CN113219078B - Rapid screening method for 17 organic antibacterial agents in household paper products - Google Patents
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Abstract
The invention discloses a method for rapidly screening 17 organic antibacterial agents in a household paper product, which comprises the following steps: the method comprises the steps of taking a household paper product as a research object, adopting acetonitrile water solution as an extracting agent, heating in a water bath and carrying out ultrasonic extraction, salting out anhydrous magnesium sulfate and ammonium sulfate, degreasing by acetonitrile saturated normal hexane, purifying by an anhydrous sodium sulfate and graphitized carbon black mixed purification column, carrying out rotary evaporation concentration to dryness, and screening by using UPLC/MS-IT-TOF after redissolving by a methanol water solution. An ion trap-time-of-flight mass spectrum screening database of 17 organic antibacterial agents in the household paper products is established to obtain mass spectrum fragmentation characteristics and MS 1 、MS 2 The accurate mass number m/z, and the quasi-targeting screening technology can be used for rapidly screening and accurately identifying 17 organic antibacterial agents in the domestic paper product without standard substances (control substances).
Description
Technical Field
The invention belongs to the technical field of paper product quality safety detection, and particularly relates to a method for quickly screening 17 organic antibacterial agents in paper products for daily use, in particular to a method for quickly screening 17 organic antibacterial agents in paper products for food contact and paper products for human body contact.
Background
The daily-use paper products are inseparable from our lives, closely contact our skins or directly contact our foods, and in the production, processing and storage processes of the paper products, organic antibacterial agents are often used for preventing the paper products from mildewing, inhibiting and killing bacteria, mold, algae and the like, so that the effects of antisepsis and bacteriostasis are achieved, and the storage and service life of the paper products are prolonged. Common organic antibacterial agents comprise benzimidazoles, quaternary ammonium salts (such as benzalkonium chloride and the like), isothiazolinones and the like, and due to the lack of complete detection standards, some organic antibacterial agents are not listed in regulatory projects, so that potential safety hazards may exist in the quality of the household paper. The organic antibacterial agent has no obvious harm to the environment and human body in a short time, but the organic antibacterial agent is accumulated to a certain content in a long time, and the harm to the human body and the environment cannot be ignored.
Currently, the common detection methods of the organic antibacterial agent mainly comprise High Performance Liquid Chromatography (HPLC), gas Chromatography (GC), high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and gas chromatography/mass spectrometry combined method (GC-MS). The GC-MS method has high sensitivity, is suitable for detecting low-content organic antibacterial agents, but is not suitable for detecting quaternary ammonium salts in the experiment. The HPLC method can simultaneously detect a plurality of organic antibacterial agents without derivation, has strong universality and low maintenance cost, is easily interfered by impurities in a sample, has weak qualitative identification capability and has high requirements on liquid chromatography separation conditions.
Literature reports on the detection of organic antibacterial agents in paper products have focused mainly on food-contact paper products and less reports have been made on paper products for daily use. Many types of organic antibacterial agents are possibly added into the domestic paper product, and if each type of antibacterial agent is detected, the defects of low efficiency, high cost and the like exist, and a detection method for simultaneously and rapidly screening a plurality of organic antibacterial agents such as benzimidazoles, quaternary ammonium salts (such as benzalkonium chloride and the like), isothiazolinones and the like at high flux is not reported in documents.
Disclosure of Invention
The invention aims to provide a method for quickly screening 17 organic antibacterial agents in a household paper product, which has simple, accurate and reliable pretreatment and is suitable for quickly screening and identifying the 17 organic antibacterial agents in the household paper product.
