Method for detecting cetriammonium bromide in mixed solution by utilizing HPLC (High Performance Liquid Chromatography)

Abstract

The invention relates to a method for detecting cetriammonium bromide in a mixed solution by utilizing HPLC (High Performance Liquid Chromatography). The method comprises the following detection steps: step 1) preparing a solvent, a blank solution, a system applicability solution and a test solution; 2) based on chromatographic conditions, performing sample injection detection by adopting a high performance liquid chromatograph with an evaporative light scattering detector, separating cetrimonium bromide in a sample by virtue of a chromatographic column, and detecting the separated cetrimonium bromide by virtue of a detector, so as to obtain a blank solution spectrogram, a standard series solution spectrogram and a test solution spectrogram; and (3) carrying out qualitative and quantitative analysis on the cetrimonium bromide according to the obtained standard series solution spectrogram and the test solution spectrogram. The method for detecting the cetriammonium bromide in the mixed liquid by the high performance liquid chromatography has the advantages of high accuracy, high precision, good reproducibility and good separation degree.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection of sanitary products, and in particular to a method for detecting cetrimonium bromide in a mixed solution by utilizing HPLC. Background Art

[0002] Cetrimonium bromide, also known as hexadecyltrimethylammonium bromide, is a cationic surfactant that has good compatibility with cationic, nonionic and amphoteric surfactants. It has excellent penetration, softening, emulsifying, antistatic, biodegradable and bactericidal properties. This product has good chemical stability, heat resistance, light resistance, pressure resistance, strong acid and alkali resistance, these characteristics make it stable in a variety of environments, not easy to decompose or denature, these characteristics make cetrimonium bromide compatible with other ingredients in cosmetic formulas, will not produce adverse reactions or affect the stability of the product.

[0003] Existing technology: GB / T 26369-2020 "Hygiene requirements for quaternary ammonium disinfectants", the test method in this national standard:

[0004] Method 1 is applicable to the determination of benzethonium chloride in raw materials, single and compound chemical disinfectants;

[0005] Method 2 is applicable to the determination of benzalkonium chloride content in raw materials, single and compound chemical disinfectants, and is also applicable to the determination of benzalkonium bromide content. It is also applicable to the simultaneous determination of these substances in compound chemical disinfectants prepared by mixing chlorhexidine acetate with benzalkonium chloride or benzalkonium bromide.

[0006] Method three is applicable to the determination of benzethonium chloride in raw materials, single and compound chemical disinfectants, the simultaneous determination of benzethonium chloride and polyhexamethylene biguanide in single and compound chemical disinfectants, and the simultaneous determination of benzethonium chloride and chlorhexidine acetate in compound chemical disinfectants.

[0007] Method 4 is applicable to the determination of benzalkonium bromide content in raw materials or single chemical disinfectants, and is also applicable to the determination of benzalkonium chloride or other anion-substituted quaternary ammonium salts in raw materials or single chemical disinfectants.

[0008] Journal 1: Flavors, Fragrances and Cosmetics, Issue 3, June 2012, "Quantitative Determination of Cetrimonium Bromide in Wash-off Cosmetics by High Performance Liquid Chromatography"_Leng Taohua

[0009] Journal 2: Analysis Laboratory, Vol. 34, No. 2, February 2015, "Determination of Five Quaternary Ammonium Preservatives in Cosmetics by High Performance Liquid Chromatography"_Jie Nan

[0010] Journal 3: Chin J Biologicals October 2020, Vol.33No.10

[0011] In summary, there is no detection method for cetrimonium bromide in the above national standard for quaternary ammonium salts, which is lacking; the peak time of cetrimonium bromide in the detection method in the journal is too early, which is easily interfered during the sample detection process and overlaps with the peaks of other components in the formula, resulting in abnormal peak shape and inaccurate results; the existing technologies in the journal all use the 208nm wavelength of the ultraviolet detector, which is relatively low and is greatly affected by the solvent and mobile phase. Summary of the invention

[0012] The object of the present invention is to overcome the deficiencies in the prior art and provide a method for detecting cetrimonium bromide in a mixed solution by using HPLC. The method has high sensitivity, good durability and good reproducibility, can make the detection of cetrimonium bromide in the solution not be interfered by impurity peaks, has perfect peak shape and accurate integration.

