Method for determining the content of benzalkonium chloride homologues in disinfection products by reversed phase liquid chromatography

Abstract

The invention discloses a method for determining the content of benzalkonium chloride homologues in disinfection products by reverse phase liquid chromatography, which comprises the following steps: step 1, preparing a detection instrument; step 2, preparing a detection reagent; step 3, preparing a standard solution; step 4, pre-treating a sample; and step 5, analyzing the determined solution by liquid chromatography. The invention provides a method for determining the content of benzalkonium chloride homologues in disinfection products by reverse phase liquid chromatography, which is not only simple to operate, fast and accurate, and has good stability, but also solves the problem of possible damage to the chromatographic column by the sweeping agent triethylamine and the appearance of ghost peaks in the standard product and the sample.

Description

Technical Field

[0001] The invention relates to a method for determining the content of benzalkonium chloride homologues in disinfection products by reverse phase liquid chromatography. Background Art

[0002] Benzalkonium chloride (BAC) is a mixture of dimethylbenzyl ammonium chloride substituted by fatty alkyl groups. There are mainly three ammonium chloride homologues, namely dodecyl dimethylbenzyl ammonium chloride (DDBAC, molecular formula: C 21 H 38 Cl N), tetradecyl dimethyl benzyl ammonium chloride (TDBAC, molecular formula: C 23 H 42 Cl N), hexadecyl dimethylbenzyl ammonium chloride (HDBAC, molecular formula: C 25 H 46 Cl N) [1] It is a cationic surfactant and a non-oxidizing fungicide. It has a broad-spectrum and highly effective ability to kill bacteria and algae. It also has certain dispersing and penetrating effects. It is widely used in disinfection, medicine, antiseptic, insect control, textile printing and dyeing, industrial water treatment and other fields. Currently, the composite quaternary ammonium salt disinfection products on the market mostly use benzalkonium chloride as the active ingredient, but some products also add other types of composite ingredients.

[0003] Testing has found that the content of benzalkonium chloride in many disinfection products is around 0.1% or below. According to the 2002 edition of the "Technical Specifications for Disinfection", the sampling volume needs to be equivalent to 0.5g of benzalkonium chloride, which means that about 500ml of sample is needed. For parallel determination, at least 1000ml of sample is needed, which is a huge sampling volume. Secondly, the "Technical Specifications for Disinfection" contains benzalkonium chloride (chlorhexidine, C 22 H 40 The determination of the content of benzalkonium chloride (Cl N) was not clear, nor was the specific composition and proportion of benzalkonium chloride. In fact, benzalkonium chloride is a mixture, with three specific components, namely dodecyl dimethyl benzyl ammonium chloride, tetradecyl dimethyl benzyl ammonium chloride, and hexadecyl dimethyl benzyl ammonium chloride. However, the 2002 edition of the Technical Specifications for Disinfection only used an average molecular weight of benzalkonium chloride (chlorhexidine, C 22 H 40Cl N) to replace two or more mixtures, and titration cannot distinguish these homologues. Through testing, it was found that the proportions of several homologues of benzalkonium chloride in products from different manufacturers are different. The average molecular weight is used uniformly, which sometimes produces relatively large errors. Liquid chromatography can qualitatively and quantitatively analyze these two homologues, or even three homologues, respectively, and the sampling volume is small, which is suitable for laboratory qualitative and quantitative analysis and corrects the errors caused by titration analysis. In addition, liquid chromatographs are more common than capillary electrophoresis instruments in the method of GB 26369-2020 "Hygiene Standard for Quaternary Ammonium Disinfectants".

[0004] At present, the methods for determining benzalkonium chloride include titration, ultraviolet spectrophotometry, ion chromatography, capillary electrophoresis, [ , gas chromatography-mass spectrometry, liquid chromatography, liquid chromatography-mass spectrometry, nanoelectrospray-mass spectrometry ] Etc. The United States only stipulates the content of two homologues of benzalkonium chloride and the determination method of normal liquid chromatography in the determination technology of benzalkonium chloride homologues. my country already has standard methods for the detection of cosmetics by polar chromatography column-liquid chromatography, the detection of cosmetics and toothpaste products by normal chromatography column-liquid chromatography, and the determination of benzalkonium chloride in disinfection products (GB 26369-2020) by capillary electrophoresis. The content range of benzalkonium chloride in disinfection products is relatively wide (from almost 100% pure products to concentrations of 0.1% or even below), but the reversed-phase liquid chromatography standardized method for the detection of disinfection products with benzalkonium chloride as the active ingredient has not yet been found. Summary of the invention

[0005] The invention provides a method for determining the content of benzalkonium chloride homologues in disinfection products by reverse phase liquid chromatography. The method is not only simple, fast and accurate in operation and has good stability, but also solves the problems of possible damage of the sweep agent triethylamine to the chromatographic column and the appearance of ghost peaks in standard products and samples.

