Detection method for simultaneously and rapidly detecting four bacteriostatic agents in hand sanitizer

Abstract

The invention discloses a detection method for simultaneously and rapidly detecting four bacteriostatic agents in a hand sanitizer, and relates to the technical field of analytical chemistry detection, and the detection method specifically comprises the following steps: S1, MMIP synthesis: preparing a Fe3O4atSiO2 magnetic carrier and constructing a molecular imprinting layer; s2, pretreatment: sample extraction, MMIP enrichment and purification, and in-situ derivatization are carried out; s3, drawing a standard curve; and S4, detection: carrying out UHPLC-MS / MS analysis, and calculating the contents of the four bacteriostatic agents in the to-be-detected hand sanitizer sample according to the standard curve. The four types of bacteriostatic agents are triclocarban, 4-chloro-3-cresol, dichlorphenol and benzyl chlorophenol. The method disclosed by the invention is short in pretreatment time, high in sensitivity, strong in anti-interference performance, good in selectivity, high in recovery rate and good in stability, and fills the defect of detection methods of hand sanitizer product standards in the aspect of antibacterial preservatives at present in China.

Description

Technical Field

[0001] The present invention relates to the technical field of analytical chemistry detection, and particularly relates to a detection method for simultaneously and rapidly detecting 4 bacteriostatic agents in hand sanitizer. Background Art

[0002] With the increasing attention of people to health and safety, the use of bacteriostatic hand sanitizers is becoming increasingly widespread. Triclocarban (TCC), p-Choro-m-cresol (PCMC), Dichlorophen (DCP), and Benzylchlorophen (BCP) are common bacteriostatic components in hand sanitizers, which can effectively inhibit the growth of bacteria and fungi. However, if these compounds are used in excess, they may cause skin irritation, allergic reactions, and even interfere with the human endocrine system or cause environmental residue pollution.

[0003] Traditional detection methods for bacteriostatic agents, such as high-performance liquid chromatography (HPLC) and gas chromatography (GC), have limitations such as long analysis time, insufficient sensitivity, or complex pretreatment, and are difficult to meet the requirements for simultaneously detecting multiple bacteriostatic agents. Ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) has become an ideal choice for multi-component analysis in complex matrices due to its high separation efficiency, high sensitivity, and high selectivity.

[0004] At present, there have been reports on the detection of single or a few bacteriostatic agents in hand sanitizers, but there are still few methods for simultaneously detecting the above 4 bacteriostatic agents, especially methods that combine rapid pretreatment and high sensitivity are urgently needed to be developed. In addition, the matrix of hand sanitizer is complex (containing surfactants, fragrances, etc.), which is easy to interfere with the analysis of target substances, and the sample extraction and purification steps need to be optimized. Therefore, this study aims to establish a simultaneous and rapid detection method based on UPLC-MS / MS, combined with efficient sample pretreatment technology, to achieve synchronous quantitative analysis of 4 bacteriostatic agents in hand sanitizer, and provide reliable technical support for market supervision and product research and development. Summary of the Invention

[0005] The present invention addresses the above problems and provides a detection method for simultaneously and rapidly detecting 4 bacteriostatic agents in hand sanitizer.

[0006] In order to achieve the above object, the technical solutions adopted by the present invention are as follows: The present invention provides a detection method for simultaneously and rapidly detecting 4 bacteriostatic agents in hand sanitizer, specifically including the following steps: S1: MMIP synthesis: including the preparation of Fe3O4@SiO2 magnetic carriers and the construction of a molecularly imprinted layer; S2: Pretreatment: including sample extraction, MMIP enrichment and purification, and in-situ derivatization; S3: Plot the standard curve; S4: Detection: Perform UHPLC-MS / MS analysis and calculate the contents of 4 bacteriostatic agents in the hand sanitizer sample to be measured according to the standard curve; The 4 bacteriostatic agents are triclocarban (TCC), 4-chloro-3-methylphenol (PCMC), dichlorophenol (DCP), and benzylchlorophenol (BCP).

