A method for rapid simultaneous detection of four antibacterial agents in hand sanitizer

Abstract

The present invention discloses a method for simultaneously and rapidly detecting four antibacterial agents in hand sanitizer, relating to the field of analytical chemistry detection technology. The method specifically comprises the following steps: S1: MMIP synthesis, including Fe3O4@SiO2 magnetic carrier preparation and molecular imprinting layer construction; S2: pretreatment, including sample extraction, MMIP enrichment and purification, and in situ derivatization; S3: drawing a standard curve; S4: detection, performing UHPLC-MS / MS analysis, and calculating the content of four antibacterial agents in the hand sanitizer sample to be tested based on the standard curve; the four antibacterial agents are triclocarban, 4-chloro-3-cresol, dichlorophen, and benzylchlorophen. The method of the present invention has a short pretreatment time, high sensitivity, strong anti-interference, good selectivity, high recovery rate, and good stability, filling the gap in the current detection method for antibacterial preservatives in my country's hand sanitizer product standards.

Description

Technical Field

[0001] The present invention relates to the technical field of analytical chemistry detection, and in particular to a detection method for simultaneously and rapidly detecting four antibacterial agents in hand sanitizer. Background Art

[0002] With increasing attention to hygiene and safety, the use of antibacterial hand sanitizers is becoming increasingly widespread. Triclocarban (TCC), p-Choro-m-cresol (PCMC), dichlorophen (DCP), and benzylchlorophen (BCP) are common antibacterial ingredients in hand sanitizers, effectively inhibiting bacterial and fungal growth. However, excessive use of these compounds can cause skin irritation, allergic reactions, and even disrupt the human endocrine system or lead to residual environmental contamination.

[0003] Traditional methods for detecting antibacterial agents, such as high-performance liquid chromatography (HPLC) and gas chromatography (GC), have limitations such as long analysis times, insufficient sensitivity, or complex pretreatment, making them difficult to meet the demand for simultaneous detection of multiple antibacterial agents. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS), with its high separation efficiency, high sensitivity, and high selectivity, is an ideal choice for multi-component analysis in complex matrices.

[0004] While methods for detecting single or a few antibacterial agents in hand sanitizers have been reported, simultaneous detection of the four antibacterial agents mentioned above remains limited. In particular, methods that combine rapid pretreatment with high sensitivity are urgently needed. Furthermore, the complex matrix of hand sanitizers (including surfactants and fragrances) can easily interfere with target analysis, necessitating optimization of sample extraction and cleanup procedures. Therefore, this study aimed to develop a rapid simultaneous detection method based on UPLC-MS / MS, combined with efficient sample pretreatment techniques, to achieve simultaneous quantitative analysis of the four antibacterial agents in hand sanitizers, providing reliable technical support for market regulation and product development. Summary of the Invention

[0005] In response to the above problems, the present invention provides a method for simultaneously and rapidly detecting four antibacterial agents in hand sanitizer.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] The present invention provides a method for simultaneously and rapidly detecting four antibacterial agents in hand sanitizer, which specifically comprises the following steps:

[0008] S1: MMIP synthesis: including the preparation of Fe3O4@SiO2 magnetic carrier and the construction of molecular imprinting layer;

[0009] S2: Pretreatment: including sample extraction, MMIP enrichment and cleanup, and in situ derivatization;

[0010] S3: draw the standard curve;

[0011] S4: Detection: Perform UHPLC-MS / MS analysis and calculate the content of four antibacterial agents in the hand sanitizer samples according to the standard curve;

[0012] The four antibacterial agents are triclocarban TCC, 4-chloro-3-methylphenol PCMC, dichlorophenol DCP and benzylchlorophenol BCP.

[0013] Furthermore, the synthesis of MMIP in step S1 specifically includes the following steps:

[0014] S11: Preparation of Fe3O4@SiO2 magnetic carrier:

[0015] Preparation of Fe3O4 nanoparticles: FeCl3·6H2O and FeSO4·7H2O were dissolved in ultrapure water and deoxygenated with nitrogen for 30 minutes to obtain an iron salt solution. Under nitrogen protection, the iron salt solution was heated to 80°C and mechanically stirred at 500 rpm. 25% NH4OH was added dropwise to a pH of 11±0.2. The mixture was reacted at 80°C for 30 minutes. After magnetic separation, the precipitate was washed three times with ultrapure water and once with 0.02M NaCl, and dried under vacuum at 40°C for 6 hours.

[0016] SiO2 coating: Fe3O4 was dispersed in a mixed solvent of ethanol and water, TEOS was added dropwise at 0.5 mL / min, and 28% NH4OH was added dropwise until the pH reached 9.5±0.1. The mixture was stirred mechanically at 300 rpm at 25°C for 6 h. Fe3O4@SiO2 was collected by magnetic separation, washed three times with ethanol, and dried in a vacuum at 60°C for 12 h.

