A method for detecting bacteriostatic agents in atropine sulfate eye drops

Abstract

The present invention relates to the technical field of pharmaceutical analysis, and specifically relates to a method for detecting bacteriostatic agents in atropine sulfate eye drops. The present invention dilutes the sample of the to-be-detected atropine sulfate eye drops with an aqueous sodium chloride solution, and then performs HPLC detection. Using an aqueous solution of potassium dihydrogen phosphate - sodium heptanesulfonate as mobile phase A and acetonitrile as mobile phase B, isocratic elution (mobile phase A 80 - 88 v / v%) is adopted, which can realize the detection of four bacteriostatic agents, namely 4-hydroxybenzoic acid, benzoic acid, benzyl alcohol and sorbic acid, in atropine sulfate eye drops. It has high precision, is accurate and reliable, has a short detection period, strong specificity, high sensitivity, can detect bacteriostatic agent components that cannot be detected by existing detection methods, provides an accurate, comprehensive and reliable detection method for the detection of bacteriostatic agents in atropine sulfate eye drops, makes up for the deficiencies of existing detection methods, can effectively control the types and quantities of bacteriostatic agents in atropine sulfate eye drops, and ensures the safety of drug use.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical analysis, and particularly relates to a method for detecting bacteriostatic agents in atropine sulfate eye drops. Background Art

[0002] Atropine has a wide range of clinical applications, such as relieving smooth muscle spasm, inhibiting glandular secretion, dilating pupils, premedication for anesthesia, and anti-shock. At present, the effect of atropine in delaying myopia progression has been confirmed. Research shows that low-concentration atropine sulfate eye drops have good clinical effects in the treatment of adolescent myopia, can effectively delay the increase in diopter and eye axis of patients, and the incidence of adverse reactions is extremely low, and it is relatively safe for long-term use. Appropriate bacteriostatic agents are usually added to multi-dose packaged eye drops to prevent microbial contamination during use. However, if used for a long time, the bacteriostatic agents in the eye drops can also cause ocular surface damage. Therefore, strictly controlling the concentration of bacteriostatic agents in eye drops is of great significance for the quality of atropine sulfate eye drops. The fourth part of the Chinese Pharmacopoeia 2020 edition (General Rule 0105) stipulates that single-dose eye preparations generally should not contain bacteriostatic agents, multi-dose eye preparations generally should contain appropriate bacteriostatic agents, and bacteriostatic agents with low safety risks should be selected as much as possible. The product label should indicate the type and labeled amount of bacteriostatic agents. Unless otherwise specified, when determining the prescription of the preparation, the bacteriostatic efficacy of this prescription should comply with the relevant regulations on bacteriostatic efficacy.

[0003] Atropine sulfate eye drops are products for teenagers who need to relieve and control myopia. In view of the usage and quality status of hospital-prepared atropine sulfate eye drops, in order to ensure effective quality control of the research and development and production quality of atropine sulfate eye drop preparations and ensure the safety and effectiveness of drugs, there is an urgent need to provide a method for determining bacteriostatic agents in atropine sulfate eye drop preparations.

[0004] The existing technology "Determination of the content of atropine sulfate in atropine sulfate eye drops by HPLC method" (Zhang Ke, Sheng Xin, Lan Wen. Determination of the content of atropine sulfate in atropine sulfate eye drops by HPLC method [J]. Chinese Pharmacist, 2015, 18(07): 1243-1245.) uses phosphate buffer solution (pH = 3.0 ± 0.1) as mobile phase A and acetonitrile as mobile phase B. The flow rate of the mobile phase is 1.0 mL / min, and the gradient elution program is as follows: 0-15 min, the volume fraction of mobile phase A is 82%, 30 min, the volume fraction of mobile phase A is 75%, and 35 min, the volume fraction of mobile phase A is 82%. However, the above method uses gradient elution, and there are large errors in the detection of each component at the stage where the baseline is not stable. Moreover, the existing detection methods cannot accurately and comprehensively detect 4 kinds of bacteriostatic agents, cannot effectively control the quality of atropine sulfate eye drops, and cannot guarantee the safety of drug use. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a method for detecting bacteriostatic agents in atropine sulfate eye drops, and the detection method provided by the present invention can accurately and comprehensively detect four bacteriostatic agents, namely 4-hydroxybenzoic acid, benzoic acid, benzyl alcohol, and sorbic acid, in atropine sulfate eye drops.