[0007] Furthermore, the synthesis of MMIP in step S1 specifically includes the following steps: S11: Preparation of Fe3O4@SiO2 magnetic carrier: Preparation of Fe3O4 nanoparticles: Dissolve FeCl3·6H2O and FeSO4·7H2O in ultrapure water, deoxygenate by passing nitrogen for 30 min to obtain an iron salt solution; Under N2 protection, heat the iron salt solution to 80 °C, mechanically stir at 500 rpm, add 25% NH4OH dropwise until pH = 11 ± 0.2, react at 80 °C for 30 min, wash the precipitate by magnetic separation 3 times with ultrapure water, then wash once with 0.02M NaCl, and vacuum dry at 40 °C for 6 h to obtain; SiO2 coating: Take Fe3O4 and disperse it in an ethanol-water mixed solvent, add TEOS dropwise at 0.5 mL / min, add 28% NH4OH dropwise until pH = 9.5 ± 0.1, react at 25 °C with mechanical stirring at 300 rpm for 6 h, collect Fe3O4@SiO2 by magnetic separation, wash 3 times with ethanol, and vacuum dry at 60 °C for 12 h; S12: Construction of the molecular imprinting layer: Pre-assembly solution: Dissolve the template dichlorophenol standard and the monomer methacrylic acid in acetonitrile, ultrasonicate at 25 °C for 30 min, and let stand at 4 °C for 12 h to complete pre-polymerization; Polymerization reaction: Add the cross-linking agent EGDMA, the initiator AIBN, and Fe3O4@SiO2, bubble nitrogen for 15 min to deoxygenate, heat in a water bath at 60 °C, and magnetically stir at 200 rpm under nitrogen protection for 12 h; Template elution: Collect the polymer by magnetic separation, perform Soxhlet extraction with a methanol-acetic acid eluent for 48 h, change the eluent every 12 h until no template residue is detected by HPLC, vacuum dry at 40 °C for 12 h, and sieve to obtain MMIP.

[0008] Furthermore, the pre-treatment in step S2 specifically includes the following steps: S21: Sample extraction: Take the hand sanitizer sample into a centrifuge tube, add methanol-water (8:2, v / v) mixed solvent, immediately vortex for 2 min (to avoid emulsification caused by too high local solvent concentration), perform ultrasonic-assisted extraction at 40 °C and 300 W for 10 min to promote the release of the target, centrifuge at 8000 rpm for 5 min, and transfer the supernatant to a new centrifuge tube; add the methanol-water mixed solvent to the residue again, repeat the vortexing, ultrasonic, and centrifugation steps, and combine the two supernatants to obtain the sample extract; slowly blow to near dry with nitrogen in a 40 °C water bath, add acetonitrile to redissolve the residue, vortex for 1 min, and filter through a 0.22 μm organic filter membrane to obtain the sample extract for purification; S22: MMIP enrichment and purification: S221: Activate MMIP: Place MMIP in a centrifuge tube, add methanol, vortex for 1 min, magnetically separate and discard the supernatant, repeat once; then equilibrate 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 at 1200 rpm for 5 min, magnetically separate and discard the supernatant; add acetonitrile-water (1:9, v / v) mixed solvent, vortex for 2 min, magnetically separate and discard the supernatant, repeat 2 times (to remove polar interfering substances such as surfactants (e.g., SLS) and glycerol); S223: Elution of the target: Add methanol - acetic acid (9:1, v / v) eluent, vortex for 3 min, magnetically separate, and collect the eluate; add the eluent again, combine the two eluates, and set aside; S23: In-situ derivatization: S231: Pretreatment: Blow the eluate to near dry with nitrogen at 40 °C, redissolve the residue with 2 volumes of acetonitrile, and transfer to a glass derivatization bottle; S232: Derivatization reaction: Sequentially add acetic anhydride and pyridine, immediately vortex for 10 s to mix evenly, react in a 70 °C water bath in the dark for 15 min; S233: Terminate the reaction: Take out the derivatization bottle, immediately cool it on ice for 2 min, blow to near dry with nitrogen at 40 °C to remove unreacted acetic anhydride and pyridine (to avoid mass spectrometry signal suppression); add 0.1% formic acid water - acetonitrile mixed solvent (95:5, v / v), vortex for 1 min, filter through a 0.22 μm nylon filter membrane, and the filtrate is directly used for UHPLC - MS / MS analysis.

[0009] Furthermore, in the step S11: Prepare Fe3O4 nanoparticles: The molar ratio of FeCl3·6H2O to FeSO4·7H2O is 2:1; SiO2 coating: The solid solution ratio of Fe3O4, mixed solvent to TEOS is 100 mg:100 mL:1 mL; the volume ratio of ethanol to water in the mixed solvent is 4:1.

[0010] Further, in the step S12: Pre-assembled solution: The dosage ratio of the template, monomer, and acetonitrile is 0.1 mmol: 0.4 mmol: 20 mL; Polymerization reaction: The dosage ratio of the template, cross-linking agent, initiator, and Fe3O4@SiO2 is 0.1 mmol: 1 mmol: 10 mg: 50 mg; Template elution: The volume ratio of methanol and acetic acid in the eluent is 9:1.

