Method for Determining Residues of Fifteen Kinds of Quaternary Ammonium Salt Disinfectants in Dairy Products

Abstract

The present invention belongs to the field of detection technology, and particularly relates to a dispersive solid-phase extraction purification-ultra-high performance liquid chromatography-tandem mass spectrometry detection method for the residues of fifteen quaternary ammonium salt disinfectants in dairy products. The present invention comprises the following steps: 1), preparing a sample solution of the dairy product to be tested; 2), preparing a standard solution: preparing a mixed standard series working solution by mixing fifteen QACs standard substances; 3), injecting the mixed standard series working solution into a liquid chromatography-mass spectrometry instrument, determining the peak positions of the fifteen QACs and their qualitative and quantitative ion pairs, and making a standard curve equation; 4), taking the sample solution of the dairy product to be tested obtained in step 1) and measuring the peak areas of each QAC in the sample solution and their quantitative and qualitative ion pairs according to the method in step 3), performing qualitative analysis according to the peak time of each peak and the abundance ratio of the qualitative and quantitative ion pairs, and calculating according to the standard curve equation obtained in step 3) to obtain the contents of each QAC in the dairy product to be tested.

Description

Technical Field

[0001] The present invention belongs to the technical field of detection, and particularly relates to a dispersive solid-phase extraction purification-ultra high performance liquid chromatography-tandem mass spectrometry detection method for the residues of fifteen quaternary ammonium salt disinfectants in dairy products. Background Technique

[0002] Dairy products are mainly made from animal milk and come in a wide variety, mainly including sterilized milk, pasteurized milk, fermented milk, formulated milk, yogurt, pure milk, cheese, and milk-containing beverages, etc. With the improvement of living standards, people's quality requirements for dairy products are gradually increasing; however, due to its rapid development, there are still some imperfections in management and technology, and the quality of dairy products will inevitably be affected.

[0003] During the production and processing of dairy products, they are prone to microbial contamination. To meet the relevant hygiene requirements, disinfectants are often used to disinfect milking utensils, milk containers, production equipment, etc. The commonly used chemical disinfectants in dairy production mainly include peroxides, chlorine compounds, iodides, and quaternary ammonium salt compounds, etc. Traditional disinfection and sterilization fine chemicals are mainly chlorine-containing preparations and peroxy compounds. Although they have good bactericidal efficiency, they are not stable in storage, have a short shelf life, and have many unsafe factors. For example, peroxides will explode in containers during storage, can burn human skin, and are irritating to the eyes, etc.

[0004] Quaternary ammonium compounds (QACs) are a type of surfactant containing quaternary nitrogen atoms, and their structures and properties vary according to the nature of the groups attached to the nitrogen atoms. QACs have dual characteristics of sterilization and antibacterial, and are often used as disinfectants and cleaners in industrial and commercial disinfection formulations. Their disinfection effect is achieved by destroying the outer membrane of bacteria, causing the contents of the bacteria to leak out and leading to cell death. Due to the advantages of easy use and low price, QACs are commonly used for the disinfection during the extraction, storage of raw milk, and the production process of dairy products.

[0005] With the in-depth study of QACs, their toxic side effects have also received increasing attention. Some studies have found that QACs are highly toxic to some aquatic organisms (such as fish, algae, protozoa, and many aquatic microorganisms). Recent studies have shown that some QACs, such as benzalkonium chloride, can irritate the skin and eyes, causing diseases such as human asthma, skin allergic reactions, and vision loss. In the food industry, the use of chemical disinfectants such as QACs to eliminate residues caused by microorganisms may indirectly contaminate food, thus triggering a series of related food safety risks. In the case of improper cleaning operations, especially when water rinsing is insufficient, the residues of these compounds will transfer to the food surface, thus becoming a food pollution source and potentially posing a health hazard to consumers. Some studies have shown that quaternary ammonium salt disinfectants can remain on the surface of wiped containers for more than 6 days. The residual QACs will also combine with metal ions such as calcium and magnesium in milk to produce precipitates, and antagonize with anions in milk, reducing the quality of dairy products. The European Food Safety Authority stipulates that the upper limit of the residue of this type of disinfectant in food is 0.1 mg / kg.

[0006] Although there have been many research reports on the analysis and detection of QACs at home and abroad, there are still many problems that have not been solved, especially in complex food matrices. The analysis and detection of trace QACs is considered a very difficult and challenging task, mainly due to the high adsorption properties of QACs. First, in the process of sample preparation and treatment, if the laboratory utensils are not properly selected, the target substances will be directly adsorbed on the surface of laboratory consumables and equipment, resulting in serious losses of QACs during the pretreatment process. Second, due to the high adsorption of QACs in the ecological environment, there are relatively few free forms of these compounds. In addition, due to the complexity of food matrices, especially dairy products, which contain a large amount of proteins, fats, sugars, and various formulated nutrients, etc., these may all interfere with the qualitative and quantitative detection of trace substances. Therefore, it is necessary to develop a simple, efficient, and accurate pretreatment method to optimize the extraction and purification process of QACs in dairy products.

[0007] The invention of CN114965812A, "A method for simultaneously determining the residues of 15 quaternary ammonium salt disinfectants in livestock and poultry meat", discloses a method for simultaneously determining the residues of 15 quaternary ammonium salt disinfectants in livestock and poultry meat, including the following steps: sample pretreatment, the sample is dispersed in water, added with a formic acid-modified acetonitrile-ethyl acetate extraction agent for ultrasonic extraction, added with anhydrous sodium sulfate and then centrifuged, the supernatant is taken, concentrated, added with methanol for dissolution and then centrifuged for standby; preparing a standard solution of 15 quaternary ammonium salt disinfectants; determining the sample by liquid chromatography-mass spectrometry. This method has simple and inexpensive pretreatment, good recovery rate, high precision, and low matrix effect, and is suitable for the determination of the residues of 15 quaternary ammonium salt disinfectants in livestock and poultry meat. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a simple, qualitative and quantitative method with high sensitivity for determining the residues of fifteen quaternary ammonium salt disinfectants in dairy products.

[0009] To solve the above technical problem, the present invention provides a method for determining the residues of fifteen quaternary ammonium salt disinfectants in dairy products (dispersive solid-phase extraction purification-ultra-high performance liquid chromatography-tandem mass spectrometry method for detecting the residues of fifteen quaternary ammonium salt disinfectants in dairy products), comprising the following steps:

[0010] 1). Prepare a sample solution of the dairy product to be tested;

[0011] 2). Prepare a standard solution:

[0012] Prepare a mixed standard series working solution by mixing fifteen QACs standard substances;

[0013] 3). Inject the mixed standard series working solution into the liquid chromatography-mass spectrometry instrument to determine the peak positions of the fifteen QACs and their qualitative and quantitative ion pairs. Taking the abundance of the quantitative ion pair as the ordinate and the concentration as the abscissa, prepare a standard curve equation;

[0014] 4). Take the sample solution of the dairy product to be tested obtained in step 1) and determine the peak areas of each QAC in the sample solution and their quantitative and qualitative ion pairs according to the method in step 3). Conduct qualitative analysis based on the respective peak times and the abundance ratios of the qualitative and quantitative ion pairs, and calculate according to the standard curve equation obtained in step 3) to obtain the contents of each QAC in the dairy product to be tested.

[0015] As an improvement to the method for determining the residues of fifteen quaternary ammonium salt disinfectants in dairy products of the present invention, step 1) comprises the following steps:

[0016] 1.1. Thoroughly mix the dairy product to be tested (stored refrigerated) as a sample;

[0017] When the dairy product to be tested is solid or semi-solid, weigh 5-20 g (preferably 10 g) of the sample, accurate to 0.01 g, place it in a stoppered centrifuge tube, add 10-30 ml (preferably 20 ml) of deionized water, shake in a water bath at 60±10°C until completely dissolved, cool to room temperature, and use it as a sample solution for standby;

[0018] When the dairy product to be tested is liquid, let it stand and return to room temperature, and use it as a sample solution for standby;

[0019] 1.2. Precisely take 1.0 - 3.0 mL (preferably 2.0 mL) of the sample solution obtained in step 1.1 into a 25 mL polytetrafluoroethylene centrifuge tube. Precisely add 5 - 15 mL (preferably 10 mL) of an acetonitrile - methanol mixed solution containing 0.1 - 0.3% (preferably 0.2%) formic acid. After vortex - shaking for 2 - 10 min (preferably 5 min), add 2 - 6 g (preferably 4 g) of anhydrous magnesium sulfate, shake and extract for 5 - 15 min (preferably 10 min), and centrifuge at 5000 - 10000 r / min (preferably 8000 r / min) on a high - speed centrifuge for 2 - 10 min (preferably 5 min).