[0017] S12: Molecularly imprinted layer construction:

[0018] Pre-assembly solution: Dissolve the template dichlorophenol standard and monomer methacrylic acid in acetonitrile, sonicate at 25°C for 30 minutes, and let stand at 4°C for 12 hours to complete pre-polymerization;

[0019] Polymerization reaction: add crosslinker EGDMA, initiator AIBN, and Fe3O4@SiO2, deoxygenate by nitrogen bubbling for 15 min, incubate in a 60°C water bath, and react with magnetic stirring at 200 rpm under nitrogen protection for 12 h;

[0020] Template elution: The polymer was collected by magnetic separation and Soxhlet extraction was performed with methanol-acetic acid eluent for 48 h, with the eluent replaced every 12 h until no template residue was detected by HPLC. The polymer was then dried under vacuum at 40 °C for 12 h and sieved to obtain MMIP.

[0021] Furthermore, the pre-processing in step S2 specifically includes the following steps:

[0022] S21: Sample extraction:

[0023] The hand sanitizer sample was placed in a centrifuge tube, and a methanol-water (8:2, v / v) mixed solvent was added. The sample was immediately vortexed for 2 minutes (to avoid emulsification caused by excessive local solvent concentration), and ultrasonic extraction was performed at 40°C and 300W for 10 minutes to promote the release of the target compound. The sample was centrifuged at 8000 rpm for 5 minutes, and the supernatant was transferred to a new centrifuge tube. The methanol-water mixed solvent was added to the residue again, and the vortexing, ultrasonication, and centrifugation steps were repeated. The two supernatants were combined to obtain the sample extract. The sample was slowly blown to near dryness with nitrogen in a 40°C water bath, and acetonitrile was added to redissolve the residue. The sample was vortexed for 1 minute and filtered through a 0.22 μm organic filter membrane to obtain the sample extract for purification.

[0024] S22: MMIP enrichment and purification:

[0025] S221: Activation of MMIP: Place MMIP in a centrifuge tube, add methanol, vortex for 1 min, magnetically separate and discard the supernatant, repeat once; then balance with acetonitrile, vortex for 1 min, magnetically separate and discard the supernatant;

[0026] S222: Adsorption-Washing: Add the sample extract for cleanup to the activated MMIP, vortex at 1200 rpm for 5 minutes at room temperature, and magnetically separate and discard the supernatant. Add an acetonitrile-water (1:9, v / v) solvent mixture, vortex for 2 minutes, and magnetically separate and discard the supernatant. Repeat twice (to remove polar interferences such as surfactants (e.g., SLS) and glycerol).

[0027] S223: Elution of target compound: Add methanol-acetic acid (9:1, v / v) eluent, vortex for 3 minutes, perform magnetic separation, and collect the eluent; add the eluent again, combine the two eluents, and set aside;

[0028] S23: In situ derivatization:

[0029] S231: Pretreatment: Blow the eluate to near dryness with nitrogen at 40°C, reconstitute the residue with 2 volumes of acetonitrile, and transfer to a glass derivatization bottle;

[0030] S232: Derivatization reaction: Add acetic anhydride and pyridine in sequence, immediately vortex and mix for 10 seconds, and react in a 70°C water bath in the dark for 15 minutes;

[0031] S233: Termination of the reaction: Remove the derivatization vial and immediately cool it on ice for 2 minutes. Purify it with nitrogen at 40°C until it is nearly dry to remove unreacted acetic anhydride and pyridine (to avoid mass spectrometry signal suppression). Add a 0.1% formic acid-acetonitrile mixed solvent (95:5, v / v), vortex for 1 minute, and filter it through a 0.22 μm nylon filter. The filtrate is directly used for UHPLC-MS / MS analysis.

[0032] Furthermore, in step S11:

[0033] Preparation of Fe3O4 nanoparticles: the molar ratio of FeCl3·6H2O to FeSO4·7H2O was 2:1;

[0034] SiO2 coating: the solid solution ratio of Fe3O4, mixed solvent and TEOS is 100 mg:100 mL:1 mL; the volume ratio of ethanol to water in the mixed solvent is 4:1.

[0035] Furthermore, in step S12:

[0036] Pre-assembly solution: the ratio of template, monomer and acetonitrile is 0.1mmol:0.4mmol:20mL;

[0037] Polymerization reaction: the ratio of template, cross-linker, initiator and Fe3O4@SiO2 is 0.1mmol:1mmol:10mg:50mg;

[0038] Template elution: The volume ratio of methanol and acetic acid in the eluent was 9:1.

[0039] Furthermore, in step S21, the usage ratio of the hand sanitizer sample, the mixed solvent and acetonitrile is 1.0 g:15 mL:2 mL; and the volume ratio of methanol to water in the mixed solvent is 8:2.