[0006] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides a method for detecting bacteriostatic agents in atropine sulfate eye drops, comprising the following steps:

[0008] Dilute the sample of atropine sulfate eye drops to be tested with an aqueous sodium chloride solution to obtain a sample solution to be tested;

[0009] Perform high performance liquid chromatography (HPLC) detection on the sample solution to be tested to obtain the detection result of the bacteriostatic agent in atropine sulfate eye drops;

[0010] The mobile phase A used in the HPLC detection is an aqueous solution of potassium dihydrogen phosphate - sodium heptanesulfonate, the mobile phase B is acetonitrile, the elution method is isocratic elution, and the volume fraction of the mobile phase A during the isocratic elution process is 80 - 88%;

[0011] The bacteriostatic agent includes one or more of 4-hydroxybenzoic acid, benzoic acid, benzyl alcohol, and sorbic acid.

[0012] Preferably, the concentration of the aqueous sodium chloride solution is 0.085 - 0.095 g / L.

[0013] Preferably, the concentration of atropine sulfate in the sample solution to be tested is 0.95 - 0.105 mg / mL.

[0014] Preferably, in the mobile phase A, the concentration of the potassium dihydrogen phosphate solution is 30 - 80 mmol / L, the concentration of sodium heptanesulfonate is 1.5 - 3.5 mmol / L, and the pH value is 4.5 - 5.5.

[0015] Preferably, the pH value of the mobile phase A is adjusted with triethylamine.

[0016] Preferably, the conditions for the HPLC detection include: the chromatographic column is an octadecylsilane-bonded silica gel chromatographic column, the column temperature is 25 - 35 °C, the flow rate of the mobile phase is 0.8 - 1.2 mL / min, the injection volume is 10 - 30 μL, and the detection wavelength is 225 nm.

[0017] The present invention dilutes the sample of atropine sulfate eye drops to be tested with an aqueous sodium chloride solution, uses an aqueous solution of potassium dihydrogen phosphate - sodium heptanesulfonate as mobile phase A and acetonitrile as mobile phase B, and adopts isocratic elution (the volume fraction of mobile phase A is 80 - 88%). The present invention adopts the isocratic elution method, which has low baseline noise and can improve the unstable baseline condition of gradient elution in the existing method. The separation between (atropine sulfate) and adjacent impurities is optimal, and it can accurately and comprehensively detect four bacteriostatic agents, namely 4 - hydroxybenzoic acid, benzoic acid, benzyl alcohol, and sorbic acid, in atropine sulfate eye drops. Moreover, the detection has high precision, is accurate and reliable, has a short detection period, strong specificity, is simple to operate, saves time and effort, can detect bacteriostatic agent components that cannot be detected by existing detection methods, provides an accurate, comprehensive, and reliable detection method for the detection of bacteriostatic agents in atropine sulfate eye drops, makes up for the deficiencies of existing detection methods, can effectively control the types and quantities of bacteriostatic agents in atropine sulfate eye drops, and ensures the safety of drug use. Description of the Drawings

[0018] Figure 1 The HPLC chromatogram of a 0.09 g / L aqueous sodium chloride solution;

[0019] Figure 2 The HPLC chromatogram of the system suitability solution;

[0020] Figure 3 The HPLC chromatogram of the test solution 1;

[0021] Figure 4 The HPLC chromatogram of the test solution 2;

[0022] Figure 5 The HPLC chromatogram of the test solution 3. Detailed Embodiments

[0023] The present invention provides a method for detecting bacteriostatic agents in atropine sulfate eye drops, which includes the following steps:

[0024] Dilute the sample of atropine sulfate eye drops to be tested with an aqueous sodium chloride solution to obtain a test sample solution;