[0006] The present invention adopts the following technical scheme: a method for determining the content of benzalkonium chloride homologues in disinfection products by reverse phase liquid chromatography, which comprises the following steps: step one, preparing a detection instrument: the detection instrument comprises an Agilent 1200 liquid chromatograph, a liquid chromatographic column, an ultrasonic extractor, a 0.45 μm polysulfone ether aqueous phase filter membrane and an electronic analytical balance, and the liquid chromatograph is equipped with a diode array detector; step two, preparing a detection reagent: the detection reagent comprises acetonitrile, triethylamine, glacial acetic acid, ammonium acetate, dodecyl dimethyl benzyl ammonium chloride with a purity of ≥98%, tetradecyl dimethyl benzyl ammonium chloride with a purity of ≥98%, hexadecyl dimethyl benzyl ammonium chloride with a purity of ≥98%, and ultrapure water with a resistivity of 18.2 MΩ.cm; step three, preparing a standard solution: using an electronic analytical balance to accurately weigh 0.02 of each of the three component reference substances of benzalkonium chloride, dodecyl dimethyl benzyl ammonium chloride, tetradecyl dimethyl benzyl ammonium chloride and hexadecyl dimethyl benzyl ammonium chloride, calculated as the pure product. g, accurate to 0.0001 g, dissolve in water and transfer to a 10 mL volumetric flask, dilute to the mark and shake well. The concentration of the three components of benzalkonium chloride in this solution is 2 mg / mL. Store at 4°C in a sealed container and away from light. When used, transfer an appropriate amount of the above standard stock solution and dilute with water to prepare a series of standard working solutions with concentrations of 5 mg / L, 10 mg / L, 20 mg / L, 100 mg / L, and 500 mg / L respectively; Step 4, sample pretreatment: Use an electronic analytical balance to accurately weigh 1 g of the disinfected product in a 10 mL volumetric flask, add water to dilute to the mark, shake well, use an ultrasonic extractor to ultrasonically extract for 5 min, let it stand, the weight of the disinfected product is accurate to 0.0001g, and pass 0.45 µm polysulfone ether aqueous phase filter membrane, and then test on the machine. Since the content of benzalkonium chloride in disinfection products ranges from constant to trace amounts, the sampling amount is operated according to the trace amount. The constant sampling amount can be reduced, or it can be diluted and then measured on the machine; step five, use liquid chromatography to analyze the measured solution, select the mobile phase, the mobile phase is acetonitrile-0.5 g / L ammonium acetate buffer solution, acetonitrile-0.5 g / L ammonium acetate buffer solution containing 0.1% triethylamine, glacial acetic acid to adjust pH=5.0, after adding the mobile phase to the liquid chromatography column, the sample on the liquid chromatography column is detected by the DAD detector, and then chromatographic analysis is performed, and finally isocratic elution is performed for chromatographic separation.

[0007] The liquid chromatography column in step 1 and step 5 of the present invention is Waters BEH C 18 Chromatographic column, Agilent XDB-C 18 Chromatographic column or C 18 Chromatographic columns, including Waters BEH C 18 Column size: 250 mm × 4.6 mm, 5 μm; Agilent XDB-C 18The size of the chromatographic column is 250 mm × 4.6 mm, 5 μm; C 18 The size of the chromatographic column is 250 mm × 4.6 mm, 5 μm.

[0008] The electronic analytical balance in step one of the present invention is a CP225D electronic analytical balance. The CAS number of dodecyl dimethyl benzyl ammonium chloride in step two of the present invention is 139-07-1; the CAS number of tetradecyl dimethyl benzyl ammonium chloride is 139-08-2; the CAS number of hexadecyl dimethyl benzyl ammonium chloride is 122-18-9. The wavelength of the DAD detector in step five of the present invention is 262nm, the column temperature is 40°C, the flow rate is 1.0 mL / min, isocratic elution, and the injection volume is 20 μL. The preparation process of acetonitrile-0.5 g / L ammonium acetate buffer solution in step five of the present invention is as follows: weigh 0.5 g of ammonium acetate and dissolve it in 200 mL of water, add 1 mL of triethylamine, transfer to a 1000 mL volumetric flask, dilute to the scale, adjust the pH to 5.0 with glacial acetic acid, pass through a 0.45 μm aqueous phase filter membrane, and the volume ratio is 70:30.

