CN113484436A - Method for simultaneously determining content of five forbidden substances in bacteriostatic paper and application thereof - Google Patents
Method for simultaneously determining content of five forbidden substances in bacteriostatic paper and application thereof Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N30/8624—Detection of slopes or peaks; baseline correction
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Abstract
The invention belongs to the technical field of detection, and particularly relates to a method for simultaneously determining the content of five bacteriostatic forbidden substances in bacteriostatic paper and application thereof. The method comprises the steps of preparation of a series of mixed standard working solutions, sample pretreatment, drawing of a standard working curve, content determination of each target compound in a sample and result calculation. The method is verified to have higher specificity and anti-interference capability, has good linear relation for the determination of each target compound, has the detection limit of 0.01-10.00 ng/mL and the quantitative limit of 0.02-26.00 ng/mL, and is beneficial to the rapid sensitive quantitative detection of five halogenated ultra-trace components in a complex mixed system; in addition, the sample pretreatment is simple, and the recovery rate and the reproducibility are better; compared with a single target compound detection method, the method can improve the daily analysis sample amount by at least 3-4 times, and is suitable for large-batch sample analysis.
Description
Technical Field
The invention belongs to the technical field of detection, and particularly relates to a method for simultaneously determining the content of five bacteriostatic forbidden substances in bacteriostatic paper and application thereof.
Background
Nowadays, the science and technology are developed more and more, and the quality of life is higher and more, many consumers require that paper in different occasions not only is aseptic, but also has the antibacterial effect. These bacteriostatic papers mainly comprise: bacteriostatic napkin paper, oil absorbing paper, bacteriostatic paper extraction, bacteriostatic medical paper and the like.
The bacteriostatic agent is the core of the bacteriostatic material. The bacteriostatic agent mainly comprises the following components in percentage by weight: inorganic bacteriostatic agent, organic bacteriostatic agent, natural bacteriostatic agent. However, some suppliers may misuse cheap bacteriostatic agents whose safety cannot be guaranteed due to economic cost and the like. If the supplier does not really disclose the bacteriostatic components/components used, the related industry also lacks a determination method specific to a plurality of bacteriostatic forbidden substances in bacteriostatic paper, which causes the consumer to be exposed to the safety risk caused by the possible misuse of bacteriostatic agents by the material supplier.
In 2016, 9 months, the Food and Drug Administration (FDA) banned, banned messages of bacteriostatic soap, being overwhelmed, and disconcerting one time disconcerting. The FDA prohibits the use of halocarban, triclocarban, triclosan, hexachlorophene, tribromosalen and other antibacterial compounds in the antibacterial soap.
Halocarban, CAS number: 369-77-7; chinese alias: fluoromethylphenylurea; halocarban; difluoride diphenyloxide urea; english name: haloarban; the chemical structural formula is shown in figure 1, and the molecular formula is C14H9Cl2F3N2O, molecular weight 348.0044. Because halocarban has antifungal, disinfectant and antiseptic effects, it is often used as a bacteriostatic for deodorants, soaps and hand lotions for medical workers. In addition, the cationic surfactant is also applied to laundry detergent. However, due to the reproductive toxicity of halocarbn, the U.S. Food and Drug Administration (FDA) declared a ban on the continued use of halocarbn in antibacterial soaps in 2016. The European Chemicals Agency (ECA) in the list of substances released in 2016 lists halocasan as a suspected carcinogen, suspected of harming aquatic environments, and possibly having persistent and reproductive toxicity in the environment. The argentina-related agency banned the use of halocarb for antimicrobial products (such as personal care products, cosmetics, and/or perfume products) registered with argentina via resolution 13832/2016.
Triclocarban, CAS No.: 101-20-2; chinese alias: trichloro-plaque; kangjiexin; skin care is carried out; trichlorocarba; trichlorophenylurea; 3,4,4' -trichloro-diphenyl, etc.; english name: triclocarban (TCC for short); the chemical structural formula is shown in figure 2, and the molecular formula is C13H9Cl3N2O, molecular weight 313.978046. Triclocarban as an antibacterial agent is widely used in deodorants, hand sanitizers, toothpastes, shampoos, perfumesIn addition, the soap, shaving cream and other personal care products can also be widely applied to medical disinfection instruments and textile antibacterial and mildew-proof. However, the results of recent studies at home and abroad show that triclocarban has high lipophilicity and can be accumulated and accumulated in vivo; is a novel endocrine disruptor, and may cause diseases including cancer, reproductive dysfunction and dysplasia. The technical specification for cosmetic safety (2015 edition) of China stipulates that the maximum allowable concentration of triclocarban as a preservative used in cosmetics is 0.2%. In 2016, 9 months, the U.S. Food and Drug Administration (FDA) announced a ban on triclocarban in antibacterial hand washes/soaps.
Triclosan, CAS No.: 3380-34-5; chinese alias: dichlorophenoxy chlorophenol; triclosan; acrylic acid en; trichlorosand, and the like; english name: triclosan (abbreviated as TCS). The chemical structural formula is shown in figure 3, and the molecular formula is C12H7Cl3O2Molecular weight is 287.951163; is a specific bactericide variety recognized internationally. The high-purity toothpaste is widely applied to daily chemicals such as high-efficiency medical soaps (toilet soaps and sanitary washing liquids), bromhidrosis (dermatophytosis mists), disinfection hand washing liquids, wound disinfection sprays, medical instrument disinfectants, sanitary facial cleansers (creams), air fresheners, refrigerator deodorizers and the like, is also used for finishing sanitary fabrics and performing antiseptic treatment on plastics, and can be used for treating curative effect toothpaste and gargle for treating gingivitis, periodontitis, oral ulcer and the like. At present, studies show that triclosan has potential endocrine disrupting effects on animals and has certain genetic toxicity. The technical Specification for cosmetic safety (2015 edition) in China stipulates: triclosan is used as a preservative in cosmetic products at a maximum allowable concentration of 0.3%. On 22 months 5 2014, minnesota in the united states has led to a ban on the sale of any triclosan-containing cleansing or personal care consumer products. On day 16/6 2014, the european chemical authority has publicly consulted triclosan as a replacement candidate under biocide product regulations. In 2016, 9 months, the U.S. food and drug administration announced that triclosan is prohibited from being used in antimicrobial hand sanitizers/soaps.