The above purpose of the invention is realized by the following technical scheme: a method for rapidly screening 17 organic antibacterial agents in household paper products comprises the following steps:
(1) Extraction: selecting a household paper product, crushing, uniformly mixing, placing the household paper product into a container, adding an extracting solution into the container, shaking the mixture uniformly, placing the mixture into a water bath, carrying out ultrasonic extraction, taking out the container, cooling the container to room temperature, carrying out suction filtration, transferring a filtrate into a centrifuge tube a, transferring a residual paper product sample into a centrifuge tube b for later use, adding anhydrous magnesium sulfate and ammonium sulfate into the centrifuge tube a, carrying out vortex extraction and centrifugation, collecting a supernatant into a centrifuge tube c, adding acetonitrile into the centrifuge tube b, carrying out repeated extraction once, carrying out water bath ultrasonic extraction and suction filtration, transferring a filtrate into the centrifuge tube a, carrying out vortex extraction and centrifugation, taking the supernatant, and combining the supernatant into the centrifuge tube c to obtain a liquid to be purified;
(2) Purifying: adding acetonitrile saturated normal hexane into a centrifuge tube c, carrying out vortex extraction, removing the upper layer of normal hexane, passing all the residual liquid through an anhydrous sodium sulfate and graphitized carbon black mixed purification column, collecting effluent liquid, leaching the mixed purification column with acetonitrile, combining and collecting the effluent liquid, carrying out rotary evaporation concentration under the water bath condition until the effluent liquid is dry, dissolving the residue with a methanol aqueous solution, carrying out ultrasonic treatment, and filtering with an organic phase filter membrane to prepare a liquid to be detected;
(3) Preparing a standard substance: taking 17 organic antibacterial agent standards, dissolving the standards with acetonitrile to prepare a standard stock solution, and diluting the standard stock solution with a methanol aqueous solution to prepare a mixed standard working solution;
(4) Establishing an ultra-high performance liquid chromatography method: with C 18 Taking the chromatographic column as a separation column, and taking methanol, acetonitrile, ammonium acetate solution or ammonium formate solution as a mobile phase to carry out gradient elution so as to establish an ultra-high performance liquid chromatography separation method;
(5) Establishing a screening database: optimizing parameters of the ion trap-time-of-flight mass spectrometer and combining the method established in the step (4), establishing an ultra-high performance liquid chromatography-ion trap-time-of-flight mass spectrometer (UPLC/MS-IT-TOF) detection method, and measuring the standard solution in the step (3) to obtain the quasi-molecular ions (MS) of 17 organic antibacterial agents 1 ) And fragment ion (MS) 2 ) The accurate mass number of the antibacterial agent is obtained, and a screening database of 17 organic antibacterial agents is established;
(6) Screening: using the ultra-high performance liquid chromatography-ion trap-flight time to the liquid to be detected prepared in the step (2)MS of the obtained sample as determined by Mass Spectrometry (UPLC/MS-IT-TOF) 1 And MS 2 And (4) qualitatively confirming the data information with the screening database and molecular Formula prediction software (Formula Predictor) established in the step (5) to obtain the information of the 17 organic antibacterial agents in the household paper products.
The invention adopts ultra-high performance liquid chromatography-ion trap-time of flight mass spectrometry (UPLC/MS-IT-TOF) to establish a high-flux rapid screening analysis method for 17 organic antibacterial agents in the paper products for daily use, forms a quasi-targeting screening technology, can realize screening and identification of the 17 organic antibacterial agents in the paper products for daily use under the condition of no standard substance (reference substance), and provides a novel detection means for quality monitoring of the paper products.
In the quick screening method for the 17 organic antibacterial agents in the living paper product, the method comprises the following steps:
preferably, the household paper products in the step (1) comprise paper products for food contact and paper products for human body contact, and the paper products for food contact comprise paper cups, kitchen absorbent paper and the like; the paper product for human body contact comprises a paper diaper (paper diaper), a wet tissue, a facial tissue and the like; cutting the samples of the household paper products into the size of about 5mm multiplied by 5mm, mixing evenly and weighing the samples.
Preferably, the extracting solution in the step (1) is an acetonitrile aqueous solution, the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 1:1-2:1, the ratio of the mass of the paper product for daily use to the volume of the extracting solution is 1g.
The application finds that:
the 17 organic antibacterial agents are all easily soluble in acetonitrile and can be directly extracted by using the acetonitrile, but because the paper products for daily use contain more organic impurities and inorganic impurities, the sample screening data directly extracted by using the acetonitrile shows that the impurities are more, under the condition of specific instrument parameters, the number of ions needing to be scanned in unit time is more, and the detection sensitivity and the resolution capability of the instrument on target compounds are reduced.
When the sample was extracted with aqueous acetonitrile, the impurities in the extract were significantly reduced. The volume ratio of the acetonitrile to the water is 1:1-2:1, and the extraction recovery rate can be ensured to reach more than 70%.
The ultrasonic extraction of the sample is carried out for 15-30 min under the condition of 30-40 ℃ water bath, the dissolution of the target compound into the extracting solution can be accelerated, and the extraction efficiency is improved.
In fact, 17 organic antibacterial agents are also easily soluble in methanol, but methanol is not selected because methanol is miscible with water, methanol cannot be separated from water after anhydrous magnesium sulfate and ammonium sulfate are added, and subsequent testing steps cannot be carried out.
Preferably, the dosage relation among the extracting solution, the anhydrous magnesium sulfate and the ammonium sulfate in the step (1) is 20-30 mL: 4-6 g: 3-5 g; the rotating speed of the vortex mixer is 1500-1800 r/min during vortex extraction, and the vortex extraction time is 5-10 min; the rotating speed of the centrifugal machine is 6500-9000 r/min during centrifugation, and the centrifugation time is 3-5 min.
The anhydrous magnesium sulfate and the ammonium sulfate are dissolved in water to be acidic, and have double functions of salting out and adjusting the pH value, so that the extraction recovery rate is improved. Under the acidic condition, the target compound is converted into a molecular state and is easier to be extracted by acetonitrile; under the salting-out action, the water phase and the acetonitrile can be layered, and quaternary ammonium salt antibacterial agents such as benzalkonium chloride and the like which are easy to dissolve in water can be promoted to be transferred from the water phase to the acetonitrile phase, so that the effect of improving the extraction recovery rate is achieved.
