A method for determining the amount of chlorhexidine acetate in a disinfectant

Abstract

The application belongs to the technical field of detection, and particularly relates to a method for determining the content of chlorhexidine acetate in a disinfectant. The method for determining the content of chlorhexidine acetate in the disinfectant comprises the following steps: (1) dispersing the sample with glacial acetic acid, releasing chlorhexidine acetate combined with sample matrix components, and extracting with a methanol aqueous solution; and (2) taking a tetrabutylammonium bromide-acetic acid-acetonitrile solution as a mobile phase, and detecting with a high-performance liquid chromatograph. The method for detecting chlorhexidine acetate is simple in operation, good in reproducibility, complete in separation from impurities, high in accuracy, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, specifically relating to a method for determining the content of chlorhexidine acetate in disinfectants. Background Technology

[0002] Chlorhexidine acetate, also known as chlorhexidine acetate, is a biguanide organic compound with the chemical formula C. 22 H 30 Cl2N 10 ·2C2H4O2 or C 26 H 38 Cl2N 10 O4. Chlorhexidine acetate is a broad-spectrum antibacterial drug with antibacterial, disinfectant, topical therapeutic, and antiseptic effects, widely used in medical and health, daily cleaning, and industrial fields. Currently, chlorhexidine acetate is available in various dosage forms on the market, including solutions, gels, powders, and creams. To suit different usage scenarios and needs, some products also contain plant-based ingredients to enhance disinfection effects, reduce irritation, and increase environmental friendliness. With the widespread use of chlorhexidine acetate disinfectants, to protect consumer rights and regulate market order, it is essential to ensure product quality. This prevents situations where the chlorhexidine acetate content is too low, failing to achieve the purpose of treating diseases or sterilization, or too high, leading to safety risks and toxic side effects. Therefore, accurate determination of the chlorhexidine acetate content in products is necessary.

[0003] The main methods for determining chlorhexidine acetate content include titration, spectrophotometry, atomic absorption spectrometry, capillary electrophoresis, and liquid chromatography. However, based on feedback from many manufacturers over a long period, the currently reported methods are only suitable for determining the chlorhexidine acetate content in samples with relatively simple matrix components. The determination of chlorhexidine acetate content in disinfectants with complex matrices, such as those containing plant components or multiple excipients, has always been a challenge in the industry, with situations where the test results do not match the actual input or even fail to detect the acetate.

[0004] Therefore, it is necessary to establish a more widely applicable method for determining the content of chlorhexidine acetate to ensure the accuracy of the detection results of chlorhexidine acetate content in samples, and to provide strong technical support for improving the quality of disinfection products, protecting consumer rights, and regulating market order. Summary of the Invention

[0005] To overcome the shortcomings and deficiencies of existing detection methods, the present invention aims to provide a method for accurately determining the content of chlorhexidine acetate in disinfectants.

[0006] The objective of this invention is achieved through the following solution:

[0007] A method for determining the chlorhexidine acetate content in a disinfectant includes the following steps:

[0008] (1) Preparation of standard solutions: Weigh chlorhexidine acetate standard into a 25 mL volumetric flask, dissolve it in methanol aqueous solution and dilute to the mark, shake well to obtain the standard stock solution; dilute the standard stock solution with methanol aqueous solution containing glacial acetic acid to prepare a series of standard working solutions, shake well, filter into injection vials, and wait for testing;

[0009] (2) Preparation of sample solution: Weigh the detoxifying agent into a 50mL volumetric flask, add glacial acetic acid, shake to completely disperse the sample, add methanol and water, extract by ultrasonication for 5-10 minutes, cool to room temperature, and then dilute to the mark with methanol-water solution. Shake well to obtain the sample solution, filter into a sample vial, and wait for testing.

[0010] (3) Use high performance liquid chromatography to determine the standard working solution of step (1) and the sample solution of step (2) and calculate the content of chlorhexidine acetate in the sample.

[0011] Preferably, the specific method for determining the standard working solution from step (1) and the sample solution from step (2) using a high-performance liquid chromatograph in step (3) includes the following steps:

[0012] a. Inject the standard working solution from step (1) into the high performance liquid chromatograph for determination. Perform regression analysis with the concentration of the standard working solution as the abscissa and the peak area of ​​the standard working solution as the ordinate to obtain the standard curve equation of chlorhexidine acetate.