Hexachlorophene, CAS number: 70-30-4; chinese alias: hexachlorophene; sterile phenol, and the like;english name: hexachlorophene; the chemical structural formula is shown in figure 4, and the molecular formula is C13H6Cl6O2Molecular weight is 403.8499; it is effective on gram-positive bacteria under alkaline conditions. Since the 50 s of the 20 th century, hexachlorophene has been widely added as a bactericide in commercial products such as soap, shampoo, hair tonic, acne medicine, deodorant, toothpaste, vaginal cleanser, shaving cream, baby powder, etc. in western europe and the united states. Emulsions of 3% hexachlorophene are also commonly used in the united states for surgical hand washing, baby bathing, post-operative wound care, burn care, and the like. However, recent research results at home and abroad show that: the chlorophenols have estrogen effect, mutagenicity, carcinogenicity, and other high toxicity. The excessive intake of hexachlorophene in organisms can inhibit the central system, which causes nervous system disorder, and the serious symptoms are loss of consciousness, respiratory depression and the like. Hexachlorophene is a prohibited component for cosmetics in accordance with technical standards for cosmetic safety (2015 edition) and regulations for cosmetics in European Union in China. In 2016, 9 months, the U.S. Food and Drug Administration (FDA) announced a ban on hexachlorophene in antibacterial hand washes/soaps.
Tribromosalen, CAS number: 87-10-5; chinese alias: tribromolucian; tribromosalicylanilide; 3,4', 5-tribromosalicylanilide; 3,4', 5-tribromosalicylic acid aniline; 3, 5-dibromo-salicyloyl-p-bromoaniline, and the like; english name: tribromsalan; the chemical structural formula is shown in figure 5, and the molecular formula is C13H8Br3NO2Molecular weight is 446.8105; has the characteristics of stable chemical property, broad antibacterial spectrum and high efficiency, and is widely applied to mildew-proof treatment of chemical products and agricultural feeds. However, after the human body intakes the substances, menstrual blood is circularly distributed on the surface of the skin, and light allergic reaction can occur when the human body is exposed to sunlight, so that sunlight urticaria or contact photoallergic dermatitis is caused, and the health of the human body is damaged. Tribromosalen is regarded as a forbidden component of cosmetics in technical specifications for cosmetic safety (2015 edition) and regulations for cosmetics in European Union in China. In 2016, 9 months, the U.S. Food and Drug Administration (FDA) announced a ban on the use of tribromosalen in antimicrobial hand sanitizers/soaps.
At present, a method for simultaneously measuring the contents of various bacteriostatic forbidden substances such as halocarban, triclocarban, triclosan, hexachlorophene, tribromosalen and the like in bacteriostatic paper belongs to the technical blank.
Arno h.a. heyn a et al established a method for measuring halocarb in antibacterial soap by gas-liquid chromatography (GLC) in 1982 (International Journal of environmental Analytical chemistry, 1982,11(2): 131-.
The existing method for respectively measuring hexachlorophene and tribromosalen is mainly suitable for detecting related substances in cosmetics and foods and is not suitable for detecting related substances in paper substrates.
The method for measuring triclosan and triclocarban in food packaging paper by using high performance liquid chromatography (a food safety guide & periodicals 2020,12,139- & lt140- & gt) established by Xutengyang team adopts a diode array detector, the linear ranges of the detector are respectively 0.2-100 mg/L and 0.05-100 mg/L, the quantitative limits are respectively 0.04mg/kg and 0.01mg/kg, and the sensitivity of the method is not high; and the pretreatment needs rotary evaporation, volume fixing and the like, the procedure is complicated, and the stability of the compound can be influenced, so that the accuracy of the content measurement value of the target compound is influenced.
Therefore, no method for determining halocarban by using ultra-performance liquid chromatography-mass spectrometry (UPLC-ESI-MS/MS) is reported at home and abroad at present; there are also no reported methods for determining halocarban, hexachlorophene, tribromosalen in paper substrates; and no complete and quick method for simultaneously measuring the contents of five halogenated bacteriostasis forbidden substances, namely halocarban, triclocarban, triclosan, hexachlorophene and tribromosalen in the bacteriostasis paper is reported.
Therefore, in the present stage, it is necessary to establish a simple, fast, efficient, sensitive and wide-application method capable of simultaneously determining the content of five halogenated bacteriostatic forbidden substances, namely halocarban, triclocarban, triclosan, hexachlorophene and tribromosalen, in the bacteriostatic paper, so as to provide technical support for the safety risk assessment of the bacteriostatic paper and provide technical support for the bacteriostatic paper consumers to enjoy the safety right.
The present invention has been made to solve the above problems.
Disclosure of Invention
The invention aims to provide a method for simultaneously measuring the content of five halogenated bacteriostasis forbidden substances, namely halocarban, triclocarban, triclosan, hexachlorophene and tribromosalen in bacteriostatic paper, so as to overcome the defects of the prior art, provide technical support for safety risk evaluation of bacteriostatic paper and provide technical support for consumers to enjoy safety rights.
The invention provides a method for simultaneously measuring the content of five bacteriostatic forbidden substances in bacteriostatic paper, wherein the five bacteriostatic forbidden substances are five target compounds of halocarban, trichlorocarban, triclosan, hexachlorophene and tribromosaran, and the method comprises the following steps:
step S1, preparing a series of mixed standard working solutions containing five bacteriostatic forbidden substances: preparing a series of mixed standard working solutions simultaneously containing halocarban, triclocarban, triclosan, hexachlorophene or tribromosalen by using 80% vol methanol aqueous solution;
for the same mixed standard working solution, the concentrations of halocarban, trichlorocarban, hexachlorophene and tribromosalen are the same, and the concentration of triclosan is 8-12 times of the corresponding concentrations of halocarban, trichlorocarban, hexachlorophene and tribromosalen;
step S2, sample pretreatment: cutting the bacteriostatic paper sample to obtain a sample, wherein the sample comprises a single side; cutting the cut sample into pieces of 0.5cm multiplied by 0.5cm, mixing uniformly, and placing in a clean sealed bag for later use; weighing a sample to be detected, placing the sample in a triangular flask with a plug, adding V mL of chromatographic pure methanol, and performing ultrasonic extraction for 20-40 min; standing for 3-7 min, transferring 2-3 mL of extract liquor into a centrifuge tube, and centrifuging for 3-7 min; transferring 1mL of supernatant, filtering the supernatant through a 0.22-micrometer organic phase filter membrane to obtain a sample filtrate to be detected, and detecting the ultra-high performance liquid chromatography-electrospray ionization source quadrupole mass spectrometry (UPLC-ESI-MS/MS) by ultra-high performance liquid chromatography-electrospray ionization source quadrupole mass spectrometry;
step S3, blank experiment: under the condition that a paper sample to be detected is not added, executing the step S2, obtaining blank sample filtrate to be detected, and waiting for ultra performance liquid chromatography-electrospray ionization source quadrupole mass spectrometry (UPLC-ESI-MS/MS) detection;
step S4, drawing a standard working curve: measuring the peak area of each target compound in the series of mixed standard working solutions in the step S1 by adopting ultra performance liquid chromatography-electrospray ionization source quadrupole mass spectrometry (UPLC-ESI-MS/MS), and performing linear regression to draw a standard working curve by taking the concentration of each target compound as a horizontal coordinate and the peak area of the target compound as a vertical coordinate;
step S5, determining the content of each target compound in the sample: determining S2 peak areas of the target compounds in the filtrate of the sample to be detected by adopting ultra performance liquid chromatography-electrospray ionization source quadrupole rod mass spectrometry (UPLC-ESI-MS/MS), and calculating the concentration C of the target compounds in the filtrate of the sample to be detected according to the standard working curve in the step S4;
step S6, measuring the content of each target compound in the blank sample: measuring S3 peak area of each target compound in the blank sample filtrate to be measured by adopting ultra performance liquid chromatography-electrospray ionization source quadrupole mass spectrometry (UPLC-ESI-MS/MS), and calculating the concentration C of each target compound in the blank sample filtrate to be measured according to the standard working curve in the step S40；
Step S7, calculating the content result of each target compound in the bacteriostatic paper sample: calculating the content of each target compound in the bacteriostatic paper sample to be detected according to the following formula;
in the formula (I), the compound is shown in the specification,
x represents the content of each target compound in the bacteriostatic paper sample to be detected, and the unit is mu g/kg;
c represents the concentration of the corresponding target compound in the filtrate of the sample to be detected obtained by the standard working curve, and the unit is ng/mL;
C0the concentration of the corresponding target compound in the blank sample filtrate to be detected, which is obtained from the standard working curve, is expressed in ng/mL;
v represents the volume of the extract in mL;
m represents the mass of the bacteriostatic paper sample to be detected, and the unit is g;
:; if the concentration of the target compound in the sample solution to be detected does not exceed the maximum concentration of the mixed standard working solution, taking 1 as the dilution factor; if the concentration of the target compound in the sample solution to be tested exceeds the maximum concentration of the mixed standard working solution, adjusting and diluting the solution to be tested by using methanol, and then measuring, if the solution is diluted to 2 times, taking a dilution factor of 2, and so on;
taking the arithmetic mean value of the multiple parallel measurement results as a final measurement result, and accurately measuring the result to 0.01 mu g/kg;
the relative mean deviation of multiple parallel measurements should be less than 10%.
