed 5 μg / ml.

[0030] This invention exhibits good specificity, linearity, repeatability, intermediate precision, accuracy, stability, and robustness. The relative standard deviation of sample repeatability is less than 10%, and the detection limit is 4.22% of the sample. It is applicable to the content of 1,4-naphthoquinone and 1,2-naphthoquinone in cefaclor raw material. 1,4-naphthoquinone shows good linearity in the range of 6.3895 ng / ml to 63.8947 ng / ml, with the linear equation: Y = 550.22X + 1599.9 and a correlation coefficient (R) = 0.9997 (R ≥ 0.998). Furthermore, this method is simple, reliable, and highly operable. Attached Figure Description

[0031] Figure 1 Example 2: Standard curve of 1,4-naphthoquinone in linear relationship experiment. Detailed Implementation

[0032] The embodiments of the technical solution of the present invention will be described in detail below with reference to the examples. The following embodiments are only used to illustrate the technical solution of the present invention more clearly, and are therefore only examples, and should not be used to limit the scope of protection of the present invention.

[0033] It should be noted that, unless otherwise specified, the experimental methods used in the implementation examples are all conventional methods; and the materials and reagents used are all commercially available unless otherwise specified.

[0034] In this invention, unless otherwise stated, the numerical range "a~b" represents an abbreviation of any combination of real numbers between a and b, where a and b are both real numbers. For example, the numerical range "0~5" means that all real numbers between "0~5" have been listed herein, and "0~5" is simply an abbreviation of these numerical combinations.

[0035] In this invention, unless otherwise specified, all embodiments and preferred embodiments mentioned herein can be combined to form new technical solutions.

[0036] In this invention, unless otherwise specified, all steps mentioned herein may be performed sequentially or randomly, but are preferably performed sequentially; for example, the method includes steps (1) and (2), indicating that the method may include steps (1) and (2) performed sequentially, or may include steps (2) and (1) performed sequentially; for example, the method may also include step (3), indicating that step (3) may be added to the method in any order, for example, the method may include steps (1), (2) and (3), or may include steps (1), (3) and (2), or may include steps (3), (1) and (2), etc.

[0037] In this invention, unless otherwise specified, the specific values ​​and substances in the embodiments of this invention can be combined with other features in the description of this invention; for example, if the specification mentions that the reaction temperature is 10 to 100°C, while the embodiment mentions that the reaction temperature is 20°C, then it can be considered that this invention has specifically disclosed the range of 10 to 20°C, or the range of 20 to 100°C, and this range can be combined with other features in the description to form a new technical solution.

[0038] Preparation of test solution: Weigh 60 mg of cefaclor accurately, place it in a 10 ml volumetric flask, add 5 ml of 0.5% ammonia water to dissolve it, dilute to the mark with acetonitrile, and shake well;

[0039] The preparation steps for the reference stock solution are as follows: Take an appropriate amount of 1,4-naphthoquinone reference standard, accurately weigh it, add acetonitrile to dissolve it, and quantitatively dilute it to prepare a solution containing approximately 300 ng per ml.

[0040] Preparation of reference solution: Accurately measure 1 ml of the reference stock solution and place it in a 10 ml volumetric flask. Dilute to the mark with the solution and shake well.

[0041] Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase (GL Sciences Inertsustain C18, 250 mm × 4.6 mm, 5 μm or equivalent column); 0.1% formic acid aqueous solution was used as mobile phase A, and methanol was used as mobile phase B, with gradient elution according to Table 1; the flow rate was 1.0 mL per minute; the column temperature was 40 °C; the injection volume was 20 μL; and the detection wavelength was 250 nm.

[0042] Table 1

[0043]

[0044] Determination method: Inject the test solution and the reference solution into the liquid chromatograph separately and record the chromatograms.

[0045] Example 1;

[0046] Specificity experiment

[0047] Blank solution: 0.5% ammonia-acetonitrile (50:50) solution.

[0048] Reference solution: Accurately measure 1 ml of the reference stock solution and place it in a 10 ml volumetric flask. Dilute to the mark with 0.5% ammonia-acetonitrile (50:50) solution and shake well.

[0049] Test solution: Weigh 60 mg of cefaclor accurately, place it in a 10 ml volumetric flask, add 5 ml of 0.5% ammonia water to dissolve it, dilute to the mark with acetonitrile, and shake well.

[0050] Take 10 μl each of the blank solution, reference solution and test solution, and perform detection according to the above chromatographic conditions.

[0051] The experimental results show that the test solution and the control solution do not interfere with each other, which meets the requirements.