[0011] Further, in the step S21, the dosage ratio of the hand sanitizer sample, mixed solvent, and acetonitrile is 1.0 g: 15 mL: 2 mL; the volume ratio of methanol and water in the mixed solvent is 8:2.

[0012] Further, in the step S22: The mass ratio of MMIP to the hand sanitizer sample is 1:100; the dosage ratio of MMIP, methanol, acetonitrile, mixed solvent, and eluent is: 10 mg: 1 mL: 1 mL: 2 mL: 1.5 mL; The volume ratio of acetonitrile and water in the mixed solvent is 1:9; The volume ratio of methanol and acetic acid in the eluent is 9:1.

[0013] Further, in the step S23: The volume ratio of acetonitrile, acetic anhydride, pyridine, and mixed solvent is 10: 5: 2: 20; the volume ratio of 0.1% formic acid in water and acetonitrile in the mixed solvent is 95:5.

[0014] Further, in the 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 in water, phase B is acetonitrile; elution method: gradient elution, elution program: 0 min, 5% B; 0 - 2.0 min, phase B linearly changes from 5% to 95%; 2 - 5 min, 95% B; 5 - 7 min, phase B linearly changes from 95% to 5%; 7 - 9 min, 5% B.

[0015] Further, in the step S4, the mass spectrometry conditions are as follows: ion source: electrospray ionization source (ESI-); scanning mode: negative ion scanning; spray voltage: -4500 V; ion source temperature: 500 °C; nebulizing 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; qualitative 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; qualitative ion pair: m / z 231.0→119.0, collision energy: 25 eV.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: The method for simultaneously and rapidly detecting four bacteriostatic agents (triclocarban, 4-chloro-3-methylphenol, dichlorophenol, and benzyl chlorophenol) in hand sanitizer based on the MMIP-in-situ derivatization-UHPLC-MS / MS coupling technology of the present invention has a short pretreatment time, high sensitivity, strong anti-interference ability, good selectivity, high recovery rate, and good stability, filling the gap in the detection method for bacteriostatic and preservative agents in the current hand sanitizer product standards in China, and also providing new technical support for ensuring the quality and safety of hand sanitizer products. Description of the Drawings

[0017] Figure 1 is the transmission electron microscope image of MMIP in Example 1 of the present invention; Figure 2 is the TCC standard curve (0.5~200 μg / mL) of Example 1 of the present invention; Figure 3 is the PCMC-ac standard curve (0.5~200 μg / mL) of Example 1 of the present invention; Figure 4 is the DCP-ac standard curve (0.5~200 μg / mL) of Example 1 of the present invention; Figure 5 is the BCP-ac standard curve (0.5~200 μg / mL) of Example 1 of the present invention; Figure 6They are the total ion 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 implementation manners

[0018] In order to make the objectives and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0019] For the instruments, reagents, materials, etc. involved in the following embodiments, unless otherwise specified, they are all conventional instruments, reagents, materials, etc. existing in the prior art and can be obtained through regular commercial channels. For the experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, they are all conventional experimental methods, detection methods, etc. existing in the prior art.

[0020] Reagents: Triclocarban (TCC, ≥98%), 4-chloro-3-methylphenol (PCMC, ≥97%), dichlorophenol (DCP, ≥98%), benzylchlorophenol (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 King children's hand sanitizer and Safeguard antibacterial hand sanitizer, numbered K1 and K2 respectively).

[0021] In the following embodiments, the magnetic field strength for magnetic separation is 0.5 T, and the separation time is 1 min.