Preferably, in the step S4, the step S5, and the step S6, the liquid chromatography conditions of the hplc-electrospray ionization source quadrupole mass spectrometry method include: the chromatographic column is 100mm × 2.1mm, 1.8 μm ACQUITY UPLC HSS T3 chromatographic column; the sample injection amount is 5 mu L; the flow rate is 0.3 mL/min; the column temperature was 40 ℃; the injector temperature was 20 ℃; mobile phase a was methanol and mobile phase B was 0.1% vol formic acid in water; the gradient elution conditions included: 0-1.0 min, the proportion of the mobile phase A is 80-85 vol%; 1.0-2.0 min, wherein the proportion of the mobile phase A is 85% vol; 2.0-4.0 min, wherein the proportion of the mobile phase A is 85-90% vol; 4.0-6.0 min, wherein the proportion of the mobile phase A is 90% vol; 6.0-6.5 min, the proportion of the mobile phase A is 90-100% vol; 6.5min to 7.0min, the proportion of the mobile phase A is 100 percent vol to 80 percent vol; 7.0 min-8.0 min, and the proportion of the mobile phase A is 80% vol.
In the step S4, the step S5, and the step S6, the mass spectrometry conditions of the hplc-electrospray ionization source quadrupole mass spectrometry method include: the ion source is an electrospray ionization source; the scanning mode is negative ion scanning; the capillary voltage is 3 kV; the extraction taper hole voltage is 5V; the RF mass spectrum voltage is 0.5V; the ion source temperature is 120 ℃; the desolventizing gas is nitrogen, and the purity is not less than 99.99 percent; the temperature of the desolventizing gas is 500 ℃; the flow rate of the desolventizing agent is 600L/hr; the collision gas is argon; collision chamberPressure 3.80e-3mbar; the voltage of the photomultiplier is 650V; the detection mode is multi-reaction monitoring; the residence monitoring time of the ion pair was 50 ms.
Preferably, in step S1, in the series of mixed standard working solutions containing halocarb, triclocarban, triclosan, hexachlorophene or tribromosalen, the concentrations of halocarb, triclocarban, hexachlorophene and tribromosalen are respectively: 2ng/mL, 5ng/mL, 10ng/mL, 30ng/mL, 50ng/mL, 70ng/mL, 100ng/mL, 150ng/mL, 200 ng/mL; the concentration of triclosan is 8-12 times of the corresponding concentration of halocasan, triclocarban, hexachlorophene and tribromosalen; that is, the concentration of triclosan is 16-24 ng/mL, 40-60 ng/mL, 80-120 ng/mL, 240-360 ng/mL, 400-600 ng/mL, 560-840 ng/mL, 800-1200 ng/mL, 1200-1800 ng/mL, 1600-2400 ng/mL, respectively.
Preferably, in the step S4, the step S5 and the step S6, the retention time and the key parameters of multiple reaction monitoring of the hplc-electrospray ionization source quadrupole mass spectrometry method corresponding to each target compound are detailed in the following table:
note: are quantitative ions.
The second aspect of the invention provides application of the method for determining the content of five substances for inhibiting bacteria in the antibacterial paper, which is used for simultaneously and rapidly determining halocasan, triclocarban, triclosan, hexachlorophene and tribromosalen in a complex mixed system, wherein the detection limit is 0.01-10.00 ng/mL, and the quantification limit is 0.02-26.00 ng/mL.
Compared with the prior art, the invention has the following beneficial effects:
1. the determination method provided by the invention is suitable for simultaneous determination of the content of five halogenated bacteriostasis forbidden substances in the paper substrate. During specific operation, the related conditions and parameters of the ultra-high performance liquid chromatography-electrospray ionization source quadrupole mass spectrometry are configured according to the optimal liquid chromatography condition, the optimal mass spectrometry condition, and especially the optimal multi-reaction monitoring key parameter, and the content of the five halogenated bacteriostasis forbidden substances can be measured by one sample injection;
2. compared with the existing method for respectively measuring single target substances such as hexachlorophene, tribromosalen and the like and the existing method for simultaneously measuring triclosan and triclocarban, the method disclosed by the invention can be used for simultaneously measuring the content of five halogenated bacteriostatic forbidden substances by only one-time pretreatment and one-time sample introduction aiming at the same sample, so that the daily analysis sample amount can be at least improved by 3-4 times, and therefore, the method disclosed by the invention is suitable for analyzing large-batch samples;
3. the determination method provided by the invention has higher specificity and anti-interference capability, and can accurately determine the target compound in a complex mixed system even if the chromatographic peaks of the target compound cannot be completely and thoroughly separated;
4. the determination method provided by the invention has good linear relation for determining five halogenated bacteriostatic forbidden substances, the detection limit is 0.01-10.00 ng/mL, and the quantitative limit is 0.02-26.00 ng/mL, so that the rapid sensitive quantitative determination of five halogenated ultra-trace components (the ultra-trace refers to the content of the substance to be determined being less than 1 mu g/mL) in a complex mixed system is facilitated;
5. the method provided by the invention has simple sample pretreatment, the average standard recovery rate of the method is 83.35-96.69%, the relative standard deviation of the results of 6 parallel measurements is less than 10%, and the reliability of the measurement results is ensured by better recovery rate and reproducibility;
6. the determination method provided by the invention does not need to carry out chemical derivatization on the target analyte, does not need rotary evaporation, and does not influence the stability of the target compound. Therefore, the determination method provided by the invention has the characteristics of simplicity, rapidness, high efficiency, sensitivity and wide application range, can provide technical support for safety risk assessment of the bacteriostatic paper, and provides technical support for consumers of the bacteriostatic paper to enjoy safety rights.