[0013] The present invention is achieved through the following technical solutions: On the one hand, a method for detecting cetrimonium bromide in a mixed solution by using HPLC is provided, and the chromatographic conditions of the method are as follows: chromatographic column: XDB-C18 (5 μm, 4.6 mm*250 mm); column temperature: 28-32° C.;

[0014] Flow rate: 0.8-1.2 mL / min; Mobile phase: Mobile phase A: 0.08-0.12% trifluoroacetic acid; Mobile phase B: methanol;

[0015] The gradient elution run time table of mobile phases A and B is as follows:

[0016]

[0017]

[0018] The specific detection steps are as follows:

[0019] Step 1) preparing solvent, blank solution, system suitability solution and test solution;

[0020] Step 2) Based on the chromatographic conditions, a high performance liquid chromatograph with an evaporative light scattering detector is used for sample injection detection. After the cetrimide bromide in the sample is separated by a chromatographic column, the separated cetrimide bromide is detected by a detector to obtain a blank solution spectrum, a standard series solution spectrum, and a test solution spectrum;

[0021] Step 3) qualitative and quantitative analysis of cetrimide is performed on the obtained standard series solution spectra and the test solution spectra.

[0022] Furthermore, in step 1), a methanol-0.1% trifluoroacetic acid aqueous solution with a mass ratio of 60:40 is prepared as a blank solution or solvent.

[0023] Furthermore, in step 2), the parameters of the detector are: evaporator temperature: 34-36°C; atomization temperature: 34-36°C; gas flow rate: 1.28-1.32 SLM; injection volume: 8-12 μL.

[0024] Further, in step 2),

[0025] Preparation of the test solution: accurately measure an appropriate amount of the solution containing cetrimonium bromide, dilute to the scale with a solvent, ensure that the final concentration is within the detection range, and shake well;

[0026] The preparation of the standard series solution includes the following two steps:

[0027] The first step is to prepare the cetrimonium bromide reference solution: take 100 mg of cetrimonium bromide reference, accurately weigh it, put it in a 100 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and use it as the cetrimonium bromide reference stock solution;

[0028] The second step is the preparation of a standard series of solutions: accurately pipette 100uL, 200uL, 300uL, 400uL, 500uL, and 600uL of the cetrimide reference stock solution into centrifuge tubes of appropriate volumes, dilute to 1mL with solvent, shake well, and prepare a standard series of cetrimide solutions with concentrations of 100ug / mL, 200ug / mL, 300ug / mL, 400ug / mL, 500ug / mL, and 600ug / mL.

[0029] Through the above technical solution, the cetrimonium bromide reference stock solution can be accurately aspirated and a 1000uL pipette can be used to aspirate, which can significantly improve the experimental efficiency and accuracy. When testing a large number of samples, the rapid operation of the pipette can greatly improve efficiency and accuracy and reduce the risk of contamination.

[0030] Further, in step 3), a qualitative analysis is performed on the blank solution spectrum, and the peak in the blank solution spectrum is a solvent peak. During the quantitative analysis, the peak area or peak height of the solvent peak in the test solution spectrum is ignored;

[0031] Qualitative and quantitative analysis of cetrimide is performed on the spectra of the standard series solutions and the spectra of the test solution; a standard working curve is drawn with the mass concentration X of the standard solution as the abscissa and the peak area Y of cetrimide as the ordinate, and the cetrimide in the test solution is quantitatively analyzed by the external standard method using the standard working curve; and cetrimide is qualitatively analyzed using the retention time of the appearance of cetrimide in the test solution.