Preferably, the volume ratio of the acetonitrile saturated normal hexane to the extracting solution in the step (2) is 1-2: 4-6, the rotating speed of a vortex mixer during vortex extraction is 1500-1800 r/min, the vortex extraction time is 5-10 min, and the upper layer of normal hexane is discarded.
Preferably, the anhydrous sodium sulfate and graphitized carbon black purifying column described in the step (2) is prepared by mixing and grinding 8 to 12g of anhydrous sodium sulfate which is dried at 105 ℃ for 2 to 3 hours and does not contain crystal water and 100 to 300mg of graphitized carbon black for 10 minutes, and then filling the mixture into a disposable syringe needle cylinder with the volume of 10mL, wherein the outflow speed is controlled to be 1 to 2mL/min when the column is passed through the purifying column, then the purifying column is rinsed with acetonitrile with the volume of 5 to 10mL, the effluent and the rinsing liquid are collected, the column is evaporated and concentrated to be dry in a water bath with the temperature of 45 to 50 ℃ by rotary evaporation, and the volume ratio of methanol to water is 4:6 to 7:3, dissolving the residue in 1-2 mL of methanol aqueous solution, and filtering with an organic filter membrane with the aperture of 0.22 mu m to prepare the solution to be detected.
Removing nonpolar impurities dissolved out from the sample in the pretreatment process by using acetonitrile saturated normal hexane to purify the sample. Anhydrous sodium sulfate in the anhydrous sodium sulfate and graphitized carbon black mixed purification column is used for removing water in acetonitrile, the graphitized carbon black removes pigments, the outflow speed of the acetonitrile is controlled not to exceed 1-2 mL/min, so that the acetonitrile is in full contact with a filler, and full dehydration and decoloration are ensured. The target compound has better chromatographic peak shape and response value by the methanol aqueous solution with the volume ratio of the methanol to the water of 4:6-7:3.
Preferably, the 17 organic antimicrobial standards in step (3) include 2-octyl-4-isothiazolin-3-One (OIT), 1,2-benzisothiazolin-3-one (BIT), 2-methyl-4-isothiazolin-3-one (MIT), carbendazim, albendazole-2-aminosulfone, albendazole sulfoxide, albendazole sulfone, thiabendazole, fuberidazole, benzalkonium chloride, cetylpyridinium chloride, lorammonium chloride, benzethonium chloride, dequalinium chloride, sitafloxacin chloride, and triclocarban.
Preferably, the 17 organic antibacterial agent standard substances in the step (3) are respectively weighed and placed in different volumetric flasks, methanol is added for dissolution to prepare a standard stock solution, and the methanol aqueous solution in the step (2) is used for dilution to prepare a mixed standard working solution with the concentration of 50-2000 ng/mL.
The 17 organic antibacterial agents are all easily soluble in methanol and acetonitrile, but methanol is used as a solvent, so that the stability is higher, and the effective period is longer, so methanol is selected as the solvent. The target compound has better peak shape and sensitivity by diluting with methanol aqueous solution.
The invention aims to provide a rapid screening method, which can be qualitatively confirmed when the concentration of 17 organic antibacterial agents reaches 50ng/mL, when the concentration reaches 2000ng/mL, signals of partial target objects in LC/MS-IT-TOF are close to saturation, flat-top chromatographic peaks appear, and when the concentration exceeds 2000ng/mL, a sample to-be-detected liquid needs to be diluted, so that the optimal screening concentration range of the target compounds is 50-2000 ng/mL.
Preferably: the ultra-high performance liquid chromatography method established in the step (4) adopts the following parameters:
a chromatographic column: waters ACQUITY UPLC BEH C 18 Column, specification 100mm × 2.1mm,1.7 μm; flow rate: 0.2mL/min; column temperature: 25-30 ℃; sample introduction amount: 20 μ L.
The mobile phase A is: methanol + acetonitrile (volume ratio 1:9); the mobile phase B is as follows: 10mmol/L ammonium acetate solution or 10mmol/L ammonium formate solution.
The gradient elution procedure was: 0.00min: phase A, 5%, phase B, 95%;2.00min: phase A, 5%, phase B, 95%;2.10min: 35% of phase A and 65% of phase B; 10.00min: 35% of phase A and 65% of phase B; 10.10min: 50% of phase A and 50% of phase B; 20.00min: 50% of phase A and 50% of phase B; 20.10min: phase A, 90%, phase B, 10%;25.00min: phase A, 90%, phase B, 10%;25.10min: phase A, 100 percent, phase B, 0 percent; 31.00min: phase A, 100 percent, phase B, 0 percent; 31.50min: phase A, 5 percent, phase B, 95 percent; 33.00min: phase A, 5%, phase B, 95%; all the contents are volume percentage.
The methanol is taken as an organic phase mobile phase, the peak emergence time of a target compound is slow, the rapid screening is not facilitated, if acetonitrile is selected, the peak emergence time is too fast, the separation effect with an impurity peak is not ideal, and the ratio of the methanol to the acetonitrile is 1: at 9 volume ratio, the target mixture had better sensitivity and peak shape.