[0052] Example 2;

[0053] Linear Relationship Experiment

[0054] Linear solutions: Accurately measure an appropriate amount of the reference stock solution and dilute it with 0.5% ammonia-acetonitrile (50:50) solution to obtain solutions containing approximately 6.3895 ng, 18.4526 ng, 27.5827 ng, 38.5264 ng, 51.2534 ng, and 63.8947 ng of 1,4-naphthoquinone per ml, respectively. Shake well to obtain linear solutions. Perform chromatographic analysis on each linear solution. Plot a standard curve with the 1,4-naphthoquinone injection concentration (ng / ml) on the x-axis and the peak area on the y-axis, as shown below. Figure 1 The regression equation was calculated, and the results are shown in Table 2.

[0055] Table 2. Linear experimental results of 1,4-naphthoquinone

[0056]

[0057] The results above show that when the concentration of 1,4-naphthoquinone is in the range of 6.3895 ng / ml to 63.8947 ng / ml and the injection volume is 10 μl, the linear equation is Y = 550.22X + 1599.9, the correlation coefficient (R) is 0.9997, and the peak area and concentration have a good linear relationship.

[0058] Example 3;

[0059] Repeatable experiments

[0060] Test solution: Accurately weigh 60 mg of cefaclor and place it in a 10 ml volumetric flask. Add 5 ml of 0.5% ammonia solution to dissolve it, then dilute to the mark with acetonitrile and shake well. Prepare 6 parallel solutions.

[0061] The above-mentioned test solution was subjected to chromatographic detection, and the results are shown in Table 3.

[0062] Table 3 Results of repeatability tests for 1,4-naphthoquinone

[0063]

[0064] As shown in Table 3 above, 1,4-naphthoquinone was detected in all 6 test sample solutions, indicating that this method has good repeatability.

[0065] Example 3;

[0066] Precision test

[0067] Reference solution: Accurately measure 1 ml of the reference stock solution and place it in a 10 ml volumetric flask. Dilute to the mark with 0.5% ammonia-acetonitrile (50:50) solution and shake well.

[0068] The above reference solution was subjected to chromatographic detection, with 6 injections. The experimental results are shown in Table 4.

[0069] Table 4. Precision Experiment Results

[0070]

[0071] As shown in Table 4, the peak area RSD% of 1,4-naphthoquinone is 4.22, indicating that the method has good precision.

[0072] Intermediate precision experiment

[0073] Test solution: Accurately weigh 60 mg of cefaclor and place it in a 10 ml volumetric flask. Add 5 ml of 0.5% ammonia solution to dissolve it, then dilute to the mark with acetonitrile and shake well. Prepare 6 parallel solutions.

[0074] The above-mentioned test solution was subjected to chromatographic detection. Two experimenters, A and B, injected the sample three times each. The experimental results are shown in Table 5.

[0075] Table 5 Results of intermediate precision test for 1,4-naphthoquinone

[0076]

[0077] As shown in Table 5 above, 1,4-naphthoquinone was not detected when the test samples were injected at different times and by different experimenters, indicating that the method has good intermediate precision.

[0078] Example 4;

[0079] Stability test

[0080] Reference solution: Accurately measure 1 ml of the reference stock solution and place it in a 10 ml volumetric flask. Dilute to the mark with 0.5% ammonia-acetonitrile (50:50) solution and shake well.

[0081] Test solution: Weigh 60 mg of cefaclor accurately, place it in a 10 ml volumetric flask, add 5 ml of 0.5% ammonia water to dissolve it, dilute to the mark with acetonitrile, and shake well.

[0082] The reference solution and the test solution were allowed to stand at room temperature, and samples were injected at 0h, 4h, 8h, 12h, 16h and 20h respectively. The test results are shown in Table 6.

[0083] Table 6. Stability Test Results

[0084]

[0085] The table above shows that the peak area RSD of the reference solution after 20 hours of storage was 5.96%, indicating that the reference solution has good stability. None of the test solutions were detected, indicating that the test solutions also had good stability.

[0086] Example 5;

[0087] Durability test

[0088] Reference solution: Accurately measure 1 ml of the reference stock solution and place it in a 10 ml volumetric flask. Dilute to the mark with 0.5% ammonia-acetonitrile (50:50) solution and shake well.

[0089] Change the chromatographic conditions: keep other conditions unchanged, and set the flow rate to 0.9 ml per minute;

[0090] With all other conditions remaining unchanged, the flow rate is set to 1.1 ml per minute;

[0091] With all other conditions remaining unchanged, the column temperature is 43℃;

[0092] With all other conditions remaining the same, the column temperature is 37℃.