[0032] Furthermore, the filler of the XDB-C18 chromatographic column: the full name is ZORBAX RR Eclipse XDB-C18, the filler of the XDB-C18 chromatographic column is chemically modified on the C18 filler, and a layer of low-crosslinked phenyl and diphenyl is covered on the surface of the filler, with a specification of 4.6×250mm, 5μm.

[0033] Through the above technical solution, the filler of XDB-C18 chromatographic column is chemically modified on the traditional C18 filler, making it more hydrophobic and lipophilic, and also having higher capacity and separation efficiency. The surface of this filler is covered with a layer of phenyl and diphenyl with low cross-linking degree, which can improve the stability and durability of the filler, and also reduce problems such as peak tailing and nonlinear response.

[0034] Further, in step 3), the qualitative analysis and quantitative analysis process are as follows:

[0035] Qualitative analysis: According to the spectrum of the standard solution, attribute the peaks in the spectrum of the test solution to determine whether the test solution contains cetrimonium bromide;

[0036] Quantitative analysis: According to the spectra of the standard series solutions, with the concentration of cetrimonium bromide in the standard series solutions as the horizontal axis and the peak area of ​​cetrimonium bromide as the vertical axis, a standard curve is drawn, and the content of cetrimonium bromide in the test sample is calculated by the external standard method.

[0037] Beneficial Effects

[0038] The detection method of the invention has high sensitivity, good durability and good reproducibility, can make the detection of cetrimonium bromide in the prepared solution not be interfered by impurity peaks, has perfect peak shape and accurate integration. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the blank solution spectrum in Example 1 of the present invention;

[0040] Figure 2 This is a spectrum of the cetrimonium bromide standard solution in Example 1 of the present invention;

[0041] Figure 3 This is a spectrum of the sample solution in Example 1 of the present invention;

[0042] Figure 4 This is a spectrum of the cetrimonium bromide standard solution in Example 2 of the present invention;

[0043] Figure 5 This is a spectrum of the sample solution in Example 2 of the present invention;

[0044] Figure 6This is a spectrum of the cetrimonium bromide standard solution in Example 1 of the present invention;

[0045] Figure 7 This is a spectrum of the sample solution in Example 2 of the present invention;

[0046] Figure 8 The comparative example 1 is the spectrum of the test sample solution under other mobile phase and chromatographic column conditions;

[0047] Fig. 9 Comparative Example 2 is a spectrum of the test sample solution under other mobile phase and chromatographic column conditions. DETAILED DESCRIPTION

[0048] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0049] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. The experimental methods without specific conditions in the following examples are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, all percentages, ratios, proportions or parts are by weight.

[0050] Unless otherwise specified, the reagents and raw materials used in the examples and comparative examples of the present invention can be obtained through commercial channels.

[0051] Example 1

[0052] A method for detecting cetrimonium bromide in a mixed solution by using HPLC, wherein the specific operation steps are as follows;

[0053] Reagents and samples used:

[0054] Methanol: chromatographic grade, Fisher reagent;

[0055] Trifluoroacetic acid: chromatography grade, Aladdin reagent;

[0056] Cetrimonium bromide: ≥98%, Aladdin reagent;

[0057] Sample: Deyou wet wipes liquid PF009;

[0058] Instrument and parameter settings:

[0059] High performance liquid chromatograph: Aglient 1260 high performance liquid chromatograph;

[0060] Detector: 1260 Infinity II Evaporative Light Scattering Detector;

[0061] Chromatographic column: XDB-C18 (5 μm, 4.6 mm*250 mm);

[0062] The gradient elution run time table of mobile phases A and B is as follows:

[0063] Running time (min) Mobile phase A (%) Mobile phase B (%) 0 40 60 3 40 60 7 12 88 15 12 88 15.1 0 100 19 0 100 19.1 40 60 25 40 60

[0064] Column temperature: 30°C;

[0065] Flow rate: 1.0 mL / min;

[0066] Detector parameters: evaporator temperature: 35°C; atomization temperature: 35°C; gas flow rate: 1.30SLM; injection volume: 10μL;

[0067] Mobile phase: Mobile phase A: 0.10% trifluoroacetic acid; Mobile phase B: methanol;

[0068] Running time: 25min.