[0009] The invention has the following beneficial effects: after adopting the above technical method, the invention is simple and fast, has high accuracy, wide linear range, good precision and stability, is suitable for determining the content of benzalkonium chloride homologues in disinfection products, solves the possible damage of the sweep agent triethylamine to the chromatographic column through method optimization, solves the problem of ghost peaks appearing in standard products and samples, is suitable for large-scale qualitative analysis and quantitative detection of benzalkonium chloride in disinfection products by inspection and testing institutions, provides a standardized method for detecting the content of benzalkonium chloride in disinfection products in my country, has certain practical significance, is suitable for standardization and promotion, and establishes corresponding detection standard methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 It is the DAD spectrogram of benzalkonium chloride of the present invention.

[0011] Figure 2 The DAD spectra of the three homologue components of benzalkonium chloride of the present invention absorbed at ultraviolet wavelengths of 210nm and 262nm.

[0012] Figure 3 It is the LC-DAD chromatogram of the three components of benzalkonium chloride in the standard working solution of the present invention.

[0013] Figure 4 It is the LC-DAD chromatogram of two components of benzalkonium chloride in the actual sample solution of the present invention.

[0014] Figure 5 It is a typical chromatogram of the triethylamine concentration from small to large (0.05%, 0.1%, 1%) of the present invention.

[0015] Figure 6 It is a typical chromatogram of the ion pair reagent ammonium acetate of the present invention with increasing concentrations (0.3 g / L, 0.5 g / L, 5 g / L).

[0016] Figure 7 This is a typical chromatogram of the pH value from large to small (7.0, 6.0, 5.0) of the present invention. DETAILED DESCRIPTION

[0017] The present invention provides a method for determining the content of benzalkonium chloride homologues in a disinfection product by reverse phase liquid chromatography, which is characterized in that it comprises the following steps:

[0018] Step 1: Prepare the testing instruments: The testing instruments include Agilent 1200 liquid chromatograph, liquid chromatographic column, ultrasonic extractor, 0.45 μm polysulfone ether aqueous phase filter and electronic analytical balance. The liquid chromatograph is equipped with a diode array detector and the liquid chromatographic column is Waters BEH C 18 Chromatographic column, Agilent XDB-C 18 Chromatographic column or C 18 Chromatographic columns, including Waters BEH C 18 Column size: 250 mm × 4.6 mm, 5 μm; Agilent XDB-C 18 The size of the chromatographic column is 250 mm × 4.6 mm, 5 μm; C 18 The size of the chromatographic column is 250 mm × 4.6 mm, 5 μm. The Agilent XDB-C 18 Chromatographic column, Agilent 1200 liquid chromatograph, Agilent, USA, C 18 The chromatographic column was from Jiangsu Green Alliance Scientific Instrument Co., Ltd., the ultrasonic extractor was from Elma, Germany, the 0.45 μm polysulfone ether aqueous phase filter membrane was from Shanghai Anpu Scientific Instrument Co., Ltd., and the electronic analytical balance was CP225D electronic analytical balance from Sartorius, Germany;

[0019] Step 2, prepare the detection reagent: the detection reagent includes acetonitrile, triethylamine, glacial acetic acid, ammonium acetate, dodecyl dimethyl benzyl ammonium chloride with a purity of ≥98%, tetradecyl dimethyl benzyl ammonium chloride with a purity of ≥98%, hexadecyl dimethyl benzyl ammonium chloride with a purity of ≥98%, and ultrapure water with a resistivity of 18.2 MΩ.cm. The CAS number of dodecyl dimethyl benzyl ammonium chloride in step 2 is 139-07-1; the CAS number of tetradecyl dimethyl benzyl ammonium chloride is 139-08-2; the CAS number of hexadecyl dimethyl benzyl ammonium chloride is 122-18-9, acetonitrile is chromatographically pure and obtained from Merck, and ammonium acetate is analytically pure and obtained from Aladdin;