Ammonium acetate as a mobile phase additive can improve the ionization efficiency of 17 organic antibacterial agents.
Because the property difference of the 17 organic antibacterial agents is large, a gradient elution mode is selected to obtain better resolution, and under the chromatographic condition, the chromatographic peak has symmetrical peak shapes and high sensitivity.
Preferably, the ion trap-time-of-flight mass spectrometer parameters in step (5) comprise: an ion source: ESI, positive ion mode, automatic scan acquisition; heating module temperature: 150 to 200 ℃; CDL temperature: 150 to 200 ℃; flow rate of the atomizer: 1.3-1.5L/min; pressure of drying gas: 95kPa to 110kPa; ion source voltage: 3.5-4.5 kV; detector voltage: 1.6-1.7 kV; the calibration method comprises the following steps: automatically tuning the optimized voltage; calibrating the mass number by an external standard method; the collection range of the quasi-molecular ions is m/z 100-450, repeated scanning is carried out for 3-5 times, and the ion accumulation time is as follows: 20-40 msec; the secondary fragment ion collection range is m/z 60-450, repeated scanning is carried out for 3-5 times, and the ion accumulation time is as follows: 30-50 msec, and the screening range of precursor ions is as follows: m/z is 100-450; CID (induced collision dissociation) energy: 50% -70%; collision gas: 50% -60%; the collection frequency is 45.0-67.5 kHz.
And (5) setting m/z to 100-450, adopting positive and negative ions to scan simultaneously, wherein 17 ions have no characteristic ion peak in a negative ion mode and have a higher response value in a positive ion mode, and therefore, selecting the positive ion scanning mode. The ion trap-time-of-flight mass spectrometer parameter setting directly influences the minimum screening concentration of 17 organic antibacterial agents in a paper product, the minimum screening concentration requirement of 50ng/mL is met according to the set parameter range, and the response sensitivity of the target compound is higher along with the increase of the parameters of the detector voltage and the ion accumulation time.
Preferably, the 17 organic antibacterial agent screening database in the step (5) is shown in the following table 1:
TABLE 1 screening database for 17 organic antibacterial agents
Compared with the prior art, the invention has the following advantages:
(1) Heating and ultrasonic extraction: by adopting heating ultrasonic extraction, the dissolving of the organic antibacterial agent in the extracting solution can be accelerated, the complete extraction is ensured, and the conditions of insufficient accuracy, low result, poor repeatability and the like are avoided;
(2) Salting out and extracting a sample extracting solution under an acidic condition: the organic antibacterial agent is favorable to form a molecular state under an acidic condition, and the extraction rate of acetonitrile on a substance to be detected is improved under the salting-out condition of adding anhydrous magnesium sulfate and ammonium sulfate;
(3) Triple purification, impurity removal: salting out to extract the target compound in the extract (acetonitrile aqueous solution) to an acetonitrile layer, and retaining water-soluble impurities such as inorganic salts in the water phase; removing impurities such as fat-soluble pigment, nonpolar compound and the like from acetonitrile saturated normal hexane; the anhydrous sodium sulfate and graphitized carbon black are mixed and purified by a column, so that residual water and adsorption pigments in acetonitrile are removed, impurities are removed by triple purification, the content of the impurities in the liquid to be detected is reduced, and the screening sensitivity is improved;
(4) The method of the invention has high flux, accuracy, reliability, simplicity, rapidness and economy: the method has the advantages that the property difference of the 17 organic antibacterial agents is large, the same solid phase extraction column cannot meet the requirement of simultaneous extraction, in addition, when the antibacterial agent content in a sample is high, the solid phase extraction column is overloaded, so that part of target compounds are not extracted, the recovery rate is low, the antibacterial agent content condition in the sample cannot be predetermined, if the filler amount of the solid phase extraction column is increased, waste is caused when a low-content sample is detected, and therefore, the solid phase extraction method cannot meet the requirement of high-flux accurate screening of the 17 organic antibacterial agents;
(5) The method takes methanol and acetonitrile (the volume ratio is 1:9) and 10mmol/L ammonium acetate solution or 10mmol/L ammonium formate solution as mobile phases, and adopts a gradient elution program separation mode to obtain better separation degree and sensitivity, and the minimum screening concentration of 17 organic antibacterial agents in a sample solution to be tested is 50ng/mL;
(6) Establishing an ion trap-time-of-flight mass spectrum screening database of 17 organic antibacterial agents in the household paper products to obtain the mass spectrum fragmentation characteristics and MS of the 17 organic antibacterial agents 1 、MS 2 The accurate mass number of m/z, and the quasi-target screening technology can realize the screening and identification of 17 organic antibacterial agents in the household paper products under the condition of no standard substance (reference substance).