Drawings
FIG. 1 is a structural formula of halocarban according to the present invention;
FIG. 2 is a structural formula of triclocarban in accordance with the present invention;
FIG. 3 is a structural formula of triclosan according to the present invention;
FIG. 4 is a structural formula of hexachlorophene in accordance with the present invention;
figure 5 is a structural formula of tribromosalen according to the present invention;
FIG. 6 is a flowchart of an embodiment of a method for determining the contents of five halogenated bacteriostatic forbidden substances in bacteriostatic paper according to the present invention;
FIG. 7 is a halogen Carban UPLC-ESI-MS/MS total ion flow chromatogram (TIC) of a mixed standard working solution of concentration 100ng/mL according to an embodiment of the present invention;
FIG. 8 is a halogen Carban UPLC-ESI-MS/MS quantitative ion chromatogram (MRM) of a mixed standard working solution of concentration 100ng/mL according to an embodiment of the present invention;
FIG. 9 is a halogen Carban UPLC-ESI-MS/MS qualitative ion chromatogram (MRM) of a mixed standard working solution of concentration 100ng/mL according to an embodiment of the present invention;
FIG. 10 is a trichlorocarba UPLC-ESI-MS/MS total ion flux chromatogram (TIC) of a mixed standard working solution of concentration 100ng/mL according to an embodiment of the present invention;
FIG. 11 is a UPLC-ESI-MS/MS quantitative ion chromatogram (MRM) of triclocarban in a mixed standard working solution at a concentration of 100ng/mL according to an embodiment of the present invention;
FIG. 12 is a trichlorocarba UPLC-ESI-MS/MS qualitative ion chromatogram (MRM) of a 100ng/mL mixed standard working solution according to an embodiment of the present invention;
FIG. 13 is a UPLC-ESI-MS/MS total ion flow chromatogram (TIC) of triclosan in a mixed standard working solution at a concentration of 1000ng/mL according to an embodiment of the present invention;
FIG. 14 is a UPLC-ESI-MS/MS quantitative ion chromatogram (MRM) of triclosan in a mixed standard working solution at a concentration of 1000ng/mL according to an embodiment of the present invention;
FIG. 15 is a UPLC-ESI-MS/MS qualitative ion chromatogram (MRM) of triclosan in a mixed standard working solution at a concentration of 1000ng/mL according to an embodiment of the present invention;
FIG. 16 is a UPLC-ESI-MS/MS total ion flow chromatogram (TIC) of hexachlorophene in a mixed standard working solution of 100ng/mL according to an embodiment of the present invention;
FIG. 17 is a UPLC-ESI-MS/MS quantitative ion chromatogram (MRM) of hexachlorophene in a mixed standard working solution of 100ng/mL according to an embodiment of the present invention;
FIG. 18 is a UPLC-ESI-MS/MS qualitative ion chromatogram (MRM) of hexachlorophene in a mixed standard working solution of 100ng/mL according to an embodiment of the present invention;
FIG. 19 is a total ion flow chromatogram (TIC) of tribromosalen UPLC-ESI-MS/MS of a mixed standard working solution with a concentration of 100ng/mL according to an embodiment of the present invention;
FIG. 20 is a tribromosalen UPLC-ESI-MS/MS quantitative ion chromatogram (MRM) of a mixed standard working solution with a concentration of 100ng/mL according to an embodiment of the present invention;
FIG. 21 is a qualitative ion chromatogram (MRM) of tribromide-salam UPLC-ESI-MS/MS of a mixed standard working solution at a concentration of 100ng/mL, in accordance with an embodiment of the present invention.
Detailed Description
The present invention is further illustrated by the following examples, but is not limited to these examples. The experimental methods not specified in the examples are commercially available, generally, according to the conventional conditions and the conditions described in the manual, or according to the conditions recommended by the manufacturer using general-purpose equipment, materials, reagents, etc., unless otherwise specified. The starting materials required in the following examples and comparative examples are all commercially available.
Example 1
A method for simultaneously determining the content of five halogenated bacteriostasis forbidden substances in bacteriostatic paper comprises the following steps:
1 instruments, reagents and materials
The instrument comprises the following steps: waters ACQUITYTM UPLC ultra high performance liquid chromatograph in series with UPLC QUATTRO PREMI XE mass spectrometer (Waters Corp.); an ACQUITY UPLC HSS T3 column (2.1 mm. times.100 mm, 1.8 μm, Waters Corp.); P300H ultrasonic cleaner (Elma, switzerland); HYQ-2110 vortex mixer (Crystal Co., USA); METTLER TOLODO XP 504 electronic analytical balance (METTLER TOLODO, Switzerland, sensory was 0.1 mg); BCD-620WDBF refrigerator (Qingdao Haier Co., Ltd.); DIRET UV3 water purifier (Millipore, USA); a vortex oscillator with the use rotating speed of 500 r/min; a centrifuge with the use speed of 5000 r/min; pipettes of various specifications, pipette guns, volumetric flasks, triangular flasks with stoppers and centrifuge tubes.
Reagent: the reagents used in the present invention should be analytical grade or more, except for special requirements. Halocarban (Halocarban, CAS number 369-77-7; British Alfa Aesar standard, 99.7% purity); triclocarban (Triclocarban, CAS number: 101-20-2; germany dr. ehrenstorfer standard, purity 99.08%); triclosan (Triclosan, CAS number 3380-34-5; german dr. ehrenstorfer standard, purity 99.5%); hexachlorophene (Hexachlorophene, CAS number: 70-30-4; Germany Dr. Ehrenstontorfer Standard, purity 99.0%); tribromosalen (Tribrolsalan, CAS number: 87-10-5; Germany Dr. Ehrenstorfer standard, purity 97.8%); methanol (HPLC grade, Merck, germany); formic acid (LC-MS grade, welength technologies ltd, china); water, GB/T6682, first order.
Materials: bacteriostatic paper samples (comprising bacteriostatic napkins, bacteriostatic oil-absorbing papers, bacteriostatic paper extracts and the like) detected by the embodiment of the invention are purchased from the market.