[0020] In the acetonitrile - methanol mixed solution, the volume ratio of acetonitrile:methanol = 7:3 - 9:1 (preferably 8:2, V / V).

[0021] 1.3. Precisely take 5 - 8 mL (preferably 6 mL) of the supernatant obtained in step 1.2 and transfer it to a 25 mL polytetrafluoroethylene centrifuge tube. Add 200 - 500 mg (preferably 300 mg) of a purification agent and vortex - shake for 2 - 10 min (preferably 5 min).

[0022] 1.4. Precisely take 5 mL of the supernatant obtained in step 1.3 and dry it (nitrogen - blow to near - dryness in a water bath at 40 ± 5°C). Add 0.5 mL of a 60 - 80% (preferably 70%) acetonitrile aqueous solution to redissolve, vortex - mix for 0.5 - 2 min (preferably 1 min) to dissolve the residue, filter with a 0.22 μm microporous organic filter membrane, and place the filtrate in a polytetrafluoroethylene injection vial. The purpose is to perform qualitative and quantitative analysis using an ultra - high - performance liquid chromatography - tandem mass spectrometer; that is, the filtrate is used as the sample - loading solution for the dairy product to be tested.

[0023] As a further improvement of the method for determining the residues of fifteen quaternary ammonium salt disinfectants in dairy products of the present invention:

[0024] The purification agent in step 1.3 is composed of amino - propyl ethylenediamine and quaternary ammonium - propyl bonded silica gel in a weight ratio of 1:1 - 3:1 (preferably 2:1).

[0025] As a further improvement of the method for determining the residues of fifteen quaternary ammonium salt disinfectants in dairy products of the present invention:

[0026] The chromatographic conditions in step 3) (i.e., the liquid chromatographic conditions for ultra - high - performance liquid chromatography (UPLC) detection) are: flow rate: 0.3 - 0.8 mL / min (preferably 0.4 mL / min); column temperature: 20 - 50°C (preferably 40°C); injection volume: 0.5 - 5 μL (preferably 1 μL).

[0027] The mobile phase consists of mobile phase A and mobile phase B: Mobile phase A is an aqueous solution of 0.2% formic acid; Mobile phase B is a methanol-acetonitrile mixed solution containing 0.2% formic acid (methanol:acetonitrile = 60:40 by volume);

[0028] The gradient elution program is as follows: 0 - 5.5 min, 8% - 100% B; 5.5 - 7.5 min, 100% B; 7.5 - 7.6 min, 100% - 8% B; 7.6 - 10.0 min, 8% B;

[0029] The above % is volume %.

[0030] As a further improvement to the method for determining the residues of fifteen quaternary ammonium salt disinfectants in dairy products according to the present invention:

[0031] The liquid chromatography column is: Waters CORTECS C 18+ (100×2.1 mm, 1.7 μm).

[0032] As a further improvement to the method for determining the residues of fifteen quaternary ammonium salt disinfectants in dairy products according to the present invention:

[0033] The mass spectrometry conditions for step 3) (i.e., the mass spectrometry conditions for triple quadrupole tandem mass spectrometry (MS / MS) detection) are as follows: electrospray ionization source (ESI), positive ion detection mode (ESI+); multiple reaction monitoring (MRM mode); ion source temperature: 150 °C; capillary voltage: 2.0 kV; cone voltage: 20 - 25 V; desolvation gas flow rate: 600 L / h; desolvation gas temperature: 1000 °C; cone gas flow rate: 150 L / h; calibration method: automatic mass axis tuning and correction; MRM performs segmented scanning; other mass spectrometry analysis parameters are as shown in Table 1 below:

[0034] Table 1. Mass spectrometry analysis parameters of fifteen quaternary ammonium salt disinfectants

[0035]

[0036]

[0037] Note: * is the quantitative ion pair

[0038] In the present invention: all organic solvents are chromatographically pure methanol and acetonitrile.

[0039] As a further improvement to the method for determining the residues of fifteen quaternary ammonium salt disinfectants in dairy products according to the present invention, in step 2):

[0040] Using an aqueous solution of 70% acetonitrile as the solvent, a series of mixed standard working solutions with different concentrations were prepared. In each mixed standard working solution, the concentrations of the fifteen QACs were the same, and the concentrations were 0.2, 0.5, 1, 5, 10, and 20 μg / L respectively.

[0041] Specifically:

[0042] Step 2) includes the following steps:

[0043] 2.1. Using an aqueous solution of 70% (volume%) acetonitrile, dissolve the fifteen QAC standard substances respectively to prepare fifteen QAC stock solutions with a concentration of 1000 μg / mL each, and store them at -20°C.

[0044] 2.2. Take appropriate volumes of the above standard stock solutions and prepare a mixed standard stock solution with a concentration of 100 mg / L for each of the fifteen QACs using an aqueous solution of 70% acetonitrile.

[0045] 2.3. Precisely pipette appropriate volumes of the above mixed standard stock solution respectively, dilute it with an aqueous solution of 70% acetonitrile to prepare a mixed standard intermediate solution with a concentration of 1 mg / L. Then, precisely pipette appropriate volumes of the above mixed standard intermediate solution into 10 mL polytetrafluoroethylene volumetric flasks, dilute and make up the volume to prepare mixed standard working solutions with concentrations of 0.2, 0.5, 1, 5, 10, and 20 μg / L.

[0046] In the present invention, the fifteen QACs are shown in Table 2 below:

[0047] Table 2. Chemical information table of fifteen quaternary ammonium salt disinfectants

[0048]

[0049]

[0050] The present invention is a novel dispersive solid-phase extraction combined with purification - ultra-high performance liquid chromatography - tandem mass spectrometry method suitable for the detection of residues of fifteen quaternary ammonium salt disinfectants in dairy products, which simultaneously solves a series of problems of various residual adsorptions of QACs during the detection process.

[0051] The technical key points of the present invention mainly lie in:

[0052] 1. The existing food safety standards for the detection of residues of some quaternary ammonium salt disinfectants in dairy products are SN / T 4048-2014 "Determination of Quaternary Ammonium Salts in Export Foods - Liquid Chromatography-Mass Spectrometry / Mass Spectrometry" and BJS202007 "Detection of Disinfectant Residues in Infant Formula Foods". The sample pretreatment method adopted in the standard SN / T 4048-2014 is the solid phase extraction cartridge method. This method is cumbersome and time-consuming. The WCX solid phase extraction cartridge will have a strong adsorption effect on some long-chain quaternary ammonium salt disinfectants and cannot be eluted, resulting in a low recovery rate. While BJS202007 directly adopts the solvent extraction method (acetonitrile), the extraction efficiency for some long-chain quaternary ammonium salt disinfectants is very low, and there is no purification step. The complex matrix in dairy products leads to a high matrix effect in the detection results and a low recovery rate. In the chromatographic conditions adopted by the two standards, the chromatographic columns both use C18 packing materials, which will have a strong adsorption effect on some long-chain quaternary ammonium salt disinfectants and cannot be completely eluted with normal organic solvents, causing greater pollution to the entire detection system. The present invention is the development of a new detection method for the residues of some quaternary ammonium salt disinfectants in dairy products.

[0053] 2. The prior art teaches that in the process of detecting the residues of quaternary ammonium salt disinfectants, the target substances are separated from the complex matrix. Some literature uses solvents such as acetonitrile and ethyl acetate for direct extraction. However, in the present invention, it is found that if solvents such as acetonitrile or ethyl acetate are directly used for extraction, the solubility of some long-chain quaternary ammonium salt disinfectants is extremely small, and the extraction efficiency is very low. In the extraction system of the present invention, solid samples need to be first dissolved in water, and a mixed solution of acetonitrile-methanol (8:2, V / V) containing 0.2% formic acid is used as the extraction agent, which can make the accuracy of the detection of the residues of quaternary ammonium salt disinfectants in the dairy products finally obtained by the present invention higher. It not only removes the matrix influence of most macromolecular substances such as proteins on the detection results, but also ensures the solubility and stability of long-chain quaternary ammonium salt disinfectants in the subsequent experimental process (especially the water removal process).