[0025] Perform high - performance liquid chromatography detection on the test sample solution to obtain the detection results of bacteriostatic agents in atropine sulfate eye drops;

[0026] The mobile phase A used in the high - performance liquid chromatography detection is an aqueous solution of potassium dihydrogen phosphate - sodium heptanesulfonate, the mobile phase B is acetonitrile, the elution method is isocratic elution, and the volume fraction of mobile phase A during the isocratic elution process is 80 - 88%;

[0027] The bacteriostatic agents include one or more of 4 - hydroxybenzoic acid, benzoic acid, benzyl alcohol, and sorbic acid.

[0028] Unless otherwise specified, the raw materials used in the present invention are all commercially available products.

[0029] The present invention dilutes the sample solution of atropine sulfate eye drops to be measured with an aqueous sodium chloride solution to obtain a sample solution to be measured.

[0030] In the present invention, the concentration of the aqueous sodium chloride solution is preferably 0.085 - 0.095 g / L, more preferably 0.09 g / L. In the present invention, the concentration of atropine sulfate in the sample solution to be measured is preferably 0.95 - 0.105 mg / mL, more preferably 0.1 mg / mL.

[0031] After obtaining the sample solution to be measured, the present invention performs high - performance liquid chromatography detection on the sample solution to be measured to obtain the detection result of the bacteriostatic agent in the atropine sulfate eye drops; the mobile phase A used in the high - performance liquid chromatography detection is an aqueous solution of potassium dihydrogen phosphate - sodium heptanesulfonate, the mobile phase B is acetonitrile, the elution method is isocratic elution, and the volume fraction of the mobile phase A during the isocratic elution process is 80 - 88%; the bacteriostatic agent includes one or more of 4 - hydroxybenzoic acid, benzoic acid, benzyl alcohol, and sorbic acid.

[0032] In the present invention, the conditions for the high - performance liquid chromatography detection preferably include: the chromatographic column is an octadecylsilane - bonded silica gel chromatographic column; the column temperature is 25 - 35°C, more preferably 30°C; the mobile phase system includes mobile phase A and mobile phase B, the mobile phase A is an aqueous solution of potassium dihydrogen phosphate - sodium heptanesulfonate, and the mobile phase B is acetonitrile; the flow rate of the mobile phase is preferably 0.8 - 1.2 mL / min, more preferably 0.9 - 1.05 mL / min, further preferably 1 mL / min; the elution method is isocratic elution, and the volume fraction of the mobile phase A during the isocratic elution process is 80 - 88%, preferably 82 - 86%, more preferably 84 - 85%; the injection volume is 10 - 30 μL, more preferably 15 - 25 μL, further preferably 20 μL; the detection wavelength is 225 nm. In the present invention, in the mobile phase A, the concentration of the potassium dihydrogen phosphate solution is preferably 30 - 80 mmol / L, more preferably 40 - 70 mmol / L, further preferably 50 - 60 mmol / L; the concentration of sodium heptanesulfonate in the mobile phase A is preferably 1.5 - 3.5 mmol / L, more preferably 2 - 3 mmol / L, further preferably 2.5 mmol / L; the pH value of the mobile phase A is preferably 4.5 - 5.5, more preferably 4.8 - 5.2, further preferably 5.0; the pH value of the mobile phase A is preferably adjusted with triethylamine.

[0033] In the present invention, the detection result preferably includes a quantitative detection result and / or a qualitative detection result.

[0034] In the present invention, the method for qualitative detection preferably comprises the following steps: performing high performance liquid chromatography (HPLC) on the test sample solution and the reference solution of the bacteriostatic agent respectively, recording the chromatographic retention time of the bacteriostatic agent in the test sample solution and the reference solution. When a chromatographic peak with a retention time consistent with that of the bacteriostatic agent standard in the reference solution is detected in the test sample solution (with a variation range within ±2.5%), it can be determined that the corresponding compound is detected in the test sample. In the present invention, the detection conditions of the HPLC are the same as those of the aforementioned HPLC, and will not be elaborated herein.