Drawings
FIG. 1-1 to FIG. 1-17 show characteristic ions MS of 17 organic antibacterial agents in example 1 1 、MS 2 Mass spectrogramFig. 1-1: 2-octyl-4-isothiazolin-3-One (OIT), fig. 1-2:1,2-benzisothiazolin-3-one (BIT), FIGS. 1-3: 2-methyl-4-isothiazolin-3-one (MIT), fig. 1-4: fubering, fig. 1-5: carbendazim, fig. 1-6: thiabendazole, FIGS. 1-7: triclocarban, fig. 1-8: albendazole, FIGS. 1-9: albendazole-2-aminosulfone, figures 1-10: albendazole sulfone, fig. 1-11: albendazole sulfoxide, fig. 12: loran, fig. 13: benzalkonium chloride, FIGS. 1-14: sitafloxacin chloride, fig. 1-15: benzethonium chloride, FIGS. 1-16: dequalinium chloride, fig. 1-17: cetylpyridinium chloride, wherein the upper panel is MS 1 The lower diagram is MS 2 Mass spectrogram;
FIG. 2 is an ion chromatogram obtained by extracting 17 types of organic antibacterial agent standards (reference) in example 1;
FIG. 3 is an ion chromatogram of benzethonium chloride (479.9 mg/kg) extracted from sterilized wet tissue 1 of example 2;
FIG. 4 is an extracted ion chromatogram of Laura ammonium chloride (246.5 mg/kg) in sterilized wet wipe 1 of example 2;
FIG. 5 is an extraction ion chromatogram of benzalkonium chloride (200.3 mg/kg) in wet tissue of purified water in example 2;
FIG. 6 is a total ion flow chart (TIC) of the diaper (diaper) in example 3.
Detailed Description
The present invention will be further described with reference to specific embodiments, but the scope of the invention as claimed is not limited to the following embodiments.
The apparatus and equipment used in the invention: ultra-high performance liquid chromatography-ion trap-time-of-flight mass spectrometer (LC/MS-IT-TOF), configuring an ESI ion source; an ultrasonic cleaning machine; an automatic vortex mixer; a rotary evaporator; an electronic balance: the sensory amounts were 0.00001g and 0.01g, respectively.
The reagents used in the present invention: and (3) chromatographic purification: methanol, acetonitrile, n-hexane; first-stage water; and (3) analytical purification: anhydrous magnesium sulfate, anhydrous sodium sulfate, graphitized carbon black.
Example 1
The embodiment provides a method for rapidly screening 17 organic antibacterial agents in a food contact material paper product, which comprises the following steps:
1. pretreatment step
1.1 extraction
Cutting food contact materials such as paper cups and kitchen absorbent paper into the size of about 5mm multiplied by 5mm, uniformly mixing, weighing 2g (accurate to 0.01 g) of the sample, placing the sample in a conical flask, adding 15mL of water and 15mL of acetonitrile, shaking uniformly, placing the sample in an ultrasonic cleaning machine, performing water bath ultrasonic treatment at the temperature of 40 ℃ for 30min at the frequency of 50kHz, taking out the conical flask, cooling to the room temperature, performing suction filtration, transferring filtrate to a 50mL plastic centrifuge tube a, and transferring residual paper product samples to a centrifuge tube b for later use. 6g of anhydrous magnesium sulfate and 5g of ammonium sulfate are added into the centrifuge tube a, an automatic vortex mixer performs vortex extraction for 10min at 1800r/min, a centrifugal machine performs centrifugal extraction for 3min at 8000r/min, and supernatant is taken and transferred into a centrifuge tube c.
And (3) adding 20mL of acetonitrile into the centrifuge tube b, placing the centrifuge tube b into an ultrasonic cleaner, performing ultrasonic treatment for 15min in a water bath at 40 ℃ at the frequency of 50kHz, performing suction filtration, transferring the filtrate into the centrifuge tube a, performing vortex extraction for 5min at 1800r/min, centrifuging for 3min at 8000r/min, taking supernate, combining the supernate and preparing about 36mL of liquid to be purified in the centrifuge tube c.
1.2 purification
Adding 9mL of acetonitrile saturated normal hexane into a centrifuge tube c, performing vortex extraction at 1800r/min for 2min, discarding the upper layer of normal hexane, allowing the rest liquid to pass through a purification column, mixing and grinding 10g of anhydrous sodium sulfate and 200mg of graphitized carbon black for 10min, filling the mixture into a 10mL disposable syringe needle cylinder to prepare the purification column, controlling the column passing outflow speed to be 1mL/min, collecting the effluent by using a heart-shaped bottle, then leaching the purification column by using 10mL of acetonitrile, collecting the leacheate by using the heart-shaped bottle, and combining the effluent and the leacheate; concentrating to dryness by rotary evaporation in a water bath at 50 ℃, and performing concentration by using methanol and water in a volume ratio of 5:5, dissolving the residue in 1.0mL of methanol aqueous solution, filtering with an organic filter membrane with the pore diameter of 0.22 μm to prepare a solution to be detected, and screening by LC/MS-IT-TOF.