[0054] 3. The prior art teaches that in the process of detecting the residues of quaternary ammonium salt disinfectants, impurities (including proteins, fats, pigments, etc.) are removed by using organic solvents (such as acetonitrile, etc.) to remove impurities or solid phase extraction cartridges for sample purification. However, the operation is complex, the detection efficiency is low, and the reproducibility is poor. There are also cases where dispersive solid phase extraction materials such as C 18 -N, N-vinylpyrrolidone-divinylbenzene copolymer are used to remove impurities, but it is found that they have a strong adsorption effect on quaternary ammonium salt disinfectants, resulting in a large loss. By optimizing the combined use of two purification agents, namely amino-propyl ethylenediamine and quaternary ammonium propyl bonded silica gel, the sample solution finally obtained by the present invention has fewer impurities, the matrix effect is significantly reduced, and there is almost no adsorption effect on quaternary ammonium salt disinfectants, improving the detection efficiency, accuracy and precision.

[0055] 4. The prior art indicates that during the inspection of the residue of quaternary ammonium salt disinfectants, some experimental consumables and materials have a strong adsorption effect on quaternary ammonium salt disinfectants. However, no literature or standard has proposed or solved such adsorption problems. The whole-process contact material used in the present invention is polytetrafluoroethylated - amino - propyl ethylenediamine / quaternary ammonium propyl - bonded silica - Waters CORTECS C 18+ detection system. Polytetrafluoroethylation of the whole - process contact material means that consumables such as volumetric flasks, centrifuge tubes, nitrogen - blowing tubes, pipette tips, and injection vials used in the entire detection process are all made of polytetrafluoroethylene materials. By establishing the above - mentioned system, the loss of the quaternary ammonium salt disinfectant in the present invention due to adsorption during the entire experimental operation process can be minimized.

[0056] The present invention has the following remarkable effects compared with the prior art:

[0057] (1) This method uses an acetonitrile - methanol mixed solution (8:2, V / V) containing 0.2% formic acid as the extraction solvent for the residue of fifteen quaternary ammonium salt disinfectants in dairy products. Considering the different hydrophobicities and hydrophilicities of the fifteen quaternary ammonium salt disinfectants, they can be completely dissolved in the same extraction solvent system, and it is ensured that during the subsequent water - removal process (adding anhydrous magnesium sulfate), the solubility of some long - chain quaternary ammonium salt disinfectants will not be reduced, resulting in low extraction efficiency. Using the acetonitrile - methanol mixed solution (8:2, V / V) containing 0.2% formic acid as the extraction system in this method is more convenient for subsequent water - removal and purification operations compared with the extraction systems used in other methods (such as the commonly used 70% aqueous acetonitrile solution), improving the stability and operability of the entire detection system.

[0058] (2) This method uses the dispersive solid - phase extraction technology to deeply purify dairy products with complex matrices. By evaluating the adsorption of the dispersive solid - phase extraction purification materials on the fifteen quaternary ammonium salt disinfectants and the purification efficiency of the milk powder matrix, the optimized combination of two purification agents, amino - propyl ethylenediamine and quaternary ammonium propyl - bonded silica, can fully remove interfering substances such as proteins and lipids, and it is ensured that this combination of purification agents will not have an adsorption effect on the fifteen quaternary ammonium salt disinfectants. Compared with other extraction technologies, this method eliminates the matrix effect brought by complex matrices, saves detection time, and improves detection accuracy.

[0059] (3) During the entire experimental operation process of this method, by evaluating the adsorption of various experimental materials and materials on the fifteen quaternary ammonium salt disinfectants, experimental consumables such as volumetric flasks, centrifuge tubes, droppers, nitrogen - blowing tubes, and injection vials are all made of polytetrafluoroethylene materials, avoiding the phenomenon of great adsorption of quaternary ammonium salt disinfectants by using materials such as glass and causing large losses.

[0060] (4) This method utilizes the ultra-high pressure advantage unique to the ultra-high performance liquid chromatography system and the implementation of core particle chromatography technology (Cortecs C18+ chromatographic column) with surface charging technology, optimizes the operating backpressure of the high performance liquid chromatography instrument, maximizes the separation efficiency of the liquid chromatography instrument by improving the resolution and peak capacity, obtains a faster analysis speed while improving the resolution of the target compound, bonds the C18 chain after a small amount of charge on the particle surface, reduces the adsorption of quaternary ammonium salt disinfectants, combines the two chromatographic technologies, obtains higher sensitivity, greatly shortens the analysis time, and improves the analysis efficiency.

[0061] (5) The determination method disclosed in the present invention has a good linear relationship in the range of 0.2 - 20 μg / L, and the linear correlation coefficient is 0.9989. The lowest detection limit of the method is: 0.1 - 0.5 μg / kg for solids and semi-solids; 0.05 - 0.25 μg / L for liquids. The determination method disclosed in the present invention improves the detection sensitivity of quaternary ammonium salt disinfectants in dairy products.

[0062] In summary, the novel dispersive solid-phase extraction in the present invention purifies impurities in dairy products, has a simple pretreatment, less impurity interference and matrix effect, reduces the adsorption of the target compound to the lowest level throughout the experimental process, has a good recovery rate and high reproducibility. The ultra-high performance liquid chromatography-tandem mass spectrometry method described in the present invention can quickly and accurately separate, qualitatively and quantitatively analyze fifteen quaternary ammonium salt disinfectants within 10 minutes, is easy to operate, and has good sensitivity and accuracy.

[0063] In summary, the present invention uses a novel dispersive solid-phase extraction purification (dSPE) to extract and purify the residues of fifteen quaternary ammonium salt disinfectants in dairy products, and uses an ultra-high performance liquid chromatography-triple quadrupole mass spectrometry method with the advantages of high sensitivity and strong anti-interference ability to qualitatively and quantitatively analyze the residues of fifteen quaternary ammonium salt disinfectants in dairy products. The present invention well solves the problems in the process of detecting the residues of fifteen quaternary ammonium salt disinfectants in dairy products, has important practical significance for protecting human health, promoting the technological development of enterprises themselves, promoting the development of detection technologies in China, enhancing the international status of dairy product quality and safety detection technologies in China, increasing employment, driving the overall progress of the dairy product industry, and having great economic and social benefits for technological progress and industrial transformation and upgrading. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] The following further elaborates on the specific embodiments of the present invention in conjunction with the drawings.

[0065] Figure 1 It is a qualitative and quantitative ion pair diagram of fifteen quaternary ammonium salt disinfectants. In the figure, 1 - 15 respectively represent these fifteen quaternary ammonium salt disinfectants.

[0066] Figure 1 From top to bottom are the qualitative and quantitative ion chromatograms of benzyl dimethyl ammonium chloride (BAC-C8), N-decyl-N,N-dimethyl benzyl ammonium chloride (BAC-C10), dodecyl dimethyl benzyl ammonium chloride (BAC-C12), tetradecyl dimethyl benzyl ammonium chloride (BAC-C14), hexadecyl dimethyl benzyl ammonium chloride (BAC-C16), didodecyl dimethyl ammonium bromide (DDAC-C10), ditetradecyl dimethyl ammonium bromide (DDAC-C14), dihexadecyl dimethyl ammonium bromide (DDAC-C16), dioctadecyl dimethyl ammonium bromide (DDAC-C18), decyl trimethyl ammonium bromide (TDAB), dodecyl trimethyl ammonium bromide (DTAB), tetradecyl trimethyl ammonium bromide (TTAB), hexadecyl trimethyl ammonium bromide (CTAB), and octadecyl trimethyl ammonium bromide (STAB). Detailed implementation manners

[0067] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0068] 1 Reagents and materials

[0069] Unless otherwise specified, all reagents used in the analysis are of chromatographic pure grade, and all water used is of first-class water.