[0022] Example 1 This example provides a detection method for simultaneously and rapidly detecting 4 kinds of antibacterial agents in hand sanitizer, specifically including the following steps: S1: Synthesis of MMIP: S11: Preparation of Fe3O4@SiO2 magnetic carriers: Preparation of Fe3O4 nanoparticles: Dissolve 10 mmol of FeCl3·6H2O and 5 mmol of FeSO4·7H2O in 100 mL of ultrapure water, deoxygenate by purging with nitrogen for 30 min to obtain an iron salt solution; under N2 protection, heat the iron salt solution to 80 °C, mechanically stir at 500 rpm, add 25% NH4OH dropwise until the pH reaches 11 ± 0.2, react at 80 °C for 30 min, wash the precipitate by magnetic separation with ultrapure water 3 times, then wash with 0.02 M NaCl once, and dry in vacuum at 40 °C for 6 h to obtain (10 ± 2 nm, saturation magnetization 65 emu / g); SiO2 coating: Disperse 100 mg of Fe3O4 in 100 mL of an ethanol / water (4:1, v / v) mixed solvent, add 1 mL of TEOS dropwise at a rate of 0.5 mL / min, add 28% NH4OH dropwise until the pH reaches 9.5 ± 0.1, react at 25 °C with mechanical stirring at 300 rpm for 6 h, collect Fe3O4@SiO2 by magnetic separation, wash with ethanol 3 times, and dry in vacuum at 60 °C for 12 h (200 ± 20 nm); S12: Construction of the molecularly imprinted layer: Pre-assembly solution: Dissolve 0.1 mmol of the template dichlorophenol standard and 0.4 mmol of the monomer methacrylic acid in 20 mL of acetonitrile, ultrasonicate at 25 °C for 30 min, and let stand at 4 °C for 12 h to complete pre-polymerization; Although MMIP is synthesized using dichlorophenol (DCP) as the template molecule, its recognition mechanism is based on molecular structural similarity: triclocarban (TCC) and phenolic bacteriostatic agents (PCMC / DCP / BCP) both contain a benzene ring + chlorine substituent, and the amide bond (-NH-CO-) of triclocarban has a certain similarity in polarity with the phenolic hydroxyl group (-OH) of phenolic bacteriostatic agents, and the cavity of MMIP can adsorb both of them.

[0023] Polymerization reaction: Add 1 mmol of the cross-linking agent EGDMA, 10 mg of the initiator AIBN, 50 mg of Fe3O4@SiO2, purge with nitrogen for 15 min to deoxygenate, react in a 60 °C water bath with magnetic stirring at 200 rpm under nitrogen protection for 12 h; Template elution: Collect the polymer by magnetic separation, extract with a methanol - acetic acid (9:1, v / v) eluent in a Soxhlet extractor for 48 h (load about 500 mg of dry MMIP into the Soxhlet extraction thimble, block both ends with glass wool to prevent leakage, add 150 mL of the eluent to the round-bottom flask, assemble the Soxhlet extractor, heat in a 70 °C water bath for 48 h, reflux rate 6 - 8 cycles / h), change the eluent every 12 h until no template residue is detected by HPLC (LOD ≤ 0.01 μg / mL), change the eluent every 12 h until no template residue is detected by HPLC, dry in vacuum at 40 °C for 12 h, sieve, and obtain MMIP powder. See the transmission electron microscopy image in Figure 1(Stored in absolute ethanol at 4°C and dried with nitrogen before use).

[0024] S2: Pretreatment: S21: Sample extraction: Take 1.0 g of hand sanitizer sample into a 15 mL centrifuge tube, add 10 mL of methanol-water (8:2, v / v) mixed solvent, immediately vortex for 2 min (to avoid emulsification caused by too high local solvent concentration), at 40°C, ultrasonically assist extraction for 10 min at 300 W to promote the release of the target, centrifuge at 8000 rpm for 5 min, transfer the supernatant to a new centrifuge tube; add 5 mL of methanol-water mixed solvent to the residue again, repeat the vortex, ultrasonic, and centrifugation steps, combine the two supernatants (total 15 mL) to obtain the sample extract; slowly blow to nearly dry with nitrogen in a 40°C water bath, add 2 mL of acetonitrile to redissolve the residue, vortex for 1 min, filter through a 0.22 μm organic filter membrane to obtain the sample extract for purification; S22: MMIP enrichment and purification: S221: Activate MMIP: Take 10 mg of MMIP and place it in a 1.5 mL centrifuge tube, add 1 mL of methanol, vortex for 1 min, magnetically separate and discard the supernatant, repeat once; then equilibrate with 1 mL of 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 at 1200 rpm for 5 min, magnetically separate and discard the supernatant; add 2 mL of acetonitrile-water (1:9, v / v) mixed solvent, vortex for 2 min, magnetically separate and discard the supernatant, repeat 2 times (to remove polar interfering substances such as surfactants (e.g., SLS) and glycerol); S223: Target elution: Add 1 mL of methanol - acetic acid (9:1, v / v) eluent, vortex for 3 min, magnetically separate, and collect the eluate; add 0.5 mL of eluent again, combine the two eluates (total 1.5 mL) for standby; S23: In-situ derivatization: S231: Pretreatment: Blow the eluate to nearly dry with nitrogen at 40°C (about 50 μL), redissolve the residue with 2 volumes (100 μL) of acetonitrile, transfer to a glass derivatization bottle; S232: Derivatization reaction: Add 50 μL of acetic anhydride and 20 μL of pyridine in sequence, immediately vortex for 10 s to mix evenly, react in a 70°C water bath in the dark for 15 min; S233: Terminate the reaction: Take out the derivatization bottle, immediately cool it on ice for 2 min, blow to nearly dry with nitrogen at 40°C to remove unreacted acetic anhydride and pyridine; add 200 μL of 0.1% formic acid water - acetonitrile mixed solvent (95:5, v / v), vortex for 1 min, filter through a 0.22 μm nylon filter membrane, and the filtrate is directly used for UHPLC-MS / MS analysis.