[0040] Furthermore, in step S22:

[0041] The mass ratio of MMIP to hand sanitizer sample was 1:100; the dosage ratio of MMIP, methanol, acetonitrile, mixed solvent, and eluent was: 10 mg:1 mL:1 mL:2 mL:1.5 mL;

[0042] The volume ratio of acetonitrile to water in the mixed solvent is 1:9;

[0043] The volume ratio of methanol to acetic acid in the eluent is 9:1.

[0044] Furthermore, in step S23, the volume ratio of acetonitrile, acetic anhydride, pyridine, and the mixed solvent is 10:5:2:20; and the volume ratio of 0.1% formic acid water to acetonitrile in the mixed solvent is 95:5.

[0045] Furthermore, in step S4, the chromatographic conditions are: chromatographic column: Waters ACQUITY UPLC BEH C18, 2.1×100 mm, 1.7 μm; column temperature: 40°C; flow rate: 0.3 mL / min; injection volume: 2 μL; mobile phase: phase A is 0.1% formic acid water, phase B is acetonitrile; elution mode: gradient elution, elution program is: 0 min, 5% B; 0~2.0 min, phase B changes linearly from 5% to 95%; 2~5 min, 95% B; 5~7 min, phase B changes linearly from 95% to 5%; 7~9 min, 5% B.

[0046] Furthermore, in step S4, the mass spectrometry conditions are as follows: ion source: electrospray ion source (ESI-); scanning mode: negative ion scanning; spray voltage: -4500 V; ion source temperature: 500 ° C; nebulizer gas, 50 psi; auxiliary gas, 60 psi; curtain gas, 25 psi; detection mode: multiple reaction monitoring mode MRM; TCC: quantitative ion pair: m / z 315.9→160.0, collision energy: 20 eV; qualitative ion pair: m / z 315.9→125.0, collision energy: 25 eV, declustering voltage: -60 V; PCMC-ac: quantitative ion pair: m / z 169.0→89.0, collision energy: 15 eV; qualitative ion pair: m / z 169.0→109.0, collision energy: 18 eV; DCP-ac: quantitative ion pair: m / z 265.0→162.0, collision energy: 18 eV; Qualifier ion pair: m / z 265.0→127.0, collision energy: 22 eV; BCP-ac: Quantitative ion pair: m / z 231.0→91.0, collision energy: 22 eV; Qualifier ion pair: m / z 231.0→119.0, collision energy: 25 eV.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] The method of the present invention, based on the MMIP-in situ derivatization-UHPLC-MS / MS coupling technology, simultaneously and rapidly detects four antibacterial agents (triclocarban, 4-chloro-3-methylphenol, dichlorophen, and benzylchlorophen) in hand sanitizers. The method has short pretreatment time, high sensitivity, strong anti-interference, good selectivity, high recovery rate, and good stability. It fills the gap in the detection method for antibacterial preservatives in the current hand sanitizer product standards in my country, and also provides new technical support for ensuring the quality and safety of hand sanitizer products. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 is a transmission electron micrograph of the MMIP in Example 1 of the present invention;

[0050] Figure 2is the TCC standard curve (0.5-200 μg / mL) of Example 1 of the present invention;

[0051] Figure 3 is the PCMC-ac standard curve (0.5-200 μg / mL) of Example 1 of the present invention;

[0052] Figure 4 is the DCP-ac standard curve (0.5-200 μg / mL) of Example 1 of the present invention;

[0053] Figure 5 is the BCP-ac standard curve (0.5-200 μg / mL) of Example 1 of the present invention;

[0054] Figure 6 These are the total ion current chromatogram, quantitative chromatogram, and qualitative chromatogram (10 μg / mL) of the TCC, PCMC-ac, DCP-ac, and BCP-ac standard solutions of Example 1 of the present invention. DETAILED DESCRIPTION

[0055] In order to make the purpose and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0056] Unless otherwise specified, the instruments, reagents, and materials used in the following examples are all conventional instruments, reagents, and materials available in the prior art and can be obtained through regular commercial channels. The experimental methods and detection methods used in the following examples are all conventional experimental methods and detection methods available in the prior art, unless otherwise specified.

[0057] Reagents: triclocarban (TCC, ≥98%), 4-chloro-3-methylphenol (PCMC, ≥97%), dichlorophene (DCP, ≥98%), benzylchlorophene (BCP, ≥95%), tetraethyl orthosilicate (TEOS, ≥99.8%), methacrylic acid (MAA, ≥98%), ethylene glycol dimethacrylate (EGDMA, ≥98%), azobisisobutyronitrile (AIBN), methanol (HPLC grade), acetonitrile (HPLC grade), formic acid (≥99%, HPLC grade), acetic acid (analytical grade), acetic anhydride (Ac2O, purity ≥99%), pyridine (catalyst, purity ≥99%), hand sanitizer samples (commercially available Lion children's hand sanitizer and Safeguard antibacterial hand sanitizer, numbered K1 and K2, respectively).

