Method for determining content of chlorhexidine acetate in disinfectant

Abstract

The invention 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 a sample by using glacial acetic acid, releasing chlorhexidine acetate combined with matrix components of the sample, and extracting by using a methanol aqueous solution; and (2) detecting by using a high performance liquid chromatograph by taking a tetrabutylammonium bromide-acetic acid-acetonitrile solution as a mobile phase. The method for detecting chlorhexidine acetate is simple and convenient to operate, good in reproducibility, complete in impurity separation and high in accuracy, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of detection, and particularly relates to a method for determining the content of chlorhexidine acetate in a disinfectant. BACKGROUND

[0002] Chlorhexidine acetate, also known as chlorhexidine acetate, is a double guanidine organic compound, and its chemical formula is 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 disinfection, local treatment and antiseptic effect, and is widely used in medical health, daily cleaning and industrial fields. At present, the dosage forms of chlorhexidine acetate on the market are solution, gel, powder and cream, and in order to be suitable for different use scenarios and needs, some products also add plant ingredients to enhance the disinfection effect, reduce irritation and increase environmental protection. With the wide use of chlorhexidine acetate disinfectant products, in order to protect the rights and interests of consumers and regulate the market order, it is necessary to ensure the quality of the products and prevent the phenomenon of low content of chlorhexidine acetate in the products, which cannot achieve the purpose of treating diseases and sterilizing, or high content, which brings safety risks and side effects. The content of chlorhexidine acetate in the product needs to be accurately determined.

[0003] The methods for determining the content of chlorhexidine acetate mainly include titration method, spectrophotometry, atomic absorption spectrometry, capillary electrophoresis method and liquid chromatography. However, according to the feedback information from many manufacturers for a long time, the methods reported at present are only suitable for the determination of the content of chlorhexidine acetate in samples with simple matrix components. The determination of the content of chlorhexidine acetate in disinfectants with complex matrix such as plant ingredients or multiple excipients has been a difficult problem in the industry, and there is a situation that the determination result does not match the feeding or even the detection cannot be carried out.

[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 result of the content of chlorhexidine acetate in the sample, and to provide strong technical support for improving the quality of disinfectant products, protecting the rights and interests of consumers and regulating the market order. SUMMARY

[0005] In order to overcome the shortcomings and deficiencies of the existing detection methods, the purpose of the present application is to provide a method for accurately determining the content of chlorhexidine acetate in a disinfectant.

[0006] The purpose of the present application is achieved by the following scheme:

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

[0008] (1) Preparation of standard solution: weigh chlorhexidine acetate standard product into a 25 mL volumetric flask, dissolve and make up to the mark with methanol aqueous solution, shake well to obtain a 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 a sample vial for testing;

[0009] (2) Preparation of sample solution: weigh the disinfectant into a 50 mL volumetric flask, add glacial acetic acid, shake to disperse the sample completely, add methanol and water, ultrasonic extraction for 5-10 min, cool to room temperature, then make up to the mark with methanol aqueous solution, shake well to obtain the sample solution, filter into a sample vial for testing;

[0010] (3) Use high performance liquid chromatograph 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 of step (3) for determining the standard working solution of step (1) and the sample solution of step (2) using high performance liquid chromatograph comprises the following steps:

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

[0013] b. Inject the sample solution of step (2) into the high performance liquid chromatograph for determination under the same conditions, 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 to obtain the concentration of chlorhexidine acetate in the sample solution, and calculate the content of chlorhexidine acetate in the sample according to the mass of the sample represented by the sample solution.

[0014] Preferably, the methanol aqueous 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) comprises the following steps:

[0016] (1) Standard stock solution: accurately weigh 0.025 g (accurate to 0.0001 g) of chlorhexidine acetate standard product into a 25 mL volumetric flask, dissolve and make up to the mark with methanol aqueous solution containing 4% glacial acetic acid, 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) comprises the following steps: weighing 1g (accurate to 0.0001g) of the detoxification agent into a 50mL volumetric flask, adding 2mL of glacial acetic acid, then adding methanol, water in sequence, ultrasonic extraction, cooling to room temperature, and then using methanol aqueous solution to constant volume to the scale, shaking evenly, obtaining the sample solution, and filtering into a sample vial with a 0.22μm filter membrane.

[0032] Preferably, the preparation of the sample solution in step (2) comprises the following steps: weighing 1g (accurate to 0.0001g) of the detoxification agent into a 50mL volumetric flask, adding 2mL of glacial acetic acid, then adding methanol, water in sequence, ultrasonic extraction, cooling to room temperature, and then using methanol aqueous solution to constant volume to the scale, shaking evenly, obtaining the sample solution, and filtering into a sample vial with a 0.22μm filter membrane.