[0069] Step 1) Prepare the solvent, blank solution, system suitability solution, and test solution. The preparation process is as follows:

[0070] ①Solvent: methanol-0.1% trifluoroacetic acid aqueous solution (60:40)

[0071] ② Preparation of blank solution: directly take the above methanol-0.1% trifluoroacetic acid aqueous solution (60:40) as the blank solution.

[0072] ③ Sample test solution: Accurately measure an appropriate amount of a solution containing cetrimonium bromide (e.g. a certain solution, with a specification of 10 mg cetrimonium bromide in 1 ml), transfer it to an appropriate volumetric flask after calculating the content, dilute it to the scale with solvent, ensure that the final concentration is within the detection range, and shake well.

[0073] ④ Cetrimide reference solution: Take 100mg of Cetrimide reference, weigh accurately, place in a 100ml volumetric flask, add solvent to dissolve and dilute to scale, shake well, as Cetrimide reference stock solution. Accurately draw 100uL, 200uL, 300uL, 400uL, 500uL, 600uL of Cetrimide reference stock solution, place in a volumetric flask of appropriate volume, dilute to scale with solvent, shake well, and prepare Cetrimide standard series solutions with concentrations of 100ug / mL, 200ug / mL, 300ug / mL, 400ug / mL, 500ug / mL, 600ug / mL. This solution is the standard series solution.

[0074] Step 2) Based on the above chromatographic conditions, a high performance liquid chromatograph with an evaporative light scattering detector is used for sample injection detection, and a blank solution, a system suitability solution, a standard series solution and a test solution are respectively drawn by an automatic sampler, and injected into a chromatographic column of the high performance liquid chromatography, respectively; the cetrimonium bromide in the test sample is separated by a chromatographic column, and then the separated cetrimonium bromide is detected by a detector; a blank solution spectrum, a system suitability solution spectrum, a standard series solution spectrum, and a test solution spectrum are obtained;

[0075] Step 3) qualitative and quantitative analysis of cetrimide is performed on the obtained system suitability solution spectrum, standard series solution spectrum, and test solution spectrum; the mass concentration X of the standard solution is used as the abscissa, and the peak area Y of cetrimide is used as the ordinate to draw a standard working curve, and the cetrimide in the test solution is quantitatively analyzed by the external standard method using the standard working curve; the retention time of cetrimide in the test solution is used to qualitatively analyze cetrimide.

[0076] The blank solution spectrum is shown in Figure 1 ;

[0077] See the spectrum of cetrimonium bromide standard solution Figure 2 ;

[0078] Sample test solution spectrum see Figure 3 .

[0079] The retention time statistics in each spectrum are as follows:

[0080] Solution name Cetrimonium Bromide Chlorphenesin Chlorhexidine gluconate Standard solution 11.996min / / Sample test solution 11.840min 2.192min 5.054min

[0081] Example 2

[0082] Compared with Example 1:

[0083] Column temperature: 28°C;

[0084] Flow rate: 0.8 mL / min;

[0085] Detector parameters: evaporator temperature: 34°C; atomization temperature: 34°C; gas flow rate: 1.28SLM; injection volume: 8μL;

[0086] Mobile phase: Mobile phase A: 0.08% trifluoroacetic acid; Mobile phase B: methanol;

[0087] Running time: 25min.

[0088] The other steps are the same as in Example 1.

[0089] See the spectrum of cetrimonium bromide standard solution Figure 4 ;

[0090] Sample test solution spectrum see Figure 5.

[0091] The retention time statistics in each spectrum are as follows:

[0092] Solution name Cetrimonium Bromide Chlorphenesin Chlorhexidine gluconate Standard solution 13.673min / / Sample test solution 13.642min 2.661min 6.449min

[0093] Example 3

[0094] Compared with Example 1:

[0095] Column temperature: 32°C;

[0096] Flow rate: 1.2 mL / min;

[0097] Detector parameters: evaporator temperature: 36°C; atomization temperature: 36°C; gas flow rate: 1.32SLM; injection volume: 12μL;

[0098] Mobile phase: Mobile phase A: 0.12% trifluoroacetic acid; Mobile phase B: methanol;

[0099] Running time: 25min.