[0013] b. Under the same conditions, inject the sample solution from (2) into a high-performance liquid chromatograph for determination, measure the peak area of ​​chlorhexidine acetate in the sample solution, substitute the peak area of ​​the sample solution into the above standard curve equation, obtain the concentration of chlorhexidine acetate in the sample solution, and calculate the content of chlorhexidine acetate in the sample based on the mass of the sample represented by the sample solution.

[0014] Preferably, the methanol-water solution in step (1) is a methanol-water solution with a volume ratio of 1:1.

[0015] Preferably, the preparation of the standard solution in step (1) includes the following steps:

[0016] (1) Standard stock solution: Accurately weigh 0.025 g (accurate to 0.0001 g) of chlorhexidine acetate standard and place it in a 25 mL volumetric flask. Dissolve it in a methanol aqueous solution containing 4% glacial acetic acid and dilute to the mark. Shake well to obtain a 1000 mg / L standard stock solution.

[0017] (2) Accurately transfer 0.02 mL, 0.05 mL, 0.1 mL, 0.2 mL, 0.5 mL, 1.0 mL and 2.0 mL of the above standard stock solution into a 10 mL volumetric flask, dilute with methanol aqueous solution containing 4% glacial acetic acid and make up to the mark, shake well to obtain a series of standard working solutions with mass concentrations of 2 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, 50 mg / L, 100 mg / L and 200 mg / L respectively.

[0018] Preferably, the operating conditions of the high-performance liquid chromatograph in step (3) are as follows:

[0019] Chromatographic column: C18 column, 250 mm × 4.6 mm, 5 μm;

[0020] Column temperature: 30℃;

[0021] Detection wavelength: 258nm;

[0022] Mobile phase: Phase A: 0.01 mol / L tetrabutylammonium bromide + 4% glacial acetic acid solution; Phase B: acetonitrile; A + B = 82 + 18;

[0023] Flow rate: 1.0 mL / min;

[0024] Injection volume: 20 μL.

[0025] Preferably, in step (3), the content of chlorhexidine acetate in the sample is calculated based on the mass of the sample represented by the sample solution. The content of chlorhexidine acetate is expressed as a mass fraction W (%), and its calculation formula is as follows:

[0026] W= ;

[0027] In the formula: C: the mass concentration of chlorhexidine acetate in the sample solution obtained from the standard curve, in milligrams per liter;

[0028] V: Constant volume, in milliliters;

[0029] m: Sample mass, in grams.

[0030] Preferably, the ultrasonic extraction is performed using a power gradient ultrasonic mode: in the first stage, ultrasonication is performed at 200W power for 3 minutes; in the second stage, ultrasonication is performed at 400W power for 4 minutes; and in the third stage, ultrasonication is performed at 200W power for 2 minutes.

[0031] Preferably, the preparation of the sample solution in step (2) includes the following steps: weigh 1g of detoxifying agent (accurate to 0.0001g) into a 50mL volumetric flask, add 2mL of glacial acetic acid, then add methanol and water in sequence, extract by ultrasonication, cool to room temperature and then dilute to the mark with methanol-water solution, shake well to obtain the sample solution, and filter it into a sample vial using a 0.22μm filter membrane.

[0032] Preferably, in step (2), 1 g of the detoxifying agent (accurate to 0.0001 g) is weighed into a 50 mL volumetric flask, 2 mL of glacial acetic acid is added first, then 15 mL of methanol is added, the mixture is shaken for 1 min and then placed in an ultrasonic instrument and ultrasonicated at 25 °C and 300 W power for 3 min; then 15 mL of water is added, the mixture is ultrasonically extracted, cooled to room temperature and then diluted to the mark with methanol aqueous solution, shaken well, and the sample solution is obtained. The sample solution is filtered through a 0.22 μm filter membrane into a sample vial.

[0033] The disinfectant is selected from any one of the following: solution-type disinfectant, gel-type disinfectant, powder-type disinfectant, and cream-type disinfectant.

[0034] Compared with existing methods for detecting chlorhexidine acetate, the present invention has the following advantages and beneficial effects:

[0035] (1) The present invention uses tetrabutylammonium bromide-acetic acid-acetonitrile as the mobile phase. Compared with the existing standard method using potassium dihydrogen phosphate-phosphate-acetonitrile as the mobile phase, it greatly reduces the adsorption and tailing phenomenon of chlorhexidine acetate on the chromatographic column, making the chromatographic peak shape sharper and more symmetrical. At the same time, it changes the polarity and hydrophobicity of chlorhexidine acetate, improves the selectivity of separation, and makes the separation of chlorhexidine acetate from impurities more complete and the quantification more accurate.