[0035] In the present invention, the method for obtaining the quantitative detection result preferably comprises the following steps: preparing a linear reference solution; performing HPLC on the linear reference solution respectively to obtain the peak areas of the linear reference solution; using the concentration of the linear reference solution as the abscissa and the peak area of the linear reference solution as the ordinate to plot the concentration-peak area standard curve of each bacteriostatic agent reference, and calculating the regression equation; calculating the content of the bacteriostatic agent according to the regression equation and the peak area of the bacteriostatic agent in the test solution.

[0036] In the present invention, the method for preparing the linear reference solution preferably comprises the following steps: dissolving each bacteriostatic agent reference in acetonitrile respectively to obtain each reference stock solution; mixing the reference stock solutions and diluting them to obtain a linear mixed reference solution. In the present invention, the bacteriostatic agent references include 4-hydroxybenzoic acid reference, benzoic acid reference, benzyl alcohol reference or sorbic acid reference. In the present invention, the concentration of each reference stock solution (four kinds) is independently preferably 3 - 3.5 mg / mL. In the present invention, the diluent used for dilution is preferably a mixed solution of mobile phase A and mobile phase B, and the volume fraction of mobile phase A in the diluent is preferably 80 - 88%, more preferably 82 - 86%, and further preferably 84 - 85%, that is, the diluent is the mobile phase used in the isocratic elution process. In the present invention, the concentrations of 4-hydroxybenzoic acid reference, benzoic acid reference, benzyl alcohol reference and sorbic acid reference in the linear mixed reference solution are independently preferably 3 - 65 μg / mL.

[0037] The following examples are used to illustrate in detail the method for detecting bacteriostatic agents in atropine sulfate eye drops provided by the present invention, but they should not be construed as limiting the protection scope of the present invention.

[0038] In the following examples, the preparation methods of each solution are as follows:

[0039] Example 1

[0040] 1 Solution preparation

[0041] Atropine sulfate reference solution: Weigh accurately 31.62 mg of 4-hydroxybenzoic acid reference substance, place it in a 10 mL volumetric flask, dilute it to the mark with acetonitrile, and shake well.

[0042] Benzoic acid reference solution: Weigh accurately 30.06 mg of benzoic acid reference substance, place it in a 10 mL volumetric flask, dilute it to the mark with acetonitrile, and shake well.

[0043] Benzyl alcohol reference solution: Weigh accurately 32.01 mg of benzyl alcohol reference substance, place it in a 10 mL volumetric flask, dilute it to the mark with acetonitrile, and shake well.

[0044] Sorbic acid reference solution: Weigh accurately 30.18 mg of sorbic acid reference substance, place it in a 10 mL volumetric flask, dilute it to the mark with acetonitrile, and shake well.

[0045] System suitability solution (mixed reference solution): Pipette accurately 1.0 mL each of atropine sulfate reference solution, 4-hydroxybenzoic acid reference solution, benzoic acid reference solution, benzyl alcohol reference solution, and sorbic acid reference solution into the same 100 mL volumetric flask, dilute it to the mark with the diluent, and shake well. Among them, the diluent: mobile phase A: mobile phase B volume ratio = 84:16. Mobile phase A is an aqueous solution of 50 mmol / L potassium dihydrogen phosphate - 2.5 mmol / L sodium heptanesulfonate (adjust the pH value to 5.0 with triethylamine), and mobile phase B is acetonitrile.

[0046] 2 Screening of detection conditions

[0047] Using the system suitability solution as the detection object, the following detection conditions were screened.

[0048] Selection of packing material: Select an octadecylsilane chemically bonded silica gel chromatographic column (SVEA C18, 4.6 mm × 250 mm, 5 μm).

[0049] Mobile phase B: Acetonitrile.