[0070] 1.1 Methanol

[0071] 1.2 Acetonitrile

[0072] 1.3 Formic acid

[0073] 1.4 Anhydrous magnesium sulfate: Analytical grade

[0074] 1.5 Amino-propyl ethylenediamine adsorbent

[0075] 1.6 Quaternary ammonium propyl bonded silica gel adsorbent (SAX purification column)

[0076] 1.7 Quaternary ammonium salt disinfectant reference materials: Benzyl dimethyl ammonium chloride (BAC-C8), N-decyl-N,N-dimethyl benzyl ammonium chloride (BAC-C10), Dodecyl dimethyl benzyl ammonium chloride (BAC-C12), Tetradecyl dimethyl benzyl ammonium chloride (BAC-C14), Hexadecyl dimethyl benzyl ammonium chloride (BAC-C16), Didodecyl dimethyl ammonium bromide (DDAC-C10), Didodecyl dimethyl ammonium bromide (DDAC-C12), Ditetradecyl dimethyl ammonium bromide (DDAC-C14), Dihexadecyl dimethyl ammonium bromide (DDAC-C16), Dioctadecyl dimethyl ammonium bromide (DDAC-C18), Decyl trimethyl ammonium bromide (TDAB), Dodecyl trimethyl ammonium bromide (DTAB), Tetradecyl trimethyl ammonium bromide (TTAB), Hexadecyl trimethyl ammonium bromide (CTAB), Octadecyl trimethyl ammonium bromide (STAB);

[0077] 1.8 Standard stock solution: The above fifteen quaternary ammonium salt disinfectant reference materials were processed as follows: Each of the fifteen quaternary ammonium salt disinfectant reference materials was prepared into a standard stock solution with 70% (v / v) acetonitrile aqueous solution; then an appropriate amount of each of the above standard stock solutions was accurately taken and prepared into a mixed standard stock solution with a concentration of 1 mg / L for each of the fifteen quaternary ammonium salt disinfectants with 70% acetonitrile aqueous solution; that is, in the mixed standard stock solution, the following fifteen quaternary ammonium salt disinfectants: (Benzyl dimethyl ammonium chloride (BAC-C8), N-decyl-N,N-dimethyl benzyl ammonium chloride (BAC-C10), Dodecyl dimethyl benzyl ammonium chloride (BAC-C12), Tetradecyl dimethyl benzyl ammonium chloride (BAC-C14), Hexadecyl dimethyl benzyl ammonium chloride (BAC-C16), Didodecyl dimethyl ammonium bromide (DDAC-C10), Didodecyl dimethyl ammonium bromide (DDAC-C12), Ditetradecyl dimethyl ammonium bromide (DDAC-C14), Dihexadecyl dimethyl ammonium bromide (DDAC-C16), Dioctadecyl dimethyl ammonium bromide (DDAC-C18), Decyl trimethyl ammonium bromide (TDAB), Dodecyl trimethyl ammonium bromide (DTAB), Tetradecyl trimethyl ammonium bromide (TTAB), Hexadecyl trimethyl ammonium bromide (CTAB), Octadecyl trimethyl ammonium bromide (STAB)) had a concentration of 1 mg / L.

[0078] 1.9 Standard working solution: The above mixed standard stock solution was prepared into a mixed standard working solution with concentrations of 5 - 200 μg / L with 70% acetonitrile aqueous solution.

[0079] Note: The standard stock solution is stored in the dark at -20 °C in a polytetrafluoroethylene volumetric flask with a validity period of half a year. The standard working solution is stored in the dark at 4 °C with a validity period of 1 month.

[0080] 2 Instruments and Equipment

[0081] 2.1 Ultra Performance Liquid Chromatograph (UPLC).

[0082] 2.2 Triple Quadrupole Tandem Mass Spectrometer (MS / MS).

[0083] 2.3 Analytical Balance: Sensitivity of 0.0001 g and 0.01 g.

[0084] 2.4 Vortex Oscillator.

[0085] 2.5 High-Speed Centrifuge: Maximum speed can reach 10,000 r / min.

[0086] 2.7 Extractor: PTFE Centrifuge Tube, 25 mL.

[0087] 2.8 Nitrogen Blowing Tube: PTFE Nitrogen Blowing Tube, 10 mL.

[0088] 2.9 Injection Vial: PTFE Injection Vial, 0.2 mL.

[0089] 2.10 Organic Phase Filter Membrane: 0.22 um.

[0090] Example 1. A dispersive solid-phase extraction purification - ultra performance liquid chromatography - tandem mass spectrometry method for the determination of fifteen quaternary ammonium salt disinfectant residues in dairy products, which is carried out in the following steps:

[0091] 1). Preparation of the sample solution to be measured:

[0092] (1) Thoroughly mix the dairy product to be measured to obtain a test sample;

[0093] When the dairy product to be measured is solid or semi-solid, take 10 g of the test sample, accurate to 0.01 g, add 20 ml of deionized water, shake in a water bath at 60 °C until completely dissolved, and cool to room temperature for use as the test sample solution;

[0094] When the sample to be measured is a liquid sample, let it stand at room temperature for use as the test sample solution.

[0095] Solid and semi-solid samples include various milk powders, creams, cheeses, butters, milk tablets, yogurts, condensed milk, etc., and liquid samples include fresh milk, raw milk, etc.

[0096] Note: The dairy product to be measured is generally stored refrigerated at 4 °C.

[0097] (2) Accurately take 2.0 mL of the test sample solution obtained in step (1) into a 25 mL PTFE centrifuge tube, accurately add 10 mL of an acetonitrile - methanol mixed solution (8:2, V / V) containing 0.2% formic acid, and vortex shake for 5 min;

[0098] The preparation method of the acetonitrile-methanol mixed solution (8:2, V / V) containing 0.2% formic acid is as follows: First, mix acetonitrile and methanol according to a volume ratio of 8:2 to obtain an acetonitrile-methanol mixed solution, and then add 0.2 ml of formic acid to 100 ml of the acetonitrile-methanol mixed solution.

[0099] (3) Add 4 g of anhydrous magnesium sulfate to the product obtained in step (2), shake and extract for 10 min, and centrifuge at 8000 r / min for 5 min on a high-speed centrifuge.

[0100] Precisely take 6 mL of the supernatant and transfer it to a 25 mL polytetrafluoroethylene centrifuge tube; add 300 mg of the purifying agent (composed of amino-propyl ethylenediamine and quaternary ammonium propyl bonded silica gel, with a weight ratio of 2:1), and vortex and shake for 5 min;

[0101] (4) Precisely take 5 ml of the supernatant obtained after vortex shaking in step (3) and blow it to near dryness at 40 °C in a water bath with nitrogen (using a polytetrafluoroethylene nitrogen blowing tube). Add 0.5 mL of 70% (volume %) acetonitrile aqueous solution for reconstitution, vortex and mix for 1 min to dissolve the residue, filter with a 0.22 μm microporous organic filter membrane, and transfer the filtrate to a polytetrafluoroethylene injection vial; the obtained filtrate is for qualitative and quantitative analysis by ultra-high performance liquid chromatography-tandem mass spectrometry. That is, the filtrate is used as the sample loading solution for the test sample.

[0102] 2), Preparation of standard solutions

[0103] (1) Dissolve fifteen quaternary ammonium salt disinfectant reference substances with 70% acetonitrile aqueous solution respectively to prepare fifteen quaternary ammonium salt disinfectant standard stock solutions with a concentration of 1 mg / mL;

[0104] (2) Take appropriate amounts of the above fifteen standard stock solutions and prepare a mixed standard stock solution with a concentration of 100 mg / L using 70% acetonitrile aqueous solution;

[0105] (3) Precisely pipette appropriate volumes of the above mixed standard stock solution respectively, dilute it with 70% acetonitrile aqueous solution to obtain a mixed standard intermediate solution with a concentration of 1 mg / L, then precisely pipette appropriate volumes of the above mixed standard intermediate solution into a 10 mL polytetrafluoroethylene volumetric flask respectively, dilute and make up the volume with 70% acetonitrile aqueous solution to prepare mixed standard working solutions with concentrations of 0.2, 0.5, 1, 5, 10, 20 μg / L;

[0106] 3), Perform the following operations on the standard series working solutions obtained in step 2) respectively: Inject them into an ultra-high performance liquid chromatography-triple quadrupole mass spectrometer to determine the peak positions of the fifteen quaternary ammonium salt disinfectants and their qualitative and quantitative ion pairs (such as Figure 1As described above, with the abundance of the quantitative ion pair as the ordinate and the concentrations of the fifteen quaternary ammonium salt disinfectants in the solution to be tested as the abscissa, a standard curve equation is made as follows:

[0107] a) Chromatographic column: Waters Cortecs C18+, 100 mm × 2.1 mm, 1.6 μm chromatographic column or equivalent;

[0108] b) Flow rate: 0.4 ml / min;

[0109] c) Column temperature: 40 °C;

[0110] d) Injection volume: 1 μl;

[0111] e) Mobile phase:

[0112] It is composed of mobile phase A and mobile phase B;

[0113] Mobile phase A is an aqueous solution of formic acid with a volume concentration of 0.2%; mobile phase B is a methanol-acetonitrile mixed solution (60:40) containing 0.2% formic acid;

[0114] The preparation method of mobile phase B is: first mix methanol and acetonitrile according to a volume ratio of 60:40 to obtain a methanol-acetonitrile mixed solution, and then add 0.2 ml of formic acid to 100 ml of the methanol-acetonitrile mixed solution.