2 method of experiment
As shown in fig. 6, in an actual implementation process of the method for simultaneously determining the content of five halogenated bacteriostatic forbidden substances in bacteriostatic paper provided in this embodiment, the method specifically includes the following steps:
step S1, preparing a series of mixed standard working solutions containing five halogenated bacteriostatic forbidden substances, wherein the five halogenated bacteriostatic forbidden substances are five target compounds of halocarban, triclocarban, triclosan, hexachlorophene and tribromosalen;
step S1-1, preparing a single standard substance stock solution: respectively and accurately weighing 10mg (accurate to 0.1mg) of halocarb, triclocarban, triclosan, hexachlorophene and tribromosalen in a 10mL brown volumetric flask, diluting to a constant volume with 80% vol methanol water solution, and preparing a 1mg/mL stock solution of a single standard substance of halocarb, triclocarban, triclosan, hexachlorophene and tribromosalen. Storing in sealed and dark place at 0-4 deg.c for 6 months;
step S1-2, preparation of mixed standard stock solution: and (2) accurately transferring 200 mu L of halogen carban single standard substance stock solution, 200 mu L of trichlorocarban single standard substance stock solution, 200 mu L of hexachlorophene single standard substance stock solution, 200 mu L of tribromosalen single standard substance stock solution and 2000 mu L of triclosan single standard substance stock solution obtained in the step (S1-1) into the same 100mL brown volumetric flask, diluting the volume with 80% vol methanol water solution to a constant volume, and preparing a mixed standard stock solution, wherein the mass concentrations of halogen carban, trichlorocarban, hexachlorophenol and tribromosalen are the same, and are 2000ng/mL and the concentration of triclosan is 20000 ng/mL. Storing in sealed and light-proof condition at 0-4 deg.c for 3 months;
step S1-3, preparation of a series of mixed standard working solutions: respectively transferring the mixed standard stock solutions with different volumes obtained in the step S1-2 according to the actual content of the sample, performing constant volume by using 80% vol methanol water solution, and gradually diluting the mixed standard stock solutions into series of mixed standard working solutions with different concentration gradients;
illustratively, 10 μ L, 25 μ L, 50 μ L, 150 μ L, 250 μ L, 350 μ L, 500 μ L, 750 μ L and 1000 μ L of the mixed standard working solution obtained in the step S1-2 are respectively and accurately transferred into different 10mL volumetric flasks, and the volume is determined by using 80% vol methanol water solution, so that a series of mixed standard working solutions are obtained and are ready to use. The concentrations of halocasan, hexachlorophene, tribromosalen and trichlorocarban in the prepared series of mixed standard working solutions are respectively 2ng/mL, 5ng/mL, 10ng/mL, 30ng/mL, 50ng/mL, 70ng/mL, 100ng/mL, 150ng/mL and 200 ng/mL; the concentration gradient of the triclosan is 20ng/mL, 50ng/mL, 100ng/mL, 300ng/mL, 500ng/mL, 700ng/mL, 1000ng/mL, 1500ng/mL and 2000ng/mL respectively (namely, in each mixed standard working solution, the concentration of the triclosan is 10 times of the corresponding concentration of other components);
the series of mixed standard working solutions obtained in the step S1-3 include a first mixed standard working solution, a second mixed standard working solution, a third mixed standard working solution, a fourth mixed standard working solution, a fifth mixed standard working solution, a sixth mixed standard working solution, a seventh mixed standard working solution, an eighth mixed standard working solution, and a ninth mixed standard working solution; wherein, in the first mixed standard working solution, the concentrations of halocarban, hexachlorophene, tribromosalen and triclocarban are all 2ng/mL, and the concentration of triclosan is 20 ng/mL; in the second mixed standard working solution, the concentrations of halocarban, hexachlorophene, tribromosalen and trichlorocarban are all 5ng/mL, and the concentration of triclosan is 50 ng/mL; in the third mixed standard working solution, the concentrations of halocarban, hexachlorophene, tribromosalen and trichlorocarban are all 10ng/mL, and the concentration of triclosan is 100 ng/mL; in the fourth mixed standard working solution, the concentrations of halocarban, hexachlorophene, tribromosalen and triclocarban are all 30ng/mL, and the concentration of triclosan is 300 ng/mL; in the fifth mixed standard working solution, the concentrations of halocarban, hexachlorophene, tribromosalen and triclocarban are all 50ng/mL, and the concentration of triclosan is 500 ng/mL; in the sixth mixed standard working solution, the concentrations of halocarban, hexachlorophene, tribromosalen and triclocarban are all 70ng/mL, and the concentration of triclosan is 700 ng/mL; in the seventh mixed standard working solution, the concentrations of halocarban, hexachlorophene, tribromosalen and triclocarban are all 100ng/mL, and the concentration of triclosan is 1000 ng/mL; in the eighth mixed standard working solution, the concentrations of halocarban, hexachlorophene, tribromosalen and triclosan are 150ng/mL, and the concentration of triclosan is 1500 ng/mL; in the ninth mixed standard working solution, the concentrations of halocarban, hexachlorophene, tribromosalen and triclocarban are all 200ng/mL, and the concentration of triclosan is 2000 ng/mL;
step S2, sample pretreatment: cutting bacteriostatic paper samples (which should comprise a single side) with the length of 200mm and the width of 40 mm; cutting the cut samples into pieces of about 0.5cm multiplied by 0.5cm, mixing uniformly, and placing in a clean sealed bag for later use, wherein each sample is not less than 5 g; accurately weighing 1.0g of sample to be detected, accurately measuring the sample to be detected to 0.1mg, placing the sample in a 100mL triangular flask with a plug, adding 40mL of chromatographic pure methanol, and ultrasonically extracting for 30 min; standing for 5min, transferring 2-3 mL of extract liquid into a centrifuge tube, and centrifuging for 5min at the speed of 5000 r/min; transferring 1mL of supernatant, filtering by using a 0.22-micron organic phase filter membrane to obtain a sample filtrate to be detected, and detecting by using ultra-high performance liquid chromatography-electrospray ionization source quadrupole rod mass spectrometry;
step S3, blank experiment: under the condition that a paper sample to be detected is not added, executing the step S2 to obtain blank sample filtrate to be detected, and waiting for ultra performance liquid chromatography-electrospray ionization source quadrupole rod mass spectrometry detection;
step S4, drawing a standard working curve: determining the peak area of each target compound in the series of mixed standard working solutions in the step S1 by adopting ultra performance liquid chromatography-electrospray ionization source quadrupole mass spectrometry, and performing linear regression by taking the concentration of each target compound as a horizontal coordinate and the peak area of the target compound as a vertical coordinate to draw a standard working curve;
step S5, determining the content of each target compound in the sample: measuring the peak area of each target compound in the filtrate of the sample to be measured by adopting ultra performance liquid chromatography-electrospray ionization source quadrupole mass spectrometry (S2), and calculating the concentration C of each target compound in the filtrate of the sample to be measured according to the standard working curve in the step S4 (external standard method);
step S6, measuring the content of each target compound in the blank sample: measuring the peak area of each target compound in the blank sample filtrate to be measured by adopting ultra performance liquid chromatography-electrospray ionization source quadrupole mass spectrometry (S3), and calculating the concentration C of each target compound in the blank sample filtrate to be measured according to the standard working curve in the step S40(external standard method).
