Method for detecting content of antioxidant in bromfenac sodium eye drops by liquid chromatography

Abstract

The invention relates to the technical field of medical analysis, in particular to a method for detecting the content of an antioxidant in bromfenac sodium eye drops through liquid chromatography, and particularly relates to a method for detecting the content of the antioxidant anhydrous sodium sulfite of the bromfenac sodium eye drops through high performance liquid chromatography. Wherein the mobile phase A is a methanol-buffer salt solution; a mobile phase B is methanol; eluting by using a gradient mobile phase, wherein the volume ratio of the mobile phase is 100%; preparing a reference solution and a test solution by taking the mobile phase A as a diluent, then respectively injecting the reference solution and the test solution into a liquid chromatograph, and recording chromatograms; calculating the content of anhydrous sodium sulfite by adopting an external standard method; the method comprises the following specific steps: (1) preparation of a reference substance solution: taking an anhydrous sodium sulfite reference substance, and adding a diluent for dissolving to obtain the reference substance solution; and (2) preparation of a test solution: taking the bromfenac sodium eye drops, and adding a diluent to dilute the bromfenac sodium eye drops to obtain the bromfenac sodium eye drops. The method disclosed by the invention is strong in specificity, high in stability, high in precision and high in accuracy, and has practical significance on quality control of the bromfenac sodium eye drops.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, and in particular to a method for detecting the antioxidant content in bromfenac sodium eye drops using liquid chromatography. Background Technology

[0002] Bromfenac sodium eye drops are topical nonsteroidal anti-inflammatory drugs (NSAIDs) that inhibit the production of certain cellular substances that cause pain and swelling, thus achieving anti-inflammatory and analgesic effects. They are suitable for the symptomatic treatment of inflammatory diseases of the external and anterior ocular regions, including conjunctivitis, scleritis, and postoperative inflammation.

[0003] Anhydrous sodium sulfite is an excipient in bromfenac sodium eye drops. As an antioxidant, it can protect the main component, bromfenac sodium, from oxidation, thereby protecting its therapeutic effect. Therefore, it is necessary to pay attention to the relationship between the stability trend of bromfenac sodium eye drops and its antioxidant content to ensure product quality.

[0004] This invention presents a method for detecting the content of antioxidants (anhydrous sodium sulfite) using liquid chromatography, which is highly specific, reproducible, and accurate. Summary of the Invention

[0005] The purpose of this invention is to provide a method for detecting the content of anhydrous sodium sulfite using liquid chromatography, which solves the problems of poor specificity, poor repeatability, and low accuracy in existing methods, accurately detects the content of anhydrous sodium sulfite in products, and achieves quality control.

[0006] To solve one of the above-mentioned technical problems, the present invention adopts the following technical solution: a method for detecting the antioxidant content in bromfenac sodium eye drops by liquid chromatography, specifically a method for detecting the content of anhydrous sodium sulfite, an antioxidant, by high performance liquid chromatography.

[0007] The method uses octadecylsilane-bonded silica gel as a filler;

[0008] The mobile phase A consisted of a methanol-buffered salt solution (2.2 g of anhydrous disodium hydrogen phosphate and 0.85 g of tetrabutylammonium hydrogen sulfate were dissolved in 750 ml of water, and the pH was adjusted to 9.0 with 1 mol / L sodium hydroxide) (25:75); the mobile phase B consisted of methanol; and the solution was eluted with a gradient mobile phase, with the mobile phase volume ratio being 100%.

[0009] The gradient of the mobile phase is set as follows:

[0010]

[0011]

[0012] Prepare reference solution and test solution using mobile phase A as diluent, then inject them separately into the liquid chromatograph and record the chromatograms. Calculate the content of anhydrous sodium sulfite using the external standard method.

[0013] The specific steps of the method are as follows:

[0014] (1) Preparation of reference solution: Dissolve anhydrous sodium sulfite reference standard in diluent to obtain the solution;

[0015] (2) Preparation of the test solution: Take bromfenac sodium eye drops, dilute with diluent to obtain the solution;

[0016] Inject the reference solution and the test solution into the liquid chromatograph, respectively. If the test solution contains a chromatographic peak with the same retention time as anhydrous sodium sulfite, calculate the peak area using the external standard method.

[0017] The calculation formula is as follows:

[0018] In the formula:

[0019] Ai, As: Peak area of ​​the main peak in the test solution; Average peak area of ​​the main peak in the reference solution d1;

[0020] Ws, Wi: Sample volume of reference solution d1, mg; Sample volume of test sample, ml;

[0021] Vi, Vs: Dilution volume of the test solution, ml; Dilution volume of the reference solution d1, ml;

[0022] Ps: Content of the reference solution, %.