[0115] f) The gradient elution program is shown in Table 3

[0116] Table 3. Gradient elution program table

[0117]

[0118] The above % is volume %.

[0119] g) Ion source: Electrospray ionization source, positive ion detection mode (ESI+);

[0120] h) Monitoring mode: Multiple reaction monitoring mode (MRM); perform segmented scanning, as shown in Table 4 below;

[0121] i) Ion source temperature: 150 °C;

[0122] j) Capillary voltage: 2.0 KV;

[0123] k) Desolvation gas (nitrogen): Flow rate: 600 L / h; Temperature: 1000 °C;

[0124] l) Cone hole gas flow rate: 150 L / h;

[0125] m) Other mass spectrometry analysis parameters are shown in Table 4 for details.

[0126] Table 4 Mass Spectrometry Analysis Parameters of Fifteen Kinds of Quaternary Ammonium Salt Disinfectants

[0127]

[0128]

[0129]

[0130] Note: * represents the quantitative ion pair

[0131] When the ultra-high performance liquid chromatography-triple quadrupole mass spectrometry method disclosed by the present invention is in the concentration range of (0.2-20 μg / L), the linear relationships of fifteen kinds of quaternary ammonium salt disinfectants are all good, as shown in Table 5.

[0132] Table 5 Linear Relationships, Detection Limits and Quantification Limits of Fifteen Kinds of Quaternary Ammonium Salt Disinfectants

[0133]

[0134]

[0135] In Table 5, Y represents the abundance of the quantitative ion pair, and X represents the concentration of the target substance (the data unit is μg / L)

[0136] 4) Take the filtrate obtained by passing the filtrate obtained in step 1) through a 0.22 μm microporous organic filter membrane (the sample loading solution of the sample to be tested), and determine the abundance of the quantitative ion pairs of the metabolites of each quaternary ammonium salt disinfectant in the filtrate for quantitative analysis according to the method in step 3). Qualitative analysis is carried out according to the peak time and the abundance ratio of the qualitative and quantitative ion pairs. Calculate according to the standard curve equation obtained in step 3) to obtain the content c (μg / L) of each quaternary ammonium salt disinfectant in the filtrate. Finally, calculate according to the following conversion formula to obtain the content of each quaternary ammonium salt disinfectant in the sample to be tested.

[0137] Conversion formula

[0138]

[0139] In the formula:

[0140] X—the residue amount of the component to be measured in the sample (sample to be tested), in micrograms per kilogram or micrograms per liter (μg / kg (L)); c—the concentration of the component solution to be measured obtained from the standard curve, in micrograms per liter (μg / L);

[0141] V—the final volume of the sample solution (sample solution), in milliliters (mL);

[0142] m—the mass or volume of the final sample represented by the sample solution, in grams (g) for solid or semi-solid samples, and in milliliters (mL) for liquid samples;

[0143] F——Dilution factor, which is 20 for solid or semi-solid samples and 2 for liquid samples.

[0144] 5), Qualitative analysis

[0145] Corresponding to step 4), each component has 2 detection channels, and each channel corresponds to a monitoring ion pair. When detecting the sample, if peaks with the same retention time as the reference substance appear in multiple channels corresponding to a certain component, and the relative abundances of these daughter ions are the same as those of the reference substance, it can be determined that the component is detected in the sample. The abundance data of each component all meet the allowable deviation range of relative ion abundances for qualitative judgment in EU Regulation 2002 / 657 / EC, and it can be judged that the relative abundances of these daughter ions are the same. Calculated according to the score method (identification points of mass spectrometry analysis method) of the above regulations, if the above qualitative process reaches 4 points, greater than the requirement of 3 points, it indicates that the sample contains residues of this kind of quaternary ammonium salt disinfectant.

[0146] 6), Quantitative analysis

[0147] Corresponding to step 4), this method uses the external standard method for quantification. According to the content of the analyte in the sample solution, a standard working solution with a similar concentration is selected, and the standard working solution and the sample solution are injected for determination with the same volume. The response values of the fifteen quaternary ammonium salt disinfectants in the standard working solution and the sample solution to be measured should all be within the linear range.

[0148] Note 1: If the detection response value of the sample solution exceeds the linear range, the standard series working solution can be appropriately adjusted.

[0149] Note 2: Under the above chromatographic and mass spectrometric conditions, the MRM ion chromatograms of the fifteen quaternary ammonium salt disinfectants are shown in Figure 1 .

[0150] 7), Detection limit

[0151] The lowest detection concentration (LOD) is determined by 3 times the signal-to-noise ratio (S / N = 3), and the lowest quantification concentration (LOQ) is determined by 10 times the signal-to-noise ratio (S / N = 10). The detection limits of the dispersive solid-phase extraction combined with purification - ultra-high performance liquid chromatography - tandem mass spectrometry method for the fifteen quaternary ammonium salt disinfectants applicable to dairy products in this method are shown in Table 5.

[0152] Experiment 1. Sample addition recovery experiment and precision experiment

[0153] Use milk powder and raw milk that have been previously detected by SN / T 4048-2014 "Determination of Quaternary Ammonium Salts in Export Foods - Liquid Chromatography - Mass Spectrometry / Mass Spectrometry Method" and BJS 202007 "Detection of Disinfectant Residues in Infant Formula Foods" to ensure that they do not contain the fifteen quaternary ammonium salt disinfectants as blank samples.

[0154] Take 10 g of blank milk powder sample, accurate to 0.01 g, add 20 ml of deionized water, shake in a water bath at 60 °C until completely dissolved, and set aside after cooling to room temperature; place the blank fresh milk sample at room temperature for later use. Precisely take 2.0 mL of each of the above blank samples and conduct the following treatments respectively:

[0155] Add the standard mixed solution of fifteen quaternary ammonium salt disinfectants, set the added concentrations at three levels: low, medium, and high, as shown in Table 6 specifically. Set 6 replicates for each concentration. According to the pretreatment and analysis methods in Example 1 above, determine the added recoveries of fifteen quaternary ammonium salt disinfectants in two dairy product samples (the linear equations are the same as in Table 5), and the obtained results are shown in Table 6 (take the average of 6 replicates).

[0156] Taking BAC-C8 as an example, the following specific description is made:

[0157] I. When the sample to be tested is milk powder:

[0158] When the added concentration is 0.2 μg / kg, the data obtained from 6 replicates on the instrument are respectively: (1) 25389, (2) 30356, (3) 28559, (4) 29938, (5) 27727, (6) 29241. Therefore, substituting into Y = 302902X - 28108, the obtained results are respectively (1) 0.1766 μg / L, (2) 0.1930 μg / L, (3) 0.1871 μg / L, (4) 0.1916 μg / L, (5) 0.1843 μg / L, (6) 0.1893 μg / L. Then substitute into the conversion formula The finally obtained results are respectively (1) 0.1766 μg / kg, (2) 0.1930 μg / kg, (3) 0.1871 μg / kg, (4) 0.1916 μg / kg, (5) 0.1843 μg / kg, (6) 0.1893 μg / kg. Average the above 6 data. According to the calculation formula for the average recovery rate Therefore, 93.5% is obtained. The calculation formula for RSD is Therefore, 3.2% is obtained.