[0100] The other steps are the same as in Example 1.

[0101] See the spectrum of cetrimonium bromide standard solution Figure 6 ;

[0102] Sample test solution spectrum see Figure 7 .

[0103] The retention time statistics in each spectrum are as follows:

[0104] Solution name Cetrimonium Bromide Chlorphenesin Chlorhexidine gluconate Standard solution 10.875min / / Sample test solution 10.857min / 4.198min

[0105] It can be seen from the results of Examples 1-3 that the chromatographic peak separation of other substances in the mixed solution and cetrimonium bromide meets the requirements, indicating that the method of the present invention has good specificity.

[0106] Comparative Example 1:

[0107] The mobile phase A in the above method is 0.1% formic acid, the chromatographic column is a CN chromatographic column, and the other conditions are the same as "Example 1". Figure 8 .

[0108] Comparative Example 2:

[0109] The mobile phase A in the above method is 0.2% acetic acid, the chromatographic column is SB-C18 chromatographic column, and other conditions are the same as "Example 1". Fig. 9 .

[0110] Effect Example

[0111] The methods of Examples 1 to 3 and Comparative Examples 1 to 2 were used to detect the cetrimonium bromide in the Deyou wet wipes formulation PF009. The linear equation and correlation coefficient of cetrimonium bromide by high performance liquid chromatography of each embodiment are shown in the following table:

[0112] Example Linear regression equation Correlation coefficient Example 1 log(y)=1.4336log(x)-0.2765 0.99996 Example 2 log(y)=1.4058log(x)-0.3096 0.99970 Example 3 log(y)=1.4483log(x)-0.5123 0.99985 Comparative Example 1 log(y)=1.3859log(x)+0.6186 0.99450 Comparative Example 2 log(y)=1.4302log(x)-0.4128 0.99684

[0113] Comparison of the correlation coefficients of the HPLC of Examples 1 to 3 and Comparative Examples 1 to 2 shows that the detection method provided by the present invention can effectively improve the linear coefficient of the standard working curve of the liquid chromatography, thereby improving the detection accuracy of the HPLC to a certain extent.

[0114] The methods of Examples 1 to 3 and Comparative Examples 1 to 2 were used to detect the cetrimonium bromide in the Deyou wet wipes formulation PF009. The results of the cetrimonium bromide content detection by high performance liquid chromatography of each example are shown in the following table:

[0115]

[0116]

[0117] It can be seen from the test results of the cetrimonium bromide content of Examples 1 to 3 and Comparative Examples 1 to 2 that the test result of Example 1 is within the error range of <0.5%, has higher quantitative accuracy, smaller deviation, good specificity, good reproducibility, high accuracy, and is not interfered by blanks and other impurities.

[0118] The methods of Examples 1 to 3 and Comparative Examples 1 to 2 were used to detect cetrimonium bromide in Deyou wet wipes PF009. The recovery rate, standard deviation and detection limit of the high performance liquid chromatography of each example are shown in the following table:

[0119]

[0120] By comparing the added recovery rate, standard deviation and detection limit data of the high performance liquid chromatography of Examples 1 to 3 and Comparative Examples 1 to 2, it can be seen that the detection method provided by the present invention can effectively improve the added recovery rate of the high performance liquid chromatography and reduce the standard deviation value, thereby improving the detection accuracy of the high performance liquid chromatography to a certain extent. From the data results and effects, Example 1 has the best effect.