[0023] Based on any of the above technical solutions, the following further optimizations are made: the detection wavelength is 248-252nm, the column temperature is 24-26℃, the flow rate is 0.9-1.1ml / min, and the injection volume is 5μl.

[0024] Based on any of the above technical solutions, the following further optimizations are made: the detection wavelength is 250nm, the column temperature is 25℃, and the flow rate is 1.0ml / min.

[0025] Based on any of the above technical solutions, the following further optimization is made: the proportion of buffer salt solution (2.2g of anhydrous disodium hydrogen phosphate and 0.85g of tetrabutylammonium hydrogen sulfate, dissolved in 750ml of water, and the pH value adjusted to 8.9-9.1 with 1mol / L sodium hydroxide) in mobile phase A is 74%-76%, and the proportion of methanol is 26%-24%.

[0026] Based on any of the above technical solutions, the following optimization is made: the proportion of buffer salt solution (2.2g of anhydrous disodium hydrogen phosphate, 0.85g of tetrabutylammonium hydrogen sulfate, dissolved in 750ml of water, and the pH value adjusted to 9.0 with 1mol / L sodium hydroxide solution) in mobile phase A is 75%, the proportion of methanol is 30%, and elution is performed using a gradient mobile phase.

[0027] Based on any of the above technical solutions, the following optimization is made: Weigh anhydrous sodium sulfite reference standard, use mobile phase A as diluent, and prepare a reference standard solution with an anhydrous sodium sulfite concentration of 0.2 mg / ml; the test solution is prepared as follows: Take bromfenac sodium eye drops, use mobile phase A as diluent, dilute 10 times, and prepare the test solution.

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

[0029] 1. The liquid chromatography method of the present invention for detecting the content of anhydrous sodium sulfite is highly specific, stable, precise and accurate, and has practical significance for the quality control of bromfenac sodium eye drops.

[0030] 2. Enhanced specificity and effective elimination of interference: This invention optimizes chromatographic conditions (such as specific mobile phase composition, gradient elution program, and detection wavelength). Experimental verification shows that blank solution and blank excipient solution do not interfere with the detection of anhydrous sodium sulfite. The retention times of anhydrous sodium sulfite in the test solution are highly consistent with those of the reference standard (3.895 min and 3.910 min, respectively). This allows for precise differentiation between the target antioxidant component and other matrix components, solving the problems of poor specificity and susceptibility to interference from excipients or main components in existing technologies, thus ensuring the specificity of the detection results.

[0031] 3. Excellent accuracy and repeatability, high data reliability: Accuracy experiments of the method of this invention show that the average recovery rate of anhydrous sodium sulfite in the concentration range of 50%-150% reaches 99.1%, with an RSD of only 2.1%; in the repeatability experiment, the average recovery rate of 6 parallel solutions is 98.3%, with an RSD of 1.8%; the intermediate precision RSD of 2 operators is 1.7%, and all indicators meet the strict analytical requirements. Compared with the problems of large fluctuations in recovery rate and significant deviations in parallel experimental data that may exist in existing technologies, the detection results of this method are stable and reliable, and can truly reflect the actual content of anhydrous sodium sulfite in bromfenac sodium eye drops.

[0032] 4. Excellent durability and wide applicability: After fine-tuning the detection conditions (e.g., detection wavelength 248-252nm, flow rate 0.9-1.1ml / min, column temperature 24-26℃, buffer salt pH 8.9-9.1, different batches of columns), except for single mobile phase, the recovery rate of the test sample content is within the range of 95.0%-105.0% under all other conditions, and the peak area RSD of the reference standard is ≤2.0%, and the response factor recovery rate is 95.0%-105.0%. This indicates that this method is highly tolerant to changes in experimental conditions, does not require strict control of a single parameter, and is suitable for routine detection in different laboratories and with different equipment. It reduces the operational difficulty in practical applications and solves the problems of sensitivity to conditions and limited applicability of existing technologies.