[0159] When the added concentration is 0.4 μg / kg, the data obtained from 6 replicates on the instrument are respectively: (1) 79008, (2) 80157, (3) 82117, (4) 79202, (5) 80379, (6) 82575. Therefore, substituting into Y = 302902X - 28108, the obtained results are respectively (1) 0.3536 μg / L, (2) 0.3574 μg / L, (3) 0.3639 μg / L, (4) 0.3543 μg / L, (5) 0.3582 μg / L, (6) 0.3654 μg / L. Then substitute into the conversion formula The final obtained results are respectively (1) 0.3536 ug / kg, (2) 0.3574 ug / kg, (3) 0.3639 ug / kg, (4) 0.3543 ug / kg, (5) 0.3582 ug / kg, (6) 0.3654 ug / kg. Averaging the above 6 data, according to the calculation formula of the average recovery rate is Therefore, 89.7% is obtained. The calculation formula of RSD is Therefore, 1.4% is obtained.

[0160] When the added concentration is 2.0 ug / kg, the data obtained from 6 replicates on the instrument are respectively: (1) 495976, (2) 521936, (3) 533574, (4) 490056, (5) 537726, (6) 577944. Therefore, substituting into Y = 302902X - 28108, the obtained results are respectively (1) 1.7302 ug / L, (2) 1.8159 ug / L, (3) 1.8543 ug / L, (4) 1.7107 ug / L, (5) 1.8680 ug / L, (6) 2.0008 ug / L. Then substituting into the conversion formula The final obtained results are respectively (1) 1.7302 ug / kg, (2) 1.8159 ug / kg, (3) 1.8543 ug / kg, (4) 1.7107 ug / kg, (5) 1.8680 ug / kg, (6) 2.0008 ug / kg. Averaging the above 6 data, according to the calculation formula of the average recovery rate is Therefore, 91.5% is obtained. The calculation formula of RSD is , therefore, 5.8% is obtained;

[0161] II. When the sample to be tested is liquid milk:

[0162] When the added concentration is 0.1 ug / L, the data obtained from 6 replicates on the instrument are respectively: (1) 26558, (2) 26047, (3) 27089, (4) 27933, (5) 28692, (6) 28345. Therefore, substituting into Y = 302902X - 28108, the obtained results are respectively (1) 0.1805 ug / L, (2) 0.1788 ug / L, (3) 0.1822 ug / L, (4) 0.1850 ug / L, (5) 0.1875 ug / L, (6) 0.1864 ug / L. Then substituting into the conversion formula The final results are respectively (1) 0.09024 ug / L, (2) 0.08939 ug / L, (3) 0.09111 ug / L, (4) 0.09251 ug / L, (5) 0.09376 ug / L, (6) 0.09319 ug / L. Averaging the above 6 data, according to the calculation formula of the average recovery rate is Therefore, 91.7% is obtained. The calculation formula of RSD is Therefore, 1.9% is obtained;

[0163] When the added concentration is 0.2 ug / L, the data obtained from 6 replicates on the instrument are respectively: (1) 85147, (2) 85875, (3) 88168, (4) 87522, (5) 89366, (6) 89522. Therefore, substituting into Y = 302902X - 28108, the obtained results are respectively (1) 0.3739 ug / L, (2) 0.3763 ug / L, (3) 0.3839 ug / L, (4) 0.3817 ug / L, (5) 0.3878 ug / L, (6) 0.3883 ug / L, and then substituting into the conversion formula The final results are respectively (1) 0.1870 ug / L, (2) 0.1882 ug / L, (3) 0.1919 ug / L, (4) 0.1909 ug / L, (5) 0.1939 ug / L, (6) 0.1942 ug / L. Averaging the above 6 data, according to the calculation formula of the average recovery rate is Therefore, 95.5% is obtained. The calculation formula of RSD is Therefore, 1.6% is obtained;

[0164] When the added concentration is 1.0 ug / L, the data obtained from 6 replicates on the instrument are respectively: (1) 531196, (2) 527719, (3) 539729, (4) 567319, (5) 581698, (6) 598563. Therefore, substituting into Y = 302902X - 28108, the obtained results are respectively (1) 1.8465 ug / L, (2) 1.8350 ug / L, (3) 1.8747 ug / L, (4) 1.9657 ug / L, (5) 2.0132 ug / L, (6) 2.0689 ug / L, and then substituting into the conversion formula The final results are respectively (1) 0.9232 ug / L, (2) 0.9175 ug / L, (3) 0.9373 ug / L, (4) 0.9829 ug / L, (5) 1.0066 ug / L, (6) 1.0344 ug / L. Averaging the above 6 data, according to the calculation formula of the average recovery rate is Therefore, 96.7% is obtained. The calculation formula of RSD is Therefore, 5.0% is obtained.

[0165] Table 6. Recovery rates and relative standard deviations of quaternary ammonium salt disinfectants with different concentrations added to the samples (n = 6)

[0166]

[0167]

[0168]

[0169] According to Table 6, it can be known that the recovery rates of fifteen quaternary ammonium salts added at three levels of concentration in milk powder and raw milk matrices are in the ranges of 85.2 - 96.5% and 85.5 - 98.3% respectively, and the recovery rates are good.

[0170] Experiment 2. Detection of actual samples

[0171] Twenty-five batches of dairy products randomly selected in the laboratory (including 5 batches of milk powder, 5 batches of cheese, 5 batches of butter, 5 batches of raw milk, 5 batches of liquid milk), where samples No. 1 - 5 are milk powder, No. 6 - 10 are cheese, No. 11 - 15 are butter, No. 16 - 20 are raw milk, and No. 21 - 25 are liquid milk. Samples 26 (spiked with fifteen quaternary ammonium salt disinfectants at a concentration of 1.0 μg / kg in 20 g of milk powder confirmed to be blank), 27 (spiked with fifteen quaternary ammonium salt disinfectants at a concentration of 1.0 μg / kg in 20 g of cheese confirmed to be blank), 28 (spiked with fifteen quaternary ammonium salt disinfectants at a concentration of 1.0 μg / kg in 20 g of butter confirmed to be blank), 29 (spiked with fifteen quaternary ammonium salt disinfectants at a concentration of 0.5 μg / L in 2 mL of raw milk confirmed to be blank), and 30 (spiked with fifteen quaternary ammonium salt disinfectants at a concentration of 0.5 μg / L in 2 mL of liquid milk confirmed to be blank) were also set up.

[0172] According to the above operation steps, extraction and purification were carried out. Each sample was parallelized 2 times, and determined by liquid chromatography - mass spectrometry. The sample determination results are shown in Table 7.

[0173] According to Table 7, it can be known that BAC - C12 and TTAB were detected in sample 7, DDAC - C14 was detected in sample 9, BAC - C14 was detected in sample 11, DDAC - C14, DDAC - C16 and CTAB were detected in sample 13, BAC - C12 was detected in sample 15, BAC - C10 was detected in sample 17, DTAB was detected in sample 20, CTAB was detected in sample 23, and BAC - C8 was detected in sample 24.

[0174] Table 7. Determination results of actual samples (μg / kg or μg / L)

[0175]

[0176]

[0177] ND: Indicates not detected.

[0178] Verification experiment 1: The 25 batches of dairy products described in Experiment 2 were detected in accordance with SN / T 4048-2014 "Determination of quaternary ammonium salts in exported foods - Liquid chromatography - Mass spectrometry / mass spectrometry method". The results were as follows: BAC-C12 (result: 12.9 μg / kg) and TTAB (result: 3.1 μg / kg) were detected in sample 7, DDAC-C14 (result: 17.8 μg / kg) was detected in sample 9, BAC-C14 (result: 5.2 μg / kg) was detected in sample 11, DDAC-C14 (result: 11.2 μg / kg), DDAC-C16 (result: 5.5 μg / kg) and CTAB (result: 6.9 μg / kg) were detected in sample 13, BAC-C12 (result: 4.8 μg / kg) was detected in sample 15, BAC-C10 (result: 21.9 μg / L) was detected in sample 17, DTAB (result: 8.7 μg / L) was detected in sample 20, CTAB (result: 13.2 μg / L) was detected in sample 23, BAC-C8 (result: 6.2 μg / L) was detected in sample 24, and no residues of fifteen quaternary ammonium salt disinfectants were detected in other samples.