[0058] In the following examples, the magnetic field strength of the magnetic separation was 0.5 T and the separation time was 1 min.

[0059] Example 1

[0060] This embodiment provides a method for simultaneously and rapidly detecting four antibacterial agents in hand sanitizer, which specifically includes the following steps:

[0061] S1: Synthesis of MMIP:

[0062] S11: Preparation of Fe3O4@SiO2 magnetic carrier:

[0063] Preparation of Fe3O4 nanoparticles: 10 mmol FeCl3·6H2O and 5 mmol FeSO4·7H2O were dissolved in 100 mL ultrapure water and deoxygenated with nitrogen for 30 min to obtain an iron salt solution. Under nitrogen protection, the iron salt solution was heated to 80°C and mechanically stirred at 500 rpm. 25% NH4OH was added dropwise to a pH of 11±0.2. The mixture was reacted at 80°C for 30 min. After magnetic separation, the precipitate was washed three times with ultrapure water and once with 0.02 M NaCl. The resulting particles (10±2 nm, magnetic saturation intensity 65 emu / g) were obtained.

[0064] SiO2 coating: 100 mg of Fe3O4 was dispersed in 100 mL of a mixed solvent of ethanol / water (4:1, v / v). 1 mL of TEOS was added dropwise at 0.5 mL / min, followed by 28% NH4OH until the pH reached 9.5 ± 0.1. The mixture was stirred mechanically at 300 rpm at 25°C for 6 h. Fe3O4@SiO2 was collected by magnetic separation, washed three times with ethanol, and dried in a vacuum at 60°C for 12 h (200 ± 20 nm).

[0065] S12: Molecularly imprinted layer construction:

[0066] Pre-assembly solution: 0.1 mmol template dichlorophenol standard and 0.4 mmol monomer methacrylic acid were dissolved in 20 mL acetonitrile, ultrasonicated at 25°C for 30 min, and allowed to stand at 4°C for 12 h to complete pre-polymerization;

[0067] Although MMIP is synthesized using dichlorophenol (DCP) as a template molecule, its recognition mechanism is based on molecular structural similarity: triclocarban (TCC) and phenolic antibacterial agents (PCMC / DCP / BCP) both contain a benzene ring + chlorine substituent, and the amide bond (-NH-CO-) of triclocarban and the phenolic hydroxyl group (-OH) of phenolic antibacterial agents have certain similarities in polarity. The cavity of MMIP can adsorb both.

[0068] Polymerization reaction: add 1 mmol of crosslinker EGDMA, 10 mg of initiator AIBN, and 50 mg of Fe3O4@SiO2, deoxygenate by nitrogen bubbling for 15 min, and react in a 60°C water bath with magnetic stirring at 200 rpm under nitrogen protection for 12 h;

[0069] Template elution: The polymer was collected by magnetic separation and Soxhlet extraction was performed with methanol-acetic acid (9:1, v / v) as eluent for 48 h. (Approximately 500 mg of dried MMIP was placed in a Soxhlet extraction cartridge, both ends were sealed with glass wool to prevent leakage, 150 mL of eluent was added to a round-bottom flask, and the Soxhlet extractor was assembled. The extract was incubated in a 70°C water bath for 48 h at a reflux rate of 6-8 cycles / h). The eluent was replaced every 12 h until no template residue was detected by HPLC (LOD ≤ 0.01 μg / mL). The eluent was replaced every 12 h until no template residue was detected by HPLC. The extract was vacuum dried at 40°C for 12 h and sieved to obtain MMIP powder. Transmission electron microscopy images are shown in the figure. Figure 1 (Stored in anhydrous ethanol at 4°C and dried with nitrogen before use).

[0070] S2: Pre-treatment:

[0071] S21: Sample extraction:

[0072] Transfer 1.0 g of hand sanitizer sample to a 15 mL centrifuge tube, add 10 mL of a methanol-water (8:2, v / v) mixed solvent, and immediately vortex for 2 minutes (to avoid emulsification caused by excessive local solvent concentration). Perform ultrasonic-assisted extraction at 40°C and 300W for 10 minutes to promote the release of the target compound. Centrifuge at 8000 rpm for 5 minutes, and transfer the supernatant to a new centrifuge tube. Add 5 mL of the methanol-water mixed solvent to the residue again, repeat the vortexing, ultrasonication, and centrifugation steps, and combine the two supernatants (a total of 15 mL) to obtain the sample extract. Slowly blow the mixture with nitrogen gas in a 40°C water bath until it is nearly dry. Add 2 mL of acetonitrile to dissolve the residue, vortex for 1 minute, and filter through a 0.22 μm organic filter membrane to obtain the sample extract for purification.