2. Preparing a standard solution: weighing 17 organic antibacterial agent standards respectively, placing the standards into 17 volumetric flasks respectively, adding methanol for dissolving to prepare a standard stock solution, and diluting with a methanol water solution (the volume ratio is 5:5) to prepare a mixed standard working solution with the concentration of 50-2000 ng/mL.
Wherein the 17 organic antibacterial agent standard substances are respectively as follows: 2-octyl-4-isothiazolin-3-One (OIT), 1,2-benzisothiazolin-3-one (BIT), 2-methyl-4-isothiazolin-3-one (MIT), carbendazim, albendazole-2-aminosulfone, albendazole sulfoxide, albendazole sulfone, thiabendazole, fuberidazole, benzalkonium chloride, cetylpyridinium chloride, lorammonium chloride, benzethonium chloride, dequalinium chloride, sitalimmonium chloride and triclocarban.
3. Conditions of instrumental detection
3.1 liquid chromatography conditions
A chromatographic column: waters ACQUITY UPLC BEH C 18 Column, specification 100mm × 2.1mm,1.7 μm; flow rate: 0.2mL/min; column temperature: 25 ℃; sample introduction amount: 20 μ L.
The mobile phase A is: methanol + acetonitrile (volume ratio 1:9).
The mobile phase B is as follows: 10mmol/L ammonium acetate solution.
The gradient elution procedure was: 0.00min: phase A, 5%, phase B, 95%;2.00min: phase A, 5%, phase B, 95%;2.10min: 35% of phase A, 65% of phase B; 10.00min: 35% of phase A and 65% of phase B; 10.10min: 50% of phase A and 50% of phase B; 20.00min: 50% of phase A and 50% of phase B; 20.10min: phase A, 90%, phase B, 10%;25.00min: phase A, 90%, phase B, 10%;25.10min: phase A, 100 percent, phase B, 0 percent; 31.00min: phase A, 100 percent, phase B, 0 percent; 31.50min: phase A, 5 percent, phase B, 95 percent; 33.00min: 5 percent of phase A, 95 percent of phase B and 95 percent of phase A.
3.2 ion trap-time-of-flight Mass Spectrometry parameters
An ion source: ESI, positive ion mode, automatic scan acquisition; heating module temperature: 200 ℃; CDL temperature: 200 ℃; flow rate of the atomizer: 1.5L/min; pressure of drying gas: 110kPa; ion source voltage: 4.5kV; detector voltage: 1.7kV; the calibration method comprises the following steps: automatically tuning the optimized voltage; calibrating the mass number by an external standard method; excimer ion (MS) 1 ) The collection range is m/z 100-450, the scanning is repeated for 3 times, and the ion accumulation time is as follows: 40msec; second order fragment ion (MS) 2 ) The collection range is m/z 60-450, repeated scanning is carried out for 3 times, and the ion accumulation time is as follows: 50msec, precursor ion screening range:m/z is 100 to 450; CID (induced collision dissociation) energy: 60 percent; collision gas: 60 percent; acquisition frequency: 67.5kHz.
3.3 building a screening database
Adopting optimized ion trap-time-of-flight mass spectrum parameters, constructing a screening database by using 17 organic antibacterial agent standard substances, and listing MS 1 、MS 2 As shown in table 1 below. Characteristic ion diagram (MS) of 17 organic antibacterial agents 1 、MS 2 Mass spectra) are shown in FIGS. 1-1 to 1-17.
TABLE 1 database for screening 17 organic antibacterial agents
4. Analysis of results
The linear range of the 17 organic antibacterial agents is 50-2000 ng/mL, the correlation coefficients are all larger than 0.99, the standard recovery rate of the household paper products is 70-95%, and the lowest screening detection concentration of the 17 organic antibacterial agents in the sample solution to be detected is 50ng/mL.
The method of the embodiment is adopted to detect 17 organic antibacterial agents in 30 batches of food contact material paper products (20 batches of paper cups and 10 batches of kitchen absorbent paper), and the result shows that no organic antibacterial agent is detected in any sample.
FIG. 2 is an ion chromatogram extracted from a reference of 17 organic antibacterial agents.
Example 2
The paper product wet tissue for human body contact is taken as a detection object, and comprises a product label which indicates the wet tissue containing the organic antibacterial agent and does not contain the organic antibacterial agent.
1. And (4) a pretreatment step. The wet tissue of a household paper product is taken as a detection object, and the sample is cut into pieces with the size of about 5mm multiplied by 5mm and is uniformly mixed. The remaining extraction and purification steps were the same as in example 1.
2. The standard solution was prepared as in example 1.
3. The instrument test conditions were the same as in example 1.
4. And (4) analyzing results:
the results of screening of 10 test samples, including product labeling, with (5) and without (5) organic antimicrobial agents are listed in table 2 below.