[0050] Selection of ion pair reagent in mobile phase A: Using sodium octanesulfonate as the ion pair reagent, the resolution between the peaks of benzoic acid and sorbic acid is only 0.8, resulting in a decrease in the controllability of the quality standard. Through multiple experiments such as replacing sodium octanesulfonate, the ion pair reagent in the mobile phase was not discarded. By using the method of sodium heptanesulfonate ion pair reagent in potassium dihydrogen phosphate solution, the resolutions of atropine sulfate, 4-hydroxybenzoic acid, benzoic acid, benzyl alcohol, and sorbic acid are all > 1.5, meeting the detection requirements of the bacteriostatic agent in atropine sulfate eye drops.

[0051] Selection of the concentration of potassium dihydrogen phosphate aqueous solution: The elution conditions of mobile phase A with potassium dihydrogen phosphate aqueous solutions at concentrations of 20 mmol / L, 30 mmol / L, 50 mmol / L, 80 mmol / L, and 100 mmol / L were investigated. The results showed that when the concentration was in the range of 30 - 80 mmol / L, the resolution of atropine sulfate, 4-hydroxybenzoic acid, benzoic acid, benzyl alcohol, and sorbic acid was > 1.5. Atropine sulfate could be well separated from adjacent impurities and among various impurities. Among them, when the concentration was 50 mmol / L, the main peak was best separated from adjacent impurities, with a good peak shape and a better peak height of the detected bacteriostatic agent peak. Therefore, the optimal concentration of potassium dihydrogen phosphate aqueous solution was determined to be 50 mmol / L.

[0052] Selection of the pH value of mobile phase A: The elution conditions of mobile phase A with 50 mmol / L potassium dihydrogen phosphate - 2.5 mmol / L sodium heptanesulfonate aqueous solutions at pH values (adjusted with triethylamine) of 4.5, 5.0, and 5.5 were investigated. When the pH value was in the range of 4.5 - 5.5, the resolution of atropine sulfate, 4-hydroxybenzoic acid, benzoic acid, benzyl alcohol, and sorbic acid was > 1.5. Atropine sulfate could be well separated from adjacent impurities and among various impurities. Among them, when pH = 5.0, the main peak was best separated from adjacent impurities, with a good peak shape and a better peak height of the detected bacteriostatic agent peak. Therefore, the optimal pH value of mobile phase A was determined to be 5.0.

[0053] Selection of the reagent for adjusting the pH value: The elution conditions of mobile phase A with 50 mmol / L potassium dihydrogen phosphate - 2.5 mmol / L sodium heptanesulfonate aqueous solutions adjusted to pH 5.0 with phosphoric acid, sodium hydroxide, 25 - 28 wt% ammonia water, and triethylamine were investigated. The results showed that when phosphoric acid, sodium hydroxide, or 25 - 28 wt% ammonia water was used as the reagent for adjusting the pH value, the baseline was unstable, the peak height of benzoic acid was small, which affected the detection of benzoic acid and the resolution between benzyl alcohol and sorbic acid; when triethylamine was used to adjust pH = 5.0, the main peak was best separated from adjacent impurities, with a good peak shape and a better peak height of the detected bacteriostatic agent peak. Therefore, triethylamine was determined to be used for adjusting the pH value.

[0054] Selection of the proportion of the organic phase in the mobile phase: The elution conditions of the mobile phase system (50 mmol / L potassium dihydrogen phosphate - 2.5 mmol / L sodium heptanesulfonate aqueous solution): acetonitrile) with the volume fractions of the organic phase (acetonitrile, mobile phase B) being 10%, 16%, and 20% were investigated. By adjusting the proportion of the organic phase (acetonitrile) to change the separation ability of benzoic acid and sorbic acid, it was found that with the adjustment of the acetonitrile proportion, the main component eluted later, the analysis time was longer, the separation ability of benzoic acid and sorbic acid was poor, and the number of detected bacteriostatic agent peaks decreased; when the volume fraction of acetonitrile was 16%, the elution time of atropine sulfate was about 25 min, and the detection of bacteriostatic agents eluted within about 30 min, and the number of detected impurity peaks was comprehensive. Therefore, the optimal volume fraction of acetonitrile was determined to be 16%.