[0073] S22: MMIP enrichment and purification:

[0074] S221: Activation of MMIP: 10 mg of MMIP was placed in a 1.5 mL centrifuge tube, 1 mL of methanol was added, vortexed for 1 min, magnetically separated and the supernatant discarded, and repeated once; then equilibrated with 1 mL of acetonitrile, vortexed for 1 min, magnetically separated and the supernatant discarded;

[0075] S222: Adsorption-Washing: Add the sample extract for cleanup to the activated MMIP, vortex at 1200 rpm for 5 minutes at room temperature, and magnetically separate and discard the supernatant. Add 2 mL of acetonitrile-water (1:9, v / v) mixed solvent, vortex for 2 minutes, and magnetically separate and discard the supernatant. Repeat twice (to remove polar interferences such as surfactants (such as SLS) and glycerol).

[0076] S223: Elution of target compound: Add 1 mL of methanol-acetic acid (9:1, v / v) eluent, vortex for 3 min, perform magnetic separation, and collect the eluent; add 0.5 mL of eluent again, combine the two eluents (a total of 1.5 mL), and set aside;

[0077] S23: In situ derivatization:

[0078] S231: Pretreatment: The eluate was blown to near dryness (about 50 μL) with nitrogen at 40°C, and the residue was reconstituted with 2 volumes (100 μL) of acetonitrile and transferred to a glass derivatization bottle;

[0079] S232: Derivatization reaction: Add 50 μL acetic anhydride and 20 μL pyridine in sequence, immediately vortex and mix for 10 seconds, and react in a 70°C water bath in the dark for 15 minutes;

[0080] S233: Termination of the reaction: Remove the derivatization vial and immediately cool it on ice for 2 minutes. Purify it with nitrogen at 40°C until it is nearly dry to remove unreacted acetic anhydride and pyridine. Add 200 μL of a 0.1% formic acid-acetonitrile mixture (95:5, v / v) and vortex for 1 minute. Pass the solution through a 0.22 μm nylon filter membrane and use the filtrate directly for UHPLC-MS / MS analysis.

[0081] In the above-mentioned in situ derivatization reaction, phenolic antimicrobial agents (PCMC / DCP / BCP) contain active phenolic hydroxyl groups (-OH) that can undergo acetylation with acetic anhydride. However, triclocarban (TCC) does not have a phenolic hydroxyl group and does not react with acetic anhydride. Therefore, in situ derivatization is targeted at phenolic antimicrobial agents, and acetylation is used to improve their chromatographic retention and mass spectrometric sensitivity. Although triclocarban is not derivatized by acetic anhydride, it has sufficient mass spectrometric response ([MH] in negative ion mode). - peak intensity), can be directly detected by UHPLC-MS / MS.

[0082] Compared with the traditional SPE-HPLC detection method, which takes more than 2 hours for pretreatment, the method of the present invention only requires extraction, MMIP cleanup, and derivatization, with a total time of less than 50 minutes, significantly shortening the pretreatment time.

[0083] S3: Draw the standard curve:

[0084] (1) Stock solution (1000 μg / mL): Accurately weigh 10.0 mg of each standard into a 10 mL volumetric flask, dissolve it in methanol and dilute to the mark, vortex for 1 min, sonicate for 5 min to aid dissolution, and store at -20°C away from light. The shelf life is 6 months.

[0085] (2) Preparation of standard solution:

[0086] Triclocarban: Dilute the stock solution with methanol-water (8:2, v / v) to prepare TCC standard solutions at concentrations of 0.5, 1, 5, 10, 50, 100, and 200 μg / mL.

[0087] Phenolic derivatives (acetylated PCMC / DCP / BCP): Dilute the stock solution with methanol-water (8:2, v / v) to prepare working solutions of 0.5, 1, 5, 10, 50, 100, and 200 μg / mL. Transfer 1 mL of each working solution (0.5-200 μg / mL) to a glass derivatization vial, add 50 μL of acetic anhydride and 20 μL of pyridine, react at 70°C for 15 min, blow dry with nitrogen, and reconstitute to 1 mL with the initial mobile phase (0.1% formic acid in water-acetonitrile, 95:5) to obtain gradient concentrations of PCMC-ac, DCP-ac, and BCP-ac standard solutions.

[0088] (3) Establishing a standard curve

[0089] A series of concentrations of TCC standard solutions and PCMC-ac, DCP-ac, and BCP-ac standard solutions were analyzed by UHPLC-MS / MS.