[0033] The disinfectant is selected from any one of a solution type disinfectant, a gel type disinfectant, a powder type disinfectant, and a cream type disinfectant.

[0034] Compared with the prior art, the method has the following advantages and beneficial effects:

[0035] (1) The present application uses tetrabutylammonium bromide-acetic acid-acetonitrile as the mobile phase, which greatly reduces the adsorption and tailing phenomenon of chlorhexidine acetate on the chromatographic column compared with the potassium dihydrogen phosphate-phosphoric acid-acetonitrile used in the prior art standard method, makes the chromatographic peak shape more sharp and symmetrical, changes the polarity and hydrophobicity of chlorhexidine acetate, improves the selectivity of separation, makes the separation of chlorhexidine acetate and impurities more complete, and the quantification more accurate.

[0036] (2) Chlorhexidine acetate is a cationic surfactant that can easily combine with negatively charged components in the matrix. Due to the different auxiliary materials added in different dosage forms, their solubility and dispersibility are different, and the degree of combination of chlorhexidine acetate with different dosage forms is also different. The present application uses glacial acetic acid to disperse the sample, releases the chlorhexidine acetate combined with the matrix, and uses methanol aqueous solution as the extraction liquid, which has higher extraction efficiency, solves the problem that the chlorhexidine acetate in some dosage forms is not completely extracted or completely released when using the prior art standard method, resulting in inaccurate determination results or undetectable results, and provides strong technical support for improving product quality, protecting consumer rights and interests, and standardizing market order.

[0037] (3) The present application changes the single power ultrasonic method in the prior art, which is prone to sample aggregation, leading to uneven extraction or damage to the target object due to excessive power. Low power is not enough to penetrate the matrix, and high power may cause the interaction between chlorhexidine acetate and surfactants and stabilizers in the disinfectant to intensify, thereby affecting the extraction efficiency and reproducibility. The power gradient design of the present application cooperates through the three stages of dispersion-extraction-balance, which not only ensures uniform dispersion of the sample at low power, avoids the difference in dissolution caused by excessive local concentration, but also realizes efficient crushing of the matrix through medium and high power. Finally, low power balance reduces the extraction efficiency deviation of parallel samples, significantly improves the reproducibility, and improves the detection accuracy.

[0038] (4) The present application changes the traditional method of adding methanol and water mixed solvent at one time, which is prone to sample matrix aggregation due to sudden change of solvent polarity, and hydrophobic impurities are easily dissolved with the target object, affecting the separation effect. The present application adopts a step-by-step solvent addition design: first, pure methanol and glacial acetic acid are used to penetrate the matrix, avoiding aggregation caused by sudden change of polarity, and ensuring that chlorhexidine acetate is fully dissociated from the matrix; subsequently, water is added and the temperature is raised, which ensures the solubility of the target object in the mixed solvent. BRIEF DESCRIPTION OF DRAWINGS

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

[0040] Figure 2 is the high performance liquid chromatogram of the sample solution of the male antibacterial disinfectant solution of Example 1 obtained by using the method of GB / T26367-2020.

[0041] Figure 3 is the chlorhexidine acetate ultraviolet spectrum absorption curve graph.

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

[0043] Figure 5 is the high performance liquid chromatogram of the sample solution of the male antibacterial disinfectant solution of Example 1 obtained in Example 1.

[0044] Figure 6 is the standard curve graph of the chlorhexidine acetate standard solution in Example 1. DETAILED DESCRIPTION

[0045] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative effort belong to the protection scope of the present application.

[0046] Embodiment 1

[0047] Please refer to Figures 1-6 The embodiment provides a method for detecting the content of chlorhexidine acetate in a disinfectant, and specifically comprises the following steps.

[0048] (1) Preparation of a standard solution:

[0049] A standard stock solution is prepared as follows: 0.025 g (accurate to 0.0001 g) of chlorhexidine acetate standard substance is accurately weighed into a 25-mL volumetric flask, dissolved and diluted to the calibration mark with a methanol-water (1:1 by volume) solution containing 4% (v / v) glacial acetic acid, shaken well, and reserved for use.

[0050] A standard working solution is prepared as follows: 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 are accurately taken with a pipette and placed in 10-mL volumetric flasks, diluted to the calibration mark with a methanol-water solution containing 4% (v / v) glacial acetic acid, shaken well, to obtain standard series 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, filtered to a sample vial with a 0.22-μm filter membrane, and detected.