[0033] 5. High detection sensitivity and wide linear range: The method of this invention has a low limit of quantitation (LOQ) of 19.8753 μg / ml, with a retention time RSD of only 0.072% and a peak area RSD of 1.80% for six consecutive measurements, and a signal-to-noise ratio (S / N) of 10-30, enabling accurate detection of low concentrations of anhydrous sodium sulfite. Furthermore, the linear equation for anhydrous sodium sulfite exhibits excellent linearity, with a correlation coefficient r = 0.9999 within the concentration range of 19.8753-286.2276 μg / ml. Compared to the potential drawbacks of existing technologies, such as high detection limits and narrow linear ranges, this method not only meets the routine quality control requirements of products but also monitors the trace consumption changes of antioxidants during storage, providing crucial data support for the stability assessment of bromfenac sodium eye drops. Attached Figure Description

[0034] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or components are generally identified by similar reference numerals. In the drawings, the elements or components are not necessarily drawn to scale.

[0035] Figure 1 This is a specificity spectrum of the reference solution in the embodiments of this application.

[0036] Figure 2 This is a specificity spectrum of the test solution in the embodiments of this application.

[0037] Figure 3 This is a linear graph in the embodiments of this application. Detailed Implementation

[0038] The following will provide a detailed description of the method for quality control of antioxidant content in bromfenac sodium eye drops according to the present invention through examples. However, this should not be construed as limiting the scope of the claims of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. The chromatogram of the present invention is shown below. Figures 1-3 As shown in the image.

[0039] Example 1:

[0040] Column: Hypersil GOLD (4.6 mm × 250 mm, 5 μm)

[0041] Detector: UV; Detection wavelength: 250 nm; Flow rate: 1.0 ml / min; Column temperature: 25 ℃; Injection volume: 5 μl; Diluent: Mobile phase A; Mobile phase A: Methanol-buffered saline solution (dissolve 2.2 g of anhydrous disodium hydrogen phosphate and 0.85 g of tetrabutylammonium hydrogen sulfate in 750 ml of water, and adjust the pH to 9.0 with 1 mol / L sodium hydroxide) (25:75); Mobile phase B:

[0042] Methanol; eluted with a gradient mobile phase, with the mobile phase volume ratio being 100%, the gradient of the mobile phase is set as follows:

[0043] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 10 100 0 10.1 50 50 30 50 50 30.1 100 0 60 100 0

[0044] (1) Specificity and system applicability

[0045] Solution preparation:

[0046] Blank solution: diluent (mobile phase A).

[0047] Blank excipient solution: Accurately measure 2 ml of blank excipient, place it in a 20 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0048] Reference solution: Accurately weigh 20 mg of anhydrous sodium sulfite reference standard, place it in a 100 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and the solution is ready. Prepare two parallel solutions.

[0049] Test solution: Accurately measure 2 ml of bromfenac sodium eye drops, place it in a 20 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0050] result

[0051] Specific results

[0052] Solution name Component Name Retention time (minutes) blank solution / / Blank excipient solution / / Reference solution Anhydrous sodium sulfite 3.910 Test solution Anhydrous sodium sulfite 3.895

[0053] System applicability results

[0054] in conclusion

[0055] ① Blank solution and blank excipient solution do not interfere with the detection of anhydrous sodium sulfite;

[0056] ② The RSD of the peak area of ​​the reference solution 1# was 0.45% after five consecutive determinations, and the recovery rate of the response factor of the two reference solutions was 99.5%, which meets the requirements, indicating that the method has good specificity and system suitability.

[0057] (2) Solution stability

[0058] Solution preparation:

[0059] Blank solution: diluent (mobile phase A).

[0060] Reference solution: Accurately weigh 20 mg of anhydrous sodium sulfite reference standard, place it in a 100 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and the solution is ready. Prepare two parallel solutions.

[0061] Test solution: Accurately measure 2 ml of bromfenac sodium eye drops, place it in a 20 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0062] result

[0063] Stability results of reference solution

[0064]

[0065]

[0066] Stability results of the test solution

[0067]

[0068] in conclusion

[0069] ① The reference solution is stable after standing for 24 hours at room temperature;

[0070] ②The test solution is stable after standing for 12 hours at room temperature.

[0071] (3) Limit of quantitation

[0072] Solution preparation

[0073] Blank solution: diluent (mobile phase A).

[0074] Reference solution: Accurately weigh 20 mg of anhydrous sodium sulfite reference standard, place it in a 100 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and the solution is ready.

[0075] Limit of Quantitation Solution: Accurately measure 2 ml of the reference solution, place it in a 20 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0076] result

[0077] Quantitative limit results

[0078]

[0079]

[0080] Conclusion: The limit of quantitation was 19.8753 μg / ml. After six consecutive measurements, the RSD of the retention time of the anhydrous sodium sulfite peak was 0.072%, the RSD of the peak area was 1.80%, and the S / N value was between 10 and 30.