[0179] Verification experiment 2: The 25 batches of dairy products described in Experiment 2 were detected in accordance with BJS 202007 "Detection of disinfectant residues in infant formula foods". The results were as follows: BAC-C12 (result: 13.1 μg / kg) and TTAB (result: 3.7 μg / kg) were detected in sample 7, DDAC-C14 (result: 16.9 μg / kg) was detected in sample 9, BAC-C14 (result: 5.3 μg / kg) was detected in sample 11, DDAC-C14 (result: 10.5 μg / kg), DDAC-C16 (result: 6.1 μg / kg) and CTAB (result: 6.3 μg / kg) were detected in sample 13, BAC-C12 (result: 4.0 μg / kg) was detected in sample 15, BAC-C10 (result: 23.0 μg / L) was detected in sample 17, DTAB (result: 8.1 μg / L) was detected in sample 20, CTAB (result: 13.7 μg / L) was detected in sample 23, BAC-C8 (result: 6.6 μg / L) was detected in sample 24, and no residues of fifteen quaternary ammonium salt disinfectants were detected in other samples.

[0180] According to the comparison of Table 7 with the results of the above Verification Experiment 1 and Verification Experiment 2, it can be known that the detection result accuracy of the present invention is high.

[0181] It should be noted that: SN / T 4048-2014 used in Verification Experiment 1 has the deficiencies of cumbersome experimental operation and time-consuming compared with the detection method of the present invention, and BJS202007 used in Verification Experiment 2 has the deficiencies of incomplete purification and relatively large matrix interference compared with the detection method of the present invention. Neither of the two standards mentions how to avoid the loss problem caused by adsorption of quaternary ammonium salt disinfectants during the experiment.

[0182] Comparative Example 1-1: Modify the (2) in step 1) of Example 1 "Precisely add 10 mL of acetonitrile-methanol mixed solution (8:2, V / V) containing 0.2% formic acid" to "Precisely add 10 mL of acetonitrile"; the rest is the same as Example 1.

[0183] Change the method used in Experiment 1 from "Example 1" to "Comparative Example 1-1", and only set the addition concentration of fifteen quaternary ammonium salt disinfectants at a high level. The obtained results are as follows:

[0184] In two matrices of milk powder and liquid milk (raw milk), the recoveries of benzalkonium chloride (BAC-C16), didodecyldimethylammonium bromide (DDAC-C12), ditetradecyldimethylammonium bromide (DDAC-C14), distearyldimethylammonium bromide (DDAC-C18), tetradecyltrimethylammonium bromide (TTAB), cetyltrimethylammonium bromide (CTAB) and stearyltrimethylammonium bromide (STAB) are lower than 50%, and the sensitivity and accuracy are significantly reduced.

[0185] Comparative Example 1-2: Modify the (2) in step 1) of Example 1 "Precisely add 10 mL of acetonitrile-methanol mixed solution (8:2, V / V) containing 0.2% formic acid" to "Precisely add 10 mL of acetonitrile-ethyl acetate (1:1, V / V) containing 0.2% formic acid"; the rest is the same as Example 1.

[0186] Change the method used in Experiment 1 from "Example 1" to "Comparative Example 1-2", and only set the addition concentration of fifteen quaternary ammonium salt disinfectants at a high level. The obtained results are as follows:

[0187] In two matrices of milk powder and liquid milk, the recoveries of fifteen quaternary ammonium salt disinfectants are all lower than 50%, and the sensitivity and accuracy are significantly reduced.

[0188] Comparative Example 2-1: The use of the purifying agent in step (3) of Example 1 was cancelled, that is, step (4) was changed to "Take 5 ml of the supernatant obtained by centrifugation in step (3) and blow it to near dryness in a 40 °C water bath with nitrogen"; the rest was the same as Example 1.

[0189] Change the method used in Experiment 1 from "Example 1" to "Comparative Example 2-1", and only set the addition concentration of fifteen quaternary ammonium salt disinfectants at a high level. The obtained results were:

[0190] In the two matrices of milk powder and raw fresh milk, the recovery rates of didodecyldimethylammonium bromide (DDAC-C14), dihexadecyldimethylammonium bromide (DDAC-C16), dioctadecyldimethylammonium bromide (DDAC-C18), tetradecyltrimethylammonium bromide (TTAB), cetyltrimethylammonium bromide (CTAB), and octadecyltrimethylammonium bromide (STAB) were lower than 70%, and the sensitivity and accuracy were significantly reduced.

[0191] Comparative Example 2-2: Change "(3) Add 300 mg of purifying agent (aminopropyl ethylenediamine and quaternary ammonium propyl bonded silica gel, weight ratio 2:1)" in step 1) of Example 1 to "Add 300 mg of purifying agent (aminopropyl ethylenediamine and C18, weight ratio 2:1)"; the rest was the same as Example 1.

[0192] Change the method used in Experiment 1 from "Example 1" to "Comparative Example 2-2", and only set the addition concentration of fifteen quaternary ammonium salt disinfectants at a high level. The obtained results were:

[0193] In the two matrices of milk powder and liquid milk, the recovery rates of hexadecylbenzyldimethylammonium chloride (BAC-C16), didodecyldimethylammonium bromide (DDAC-C12), didodecyldimethylammonium bromide (DDAC-C14), dihexadecyldimethylammonium bromide (DDAC-C16), dioctadecyldimethylammonium bromide (DDAC-C18), tetradecyltrimethylammonium bromide (TTAB), cetyltrimethylammonium bromide (CTAB), and octadecyltrimethylammonium bromide (STAB) were all lower than 40%, and the sensitivity and accuracy were significantly reduced.

[0194] Comparative Example 2-3: Change "(3) Add 300 mg of purifying agent (aminopropyl ethylenediamine and quaternary ammonium propyl bonded silica gel, weight ratio 2:1)" in step 1) of Example 1 to "Add 300 mg of purifying agent (aminopropyl ethylenediamine and quaternary ammonium propyl bonded silica gel, weight ratio 1:1)"; the rest was the same as Example 1.

[0195] Change the method used in Experiment 1 from "Example 1" to "Comparative Example 2-3", and only set the addition concentration of fifteen quaternary ammonium salt disinfectants at a high level. The obtained results were:

[0196] In both milk powder and liquid milk matrices, the recoveries of dodecyl dimethyl ammonium bromide (DDAC-C12), tetradecyl dimethyl ammonium bromide (DDAC-C14), hexadecyl dimethyl ammonium bromide (DDAC-C16), octadecyl dimethyl ammonium bromide (DDAC-C18), tetradecyl trimethyl ammonium bromide (TTAB), cetyl trimethyl ammonium bromide (CTAB), and octadecyl trimethyl ammonium bromide (STAB) were all lower than 80%, and the sensitivity and accuracy were significantly reduced.

[0197] Comparative Example 3: Change the polytetrafluoroethylene centrifuge tube, polytetrafluoroethylene nitrogen blowing tube, and polytetrafluoroethylene sample injection vial in step 1) of Example 1 to glass centrifuge tubes, glass nitrogen blowing tubes, and glass sample injection vials; the rest is the same as Example 1.

[0198] Change the method used in Experiment 1 from "Example 1" to "Comparative Example 3", and only set the addition concentration of fifteen quaternary ammonium salt disinfectants at a high level. The obtained results are as follows:

[0199] In both milk powder and liquid milk matrices, the recoveries of fifteen quaternary ammonium salt disinfectants were all lower than 30%, and the sensitivity and accuracy were significantly reduced.

[0200] Comparative Example 4: Change "Waters Cortecs C18+, 100mm×2.1mm, 1.6um" used in Example 1 to "ordinary C18 chromatographic column"; and use the corresponding linear equation obtained; the rest is the same as Example 1.

[0201] Change the method used in Experiment 1 from "Example 1" to "Comparative Example 4", and only set the addition concentration of fifteen quaternary ammonium salt disinfectants at a high level. The obtained results are as follows:

[0202] In both milk powder and liquid milk matrices, serious residues appeared in the chromatographic peaks of dodecyl dimethyl ammonium bromide (DDAC-C12), tetradecyl dimethyl ammonium bromide (DDAC-C14), hexadecyl dimethyl ammonium bromide (DDAC-C16), octadecyl dimethyl ammonium bromide (DDAC-C18), tetradecyl trimethyl ammonium bromide (TTAB), cetyl trimethyl ammonium bromide (CTAB), and octadecyl trimethyl ammonium bromide (STAB), and the sensitivity and accuracy were significantly reduced.