[0090] The chromatographic conditions were as follows: chromatographic column: Waters ACQUITY UPLC BEH C18, 2.1×100 mm, 1.7 μm; column temperature: 40°C; flow rate: 0.3 mL / min; injection volume: 2 μL; mobile phase: phase A is 0.1% formic acid water, phase B is acetonitrile; elution mode: gradient elution, elution program: 0 min, 5% B; 0~2.0 min, phase B changes linearly from 5% to 95%; 2~5 min, 95% B; 5~7 min, phase B changes linearly from 95% to 5%; 7~9 min, 5% B. The mass spectrometry conditions were as follows: ion source: electrospray ion source (ESI-); scan mode: negative ion scan; spray voltage: -4500 V; ion source temperature: 500°C; nebulizer gas, 50 psi; auxiliary gas, 60 psi; curtain gas, 25 psi; detection mode: multiple reaction monitoring (MRM); TCC: quantitative ion pair: m / z 315.9→160.0, collision energy: 20 eV; qualitative ion pair: m / z 315.9→125.0, collision energy: 25 eV, declustering voltage: -60 V; PCMC-ac: quantitative ion pair: m / z 169.0→89.0, collision energy: 15 eV; qualitative ion pair: m / z 169.0→109.0, collision energy: 18 eV; DCP-ac: quantitative ion pair: m / z 265.0→162.0, collision energy: 18 eV; Qualifier ion pair: m / z 265.0→127.0, collision energy: 22 eV; BCP-ac: Quantitative ion pair: m / z 231.0→91.0, collision energy: 22 eV; Qualifier ion pair: m / z 231.0→119.0, collision energy: 25 eV.

[0091] With the peak area as the ordinate and the concentration as the abscissa, a standard curve was drawn, see Table 1. Figure 2-6 The results showed that TCC, PCMC-ac, DCP-ac, and BCP-ac had good linear relationships in the range of 0.5~200μg / mL.

[0092] Table 1 Standard curve

[0093]

[0094] Experimental Example 1 Limit of Detection (LOD) and Limit of Quantification (LOQ)

[0095] Blank samples (hand sanitizer without the target substance) were spiked at concentrations of 0.001, 0.002, 0.005, 0.01, and 0.02 μg / mL and processed according to the pretreatment procedure. Each concentration was repeated six times (n=6), and the signal-to-noise ratio (S / N) was calculated. The LOD was defined as the lowest concentration with an S / N ≥ 3, and the LOQ was defined as the lowest concentration with an S / N ≥ 3 and an RSD ≤ 20%. The results are shown in Table 2.

[0096] Table 2 LOD and LOQ

[0097]

[0098] Experimental Example 2 Spiked Recovery and Precision

[0099] Blank samples were spiked at three levels (0.1, 1, and 10 μg / mL). Following the same treatment as above, the assay was repeated six times for each level (n=6), and the spiked recoveries and precision were calculated. Table 3 shows that the average recoveries for the four antibacterial agents at the three spiked levels ranged from 94.2% to 108.2%, with RSDs less than 10%. This demonstrates that the proposed method has excellent recovery and precision, and demonstrates that the method is accurate, stable, and reliable.

[0100] Table 3 Spiked recovery and precision

[0101]

[0102] Experimental Example 3 Anti-interference Experiment

[0103] Take a blank sample, add 1 μg / mL of target compound and various interfering substances (the concentration of interfering substances is the amount commonly used in actual products), perform pretreatment process and UHPLC-MS / MS analysis, and calculate the recovery rate.

[0104] As shown in Table 4, the recovery rates of the interfering substances in the four antibacterial agents were 92.5% to 106.2%, indicating that the method of the present invention is not easily interfered by other components in hand sanitizer and has good selectivity.

[0105] Table 4 Anti-interference results

[0106]

[0107] Experimental Example 5 Sample Testing

[0108] Take 2 μL of the filtrate for UHPLC-MS / MS analysis and calculate the contents of TCC, PCMC-ac, DCP-ac, and BCP-ac in the hand sanitizers to be tested (K1, K2) according to the standard curve:

[0109] According to the standard curve obtained in step S3 and the peak areas of TCC, PCMC-ac, DCP-ac, and BCP-ac in the sample solution to be tested, the concentrations of TCC, PCMC-ac, DCP-ac, and BCP-ac in the filtrate (μg / mL, n=6) were calculated and recorded as C TCC 、C PCMC-ac 、C DCP-ac 、C BCP-ac ;

[0110] The concentration of each antibacterial agent in the filtrate was further calculated as follows:

[0111] DF=(V 净化 / V 提取 )×(V 复溶 / V 洗脱 ) = (2mL / 15mL) × (0.1mL / 1.5mL) = 1 / 112.5;

[0112] For TCC:

[0113] C TCC =C TCC-滤液 × (1 / DF) = C TCC-滤液 ×112.5 (μg / mL);

[0114] For PCMC:

[0115] C PCMC =C PCMC-ac × (M PCMC / M PCMC-ac ) × (1 / DF) = C PCMC-ac × (142 / 185) × 112.5 = C PCMC-ac ×0.768×112.5 (μg / mL);

[0116] For DCP:

[0117] C DCP =C DCP-ac × (M DCP / M DCP-ac ) × (1 / DF) = C DCP-ac × (269 / 312) × 112.5 = C DCP-ac ×0.862×112.5 (μg / mL);

[0118] For BCP:

[0119] C BCP =C BCP-ac × (M BCP / M BCP-ac ) × (1 / DF) = C BCP-ac × (218 / 261) × 112.5 = C BCP-ac ×0.835×112.5 (μg / mL);

[0120] Based on this: calculate the contents of TCC, PCMC, DCP, and BCP in the hand sanitizer samples to be tested. The results are shown in Table 5.