5 batches of labels indicate that the wet tissues containing the organic antibacterial agents all detect the corresponding organic antibacterial agents, the content is 246.5-1000 mg/kg, wherein 1 batch of labels indicate that the added benzethonium chloride is benzethonium chloride, but the actually detected wet tissues contain both benzethonium chloride and loracarbenium chloride. The 5-batch label does not indicate the wet tissue containing the organic antibacterial agent, wherein the organic antibacterial agent is not detected in 3 batches; detecting benzalkonium chloride and benzethonium chloride with the content of 200.3mg/kg and 232.4mg/kg respectively for 1 batch; benzalkonium chloride was detected in 1 lot at a concentration of 110.5mg/kg. The ion chromatograms of some positive samples are shown in fig. 3, 4 and 5.
TABLE 2 Wet tissue sample screening results
Example 3
In the present example, a paper diaper (diaper) for human body contact and a sanitary napkin were used as the test objects.
1. And (4) a pretreatment step. Taking a paper diaper (paper diaper) and a sanitary towel as detection objects, cutting the paper diaper (paper diaper) and the sanitary towel from the top surface to 5mm multiplied by 5mm, and mixing uniformly. The remaining extraction and purification steps were the same as in example 1.
2. The standard solution was prepared as in example 1.
3. The instrument test conditions were the same as in example 1.
4. And (4) analyzing results:
when 10 batches of the paper diaper (diaper) and the sanitary towel sample are detected, the organic antibacterial agent is not detected, and the products can be sterilized by other methods.
Fig. 6 is a total ion flow chart (TIC) of a diaper (pant diaper) sample.
The present invention has been described above by referring to a part of specific embodiments, and it should be noted that the above-mentioned specific embodiments are only used for further description of the present invention and do not represent a limitation to the scope of the present invention. Other insubstantial modifications and adaptations of the present invention can be made without departing from the scope of the present invention.
Claims (6)
1. A method for rapidly screening 17 organic antibacterial agents in household paper products is characterized by comprising the following steps:
(1) Extraction: selecting a household paper product, crushing, uniformly mixing, placing the household paper product into a container, adding an extracting solution into the container, shaking uniformly, placing the container into a water bath, performing ultrasonic extraction, taking out the container, cooling to room temperature, performing suction filtration, transferring filtrate into a centrifuge tube a, transferring a residual paper product sample into a centrifuge tube b for later use, adding anhydrous magnesium sulfate and ammonium sulfate into the centrifuge tube a, performing vortex extraction and centrifugation, collecting supernatant into a centrifuge tube c, adding acetonitrile into the centrifuge tube b, performing repeated extraction once, performing water bath ultrasonic extraction and suction filtration, transferring filtrate into the centrifuge tube a, performing vortex extraction and centrifugation, taking the supernatant, and combining the supernatant into the centrifuge tube c to prepare a liquid to be purified;
(2) Purification: adding acetonitrile saturated normal hexane into a centrifugal tube c, performing vortex extraction, removing the upper normal hexane layer, passing all residual liquid through an anhydrous sodium sulfate and graphitized carbon black mixed purification column, collecting effluent liquid, leaching the mixed purification column with acetonitrile, combining the collected effluent liquid, performing rotary evaporation concentration under a water bath condition until the residual is dried, dissolving the residue with a methanol aqueous solution, performing ultrasonic treatment, and filtering with an organic phase filter membrane to prepare a liquid to be detected;
(3) Preparing a standard substance: taking 17 organic antibacterial agent standards, dissolving the standards with acetonitrile to prepare a standard stock solution, and diluting the standard stock solution with a methanol aqueous solution to prepare a mixed standard working solution;
(4) Establishing an ultra-high performance liquid chromatography method;
（5）establishing a screening database: optimizing the parameters of the ion trap-time-of-flight mass spectrometer and combining the method established in the step (4), establishing an ultra-high performance liquid chromatography-ion trap-time-of-flight mass spectrometer detection method, and measuring the standard solution in the step (3) to obtain the excimer ions (MS) of 17 organic antibacterial agents 1 ) And fragment ion (MS) 2 ) The accurate mass number of the antibacterial agent is obtained, and a screening database of 17 organic antibacterial agents is established;
(6) Screening: measuring the liquid to be measured prepared in the step (2) by using an ultra-high performance liquid chromatography-ion trap-time-of-flight mass spectrometer to obtain the MS of the sample 1 And MS 2 Carrying out qualitative confirmation on the data information and the screening database and molecular formula prediction software established in the step (5) to obtain information of the 17 organic antibacterial agents in the household paper products;
the extracting solution in the step (1) is an acetonitrile aqueous solution, the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 1;