[0055] Selection of mobile phase gradient: Gradient elution was carried out, and it was found that the baseline was not stable. Isocratic elution was used, and the baseline was more stable than that of gradient elution. Isocratic elution was carried out with the volume fraction of mobile phase B being 16%, and atropine sulfate could be well separated from adjacent impurities and between various impurities.

[0056] Selection of detection wavelength: Considering that the main purpose of the detection in the present invention is to examine the content of bacteriostatic agents in drugs, since the detection wavelengths required for detecting the components of bacteriostatic agents in the subsequent verification of the present invention are 224 nm (for determining benzyl alcohol and benzoic acid), 259 nm for 4-hydroxybenzoic acid, and 264 nm for sorbic acid, considering the influence of the wavelength of 225 nm on differentiating the atropine sulfate peak, 225 nm was selected as the detection wavelength.

[0057] Selection of diluent: The reference solution used the mobile phase (mobile phase A: mobile phase B volume ratio = 84:16) as the diluent, and the test solution used 0.09 g / L sodium chloride aqueous solution as the diluent. Considering the possible influence of the diluent of the test solution of atropine sulfate eye drops on stability, 0.09 g / L sodium chloride aqueous solution in the eye drop prescription was selected as the diluent for the test solution. For the reference substance, according to its solubility, it was first dissolved in acetonitrile and then diluted with the mobile phase, which was convenient for experimental operation and did not produce a solvent effect. Therefore, the diluent used was the mobile phase (i.e., mobile phase A: mobile phase B volume ratio = 84:16).

[0058] Flow rate of mobile phase: 1.0 mL / min.

[0059] Column temperature: 30 °C.

[0060] System suitability requirements: In the chromatogram of the system suitability solution, HPLC analysis was performed on the mixed reference solution, and the resolution of each component peak was greater than 1.5.

[0061] Limit: Calculated by the external standard method, the content limits of 4-hydroxybenzoic acid, benzyl alcohol, benzoic acid, and sorbic acid are 0.3%.

[0062] Example 2

[0063] HPLC detection conditions: The chromatographic column was an octadecylsilane-bonded silica gel chromatographic column (SVEAC18, 4.6 mm × 250 mm, 5 μm); the column temperature was 30 °C; mobile phase A was 50 mmol / L potassium dihydrogen phosphate - 2.5 mmol / L sodium heptanesulfonate aqueous solution (adjusted to pH 5.0 with triethylamine), mobile phase B was acetonitrile; the elution method was isocratic elution, mobile phase A: mobile phase B volume ratio = 84:16; the flow rate of the mobile phase was 1.0 mL / min; the injection volume was 20 μL; the detection wavelength was 225 nm.

[0064] Diluent: Mobile phase A: Mobile phase B volume ratio = 84:16. Mobile phase A is an aqueous solution of 50 mmol / L potassium dihydrogen phosphate - 2.5 mmol / L sodium heptanesulfonate (pH adjusted to 5.0 with triethylamine), and mobile phase B is acetonitrile.

[0065] Method Validation

[0066] 1. Specificity

[0067] 1.1 Negative Test

[0068] Blank excipient solution: Weigh other excipients except the bacteriostatic agent and the main component (atropine sulfate) according to the prescription ratio of atropine sulfate eye drops to prepare a blank excipient solution with the concentration of each component equivalent to that of the test solution at 0.1 mg / mL.

[0069] Blank solution: Mobile phase (Mobile phase A: Mobile phase B volume ratio = 84:16, i.e., diluent).

[0070] Inject the blank excipient solution, blank solution, and 0.09 g / L sodium chloride aqueous solution into the liquid chromatograph for HPLC detection respectively, record the chromatogram, Figure 1 is the HPLC chromatogram of 0.09 g / L sodium chloride aqueous solution. The blank excipient, blank solution, and 0.09 g / L sodium chloride aqueous solution do not interfere with the detection of the bacteriostatic agent.