[0121] Table 5 Contents of four antibacterial agents in the tested samples

[0122]

[0123] The "Safety Technical Specifications for Cosmetics" stipulates that triclocarban is prohibited (not detectable), the maximum allowable concentration of 4-chloro-3-methylphenol in rinse-off products is 0.5% (5000 μg / mL), the maximum allowable concentration of dichlorophen in rinse-off products is 0.3% (3000 μg / mL), and the maximum allowable concentration of benzylchlorophen in rinse-off products is 0.1% (1000 μg / mL). Based on this, the contents of the four antibacterial agents in the two hand sanitizer samples tested in this example were all within the specified limits and met safety regulations.

[0124] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for simultaneously and rapidly detecting four antibacterial agents in hand sanitizer, characterized in that: The specific steps include: S1: MMIP synthesis: including the preparation of Fe3O4@SiO2 magnetic carrier and the construction of molecular imprinting layer; S11: Preparation of Fe3O4@SiO2 magnetic carrier; S12: Molecularly imprinted layer construction: Pre-assembly solution: Dissolve the template dichlorophenol standard and the monomer methacrylic acid in acetonitrile, sonicate at 25°C for 30 minutes, and let stand at 4°C for 12 hours to complete pre-polymerization; the ratio of the template, monomer, and acetonitrile is 0.1 mmol:0.4 mmol:20 mL; Polymerization reaction: Add crosslinker EGDMA, initiator AIBN, and Fe3O4@SiO2, deoxygenate with nitrogen bubbling for 15 minutes, and react in a 60°C water bath with magnetic stirring at 200 rpm under nitrogen protection for 12 hours. The ratio of template, crosslinker, initiator, and Fe3O4@SiO2 is 0.1 mmol:1 mmol:10 mg:50 mg. Template elution: The polymer was collected by magnetic separation and extracted with Soxhlet eluent for 48 hours, with the eluent replaced every 12 hours until no template residue was detected by HPLC. The polymer was then dried under vacuum at 40°C for 12 hours and sieved to obtain MMIP. The volume ratio of methanol to acetic acid in the eluent was 9:

1. S2: Pretreatment: including sample extraction, MMIP enrichment and cleanup, and in situ derivatization; S21: Sample extraction: The hand sanitizer sample was placed in a centrifuge tube, and a methanol-water mixture was added. The sample was immediately vortexed for 2 minutes, ultrasonically extracted at 40°C, 300W for 10 minutes, and centrifuged at 8000 rpm for 5 minutes. The supernatant was transferred to a new centrifuge tube. The methanol-water mixture was added to the residue again, and the vortexing, ultrasonication, and centrifugation steps were repeated. The two supernatants were combined to obtain the sample extract. The sample was slowly blown to near dryness with nitrogen in a 40°C water bath. Acetonitrile was added to redissolve the residue, vortexed for 1 minute, and filtered through a 0.22 μm organic filter membrane to obtain the sample extract for purification. S22: MMIP enrichment and purification: S221: Activation of MMIP: Place MMIP in a centrifuge tube, add methanol, vortex for 1 min, magnetically separate and discard the supernatant, repeat once; then balance with acetonitrile, vortex for 1 min, magnetically separate and discard the supernatant; S222: Adsorption-washing: Add the sample extract for purification to the activated MMIP, vortex at room temperature and 1200 rpm for 5 minutes, magnetically separate and discard the supernatant; add acetonitrile-water mixed solvent, vortex for 2 minutes, magnetically separate and discard the supernatant, and repeat twice; S223: Elution of target: add methanol-acetic acid eluent, vortex for 3 minutes, magnetically separate, and collect the eluent; add eluent again, combine the two eluents, and set aside; S23: In situ derivatization: S231: Pretreatment: Blow the eluate to near dryness with nitrogen at 40°C, reconstitute the residue with 2 volumes of acetonitrile, and transfer to a glass derivatization bottle; S232: Derivatization reaction: Add acetic anhydride and pyridine in sequence, immediately vortex and mix for 10 seconds, and react in a 70°C water bath in the dark for 15 minutes; S233: Termination of the reaction: Remove the derivatization bottle, immediately place it on ice for 2 min, and blow it with nitrogen at 40°C until it is almost dry; A 0.1% formic acid-acetonitrile mixed solvent was added, vortexed for 1 min, and filtered through a 0.22 μm nylon filter membrane. The filtrate was directly used for UHPLC-MS / MS analysis; S3: draw the standard curve; S4: Detection: Perform UHPLC-MS / MS analysis and calculate the content of four antibacterial agents in the hand sanitizer samples according to the standard curve; The chromatographic conditions for the UHPLC-MS / MS analysis were as follows: chromatographic column: Waters ACQUITY UPLC BEH C18, 2.1×100 mm, 1.7 μm; Column temperature: 40°C; Flow rate: 0.3 mL / min; injection volume: 2 μL; mobile phase: phase A is 0.1% formic acid water, phase B is acetonitrile; Elution method: gradient elution, elution program: 0 min, 5% B; 0-2.0 min, linear change of B phase from 5% to 95%; 2-5 min, 95% B; 5-7 min, linear change of B phase from 95% to 5%; 7-9 min, 5% B; The four antibacterial agents are triclocarban, 4-chloro-3-methylphenol, dichlorophen and benzylchlorophen.