Each sample was run in parallel twice per step S5 and a set of blanks per batch per step S3.
In the step S4, the step S5, and the step S6, the liquid chromatography conditions of the hplc-electrospray ionization source quadrupole mass spectrometry method include: the chromatographic column is 100mm × 2.1mm, 1.8 μm ACQUITY UPLC HSS T3 chromatographic column; the sample injection amount is 5 mu L; the flow rate is 0.3 mL/min; the column temperature was 40 ℃; the injector temperature was 20 ℃; mobile phase a was methanol and mobile phase B was 0.1% vol formic acid in water; the gradient elution conditions included: 0-1.0 min, the proportion of the mobile phase A is 80-85 vol%; 1.0-2.0 min, wherein the proportion of the mobile phase A is 85% vol; 2.0-4.0 min, wherein the proportion of the mobile phase A is 85-90% vol; 4.0-6.0 min, wherein the proportion of the mobile phase A is 90% vol; 6.0-6.5 min, the proportion of the mobile phase A is 90-100% vol; 6.5min to 7.0min, the proportion of the mobile phase A is 100 percent vol to 80 percent vol; 7.0 min-8.0 min, and the proportion of the mobile phase A is 80% vol (see table 1).
TABLE 1 gradient elution conditions
In the step S4, the step S5, and the step S6, the mass spectrometry conditions of the hplc-electrospray ionization source quadrupole mass spectrometry include: the ion source is an electrospray ionization source (ESI); the scanning mode is negative ion scanning (ESI)-) (ii) a The capillary voltage is 3 kV; the extraction taper hole voltage is 5V; the RF mass spectrum voltage is 0.5V; the ion source temperature is 120 ℃; the desolventizing gas is nitrogen, and the purity is not less than 99.99 percent; the temperature of the desolventizing gas is 500 ℃; the flow rate of the desolventizing agent is 600L/hr; the collision gas is argon; the pressure in the collision chamber was 3.80e-3mbar; the voltage of the photomultiplier is 650V; the detection mode is multi-reaction monitoring (MRM); the residence monitoring time of the ion pair was 50 ms.
In the Multiple Reaction Monitoring (MRM), since there is no mass spectrometry method reported for halocarbn at present, mass spectrometry conditions thereof need to be grope optimized on the basis of analysis of the structure. Considering that two C-N bonds on carbamido in halocarbn molecules are active, after the halocarbn and Ar2 generate ion-molecule collision reaction in a mass spectrometer collision pool, the two C-N bonds are easy to break, and in different fragments containing benzene rings, lone-pair electrons on N atoms connected with the benzene rings are easy to form conjugated structures with the benzene rings, and the different formed ion fragments are presumed to be easy to carry negative charges. From this, the negative ion mode (ESI) was calculated-) The parent ion and the daughter ion which may appearRelative accurate mass of the particles to predict anion scan pattern (ESI)-) The lower halocarban should present m/z 347/194 and m/z 347/126 ion pairs. The results show that in negative ion scanning mode (ESI)-) The parent ion scan was performed on halocarban and indeed the m/z 346.75 ion peak (as shown in figure 7) appeared; successively in negative ion mode (ESI)-) Next, a daughter ion scan was performed, and indeed two daughter ion peaks, m/z 193.94 and m/z 126.03, appeared, and the ion peaks were stable (as shown in FIGS. 8 and 9). Positive ion mode (ESI)+) Scanning, no expected ion pairs are present, while other fragment ions present are unstable. Therefore, mass spectrometry of halocarban can be performed in electrospray negative ion mode (ESI)-) Multiple Reaction Monitoring (MRM) was performed.
In addition, the mass spectrometric conditions of Multiple Reaction Monitoring (MRM) of other target compounds also need to be optimized according to the structure of each corresponding compound and the actual condition of the instrument, and the retention time and the key parameters of Multiple Reaction Monitoring (MRM) corresponding to each target compound after optimization are detailed in table 2.
TABLE 2 UPLC-ESI-MS/MS Retention time and Multiple Reaction Monitoring (MRM) parameters for each target compound
Note: are quantitative ions.
FIGS. 7 to 9 are UPLC-ESI-MS/MS total ion flow chromatogram (TIC), quantitative ion chromatogram (MRM) and qualitative ion chromatogram (MRM) of halocasan, respectively; FIGS. 10 to 12 are respectively a UPLC-ESI-MS/MS total ion flow chromatogram (TIC), a quantitative ion chromatogram (MRM) and a qualitative ion chromatogram (MRM) of triclocarban; FIGS. 13-15 are UPLC-ESI-MS/MS total ion flow chromatogram (TIC), quantitative ion chromatogram (MRM), and qualitative ion chromatogram (MRM), respectively, of triclosan; FIGS. 16 to 18 are UPLC-ESI-MS/MS total ion flow chromatogram (TIC), quantitative ion chromatogram (MRM) and qualitative ion chromatogram (MRM) of hexachlorophene, respectively; FIGS. 19-21 are UPLC-ESI-MS/MS total ion flow chromatogram (TIC), quantitative ion chromatogram (MRM), and qualitative ion chromatogram (MRM), respectively, of tribromosalen.
In an embodiment of the method for determining the content of five halogenated bacteriostatic forbidden substances in the bacteriostatic paper, the step S4 specifically includes:
step S4-1, adopting ultra performance liquid chromatography-electrospray ionization source quadrupole mass spectrometry to analyze and measure the series of mixed standard working solutions in the step S1-3 to obtain the peak area of each target compound, taking the concentration of each target compound as the abscissa and the peak area of the target compound as the ordinate, and performing linear regression to draw a standard working curve, wherein the square R of the correlation coefficient of the standard working curve is2≥0.95；
Step S4-2, determining a detection Limit (LOD) of the method from the concentration of each target compound corresponding to a signal-to-noise ratio of 3(S/N — 3);
in step S4-3, the limit of quantitation (LOQ) of the method is determined based on the concentration of each target compound corresponding to a signal-to-noise ratio of 10 (S/N: 10).
Linear regression equation, linear range, square of correlation coefficient (R) for each target compound2) The detection limits and the quantification limits are shown in table 3.
TABLE 3 regression equation, Linear Range, Square of correlation coefficient (R) for the target Compound2) Detection limit and quantification limit
The result shows that the method has good linearity (R is good) within the concentration range of 2.00-200.00 ng/mL for the measurement of halocarban, trichlorocarban and hexachlorophene2Not less than 0.99), the measurement of tribromosalen is good in linearity within the concentration range of 10.00-200.00 ng/mL (R2Not less than 0.95), the determination of triclosan is 30.00EThe linearity in the concentration range of 2000.00ng/mL is good (R2Not less than 0.95). The method has lower detection limit and quantification limit on five halogenated bacteriostatic forbidden substances, namely halocarban, trichlorocarban, triclosan, hexachlorophene and tribromosalen, wherein the detection limit of the halocarban is 0.01ng/mL, and the quantification limit is 0.02 ng/mL; the detection limit of the triclocarban is 0.60ng/mL, and the quantification limit is 2.00 ng/mL; the detection limit of the triclosan is 10.00ng/mL, and the quantification limit is 26.00 ng/mL; the detection limit of hexachlorophene is 0.60ng/mL, and the quantification limit is 2.00 ng/mL; the detection limit of tribromosalen is 3.00ng/mL, and the quantification limit is 10.00 ng/mL. Therefore, the detection limit of the measurement method of the present invention is 0.01 to 10.00ng/mL, and the quantification limit is 0.02 to 26.00 ng/mL.