[0020] Step 3, prepare standard solution: use electronic analytical balance to accurately weigh 0.02 g of each of the three components of benzalkonium chloride, namely, dodecyl dimethyl benzyl ammonium chloride, tetradecyl dimethyl benzyl ammonium chloride, and hexadecyl dimethyl benzyl ammonium chloride, accurate to 0.0001 g, dissolve in water and transfer to a 10 mL volumetric flask, dilute to the mark and shake well. The concentration of the three components of benzalkonium chloride in this solution is 2 mg / mL. Store at 4°C in a sealed container away from light. When used, pipette an appropriate amount of the above standard stock solution and dilute with water to prepare a series of standard working solutions, the concentrations of which are: 5 mg / L, 10 mg / L, 20 mg / L, 100 mg / L, 500 mg / L respectively.

[0021] Step 4, sample pretreatment: accurately weigh 1 g of the disinfection product using an electronic analytical balance, add water to a 10 mL volumetric flask and dilute to the scale, shake well, use an ultrasonic extractor to ultrasonically extract for 5 min, let it stand, the weight of the disinfection product is accurate to 0.0001g, pass through a 0.45 µm polysulfone ether aqueous phase filter, and then test on the machine. Since the content of benzalkonium chloride in the disinfection product ranges from constant to trace amounts, the sampling amount is operated according to the trace amount. The constant amount can be reduced in sampling amount, or it can be diluted and then measured on the machine;

[0022] Step 5, using liquid chromatography to analyze the measured solution, select the mobile phase, the mobile phase is acetonitrile-0.5g / L ammonium acetate buffer solution, acetonitrile-0.5g / L ammonium acetate buffer solution containing 0.1% triethylamine, glacial acetic acid to adjust pH=5.0, after adding the mobile phase to the liquid chromatography column, the sample of the liquid chromatography column is detected by a diode array detector, and then chromatographic analysis is performed, and finally isocratic elution is performed for chromatographic separation, the wavelength of the diode array detector is: 262nm, the column temperature is 40°C, the flow rate is: 1.0 mL / min, isocratic elution, the injection volume is: 20 μL, and the preparation process of acetonitrile-0.5 g / L ammonium acetate buffer solution is as follows: weigh 0.5 g ammonium acetate and dissolve it in 200 mL water, add 1 mL triethylamine, transfer to a 1000 mL volumetric flask, dilute to the scale, adjust pH=5.0 with glacial acetic acid, pass through a 0.45 μm aqueous phase filter, and the volume ratio is 70:30.

[0023] The technical content of the present invention is further illustrated by specific experiments below.

[0024] 1. Experimental

[0025] 1.1 Main instruments: Agilent 1200 liquid chromatograph (Agilent, USA) equipped with diode array (DAD) detector, Waters BEH C 18 (250mm×4.6mm, 5μm) chromatographic column, Agilent XDB-C18 Chromatographic column (250mm×4.6mm, 5μm) and Jiangsu Green Alliance Scientific Instrument Co., Ltd. C 18 Chromatographic column (250mm×4.6mm, 5μm); ultrasonic extractor (Elma, Germany), 0.45 μm polysulfone ether aqueous phase filter membrane and 0.45 μm polypropylene organic filter membrane (Shanghai Anpu Scientific Instrument Co., Ltd.), electronic analytical balance (CP225D, Sartorius, Germany).

[0026] 1.2 Main reagents: acetonitrile (chromatographic grade, Merck, Germany), triethylamine, glacial acetic acid, ammonium acetate (analytical grade, Aladdin), dodecyldimethylbenzylammonium chloride (DDBAC), purity ≥98%, CAS number: 139-07-1; tetradecyldimethylbenzylammonium chloride (TDBAC), purity ≥98%, CAS number: 139-08-2; hexadecyldimethylbenzylammonium chloride (HDBAC), purity ≥98%, CAS number: 122-18-9 (LGC Labor GmbH, Germany), ultrapure water (resistivity 18.2 MΩ.cm, Veolia, France).

[0027] 1.3 Preparation of standard solution: Accurately weigh 0.02 g of each of the three components of benzalkonium chloride, namely, dodecyl dimethyl benzyl ammonium chloride, tetradecyl dimethyl benzyl ammonium chloride, and hexadecyl dimethyl benzyl ammonium chloride (pure product), to an accuracy of 0.0001 g, dissolve in water and transfer to a 10 mL volumetric flask, dilute to the mark and shake well. The concentration of the three components of benzalkonium chloride in this solution is 2 mg / mL. Store at 4°C in a sealed container away from light. When used, pipette an appropriate amount of the above standard stock solution and dilute with water to prepare a series of standard working solutions, with concentrations of 5 mg / L, 10 mg / L, 20 mg / L, 100 mg / L, and 500 mg / L, respectively.