the 17 organic antibacterial agent standards in the step (3) are 2-octyl-4-isothiazolin-3-one, 1,2-benzisothiazolin-3-one, 2-methyl-4-isothiazolin-3-one, carbendazim, albendazole-2-aminosulfone, albendazole sulfoxide, albendazole sulfone, thiabendazole, fuberidazole, benzalkonium chloride, cetylpyridinium chloride, loracarbenium chloride, benzethonium chloride, dequalinium chloride, sitalimmonium chloride and triclocarban;
the ultra-high performance liquid chromatography method established in the step (4) adopts the following parameters: c 18 A chromatographic column: waters ACQUITY UPLC BEH C 18 A column with a specification of 100mm × 2.1mm,1.7 μm; flow rate: 0.2mL/min; column temperature: 25-30 ℃; sample introduction amount: 20 mu L of the solution; the mobile phase A is: methanol and acetonitrile in a volume ratio of 1:9; the mobile phase B is as follows: 10mmol/L ammonium acetate solution or 10mmol/L ammonium formate solution; the gradient elution procedure was: 0.00min: phase A, 5%, phase B, 95%;2.00min: phase A, 5%, phase B, 95%;2.10min: 35% of phase A and 65% of phase B; 10.00min: 35% of phase A and 65% of phase B; 10.10min: 50% of phase A and 50% of phase B; 20.00min: 50% of phase A and 50% of phase B; 20.10min: phase A, 90%, phase B, 10%;25.00min: phase A, 90%, phase B, 10%;25.10min: phase A, 100 percent, phase B, 0 percent; 31.00min: phase A, 100 percent, phase B, 0 percent; 31.50min: phase A, 5%, phase B, 95%;33.00min: phase A, 5%, phase B, 95%; are all volume percentage content;
the ion trap-time-of-flight mass spectrometer parameters in step (5) comprise: an ion source: ESI, positive ion mode, automatic scan acquisition; heating module temperature: 150 to 200 ℃; CDL temperature: 150 to 200 ℃; flow rate of the atomizer: 1.3 to 1.5L/min; pressure of drying gas: 95kPa to 110kPa; ion source voltage: 3.5 to 4.5kV; detector voltage: 1.6 to 1.7kV; the calibration method comprises the following steps: automatically tuning the optimized voltage; calibrating the mass number by an external standard method; excimer ion (MS) 1 ) The collection range is m/z 100 to 450, the scanning is repeated for 3 to 5 times, and the ion accumulation time is as follows: 20msec to 40msec; second order fragment ion (MS) 2 ) The collection range is m/z 60 to 450, repeated scanning is carried out for 3 to 5 times, and the ion accumulation time is as follows: 30 msec-50 msec, and the screening range of precursor ions: m/z is 100 to 450; CID energy: 50% -70%; collision gas: 50% -60%; the collection frequency was 45.0 to 67.5kHz.
2. The method for rapidly screening 17 organic antibacterial agents in household paper products according to claim 1, which is characterized in that: the household paper product in the step (1) comprises a paper product for food contact and a paper product for human body contact, wherein the paper product for food contact comprises a paper cup or kitchen absorbent paper; the paper product for human body contact comprises a paper diaper, a wet tissue or a facial tissue.
3. The method for rapidly screening 17 organic antibacterial agents in household paper products according to claim 1, which is characterized in that: the dosage relation among the extracting solution, the anhydrous magnesium sulfate and the ammonium sulfate in the step (1) is 20 to 30mL:4 to 6g:3 to 5g; the rotation speed of a vortex mixer during vortex extraction is 1500-1800 r/min, and the vortex extraction time is 5-10min; the rotating speed of the centrifugal machine is 6500 to 9000r/min during centrifugation, and the centrifugation time is 3 to 5min.
4. The method for rapidly screening 17 organic antibacterial agents in household paper products according to claim 1, which is characterized in that: in the step (2), the volume ratio of the acetonitrile saturated normal hexane to the extracting solution is 1~2:4~6, the rotation speed of a vortex mixer during vortex extraction is 1500-1800 r/min, and the vortex extraction time is 5-10min; the anhydrous sodium sulfate and graphitized carbon black purifying column is prepared by mixing and grinding 8-12g of anhydrous sodium sulfate which is dried at 105 ℃ for 2~3 hours and does not contain crystal water and 100-300mg of graphitized carbon black for 10 minutes, and then filling the mixture into a disposable syringe needle cylinder with the volume of 10mL, wherein the outflow speed is controlled to be 1-2mL/min when the mixture passes through the purifying column, then eluting the purifying column with acetonitrile with the volume of 5-10mL, collecting the effluent and the eluent, performing rotary evaporation and concentration in a water bath at 45-50 ℃ until the mixture is dried, and performing rotary evaporation in a water bath with the volume ratio of methanol to water of 4:6~7:3, dissolving the residue in 1-2mL of methanol aqueous solution, and filtering the solution by using an organic filter membrane with the aperture of 0.22 mu m to prepare the solution to be detected.
5. The method for rapidly screening 17 organic antibacterial agents in household paper products according to claim 1, which is characterized in that: and (3) weighing 17 organic antibacterial agent standard substances respectively, putting the standard substances into different volumetric flasks, adding acetonitrile to dissolve the substances to prepare standard stock solutions, and diluting the standard stock solutions with the methanol water solution obtained in the step (2) to prepare mixed standard working solutions with the concentration of 50-2000 ng/mL.
6. The method for rapidly screening 17 organic antibacterial agents in household paper products according to claim 1, which is characterized in that: the 17 organic antibacterial agent screening database in the step (5) is shown in the following table 1:
TABLE 1 screening database for 17 organic antibacterial agents
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