[0071] 2. Separation (system suitability) test of each component

[0072] System suitability requirements: Inject the system suitability solution into the liquid chromatograph for HPLC detection, the acquisition time is 30 min, record the chromatogram, as Figure 2 shown, Figure 2 in which, 1 is 4-hydroxybenzoic acid, 2 is benzoic acid, 3 is benzyl alcohol, 4 is sorbic acid, 5 is atropine sulfate, the elution order is 4-hydroxybenzoic acid, benzyl alcohol, benzoic acid, sorbic acid, and atropine sulfate in turn, and the retention times are 3 min, 7 min, 11 min, 12 min, and 25 min respectively. The resolution of each component peak is greater than 1.5.

[0073] 3. Linearity

[0074] Precisely measure appropriate amounts of the atropine sulfate reference solution, 4-hydroxybenzoic acid reference solution, benzoic acid reference solution, benzyl alcohol reference solution, and sorbic acid reference solution prepared in Example 1 into the same 100 mL volumetric flask, dilute to the mark with the diluent, and shake well to obtain Linear-1 solution (0.1 mL of each reference solution is taken), Linear-2 solution (0.5 mL of each reference solution is taken), Linear-3 solution (1.0 mL of each reference solution is taken), Linear-4 solution (1.2 mL of each reference solution is taken), and Linear-5 solution (2.0 mL of each reference solution is taken); inject the series of reference solutions into the liquid chromatograph for HPLC detection, record the chromatogram, use the peak area y of each peak as the ordinate and the corresponding concentration x as the abscissa to plot the standard curve of each component, and calculate the regression equation. The results are shown in Table 1.

[0075] Table 1 Linear range and regression equation

[0076]

[0077] As can be seen from Table 1, in the concentration range of 3.162 - 63.24 μg / mL, the peak area of 4-hydroxybenzoic acid has a good linear relationship with the concentration, with r = 0.9998. In the concentration range of 3.006 - 60.12 μg / mL, the peak area of benzoic acid has a good linear relationship with the concentration, with r = 0.9997. In the concentration range of 3.201 - 64.02 μg / mL, the peak area of benzyl alcohol has a good linear relationship with the concentration, with r = 1.0000. In the concentration range of 3.018 - 60.36 μg / mL, the peak area of benzoic acid has a good linear relationship with the concentration, with r = 0.9998.

[0078] 4. Investigation on solution stability

[0079] Take samples of the Linear-3 solution at 0 h, 4 h, 8 h, 12 h, and 24 h when placed at room temperature, inject into the liquid chromatograph for HPLC detection, record the chromatogram, and calculate the average peak area and RSD. The results are shown in Table 2.

[0080] Table 2 Stability test of the system suitability solution

[0081]

[0082]

[0083] As can be seen from Table 2, 4-hydroxybenzoic acid, benzoic acid, benzyl alcohol, and sorbic acid in the solution are stable within 24 h at room temperature.

[0084] 5. Repeatability test

[0085] Atropine sulfate eye drops (Heze Shunyuan Medical Instrument Co., Ltd., batch number 22082201, specifications as shown in Table 3) were diluted with 0.09 g / L sodium chloride aqueous solution to obtain a test solution with an atropine sulfate concentration of 0.1 mg / mL. Six test solutions were prepared and injected into a liquid chromatograph for HPLC detection, and the chromatograms were recorded. Results of the repeatability test: The average content of 4-hydroxybenzoic acid was 0.853% (n = 6), the average content of benzoic acid was 0.142% (n = 6), and benzyl alcohol and sorbic acid were not detected, indicating that the repeatability test results of the detection method provided by the present invention are good.

[0086] 6. Detection limit and quantitation limit

[0087] The linear - 1 solution was serially diluted with the diluent, and each diluted solution was injected into a liquid chromatograph for HPLC detection. The chromatograms were recorded, and the quantitation limit and detection limit of each bacteriostatic agent were calculated.