[0028] 1.4 Sample pretreatment: Accurately weigh 1 g of disinfectant product (accurate to 0.0001 g) in a 10 mL volumetric flask, add water to dilute to the mark, shake well, extract by ultrasonic for 5 min, let stand, filter through a 0.45 µm polysulfone ether aqueous phase filter, and test on the machine. Since the content of benzalkonium chloride in disinfectant products ranges from constant to trace amounts, the sampling amount of this method is operated according to the trace amount. The constant amount can be reduced in sampling amount, or it can be diluted and then measured on the machine.

[0029] 1.5 Liquid chromatography (LC) analysis conditions: The liquid chromatography column selected was Agilent XDB-C18 column (250mm×4.6mm, 5μm), and the mobile phase was acetonitrile-0.5 g / L ammonium acetate buffer solution (containing 0.1% triethylamine, glacial acetic acid to adjust pH=5.0) with a volume ratio of 70:30. DAD detector wavelength: 262nm, column temperature: 40℃, flow rate: 1.0 mL / min, isocratic elution, injection volume: 20 μL. 0.5 g / L ammonium acetate buffer solution was prepared as follows: 0.5 g ammonium acetate was weighed and dissolved in 200 mL water, 1 mL triethylamine was added, and the solution was transferred to a 1000 mL volumetric flask, fixed to the mark, pH=5.0 was adjusted with glacial acetic acid, and filtered through a 0.45 μm aqueous filter. Other liquid chromatography conditions were set according to Table 1.

[0030] project condition UV detection wavelength 262nm Injection volume 20 μL flow 1.0 mL / min Column temperature 40 ℃ Mobile phase Acetonitrile: ammonium acetate buffer solution: = volume ratio 70:30

[0031] Note: The best test conditions can be selected according to the instrument used.

[0032] After analysis according to the above conditions, the spectrum is shown in Figure 1 .

[0033] 2 Results and discussion

[0034] 2.1 Selection and optimization of sample pretreatment methods: Since benzalkonium chloride is a cationic surfactant and is easily soluble in water, water, 70% acetonitrile aqueous solution, acetonitrile, and methanol were selected as extraction agents. It was found that the extraction effect was good and almost no effect. For liquid disinfection products, water can be directly used as the extraction agent, or ultrasonic extraction for 5 minutes can be performed. For solid or paste-like substances, ultrasonic extraction for 5 minutes is generally sufficient, and longer time has almost no effect.

[0035] 2.2 Selection and optimization of liquid chromatography conditions

[0036] 2.2.1 Selection of chromatographic columns

[0037] This study compared the Waters BEH C 18 (250mm×4.6mm, 5μm) chromatographic column, Agilent XDB-C 18 Chromatographic column (250mm×4.6mm, 5μm) and ordinary domestic chromatographic column, Jiangsu Green Alliance Scientific Instrument Co., Ltd. C 18 Chromatographic column (250mm×4.6mm, 5μm). The results were found to be relatively ideal, proving that this method can be suitable for different C 18 Reversed phase chromatography columns are generally applicable. The data in this example are obtained using an Agilent XDB-C 18 Resulted from the chromatographic column.

[0038] 2.2.2 Selection of detection wavelength

[0039] The three homologues of benzalkonium chloride have strong absorption at ultraviolet wavelengths of 210nm and 262nm. Figure 2 , using 210nm can reduce the detection limit of the product, but at the same time it will increase the uncertainty of substrate interference (many compounds have absorption at 210nm). The content of benzalkonium chloride in disinfection products is greater than 10mg / L, so for disinfection products containing benzalkonium chloride, a lower detection limit is not required. Using an ultraviolet wavelength of 262nm can fully meet the detection requirements and avoid interference from many substrates.