Further, in the specific implementation, a mixed standard working solution with a medium concentration is added after each sample injection for 20 times, and if the relative deviation of the measured value and the original value exceeds 10%, the standard working curve needs to be drawn again.
When the content of the corresponding target compound in the bacteriostatic paper sample to be detected is calculated according to the quantitative ion peak area of the target compound in the filtrate of the sample to be detected, 1 parent ion, 2 or more than 2 ionic ions are selected for each component to be detected, and under the same experimental conditions, the deviation between the relative retention time of the target compound in the filtrate of the sample to be detected and the retention time of the corresponding target compound in the mixed standard working solution is within +/-2.5%; and comparing the relative abundance of the characteristic ions of each component in the spectrogram corresponding to the filtrate of the sample to be detected with the relative abundance of the corresponding characteristic ions in the mixed standard working solution with the adjacent concentration, wherein when the relative abundance is greater than 50%, the allowable deviation is +/-20%, when the relative abundance is between 20% and 50%, the allowable deviation is +/-25%, when the relative abundance is between 10% and 20%, the allowable deviation is +/-30%, and when the relative abundance is less than 10%, the allowable deviation is +/-50%.
And if the concentration of the filtrate of the sample to be measured exceeds the maximum concentration of the mixed standard working solution, adjusting the dilution multiple of the filtrate of the sample to be measured by using methanol, and then measuring.
Step S7, calculating the content result of each target compound in the bacteriostatic paper sample: calculating the content of each target compound in the bacteriostatic paper sample to be detected by adopting the following formula,
in the formula (I), the compound is shown in the specification,
x represents the content of each target compound in the bacteriostatic paper sample to be detected, and the unit is mu g/kg;
c represents the concentration of the corresponding target compound in the filtrate of the sample to be detected obtained by the standard working curve, and the unit is ng/mL;
C0the concentration of the corresponding target compound in the blank filtrate obtained from the standard working curve is expressed in ng/mL;
v represents the volume of the extract in mL; in the embodiment, 40 is taken;
m represents the mass of the bacteriostatic paper sample to be detected, and the unit is gram;
:; if the concentration of the sample solution to be measured does not exceed the maximum concentration of the mixed standard working solution, taking 1 as the dilution factor; if the concentration of the sample solution to be measured exceeds the maximum concentration of the mixed standard working solution, adjusting and diluting the solution to be measured by using methanol, and then measuring, if the solution is diluted to 2 times, taking a dilution factor of 2, and so on; this example was taken as 1 without dilution.
The arithmetic mean of the two replicates was used as the final assay to the nearest 0.01. mu.g/kg. The relative mean deviation of the two parallel measurements should be less than 10%.
Example 2 Bidding recovery experiment-method accuracy and precision verification
Furthermore, in order to verify the accuracy and precision of the determination method of the invention, a standard addition recovery test is also carried out.
Specifically, 250mL portions of mixed standard working solutions (containing triclosan at a concentration 10 times the concentration of the other components) were prepared at concentrations of 3ng/mL (low concentration), 100ng/mL (medium concentration), and 200ng/mL (high concentration), respectively. Each concentration solution was transferred in 6 parts by 40mL of each transfer and placed in 100mL triangular flasks with stoppers containing 1.0g (to the nearest 0.1mg) of pieces (about 0.5 cm. times.0.5 cm) of base paper (the same type of paper without the target compound) for 30min by ultrasonic extraction. Standing for 5min, transferring 2-3 mL of extract into a centrifuge tube, and centrifuging at the speed of 5000r/min for 5 min. Transferring 1mL of supernatant, filtering by a 0.22 mu m organic phase filter membrane, and collecting filtrate; namely, the standard adding recovery test is divided into three groups of low concentration, medium concentration and high concentration according to the concentration of the added standard working solution, each group is provided with 6 parallel tests, and the scheme is shown in table 4.
After each filtrate was processed, the sample measurement and the content calculation were performed in step S5, step S6 and step S7 described in example 1. The recovery rates and relative standard deviations (RSD,%) of the low, medium and high concentration spiking were calculated according to the results of the measurements, and the results are shown in Table 4.
Table 4 recovery of target compound normalized to relative standard deviation (n ═ 6)
Note: the concentration of triclosan in the a, b, c mixed standard working solution was 10 times the corresponding concentration of the other compounds.
As can be seen from Table 4, although the recovery rate of the spiked standard is influenced to a certain extent due to the fact that the paper has a porous structure and can adsorb part of the spiked standard components, the recovery rate of the five halogenated target compounds is 83.35% -96.69% and is close to 100%, and the method has good recovery rate and accurate measurement result. The RSD of the three concentration-labeled recovery data is lower than 10 percent, which shows that the method has better reproducibility.
EXAMPLE 3 determination of actual samples
In a specific implementation, the content of halocarban, triclocarban, triclosan, hexachlorophene and tribromosalen in more than 20 bacteriostatic paper (comprising bacteriostatic napkins, bacteriostatic oil absorbing paper and bacteriostatic paper extraction) samples randomly purchased from the market is determined. Of more than 20 bacteriostatic paper samples purchased randomly, 2 paper samples detected to contain triclosan are 0.22mg/kg and 0.28mg/kg respectively, which are far lower than the requirement of limited monitoring (the maximum allowable concentration is 0.3%) of technical Specification for cosmetic safety, 2015 edition). No target compound was detected in any of the remaining bacteriostatic paper samples. The detection result shows that the bacteriostatic paper has good regional use condition to a certain extent.