[0051] (2) Preparation of a sample solution:

[0052] 1 g of a sample (accurate to 0.0001 g) is weighed into a 50-mL volumetric flask, 2 mL of glacial acetic acid is added first, followed by the addition of 15 mL of methanol, shaken for 1 min, and then placed in an ultrasonic instrument for ultrasonic extraction at 25°C and a power of 300 W for 3 min; subsequently, 15 mL of water is added, and the extraction is performed in a power gradient ultrasonic mode: the first stage is ultrasonic extraction at a power of 200 W for 3 min, the second stage is ultrasonic extraction at a power of 400 W for 4 min, and the third stage is ultrasonic extraction at a power of 200 W for 2 min; after cooling to room temperature, the solution is diluted to the calibration mark with a methanol-water (1:1 by volume) solution, shaken well, filtered to a sample vial with a 0.22-μm filter membrane, and detected.

[0053] (3) Determination:

[0054] An Agilent 1260 high-performance liquid chromatograph (Agilent, USA) is used to detect the sample, and the determination conditions are as follows:

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

[0056] Column temperature: 30 °C;

[0057] Detection wavelength: 258 nm;

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

[0059] Flow rate: 1.0 mL / min;

[0060] Injection volume: 20 μL.

[0061] (4) Calculation method

[0062] The 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, and the standard curve equation was y = 56.854x - 116.28, R 2 = 0.9998. The standard curve spectrum is shown in Figure 6 . The content of chlorhexidine acetate in the sample is expressed as a mass fraction W (%), and the 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: constant volume, mL;

[0066] m: sample mass, g.

[0067] According to the above method, the sample extraction, detection and calculation of the solution type disinfectant sample were carried out respectively, each sample was determined in parallel for 2 times, and the average value was taken. The results are shown in Table 1.

[0068]

[0069] Example 2

[0070] This example 2 uses the method of example 1 to determine the content of chlorhexidine acetate in gel type disinfectant. According to the above method, the sample extraction, detection and calculation of the gel type disinfectant sample were carried out respectively, each sample was determined in parallel for 2 times, and the average value was taken.

[0071] The results are shown in Table 2.

[0072] Table 2 Content of chlorhexidine acetate in gel type disinfectant

[0073]

[0074] Example 3

[0075] Example 3 The content of chlorhexidine acetate in the powder type disinfectant was determined by the method of Example 1. The sample extraction, detection and calculation were carried out according to the above method, each sample was determined in parallel for 2 times, and the average value was taken, and the results were shown in Table 3.

[0076] Table 3 Content of chlorhexidine acetate in the powder type disinfectant

[0077]

[0078] Example 4

[0079] Example 4 The content of chlorhexidine acetate in the cream type disinfectant was determined by the method of Example 1. The sample extraction, detection and calculation were carried out according to the above method, each sample was determined in parallel for 2 times, and the average value was taken, and the results were shown in Table 4.

[0080] Table 4 Content of chlorhexidine acetate in the cream type disinfectant

[0081]

[0082] From the results of Example 1, Example 2, Example 3 and Example 4, it was found that when the sample matrix was relatively simple, the content of chlorhexidine acetate in the disinfectant determined by the existing standard method and the method of the present application was basically consistent. When the auxiliary materials in the sample were relatively complex, especially the samples with natural plant ingredients added in the formula, the determination results were mostly low or undetectable by using the existing standard method. By using the method of the present application, the production manufacturers feedback the results basically consistent with the process feedstock.

[0083] The above is the preferred embodiment of the present application, it should be noted that for ordinary skilled in the art, without departing from the principles of the present application, can make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application.

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; (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) 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.

2. The method for determining the chlorhexidine acetate content in a disinfectant according to claim 1, characterized in that, The method for performing the measurement in step (3) includes the following steps: 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 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.

3. The method for determining the chlorhexidine acetate content in a disinfectant according to claim 1, characterized in that, In step (1), the methanol-water solution is a methanol-water solution with a volume ratio of 1:

1.

4. 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.

5. The method for determining the chlorhexidine acetate content in a disinfectant according to claim 1, characterized in that, The operating conditions for the high-performance liquid chromatograph in step (3) 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.

6. The method for determining the chlorhexidine acetate content in a disinfectant according to claim 5, characterized in that, In step (3), the content of chlorhexidine acetate in the sample is calculated based on the mass of the sample solution representing the sample. The content of chlorhexidine acetate is expressed as a mass fraction W (%), and its calculation formula is as follows: W= ； In the formula: C: the mass concentration of chlorhexidine acetate in the sample solution obtained from the standard curve, in milligrams per liter; V: Constant volume, in milliliters; m: Sample mass, in grams.

7. The method for determining the chlorhexidine acetate content in a disinfectant according to claim 5, 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.

8. The method for determining the chlorhexidine acetate content in a disinfectant according to claim 7, 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.

9. The method for determining the chlorhexidine acetate content in a disinfectant according to claim 8, 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.

10. 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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