[0203] Comparative Example 5-1: Change "Mobile phase B is a methanol-acetonitrile mixed solution (60:40) containing 0.2% formic acid" used in Example 1 to "Mobile phase B is acetonitrile containing 0.2% formic acid"; and use the corresponding linear equation obtained; the rest is the same as Example 1.

[0204] Change the method used in Experiment 1 from "Example 1" to "Comparative Example 5-1", and only set the addition concentrations of the fifteen quaternary ammonium salt disinfectants at a high level. The obtained results are as follows:

[0205] In the two matrices of milk powder and liquid milk, five components such as didodecyldimethylammonium bromide (DDAC-C14), dihexadecyldimethylammonium bromide (DDAC-C16), dioctadecyldimethylammonium bromide (DDAC-C18), cetyltrimethylammonium bromide (CTAB), and octadecyltrimethylammonium bromide (STAB) were not detected, and serious chromatographic peak tailing occurred, with significantly reduced sensitivity and accuracy.

[0206] Comparative Example 5-2: Change "Mobile phase B is a methanol-acetonitrile mixed solution (60:40) containing 0.2% formic acid" used in Example 1 to "Mobile phase B is methanol containing 0.2% formic acid"; and use the corresponding obtained linear equation; the rest is the same as Example 1.

[0207] Change the method used in Experiment 1 from "Example 1" to "Comparative Example 5-2", and only set the addition concentrations of the fifteen quaternary ammonium salt disinfectants at a high level. The obtained results are as follows:

[0208] In the two matrices of milk powder and liquid milk, chromatographic peak tailing occurred for six components such as cetylbenzyldimethylammonium chloride (BAC-C16), didodecyldimethylammonium bromide (DDAC-C14), dihexadecyldimethylammonium bromide (DDAC-C16), dioctadecyldimethylammonium bromide (DDAC-C18), cetyltrimethylammonium bromide (CTAB), and octadecyltrimethylammonium bromide (STAB), with significantly reduced sensitivity and accuracy, and serious residue occurred on the chromatographic column.

[0209] Comparative Example 5-3: Change "Mobile phase B is a methanol-acetonitrile mixed solution (60:40) containing 0.2% formic acid" used in Example 1 to "Mobile phase B is a methanol-acetonitrile mixed solution (50:50) containing 0.2% formic acid"; and use the corresponding obtained linear equation; the rest is the same as Example 1.

[0210] Change the method used in Experiment 1 from "Example 1" to "Comparative Example 5-3", and only set the addition concentrations of the fifteen quaternary ammonium salt disinfectants at a high level. The obtained results are as follows:

[0211] In the two matrices of milk powder and liquid milk, chromatographic peak tailing occurred for three components such as dihexadecyldimethylammonium bromide (DDAC-C16), dioctadecyldimethylammonium bromide (DDAC-C18), and octadecyltrimethylammonium bromide (STAB), and residue occurred on the chromatographic column.

[0212] Finally, it should also be noted that the above are only several specific embodiments of the present invention. It shows that the present invention is not limited to the above embodiments and there can be many variations. All variations that can be directly derived or associated by those of ordinary skill in the art from the disclosed content of the present invention should be considered within the protection scope of the present invention.

Claims

1. Method for determining residues of fifteen quaternary ammonium salt disinfectants in dairy products, characterized in that It includes the following steps: 1). Prepare the sample solution of the dairy product to be tested; It includes the following steps: 1.

1. Thoroughly mix the dairy product to be tested as a sample; When the dairy product to be tested is solid or semi-solid, weigh 5 - 20 g of the sample, add 10 - 30 ml of deionized water, shake in a water bath at 60 ± 10 °C until completely dissolved, and after cooling to room temperature, use it as the sample solution; When the dairy product to be tested is liquid, let it stand and return to room temperature, and use it as the sample solution; 1.

2. Take 1.0 - 3.0 mL of the sample solution obtained in step 1.1 into a polytetrafluoroethylene centrifuge tube, add 5 - 1 mL of acetonitrile - methanol mixed solution containing 0.1 - 0.3% formic acid, vortex and shake for 2 - 10 min, then add anhydrous magnesium sulfate, shake and extract for 5 - 15 min, and centrifuge at 5000 - 10000 r / min on a high-speed centrifuge for 2 - 10 min; In the acetonitrile - methanol mixed solution, the volume ratio of acetonitrile: methanol = 7:3 - 9:1; 1.

3. Take 5 - 8 mL of the supernatant obtained in step 1.2 and transfer it to a polytetrafluoroethylene centrifuge tube, add 200 - 500 mg of the purification agent, and vortex and shake for 2 - 10 min; The purification agent is composed of amino - propyl ethylenediamine and quaternary ammonium propyl bonded silica gel in a weight ratio of 1:1 - 3:1; 1.

4. Take 5 ml of the supernatant obtained in step 1.3 and dry it; add 0.5 mL of 60 - 80% acetonitrile aqueous solution for re - dissolution, vortex and mix for 0.5 - 2 min to dissolve the residue, filter with a 0.22 µm microporous organic filter membrane, and place the filtrate in a polytetrafluoroethylene injection vial as the sample solution of the dairy product to be tested; 2). Prepare the standard solution: Prepare a mixed standard series working solution from fifteen QACs standard substances; 3). Inject the mixed standard series working solution into the liquid chromatography - mass spectrometry instrument, determine the peak positions and qualitative and quantitative ion pairs of the fifteen QACs, use the abundance of the quantitative ion pair as the ordinate and the concentration as the abscissa to make the standard curve equation; The chromatographic conditions are: flow rate: 0.3 - 0.8 ml / min; column temperature: 20 - 50 °C; injection volume 0.5 - 5 µL; The mobile phase consists of mobile phase A and mobile phase B: mobile phase A is 0.2% formic acid aqueous solution; mobile phase B is a methanol - acetonitrile mixed solution containing 0.2% formic acid, and the volume ratio of methanol: acetonitrile = 60:40; The gradient elution program is: 0 - 5.5 min, 8% - 100% B; 5.5 - 7.5 min, 100% B; 7.5 - 7.6 min, 100% - 8% B; 7.6 - 10.0 min, 8% B; The above % is volume %. The liquid chromatography column is: Waters CORTECS C 18+ ; The fifteen QACs are: benzyl dimethyl ammonium chloride, N-decyl-N,N-dimethyl benzyl ammonium chloride, dodecyl dimethyl benzyl ammonium chloride, tetradecyl dimethyl benzyl ammonium chloride, hexadecyl dimethyl benzyl ammonium chloride, didodecyl dimethyl ammonium bromide, ditetradecyl dimethyl ammonium bromide, dihexadecyl dimethyl ammonium bromide, dioctadecyl dimethyl ammonium bromide, decyl trimethyl ammonium bromide, dodecyl trimethyl ammonium bromide, tetradecyl trimethyl ammonium bromide, hexadecyl trimethyl ammonium bromide, octadecyl trimethyl ammonium bromide; 4) Take the sample solution of the dairy product to be tested obtained in step 1) and determine the peak areas and their quantitative and qualitative ion pairs of each QAC in the sample solution according to the method in step 3). Conduct qualitative analysis based on the elution times and the abundance ratios of the qualitative and quantitative ion pairs, and calculate according to the standard curve equation obtained in step 3) to obtain the contents of each QAC in the dairy product to be tested.

2. The method for determining the residues of fifteen quaternary ammonium salt disinfectants in dairy products according to claim 1, characterized in that: The mass spectrometry conditions in step 3) are: electrospray ionization source, positive ion detection mode; multiple reaction monitoring; ion source temperature: 150 °C; capillary voltage: 2.0 kV; cone voltage: 20 - 25 V; desolvation gas flow rate: 600 L / h; desolvation gas temperature: 1000 °C; cone gas flow rate: 150 L / h; calibration method: automatic mass axis tuning and correction; segmented scanning by MRM; other mass spectrometry analysis parameters are as follows: ； * is the quantitative ion pair.

3. The method for determining the residues of fifteen quaternary ammonium salt disinfectants in dairy products according to claim 2, wherein In step 2): Use 70% aqueous acetonitrile solution as the solvent to prepare a series of mixed standard working solutions with different concentrations. In each mixed standard working solution with different concentrations, the concentrations of the fifteen QACs are the same; the concentrations are 0.2, 0.5, 1, 5, 10, 20 μg / L respectively.

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