Claims (5)
1. A method for simultaneously determining the content of five bacteriostatic forbidden substances in bacteriostatic paper is characterized in that the five bacteriostatic forbidden substances are five target compounds of halocarb, trichlorocarb, triclosan, hexachlorophene and tribromosaran, and the method comprises the following steps:
step S1, preparing a series of mixed standard working solutions containing five bacteriostatic forbidden substances: preparing a series of mixed standard working solutions simultaneously containing halocarban, triclocarban, triclosan, hexachlorophene or tribromosalen by using 80% vol methanol aqueous solution;
for the same mixed standard working solution, the concentrations of halocarban, trichlorocarban, hexachlorophene and tribromosalen are the same, and the concentration of triclosan is 8-12 times of the corresponding concentrations of halocarban, trichlorocarban, hexachlorophene and tribromosalen;
step S2, sample pretreatment: cutting the bacteriostatic paper sample to obtain a sample, wherein the sample comprises a single side; cutting the cut sample into pieces of 0.5cm multiplied by 0.5cm, mixing uniformly, and placing in a clean sealed bag for later use; weighing a sample to be detected, placing the sample in a triangular flask with a plug, adding V mL of chromatographic pure methanol, and performing ultrasonic extraction for 20-40 min; standing for 3-7 min, transferring 2-3 mL of extract liquor into a centrifuge tube, and centrifuging for 3-7 min; transferring 1mL of supernatant, filtering by using a 0.22-micron organic phase filter membrane to obtain a sample filtrate to be detected, and detecting by using ultra-high performance liquid chromatography-electrospray ionization source quadrupole mass spectrometry;
step S3, blank experiment: under the condition that a paper sample to be detected is not added, executing the step S2 to obtain blank sample filtrate to be detected, and waiting for ultra performance liquid chromatography-electrospray ionization source quadrupole rod mass spectrometry detection;
step S4, drawing a standard working curve: determining the peak area of each target compound in the series of mixed standard working solutions in the step S1 by adopting ultra performance liquid chromatography-electrospray ionization source quadrupole mass spectrometry, and performing linear regression by taking the concentration of each target compound as a horizontal coordinate and the peak area of the target compound as a vertical coordinate to draw a standard working curve;
step S5, determining the content of each target compound in the sample: determining the peak area of each target compound in the filtrate of the sample to be detected by adopting ultra-high performance liquid chromatography-electrospray ionization source quadrupole mass spectrometry (S2), and calculating the concentration C of each target compound in the filtrate of the sample to be detected according to the standard working curve in the step S4;
step S6, measuring the content of each target compound in the blank sample: measuring the peak area of each target compound in the blank sample filtrate to be measured by adopting ultra performance liquid chromatography-electrospray ionization source quadrupole mass spectrometry (S3), and calculating the concentration C of each target compound in the blank sample filtrate to be measured according to the standard working curve in the step S40；
Step S7, calculating the content result of each target compound in the bacteriostatic paper sample: calculating the content of each target compound in the bacteriostatic paper sample to be detected according to the following formula;
in the formula (I), the compound is shown in the specification,
x represents the content of each target compound in the bacteriostatic paper sample to be detected, and the unit is mu g/kg;
c represents the concentration of the corresponding target compound in the filtrate of the sample to be detected obtained by the standard working curve, and the unit is ng/mL;
C0the concentration of the corresponding target compound in the blank sample filtrate to be detected, which is obtained from the standard working curve, is expressed in ng/mL;
v represents the volume of the extract in mL;
m represents the mass of the bacteriostatic paper sample to be detected, and the unit is g;
:; if the concentration of the target compound in the sample solution to be detected does not exceed the maximum concentration of the mixed standard working solution, taking 1 as the dilution factor; if the concentration of the target compound in the sample solution to be tested exceeds the maximum concentration of the mixed standard working solution, adjusting and diluting the solution to be tested by using methanol, and then measuring, if the solution is diluted to 2 times, taking a dilution factor of 2, and so on;
taking the arithmetic mean value of the multiple parallel measurement results as a final measurement result, and accurately measuring the result to 0.01 mu g/kg;
the relative mean deviation of multiple parallel measurements should be less than 10%.
2. The method for determining the content of five bacteriostatic forbidden substances in bacteriostatic paper according to claim 1, which is characterized in that:
in step S1, 80% vol methanol water solution is used for preparing a series of mixed standard working solutions simultaneously containing halocarban, triclocarban, triclosan, hexachlorophene and tribromosalen; wherein the concentrations of halocarban, triclocarban, hexachlorophene and tribromosalen are respectively as follows: 2ng/mL, 5ng/mL, 10ng/mL, 30ng/mL, 50ng/mL, 70ng/mL, 100ng/mL, 150ng/mL, 200 ng/mL; the concentration of triclosan is 8-12 times of the corresponding concentration of halocasan, triclocarban, hexachlorophene and tribromosalen; that is, the concentration of triclosan is 16-24 ng/mL, 40-60 ng/mL, 80-120 ng/mL, 240-360 ng/mL, 400-600 ng/mL, 560-840 ng/mL, 800-1200 ng/mL, 1200-1800 ng/mL, 1600-2400 ng/mL, respectively.
3. The method for simultaneously determining the content of five bacteriostatic forbidden substances in bacteriostatic paper according to claim 1, which is characterized in that:
in the step S4, the step S5, and the step S6, the liquid chromatography conditions of the hplc-electrospray ionization source quadrupole mass spectrometry method include: the chromatographic column is 100mm × 2.1mm, 1.8 μm ACQUITY UPLC HSS T3 chromatographic column; the sample injection amount is 5 mu L; the flow rate is 0.3 mL/min; the column temperature was 40 ℃; the injector temperature was 20 ℃; mobile phase a was methanol and mobile phase B was 0.1% vol formic acid in water; the gradient elution conditions included: 0-1.0 min, the proportion of the mobile phase A is 80-85 vol%; 1.0-2.0 min, wherein the proportion of the mobile phase A is 85% vol; 2.0-4.0 min, wherein the proportion of the mobile phase A is 85-90% vol; 4.0-6.0 min, wherein the proportion of the mobile phase A is 90% vol; 6.0-6.5 min, the proportion of the mobile phase A is 90-100% vol; 6.5min to 7.0min, the proportion of the mobile phase A is 100 percent vol to 80 percent vol; 7.0 min-8.0 min, the mobile phase A accounts for 80% vol;
in the step S4, the step S5, and the step S6, the mass spectrometry conditions of the hplc-electrospray ionization source quadrupole mass spectrometry method include: the ion source is an electrospray ionization source; the scanning mode is negative ion scanning; the capillary voltage is 3 kV; the extraction taper hole voltage is 5V; the RF mass spectrum voltage is 0.5V; the ion source temperature is 120 ℃; the desolventizing gas is nitrogen, and the purity is not less than 99.99 percent; the temperature of the desolventizing gas is 500 ℃; the flow rate of the desolventizing agent is 600L/hr; the collision gas is argon; the pressure in the collision chamber was 3.80e-3mbar; the voltage of the photomultiplier is 650V; the detection mode is multi-reaction monitoring; the residence monitoring time of the ion pair was 50 ms.
4. The method of claim 1, wherein:
in the step S4, the step S5 and the step S6, the retention time and the multiple reaction monitoring key parameters of the hplc-electrospray ionization source quadrupole mass spectrometry method corresponding to each target compound are detailed in the following table:
note: are quantitative ions.
5. The application of the method for determining the content of the five substances for inhibiting the bacteria in the antibacterial paper as claimed in claim 1 is characterized in that the method is used for simultaneously and rapidly determining halocasan, triclocarban, triclosan, hexachlorophene and tribromosaran in a complex mixed system, the detection limit is 0.01-10.00 ng/mL, and the quantification limit is 0.02-26.00 ng/mL.
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