[0040] 2.2.3 Choice of mobile phase

[0041] 2.2.3.1 Changes in the concentration of the sweeping agent triethylamine: In 2012, when testing benzalkonium chloride in disinfectant wipes, the mobile phase was acetonitrile-70mmol / L ammonium acetate (containing 1% triethylamine, glacial acetic acid adjusted to pH 5.0) with a volume ratio of 72:28; it was found that there was almost no effect on the detection of DDBAC, and the effect was good. Later, when testing samples mixed with TDBAC, it was also found that there was matrix interference in the samples, and ghost peaks appeared from time to time. In addition, 1% triethylamine, obviously with a higher concentration, caused greater damage to the chromatographic column.

[0042] In addition, HPLC was used to determine three benzalkonium chloride homologues in disinfectant products. 18 The chromatographic column was used, but the liquid phase conditions of the 2015 version of the "Safety Technical Specifications for Cosmetics" were used (the mobile phase was acetonitrile-0.01 mol / L ammonium acetate buffer solution (pH=5.0) with a volume ratio of 70:30), and the tailing agent triethylamine was not added. The "Safety Technical Specifications for Cosmetics" used a cyano column, which is a normal phase chromatographic column. After repeated testing, it was found that most of the cases had serious tailing, making it difficult to achieve batch testing.

[0043] This experiment investigated the mobile phase B as 0.5 g / L ammonium acetate solution. After adding 0.05%, 0.08%, 0.1%, 0.2%, 0.5%, and 1% triethylamine as a sweep agent, the pH was adjusted to 5 with glacial acetic acid. When acetonitrile was used as mobile phase A and A:B was 70:30, the samples were tested and it was found that with the increase of triethylamine concentration, the half-peak width of the three homologues of benzalkonium chloride decreased. However, when the concentration was 0.1%, the half-peak width had reached equilibrium. Continuing to increase the concentration of triethylamine had little effect on the half-peak width. On the contrary, high concentrations of triethylamine would remain in the chromatographic column, resulting in ghost peaks and decreased column performance. The typical chromatogram is shown in Figure 3 Therefore, this experiment finally used 0.1% triethylamine as the sweep agent. After the detection, it was also convenient for flushing and protecting the chromatographic column and solved the problem of ghost peaks in the chromatogram.

[0044] 2.2.3.2 Changes in the concentration of ion-pair reagents: Since benzalkonium chloride and the filler components of the reverse-phase chromatographic column are similar, there is almost no retention on the reverse-phase chromatographic column, and an ion-pair reagent must be added for detection. This article examines mobile phase B: 0.1% triethylamine solution. After adding 0.1 g / L, 0.3 g / L, 0.5 g / L, 0.8 g / L, 1.5 g / L, and 5.0 g / L of ammonium acetate as an ion-pair reagent, the pH was adjusted to 5 with glacial acetic acid. When acetonitrile was used as mobile phase A and A:B was 70:30, the samples were tested and it was found that with the increase in the concentration of ammonium acetate, the half-peak widths of the three homologues of benzalkonium chloride decreased. However, when the concentration was 0.5 g / L, the half-peak width had reached equilibrium. Continuing to increase the concentration of ammonium acetate has little effect on the half-peak width. On the contrary, a large concentration of ammonium acetate will remain in the chromatographic column, resulting in ghost peaks and a longer flushing time of the chromatographic column after the detection. The typical chromatogram is shown in Figure 4 Therefore, 0.5 g / L ammonium acetate solution was finally used in this experiment.

[0045] 2.2.3.3 Changes in pH: This experiment examined mobile phase B: 0.5 g / L ammonium acetate solution. After adding 0.1% triethylamine as a sweep agent, glacial acetic acid was used to adjust the pH to 7.0, 6.0, 5.0, and 4.5, respectively. When acetonitrile was used as mobile phase A and A:B was 70:30, the samples were tested. When the pH value decreased, the retention time of the three homologues of benzalkonium chloride decreased, and the half-peak width also decreased. When the pH was 7 and 6, the half-peak width was wide, and when the pH was 7, the retention time of the last component was longer than 30 minutes, which had little practical significance. When the pH was 5.0 and 4.5, the three homologues could be clearly separated and eluted within 20 minutes. The half-peak width was small, which was suitable for laboratory testing. Considering that a decrease in pH would be detrimental to the chromatographic column, pH=5.0 was finally selected after comprehensive consideration. The typical chromatogram is shown in Figure 5 .