[0093] Under the above chromatographic conditions, the reference solutions were taken and subjected to chromatographic detection. The detection results are shown in Table 7.

[0094] Table 7 Durability Test Results

[0095]

[0096] As can be seen from the table above, slight changes in chromatographic conditions have no effect on the detection results, indicating that the method has good robustness.

[0097] Example 6;

[0098] Sample testing

[0099] Using the detection method for 1,4-naphthoquinone and 1,2-naphthoquinone in cefaclor raw material described in this invention, five batches of cefaclor raw material samples were tested, and the test results are shown in Table 8.

[0100] Table 8. Test results of cefaclor raw material samples

[0101]

[0102] The results in Table 8 show that the 1,4-naphthoquinone and 1,2-naphthoquinone detected in the cefaclor raw material meet the limit standards.

[0103] As can be seen from the above embodiments, the detection method of the present invention has good specificity, linearity, repeatability, intermediate precision, accuracy, stability and robustness.

[0104] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. A method for detecting 1,4-naphthoquinone and 1,2-naphthoquinone in cefaclor raw material, characterized in that, The determination of 1,4-naphthoquinone and 1,2-naphthoquinone in cefaclor active pharmaceutical ingredient by high performance liquid chromatography includes the following steps: Step (1): Preparation of test solution: Weigh 60 mg of cefaclor accurately, place it in a 10 ml volumetric flask, add 5 ml of 0.5% ammonia water to dissolve it, dilute to the mark with acetonitrile, and shake well; Step (2): Preparation of reference solution: Accurately measure 1 ml of reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with solution, and shake well; Step (3): Inject the reference solution and the test solution into the liquid chromatograph separately and record the chromatograms. The high-performance liquid chromatography conditions are as follows: Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: Methanol; Gradient elution, wherein the gradient elution conditions are as follows: 0 min, mobile phase A accounts for 90%, mobile phase B accounts for 10%; 15 min, mobile phase A accounts for 60% and mobile phase B accounts for 40%; 25 min, mobile phase A accounts for 40%, mobile phase B accounts for 60%; 28 min, mobile phase A accounted for 10%, and mobile phase B accounted for 90%; 35 min, mobile phase A accounts for 10%, mobile phase B accounts for 90%; 35.1 min, mobile phase A accounts for 90%, mobile phase B accounts for 10%; For 45 minutes, mobile phase A accounted for 90% and mobile phase B accounted for 10%.

2. The method for detecting 1,4-naphthoquinone and 1,2-naphthoquinone in cefaclor raw material according to claim 1, characterized in that, The test solution in step (1) is a freshly prepared solution for immediate use.

3. The method for detecting 1,4-naphthoquinone and 1,2-naphthoquinone in cefaclor raw material according to claim 1, characterized in that, The preparation steps for the reference stock solution in step (2) are as follows: take an appropriate amount of 1,4-naphthoquinone reference standard, accurately weigh it, add acetonitrile to dissolve it, and quantitatively dilute it to prepare a solution containing approximately 300 ng per ml.

4. The method for detecting 1,4-naphthoquinone and 1,2-naphthoquinone in cefaclor raw material according to claim 1, characterized in that, The diluting solution in step (2) is a 0.5% ammonia-acetonitrile (50:50) solution.

5. The method for detecting 1,4-naphthoquinone and 1,2-naphthoquinone in cefaclor raw material according to claim 1, characterized in that, In step (3), the chromatographic column is a GL Sciences Inertsustain C18.

6. The method for detecting 1,4-naphthoquinone and 1,2-naphthoquinone in cefaclor raw material according to claim 5, characterized in that, The chromatographic column specifications are: 250mm × 4.6mm, and the packing diameter is 5μm or equivalent.

7. The method for detecting 1,4-naphthoquinone and 1,2-naphthoquinone in cefaclor raw material according to claim 1, characterized in that, In step (3), the elution flow rate is 1.0 ml per minute; the column temperature is 40℃; the injection volume is 20 μl; and the detection wavelength is 250 nm.

8. The method for detecting 1,4-naphthoquinone and 1,2-naphthoquinone in cefaclor raw material according to claim 1, characterized in that, The 1,4-naphthoquinone was calculated by peak area using the external standard method, and the 1,2-naphthoquinone was calculated by peak area after correction of 1,4-naphthoquinone (multiplied by a correction factor of 0.3). The total amount of 1,4-naphthoquinone and 1,2-naphthoquinone shall not exceed 5 ug / ml.

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