[0036] (2) Chlorhexidine acetate is a cationic surfactant that readily binds to negatively charged components in the matrix. Due to the different excipients added to different dosage forms, its solubility and dispersibility also differ, and the degree to which chlorhexidine acetate binds to the matrix of different dosage forms also varies. This invention uses glacial acetic acid to disperse the sample and simultaneously releases the chlorhexidine acetate bound to the matrix. Methanol aqueous solution is used as the extraction liquid, resulting in higher extraction efficiency. This solves the problem that when using existing standard extraction methods, chlorhexidine acetate in some dosage forms may not be completely extracted or released, leading to inaccurate test results or undetectable results. This provides strong technical support for improving product quality, protecting consumer rights, and regulating market order.

[0037] (3) This invention addresses the contradiction in existing technologies that use single-power ultrasound, which can lead to sample agglomeration resulting in uneven extraction or damage to the target analyte due to excessive power. At low power, matrix penetration is insufficient, while at high power, local overheating can exacerbate the interaction between chlorhexidine acetate and surfactants and stabilizers in the disinfectant, thus affecting extraction efficiency and reproducibility. The power gradient design of this invention, through a three-stage synergistic approach of dispersion-extraction-equilibration, ensures uniform sample dispersion at low power, avoiding dissolution differences caused by excessively high local concentrations. It also achieves efficient matrix disruption through medium-to-high power, and finally, low-power equilibration reduces system fluctuations, decreasing the extraction efficiency deviation of parallel samples and significantly improving reproducibility. Simultaneously, it increases the extraction rate of the target analyte, avoiding low quantification due to incomplete extraction and improving detection accuracy.

[0038] (4) This invention changes the traditional method of adding a mixed solvent of methanol and water all at once, which is prone to instantaneous aggregation of the sample matrix due to sudden changes in solvent polarity, and hydrophobic impurities are easily dissolved simultaneously with the target analyte, affecting the separation effect. This invention adopts a stepwise solvent addition design: first, pure methanol and glacial acetic acid are used to permeate the matrix to avoid aggregation caused by sudden changes in polarity and to ensure that chlorhexidine acetate is fully dissociated from the matrix; then, an aqueous phase is added and the temperature is increased to ensure the solubility of the target analyte in the mixed solvent. Attached Figure Description

[0039] Figure 1 The image shows the high-performance liquid chromatogram of the chlorhexidine acetate standard solution obtained using the GB / T26367-2020 method.

[0040] Figure 2 The image shows the high-performance liquid chromatogram of the sample solution of the Tuihuanmei Men's Antibacterial Disinfectant in Example 1, obtained using the method of GB / T26367-2020.

[0041] Figure 3 This is the UV absorption curve of chlorhexidine acetate.

[0042] Figure 4 The image shows the high-performance liquid chromatogram of the chlorhexidine acetate standard solution obtained in Example 1.

[0043] Figure 5 The image shows the high-performance liquid chromatogram of the sample solution of the Tuihuanmei Men's Antibacterial Disinfectant obtained in Example 1.

[0044] Figure 6 This is the standard curve of the chlorhexidine acetate standard solution in Example 1. Detailed Implementation

[0045] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0046] Example 1

[0047] Please see Figure 1-6 This embodiment provides a method for detecting the content of chlorhexidine acetate in disinfectants, specifically including the following steps:

[0048] (1) Preparation of standard solutions:

[0049] Standard stock solution: Accurately weigh 0.025 g (accurate to 0.0001 g) of chlorhexidine acetate standard into a 25 mL volumetric flask, dissolve it in a methanol-water (1:1, volume ratio) solution containing 4% (v / v) glacial acetic acid, and dilute to the mark. Shake well and set aside.

[0050] Standard working solutions: Accurately pipette 0.02 mL, 0.05 mL, 0.1 mL, 0.2 mL, 0.5 mL, 1.0 mL, and 2.0 mL of the above standard stock solution into 10 mL volumetric flasks. Dilute with a methanol aqueous solution containing 4% (v / v) glacial acetic acid and bring to volume. Shake well to obtain a series of standard working solutions with mass concentrations of 2 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, 50 mg / L, 100 mg / L, and 200 mg / L, respectively. Filter the solutions through a 0.22 μm filter membrane into vials for analysis.