[0046] 2.2.3.4 Changes in the ratio of mobile phase: This experiment investigated mobile phase B: 0.5 g / L ammonium acetate solution, after adding 0.1% triethylamine as a sweep agent, the pH was adjusted to 5 with glacial acetic acid. When acetonitrile was used as mobile phase A, the A:B ratio was 50:50, 60:40, 65:35, 70:30, 80:20, 90:10, and the samples were tested. When the ratio of acetonitrile increased, the retention time of the three homologues of benzalkonium chloride first decreased and then increased, and the half peak width also decreased and then increased. When acetonitrile was 70%, the best result was achieved, so this experiment finally adopted A:B of 70:30.

[0047] 2.2.3.5 Change of column temperature: Under the final selected liquid phase conditions, when the column oven temperature was controlled at 25°C, 30°C, and 40°C, it was found that the half-peak width and peak height of the target compound did not change significantly. However, as the column temperature increased, the peak time of the target compound moved slightly forward, so the column oven was finally set to 40°C.

[0048] 2.3 Linear range and detection limit: Prepare mixed standard solutions of 5 mg / L, 10 mg / L, 50 mg / L, 100 mg / L and 500 mg / L with water as in 1.3 and perform the determination under the above chromatographic conditions. The typical chromatogram is shown in Figure 6 , Figure 6 1-dodecyl dimethyl benzyl ammonium chloride (DDBAC, 6.338min), 2-tetradecyl dimethyl benzyl ammonium chloride (TDBAC, 10.333min), 3-hexadecyl dimethyl benzyl ammonium chloride (HDBAC, 18.534min), the mass concentration of the component (x, mg / L) is used as the abscissa, and the corresponding mass spectrometry peak area (y) is used as the ordinate to obtain the standard curve. The results show that the three compounds have a good linear relationship within a certain range. The detection limit (LOQ) of the three homologues of benzalkonium chloride is calculated with a signal-to-noise ratio S / N≥3, which is 1.1mg / L, 1.2mg / L, and 1.5mg / L, respectively. The quantification limit (LOQ) of the three homologues of benzalkonium chloride is calculated with a signal-to-noise ratio S / N≥10: 3.5mg / L, 4.0mg / L, and 4.9mg / L, respectively (see Table 2 for details).

[0049] Table Linear range, linear equation, correlation coefficient, detection limit and quantification limit of dibenzalkonium chloride homologues

[0050]

[0051] 104.4 Precision and recovery: To compare the precision and recovery of this method, benzalkonium chloride disinfectant, compound disinfectant and disinfectant cream were spiked and recovered at three addition levels (BAC 5, 20, 100 mg / kg). Within 24 hours, the above method was used for determination, and each concentration level was determined 8 times. The recovery and relative standard deviation are listed in Table 2. As shown in Table 3, the recovery rates of the three BAC homologues were 98.8%-104.5%, and the relative standard deviations (RSD) were all less than 2%, indicating good precision. This shows that this method has good recovery and repeatability for the three benzalkonium chloride homologues in disinfectant products, and can meet the analysis requirements of actual samples.

[0052] Table 3. Experimental results of recoveries and relative standard deviations (RSD) of three benzalkonium chloride homologues (n=8)

[0053]

[0054] 2.5 Determination of actual samples: This method is used to test the samples commissioned by our unit on a daily basis. The typical chromatogram is shown in Figure 7 , Figure 7 1-dodecyldimethylbenzyl ammonium chloride (DDBAC, 6.339min), 2-tetradecyldimethylbenzyl ammonium chloride (TDBAC, 10.328min), and the content of benzalkonium chloride ranged from constant to trace amounts. Some of the test results are shown in Table 4.

[0055] Table 4. Test results of some actual samples

[0056]

[0057] 3 Conclusion

[0058] This experiment established a method for determining the content of benzalkonium chloride in disinfection products by reversed-phase liquid chromatography. This method has the advantages of small sampling volume, simple operation, rapidity, accuracy, and good stability. Through method optimization, it solves the possible damage of the chromatographic column by the sweep agent triethylamine and solves the problem of ghost peaks in standards and samples. It is suitable for large-scale qualitative analysis and quantitative detection of benzalkonium chloride in disinfection products by inspection and testing institutions. It provides a standardized method for the detection of benzalkonium chloride content in disinfection products in my country, which has certain practical significance, is suitable for standardization and promotion, and establishes corresponding standard detection methods.