[0081] (4) Linearity and Range

[0082] Solution preparation

[0083] Blank solution: diluent (mobile phase A).

[0084] Linear stock solution: Accurately weigh 50 mg of anhydrous sodium sulfite reference standard, place it in a 50 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and the solution is ready.

[0085] Linear solution 1#: Take the solution at the limit of quantitation under the limit of quantitation item, and you will get it.

[0086] Linear solutions 2#-6#: Accurately measure 2ml, 3ml, 4ml, 5ml and 6ml of the linear stock solution respectively, place them in different 20ml volumetric flasks, dilute to the mark with diluent, and shake well to obtain the solution.

[0087] Linear and range results

[0088]

[0089] Conclusion: Anhydrous sodium sulfite exhibits good linearity under this method.

[0090] (5) Repeatability

[0091] Solution preparation

[0092] Blank solution: diluent (mobile phase A).

[0093] Reference solution: Accurately weigh 20 mg of anhydrous sodium sulfite reference standard, place it in a 100 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and the solution is ready; prepare two portions in the same way as reference solutions d1 and d2.

[0094] Repeatable stock solution: Accurately weigh 100 mg of anhydrous sodium sulfite reference standard, place it in a 100 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and the solution is ready.

[0095] Repeatable solution: Accurately measure 4 ml of repeatable stock solution, place it in a 20 ml volumetric flask, add 2 ml of blank excipient, dilute to the mark with diluent, and shake well to obtain the solution; prepare 6 parallel portions as y1-y6.

[0096] result

[0097] Repeatability results

[0098]

[0099] Conclusion: Six reproducible solutions were prepared in parallel, and the average recovery rate was 98.3%, with an RSD of 1.8%, indicating that the method has good reproducibility.

[0100] (6) Accuracy

[0101] Solution preparation

[0102] Blank solution: diluent (mobile phase A).

[0103] Reference solution: Accurately weigh 20 mg of anhydrous sodium sulfite reference standard, place it in a 100 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and the solution is ready; prepare two portions in the same way as reference solutions d1 and d2.

[0104] Accuracy stock solution: Accurately weigh 100 mg of anhydrous sodium sulfite reference standard, place it in a 100 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and the solution is ready.

[0105] Accuracy solutions: Accurately measure 2 ml and 6 ml of accuracy stock solution into different 20 ml volumetric flasks, add 2 ml of blank excipient to each, dilute to the mark with diluent, and shake well to obtain the solution; prepare three parallel aliquots of each concentration as 50%-1, 50%-2, 50%-3, and 150%-1, 150%-2, 150%-3 respectively.

[0106] Take the repeatability solutions y1-y3 from the repeatability section as 100%-1, 100%-2, and 100%-3.

[0107] Accuracy results

[0108]

[0109] Conclusion: The average recovery rate (n=9) of anhydrous sodium sulfite was 99.1%, and the RSD was 2.1%, indicating that the method has high accuracy.

[0110] (7) Intermediate precision

[0111] Solution preparation

[0112] Blank solution: diluent (mobile phase A).

[0113] Reference solution: The method for preparing reference solution is described in the same section on suitability for system.

[0114] Intermediate precision stock solution: Accurately weigh 100 mg of anhydrous sodium sulfite reference standard, place it in a 100 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and the solution is ready.

[0115] Intermediate precision solution: Accurately measure 4 ml of intermediate precision stock solution, place it in a 20 ml volumetric flask, add 2 ml of blank excipient, dilute to the mark with diluent, and shake well to obtain the solution; prepare 6 parallel portions as y7-y12.

[0116] Intermediate precision results

[0117]

[0118]

[0119] Conclusion: Two researchers prepared six intermediate precision solutions. The RSD of the recovery rate of anhydrous sodium sulfite in the twelve intermediate precision solutions was 1.7%, indicating that the method has good precision.

[0120] (8) Durability

[0121] Solution preparation

[0122] Blank solution: diluent (mobile phase A).

[0123] Reference solution: The method for preparing reference solution is described in the same section on suitability for system.