[0121] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting cetrimonium bromide in a mixed solution using HPLC, characterized in that: The chromatographic conditions of the method are as follows: chromatographic column: XDB-C18 (5 μm, 4.6 mm*250 mm); column temperature: 28-32° C.; flow rate: 0.8-1.2 mL / min; mobile phase: mobile phase A: 0.08-0.12% trifluoroacetic acid; mobile phase B: methanol; The gradient elution run time table of mobile phases A and B is as follows: The specific detection steps are as follows: Step 1) preparing solvent, blank solution, system suitability solution and test solution; Step 2) Based on the chromatographic conditions, a high performance liquid chromatograph with an evaporative light scattering detector is used for sample injection detection. After the cetrimide bromide in the sample is separated by a chromatographic column, the separated cetrimide bromide is detected by a detector to obtain a blank solution spectrum, a standard series solution spectrum, and a test solution spectrum; Step 3) qualitative and quantitative analysis of cetrimide is performed on the obtained standard series solution spectra and the test solution spectra.

2. The method for detecting cetrimonium bromide in a mixed solution by HPLC according to claim 1, wherein In step 1), a methanol-0.1% trifluoroacetic acid aqueous solution with a mass ratio of 60:40 is prepared as a blank solution or solvent.

3. The method for detecting cetrimonium bromide in a mixed solution by HPLC according to claim 1, wherein In step 2), the parameters of the detector are: evaporator temperature: 34-36°C; atomization temperature: 34-36°C; gas flow rate: 1.28-1.32 SLM; injection volume: 8-12 μL.

4. The method for detecting cetrimonium bromide in a mixed solution by HPLC according to claim 1, wherein In step 2), Preparation of the test solution: accurately measure an appropriate amount of the solution containing cetrimonium bromide, dilute to the scale with a solvent, ensure that the final concentration is within the detection range, and shake well; The preparation of the standard series solution includes the following two steps: The first step is to prepare the cetrimonium bromide reference solution: take 100 mg of cetrimonium bromide reference, accurately weigh it, put it in a 100 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and use it as the cetrimonium bromide reference stock solution; The second step is the preparation of a standard series of solutions: accurately pipette 100uL, 200uL, 300uL, 400uL, 500uL, and 600uL of the cetrimide reference stock solution into centrifuge tubes of appropriate volumes, dilute to 1mL with solvent, shake well, and prepare a standard series of cetrimide solutions with concentrations of 100ug / mL, 200ug / mL, 300ug / mL, 400ug / mL, 500ug / mL, and 600ug / mL.

5. The method for detecting cetrimonium bromide in a mixed solution by HPLC according to claim 1, wherein In step 3), a qualitative analysis is performed on the blank solution spectrum, and the peak in the blank solution spectrum is a solvent peak. During the quantitative analysis, the peak area or peak height of the solvent peak in the test solution spectrum is ignored; Qualitative and quantitative analysis of cetrimide is performed on the spectra of the standard series solutions and the spectra of the test solution; a standard working curve is drawn with the mass concentration X of the standard solution as the abscissa and the peak area Y of cetrimide as the ordinate, and the cetrimide in the test solution is quantitatively analyzed by the external standard method using the standard working curve; and cetrimide is qualitatively analyzed using the retention time of the appearance of cetrimide in the test solution.

6. The method for detecting cetrimonium bromide in a mixed solution by HPLC according to claim 1, characterized in that: The filler of the XDB-C18 chromatographic column: the full name is ZORBAX RR Eclipse XDB-C18. The filler of the XDB-C18 chromatographic column is chemically modified on the C18 filler, and a layer of low-crosslinked phenyl and diphenyl is covered on the surface of the filler. The specifications are 4.6×250mm, 5μm.

7. The method for detecting cetrimonium bromide in a mixed solution by HPLC according to claim 1, characterized in that: In step 3), the qualitative analysis and quantitative analysis process are as follows: Qualitative analysis: According to the spectrum of the standard solution, attribute the peaks in the spectrum of the test solution to determine whether the test solution contains cetrimonium bromide; Quantitative analysis: According to the spectra of the standard series solutions, with the concentration of cetrimonium bromide in the standard series solutions as the horizontal axis and the peak area of ​​cetrimonium bromide as the vertical axis, a standard curve is drawn, and the content of cetrimonium bromide in the test sample is calculated by the external standard method.

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