Claims

1. A method for determining the content of benzalkonium chloride homologues in disinfectant products by reverse phase liquid chromatography, characterized in that it The following steps are involved: Step 1, prepare the testing instrument: the testing instrument includes Agilent 1200 liquid chromatograph, liquid chromatograph column, ultrasonic extractor, 0.45 μm polysulfone ether aqueous phase filter membrane and electronic analytical balance, and the liquid chromatograph is equipped with a diode array detector; Step 2, prepare the detection reagent: the detection reagent includes acetonitrile, triethylamine, glacial acetic acid, ammonium acetate, dodecyl dimethyl benzyl ammonium chloride with a purity of ≥98%, tetradecyl dimethyl benzyl ammonium chloride with a purity of ≥98%, hexadecyl dimethyl benzyl ammonium chloride with a purity of ≥98%, and ultrapure water with a resistivity of 18.2 MΩ.cm; Step 3, prepare standard solution: use electronic analytical balance to accurately weigh 0.02 g of each of the three components of benzalkonium chloride, namely, dodecyl dimethyl benzyl ammonium chloride, tetradecyl dimethyl benzyl ammonium chloride, and hexadecyl dimethyl benzyl ammonium chloride, accurate to 0.0001 g, dissolve in water and transfer to a 10 mL volumetric flask, dilute to the mark and shake well. The concentration of the three components of benzalkonium chloride in this solution is 2 mg / mL. Store at 4°C in a sealed container away from light. When used, pipette an appropriate amount of the above standard solution and dilute with water to prepare a series of standard working solutions, the concentrations of which are 5 mg / L, 10 mg / L, 20 mg / L, 100 mg / L, and 500 mg / L respectively. Step 4, sample pretreatment: accurately weigh 1 g of the disinfection product using an electronic analytical balance, add water to a 10 mL volumetric flask and dilute to the scale, shake well, use an ultrasonic extractor to ultrasonically extract for 5 min, let it stand, the weight of the disinfection product is accurate to 0.0001g, pass through a 0.45 µm polysulfone ether aqueous phase filter, and then test on the machine. Since the content of benzalkonium chloride in the disinfection product ranges from constant to trace amounts, the sampling amount is operated according to the trace amount. The constant amount can be reduced in sampling amount, or it can be diluted and then measured on the machine; Step 5, using liquid chromatography to analyze the measured solution, selecting the mobile phase, the mobile phase is acetonitrile-0.5 g / L ammonium acetate buffer solution, the acetonitrile-0.5 g / L ammonium acetate buffer solution preparation process is as follows: weigh 0.5 g ammonium acetate and dissolve it in 200 mL water, add 1 mL triethylamine, transfer to a 1000 mL volumetric flask, dilute to the scale, adjust the pH to 5.0 with glacial acetic acid, pass through a 0.45 μm aqueous filter membrane, the volume ratio is 70:30, the acetonitrile-0.5 g / L ammonium acetate buffer solution contains 0.1% triethylamine, and the glacial acetic acid is adjusted to pH=5.

0. After the mobile phase is added to the liquid chromatography column, the sample of the liquid chromatography column is detected by the DAD detector, and then chromatographic analysis is performed, and finally isocratic elution is performed for chromatographic separation. The liquid chromatography column in steps 1 and 5 is Waters BEHC 18 Chromatographic column, Agilent XDB-C 18 Chromatographic column or C 18 Chromatographic columns, including Waters BEH C 18 Column size: 250 mm × 4.6 mm, 5 μm; Agilent XDB-C 18 The size of the chromatographic column is 250 mm × 4.6 mm, 5 μm; C 18 The size of the chromatographic column is 250 mm × 4.6 mm, 5 μm.

2. The method for determining the content of benzalkonium chloride homologues in disinfection products by reverse phase liquid chromatography according to claim 1, characterized in that The electronic analytical balance in step 1 is a CP225D electronic analytical balance.

3. The method for determining the content of benzalkonium chloride homologues in disinfection products by reverse phase liquid chromatography according to claim 1, characterized in that The CAS number of dodecyl dimethyl benzyl ammonium chloride in step 2 is 139-07-1; the CAS number of tetradecyl dimethyl benzyl ammonium chloride is 139-08-2; and the CAS number of hexadecyl dimethyl benzyl ammonium chloride is 122-18-9.

4. The method for determining the content of benzalkonium chloride homologues in disinfection products by reversed phase liquid chromatography according to claim 1, characterized in that the wavelength of the DAD detector in step 5 is: 262nm, the column temperature is 40°C, the flow rate is: 1.0 mL / min, the elution is isocratic, and the injection volume is: 20 μL.

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