[0051] (2) Preparation of sample solution:

[0052] Weigh 1 g of sample (accurate to 0.0001 g) into a 50 mL volumetric flask, add 2 mL of glacial acetic acid, then add 15 mL of methanol, shake for 1 min, and place in an ultrasonic instrument. Sonicate at 25 °C and 300 W for 3 min. Then add 15 mL of water and extract using a power gradient ultrasonic mode: in the first stage, sonicate at 200 W for 3 min; in the second stage, switch to 400 W for 4 min; and in the third stage, return to 200 W for 2 min. After cooling to room temperature, dilute to the mark with methanol-water (1:1, volume ratio) solution, shake well, and filter through a 0.22 μm filter membrane into a vial for analysis.

[0053] (3) Measurement:

[0054] The samples were analyzed using an Agilent 1260 high-performance liquid chromatograph (Agilent Technologies, Inc.), under the following conditions:

[0055] Chromatographic conditions: Column: Agilent Eclipse XDB-C18, 250 mm × 4.6 mm, 5 μm;

[0056] Column temperature: 30℃;

[0057] Detection wavelength: 258nm;

[0058] Mobile phase: Phase A: 0.01 mol / L tetrabutylammonium bromide + 4% glacial acetic acid solution; Phase B: acetonitrile; A + B = 82 + 18;

[0059] Flow rate: 1.0 mL / min;

[0060] Injection volume: 20 μL.

[0061] (4) Calculation method

[0062] A standard curve was plotted with the mass concentration (x, mg / L) of the chlorhexidine acetate standard working solution as the abscissa and the corresponding peak area (y) as the ordinate. The equation of the standard curve was y = 56.854x - 116.28, R0. 2 =0.9998. See the standard curve spectrum. Figure 6 The content of chlorhexidine acetate in the sample is expressed as a mass fraction W (%), and its calculation formula is as follows:

[0063] W= ;

[0064] In the formula: C: the mass concentration of chlorhexidine acetate in the sample solution obtained from the standard curve, in milligrams per liter (mg / L).

[0065] V: Volume at constant volume, mL;

[0066] m: Sample mass, g.

[0067] The solution-type disinfectant samples were extracted, tested, and calculated according to the above method. Each sample was measured twice in parallel, and the average value was taken. The results are shown in Table 1.

[0068]

[0069] Example 2

[0070] In this Example 2, the method of Example 1 was used to determine the content of chlorhexidine acetate in gel disinfectant. The gel disinfectant samples were extracted, tested and calculated according to the above method. Each sample was measured twice in parallel and the average value was taken.

[0071] The results are shown in Table 2.

[0072] Table 2. Chlorhexidine acetate content in gel-type disinfectants

[0073]

[0074] Example 3

[0075] This embodiment uses the method of Example 1 to determine the content of chlorhexidine acetate in powder disinfectant. Samples of powder disinfectant were extracted, tested, and calculated according to the above method. Each sample was measured twice in parallel, and the average value was taken. The results are shown in Table 3.

[0076] Table 3. Chlorhexidine acetate content in powder disinfectants

[0077]

[0078] Example 4

[0079] This embodiment uses the method of Example 1 to determine the content of chlorhexidine acetate in a cream-type disinfectant. Samples of the cream-type disinfectant were extracted, tested, and calculated according to the above method. Each sample was measured twice in parallel, and the average value was taken. The results are shown in Table 4.

[0080] Table 4. Chlorhexidine acetate content in cream-type disinfectants

[0081]

[0082] The results from Examples 1, 2, 3, and 4 show that when the sample matrix is ​​relatively simple, the results of determining the chlorhexidine acetate content in the disinfectant using existing standard methods and the method of this invention are basically consistent. However, when the sample contains complex excipients, especially samples with added natural plant ingredients, the existing standard methods often result in low or undetectable levels. When the method of this invention is used, the results reported by the manufacturers are basically consistent with their process inputs.

[0083] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for determining the chlorhexidine acetate content in a disinfectant, characterized in that, Includes the following steps: (1) Preparation of standard solutions: Weigh chlorhexidine acetate standard into a 25 mL volumetric flask, dissolve it in methanol aqueous solution and dilute to the mark, shake well to obtain the standard stock solution; dilute the standard stock solution with methanol aqueous solution containing glacial acetic acid to prepare a series of standard working solutions, shake well, filter into injection vials, and wait for testing; the methanol aqueous solution is a methanol-water solution with a volume ratio of 1:

1. (2) Preparation of sample solution: Weigh the detoxifying agent into a 50mL volumetric flask, add glacial acetic acid, shake to completely disperse the sample, add methanol and water, extract by ultrasonication for 5-10 minutes, cool to room temperature, and then dilute to the mark with methanol-water solution. Shake well to obtain the sample solution, filter into a sample vial, and wait for testing. (3) The standard working solution from step (1) and the sample solution from step (2) are measured using a high-performance liquid chromatograph, specifically including: a. Inject the standard working solution from step (1) into the high performance liquid chromatograph for determination. Perform regression analysis with the concentration of the standard working solution as the abscissa and the peak area of ​​the standard working solution as the ordinate to obtain the standard curve equation of chlorhexidine acetate. b. Under the same conditions, inject the sample solution in (2) into the high performance liquid chromatograph for determination, measure the peak area of ​​chlorhexidine acetate in the sample solution, substitute the peak area of ​​the sample solution into the above standard curve equation, obtain the concentration of chlorhexidine acetate in the sample solution, and calculate the content of chlorhexidine acetate in the sample based on the mass of the sample represented by the sample solution. The operating conditions for high performance liquid chromatography are as follows: Chromatographic column: C18 column, 250 mm × 4.6 mm, 5 μm; Column temperature: 30℃; Detection wavelength: 258nm; Mobile phase: Phase A: 0.01 mol / L tetrabutylammonium bromide + 4% glacial acetic acid solution; Phase B: acetonitrile; A + B = 82 + 18; Flow rate: 1.0 mL / min; Injection volume: 20 μL; Calculate the chlorhexidine acetate content in the sample based on the mass of the sample solution representing the specimen. The chlorhexidine acetate content is expressed as a mass fraction. W (%) indicates that its calculation formula is as follows: ， In the formula: C : The mass concentration of chlorhexidine acetate in the sample solution obtained from the standard curve, in milligrams per liter; V Volume at constant volume, in milliliters; m Sample mass, in grams.

2. The method for determining the chlorhexidine acetate content in a disinfectant according to claim 1, characterized in that, In step (1).

3. The method for determining the chlorhexidine acetate content in a disinfectant according to claim 1, characterized in that, The preparation of the standard solution in step (1) includes the following steps: (1) Standard stock solution: Accurately weigh 0.025 g of chlorhexidine acetate standard into a 25 mL volumetric flask, dissolve it in a methanol aqueous solution containing glacial acetic acid and dilute to the mark, shake well to obtain a 1000 mg / L standard stock solution; (2) Accurately transfer 0.02 mL, 0.05 mL, 0.1 mL, 0.2 mL, 0.5 mL, 1.0 mL and 2.0 mL of the above standard stock solution into a 10 mL volumetric flask, dilute with methanol aqueous solution containing glacial acetic acid and make up to the mark, shake well to obtain a series of standard working solutions with mass concentrations of 2 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, 50 mg / L, 100 mg / L and 200 mg / L respectively.

4. The method for determining the chlorhexidine acetate content in a disinfectant according to claim 3, characterized in that, The ultrasonic extraction process is as follows: extraction is performed using a power gradient ultrasonic mode: in the first stage, ultrasonication is performed at 200W power for 3 minutes; in the second stage, ultrasonication is performed at 400W power for 4 minutes; and in the third stage, ultrasonication is performed at 200W power for 2 minutes.

5. The method for determining the chlorhexidine acetate content in a disinfectant according to claim 4, characterized in that, The preparation of the sample solution in step (2) includes the following steps: weigh 1g of detoxifying agent into a 50mL volumetric flask, add 2mL of glacial acetic acid, then add methanol and water in sequence, extract by ultrasonication, cool to room temperature, and then dilute to the mark with methanol-water solution. Shake well to obtain the sample solution, and filter it into a sample vial using a 0.22μm filter membrane.

6. The method for determining the chlorhexidine acetate content in a disinfectant according to claim 5, characterized in that, In step (2), 1g of the detoxifying agent is weighed into a 50mL volumetric flask, 2mL of glacial acetic acid is added first, then 15mL of methanol is added, the mixture is shaken for 1min and then placed in an ultrasonic instrument and ultrasonicated at 25℃ and 300W power for 3min; then 15mL of water is added, the mixture is ultrasonically extracted, cooled to room temperature and then diluted to the mark with methanol aqueous solution, shaken well, and the sample solution is obtained. The sample solution is filtered through a 0.22μm filter membrane into a sample vial.

7. The method for determining the chlorhexidine acetate content in a disinfectant according to claim 1, characterized in that, The disinfectant is selected from any one of the following: solution-type disinfectant, gel-type disinfectant, powder-type disinfectant, and cream-type disinfectant.

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