2. The method for simultaneously and rapidly detecting four antibacterial agents in hand sanitizer according to claim 1, wherein: In step S11, the preparation method of Fe3O4@SiO2 magnetic carrier is specifically as follows: Preparation of Fe3O4 nanoparticles: FeCl3·6H2O and FeSO4·7H2O were dissolved in ultrapure water and deoxygenated with nitrogen for 30 minutes to obtain an iron salt solution. Under nitrogen protection, the iron salt solution was heated to 80°C and mechanically stirred at 500 rpm. 25% NH4OH was added dropwise to a pH of 11±0.

2. The mixture was reacted at 80°C for 30 minutes. After magnetic separation, the precipitate was washed three times with ultrapure water and once with 0.02M NaCl, and dried under vacuum at 40°C for 6 hours. SiO2 coating: Fe3O4 was dispersed in a mixed solvent of ethanol and water, TEOS was added dropwise at 0.5 mL / min, and 28% NH4OH was added dropwise until the pH was 9.5±0.

1. The reaction was carried out at 25°C and 300 rpm with mechanical stirring for 6 h. Fe3O4@SiO2 was collected by magnetic separation, washed three times with ethanol, and dried in a vacuum at 60°C for 12 h.

3. A method for simultaneously and rapidly detecting four antibacterial agents in hand sanitizer according to claim 2, characterized in that: In the step S11: Preparation of Fe3O4 nanoparticles: the molar ratio of FeCl3·6H2O to FeSO4·7H2O was 2:1; SiO2 coating: the solid solution ratio of Fe3O4, mixed solvent and TEOS is 100 mg:100 mL:1 mL; the volume ratio of ethanol to water in the mixed solvent is 4:

1.

4. The method for simultaneously and rapidly detecting four antibacterial agents in hand sanitizer according to claim 1, wherein: In step S21, the usage ratio of the hand sanitizer sample, the mixed solvent, and acetonitrile is 1.0 g:15 mL:2 mL; and the volume ratio of methanol to water in the mixed solvent is 8:

2.

5. The method for simultaneously and rapidly detecting four antibacterial agents in hand sanitizer according to claim 1, wherein: In the step S22: The mass ratio of MMIP to hand sanitizer sample was 1:100; the dosage ratio of MMIP, methanol, acetonitrile, mixed solvent, and eluent was: 10 mg:1 mL:1 mL:2 mL:1.5 mL; The volume ratio of acetonitrile to water in the mixed solvent is 1:9; In the methanol-acetic acid eluent, the volume ratio of methanol to acetic acid is 9:

1.

6. The method for simultaneously and rapidly detecting four antibacterial agents in hand sanitizer according to claim 1, wherein: In step S23, the volume ratio of acetonitrile, acetic anhydride, pyridine, and the mixed solvent is 10:5:2:20; and the volume ratio of 0.1% formic acid water to acetonitrile in the mixed solvent is 95:

5.

7. The method for simultaneously and rapidly detecting four antibacterial agents in hand sanitizer according to claim 1, wherein: In step S4, the mass spectrometry conditions are as follows: ion source: electrospray ion source; scanning mode: negative ion scanning; spray voltage: -4500 V; ion source temperature: 500° C.; nebulizer gas, 50 psi; auxiliary gas, 60 psi; curtain gas, 25 psi; detection mode: multiple reaction monitoring mode (MRM); TCC: quantification ion pair: m / z 315.9→160.0, collision energy: 20 eV; qualifier ion pair: m / z 315.9→125.0, collision energy: 25 eV, declustering voltage: -60 V; PCMC-ac: quantification ion pair: m / z 169.0→89.0, collision energy: 15 eV; qualifier ion pair: m / z 169.0→109.0, collision energy: 18 eV; DCP-ac: quantification ion pair: m / z 265.0→162.0, collision energy: 18 eV; qualifier ion pair: m / z 265.0→127.0, collision energy: 22 eV; BCP-ac: quantification ion pair: m / z 231.0→91.0, collision energy: 22 eV; qualifier ion pair: m / z 231.0→119.0, collision energy: 25 eV.

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