[0124] Blank excipient solution: Accurately measure 2 ml of blank excipient, place it in a 20 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0125] Test solution: Accurately measure 2 ml of bromfenac sodium eye drops, place it in a 20 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0126] Durability conditions

[0127]

[0128] Durability results

[0129]

[0130] Conclusion: After fine-tuning the chromatographic conditions, the blank solvent and blank excipient did not interfere with the detection of anhydrous sodium sulfite. The RSD of the peak area of ​​anhydrous sodium sulfite on day 1 (five consecutive determinations) was ≤2.0%, and the recoveries of the response factors of the two reference solutions were both between 95.0% and 105.0%. Except for the mobile phase A ratio (methanol:buffer salt - 24: 76), where the recovery rate of anhydrous sodium sulfite relative to the standard conditions was 92.2% and not within the 95.0%-105.0% range, the recoveries of anhydrous sodium sulfite in the test solution relative to the standard conditions were all between 95.0% and 105.0% under other fine-tuned chromatographic conditions. This method has good robustness.

[0131] In summary, the method for detecting antioxidant content in bromfenac sodium eye drops established in this invention has high specificity, good repeatability, and high accuracy, and is suitable for detecting antioxidant content in bromfenac sodium eye drops.

[0132] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. 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. For those skilled in the art, any alternative improvements or transformations made to the implementation of the present invention fall within the protection scope of the present invention.

[0133] Any aspects of this invention not described in detail are well-known to those skilled in the art.

Claims

1. A method for determining the antioxidant content in bromfenac sodium eye drops using liquid chromatography, characterized in that, Specifically, the method of detecting the content of the antioxidant anhydrous sodium sulfite is carried out by high performance liquid chromatography. The method uses octadecylsilane-bonded silica gel as a filler; Wherein, mobile phase A: methanol-buffered salt solution; mobile phase B: methanol; elution is performed using a gradient mobile phase, with the mobile phase volume ratio being 100%; The reference solution and the test solution were prepared using mobile phase A as diluent, and then injected into the liquid chromatograph and the chromatograms were recorded. The content of anhydrous sodium sulfite was calculated using the external standard method. The specific steps of the method are as follows: (1) Preparation of reference solution: Dissolve anhydrous sodium sulfite reference standard in diluent to obtain the solution; (2) Preparation of the test solution: Take bromfenac sodium eye drops, dilute with diluent to obtain the solution; Inject the reference solution and the test solution into the liquid chromatograph, respectively. If the test solution contains a chromatographic peak with the same retention time as anhydrous sodium sulfite, calculate the peak area using the external standard method. The calculation formula is as follows: In the formula: Ai, As: Peak area of ​​the main peak in the test solution; Average peak area of ​​the main peak in the reference solution d1; Ws, Wi: Sample volume of reference solution d1, mg; Sample volume of test sample, ml; Vi, Vs: Dilution volume of the test solution, ml; Dilution volume of the reference solution d1, ml; Ps: Content of the reference solution, %.

2. The method for detecting the antioxidant content in bromfenac sodium eye drops by liquid chromatography according to claim 1, characterized in that: The detection wavelength is 248-252nm, the column temperature is 24-26℃, the flow rate is 0.9-1.1ml / min, and the injection volume is 5μl.

3. The method for detecting the antioxidant content in bromfenac sodium eye drops by liquid chromatography according to claim 1, characterized in that: The detection wavelength was 250 nm, the column temperature was 25 °C, and the flow rate was 1.0 ml / min.

4. The method for detecting the antioxidant content in bromfenac sodium eye drops by liquid chromatography according to claim 2, characterized in that: The proportion of buffer salt solution (2.2 g of anhydrous disodium hydrogen phosphate and 0.85 g of tetrabutylammonium hydrogen sulfate dissolved in 750 ml of water, and the pH value adjusted to 8.9-9.1 with 1 mol / L sodium hydroxide) in mobile phase A is 74%-76%, and the proportion of methanol is 26%-24%.

5. The method for detecting the antioxidant content in bromfenac sodium eye drops by liquid chromatography according to claim 1, characterized in that: The mobile phase A contains 75% buffer salt solution (2.2 g of anhydrous disodium hydrogen phosphate and 0.85 g of tetrabutylammonium hydrogen sulfate are dissolved in 750 ml of water, and the pH is adjusted to 9.0 with 1 mol / L sodium hydroxide solution) and 30% methanol. The mobile phase is eluted with a gradient mobile phase.

6. The method for detecting the antioxidant content in bromfenac sodium eye drops by liquid chromatography according to claim 1, characterized in that: Weigh anhydrous sodium sulfite reference standard and prepare a reference standard solution with an anhydrous sodium sulfite concentration of 0.2 mg / ml using mobile phase A as the diluent. The test solution is prepared as follows: take bromfenac sodium eye drops and dilute 10 times using mobile phase A to prepare the test solution.

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