[0088] The quantitation limit of 4-hydroxybenzoic acid was 0.008% (S / N = 10), and the detection limit was 0.002% (S / N = 3); the quantitation limit of benzoic acid was 0.08% (S / N = 10), and the detection limit was 0.03% (S / N = 3); the quantitation limit of benzyl alcohol was 0.02% (S / N = 10), and the detection limit was 0.007% (S / N = 3); the quantitation limit of sorbic acid was 0.04% (S / N = 10), and the detection limit was 0.002% (S / N = 3).

[0089] Through method validation studies, the detection method provided by the present invention has good specificity, accuracy, precision, and high sensitivity, and is suitable for the determination of bacteriostatic agents in atropine sulfate eye drops for hospital preparations.

[0090] Example 3

[0091] Table 3 Sample information of atropine sulfate eye drops

[0092]

[0093]

[0094] The atropine sulfate eye drops shown in Table 3 were prepared into test solutions. The atropine sulfate eye drops of Aier Eye Hospital Group Co., Ltd. were diluted with 0.09 g / L sodium chloride aqueous solution to obtain test solution 1 with an atropine sulfate concentration of 0.1 mg / mL. The atropine sulfate eye drops of Shenyang Xingqi Eye Medicine Co., Ltd. were directly used as test solution 2. The atropine sulfate eye drops of Heze Shunyuan Medical Instrument Co., Ltd. were diluted with 0.09 g / L sodium chloride aqueous solution to obtain test solution 3 with an atropine sulfate concentration of 0.1 mg / mL.

[0095] Inject each test solution into a liquid chromatograph for HPLC detection, record the chromatogram, and the test results are shown in Table 4 and Figures 3 - 5 as follows, Figure 3 The HPLC chromatogram of test solution 1, Figure 4 is the HPLC chromatogram of test solution 2, Figure 5 is the HPLC chromatogram of test solution 3.

[0096] Table 4 Determination results of atropine sulfate eye drops samples

[0097]

[0098] As can be seen from Table 4 and Figures 3 - 5 it can be known that compared with the prescriptions of the current quality standards of each enterprise, the determination results of the bacteriostatic agents provided by the detection method of the present invention have detected bacteriostatic agents not marked in a certain hospital prescription, and the doses of individual bacteriostatic agents are 1.5 times higher than the limit.

[0099] In summary, the detection method provided by the present invention can detect the contents of 4 common bacteriostatic agents in the hospital preparation atropine sulfate eye drops, and can be used for the detection of bacteriostatic agents in atropine sulfate eye drops. It not only makes up for the omissions of the existing detection technology, but also is accurate and reliable, has strong specificity, saves time and effort, and is easy to operate.

[0100] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for detecting bacteriostatic agents in atropine sulfate eye drops, characterized in that, It includes the following steps: Dilute the sample solution of atropine sulfate eye drops to be tested with an aqueous sodium chloride solution to obtain a sample solution to be tested. The concentration of the aqueous sodium chloride solution is 0.09 g / L, and the concentration of atropine sulfate in the sample solution to be tested is 0.1 mg / mL; Perform high performance liquid chromatography (HPLC) detection on the sample solution to be tested to obtain the detection result of the bacteriostatic agent in the atropine sulfate eye drops; The mobile phase A used in the HPLC detection is an aqueous solution of potassium dihydrogen phosphate - sodium heptanesulfonate. In the mobile phase A, the concentration of the potassium dihydrogen phosphate solution is 50 mmol / L, the concentration of sodium heptanesulfonate is 2.5 mmol / L, the pH value of the mobile phase A is 5.0, and the pH value of the mobile phase A is adjusted with triethylamine; the mobile phase B is acetonitrile, and the elution mode is isocratic elution. During the isocratic elution process, the volume fraction of the mobile phase A is 84%, and the volume fraction of the mobile phase B is 16%; the conditions for the HPLC detection include: the chromatographic column is an octadecylsilane-bonded silica gel chromatographic column, the column temperature is 30 °C, the flow rate of the mobile phase is 1 mL / min, the injection volume is 20 μL, and the detection wavelength is 225 nm; The bacteriostatic agents are 4-hydroxybenzoic acid, benzoic acid, benzyl alcohol and sorbic acid.

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