[0025] In the above in-situ derivatization reaction, phenolic bacteriostatic agents (PCMC / DCP / BCP) contain active phenolic hydroxyl groups (-OH), which can undergo acetylation reactions with acetic anhydride. However, triclocarban (TCC) has no phenolic hydroxyl groups and does not react with acetic anhydride. Therefore, in-situ derivatization is targeted at phenolic bacteriostatic agents, and acetylation is used to improve their chromatographic retention and mass spectrometry sensitivity. Although triclocarban will not be derivatized by acetic anhydride, it has sufficient mass spectrometry response ([M-H] peak intensity in the negative ion mode), and can be directly detected by UHPLC-MS / MS. - Peak intensity), and can be directly detected by UHPLC-MS / MS.

[0026] Compared with the traditional SPE-HPLC detection method, the total pretreatment time exceeds 2 hours. The method of the present invention only requires extraction, MMIP purification, and derivatization, and the total time is less than 50 minutes, significantly shortening the pretreatment time.

[0027] S3: Plot the standard curve: (1) Stock solution (1000 μg / mL): Accurately weigh 10.0 mg of each standard into a 10 mL volumetric flask, dissolve with methanol and make up to the mark, vortex for 1 min, sonicate for 5 min to assist dissolution, store in the dark at -20 °C, and the validity period is 6 months.

[0028] (2) Preparation of standard solutions: Triclocarban: Dilute the stock solution with methanol-water (8:2, v / v) to prepare TCC standard solutions of 0.5, 1, 5, 10, 50, 100, and 200 μg / mL.

[0029] Phenolic derivatives (PCMC / DCP / BCP acetylation): 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; take 1 mL of each concentration working solution (0.5 - 200 μg / mL) into a glass derivatization bottle, add 50 μL of acetic anhydride + 20 μL of pyridine, react at 70 °C for 15 min, blow to dry with nitrogen, and re-dissolve to 1 mL with the initial mobile phase (0.1% formic acid in water - acetonitrile, 95:5) to obtain PCMC-ac, DCP-ac, and BCP-ac standard solutions with gradient concentrations.

[0030] (3) Establish the standard curve Perform UHPLC-MS / MS analysis on the series of concentrations of TCC standard solutions and PCMC-ac, DCP-ac, and BCP-ac standard solutions respectively.

[0031] 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 was 0.1% formic acid in water, and Phase B was acetonitrile; Elution mode: Gradient elution, and the elution program was as follows: 0 min, 5% B; 0 - 2.0 min, Phase B linearly changed from 5% to 95%; 2 - 5 min, 95% B; 5 - 7 min, Phase B linearly changed from 95% to 5%; 7 - 9 min, 5% B. Mass spectrometry conditions were as follows: Ion source: Electrospray ionization source (ESI-); Scanning mode: Negative ion scanning; Spray voltage: -4500 V; Ion source temperature: 500 °C; Nebulizing 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; Qualitative 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; Qualitative ion pair: m / z231.0→119.0, Collision energy: 25 eV.

[0032] Taking the peak area as the ordinate and the concentration as the abscissa, a standard curve was plotted, as shown in Table 1. Figures 2-6 The results showed that TCC, PCMC-ac, DCP-ac, and BCP-ac all had good linear relationships in the range of 0.5 - 200 μg / mL.

[0033] Table 1 Standard curve

[0034] Experimental Example 1 Detection limit (LOD) and quantification limit (LOQ) Blank samples (hand sanitizers without the target substance) were spiked with the spiking 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 measured 6 times repeatedly (n = 6), and the signal-to-noise ratio (S / N) was calculated. The lowest concentration with S / N≥3 was taken as the LOD, and the lowest concentration with S / N≥3 and RSD≤20% was taken as the LOQ. The results are shown in Table 2.

[0035] Table 2 LOD and LOQ

[0036] Experimental Example 2 Spiked Recovery Rate and Precision Take blank samples, set 3 spiked levels (0.1, 1, 10 μg / mL) for spiking, after processing by the above method, measure each level 6 times repeatedly (n = 6), and calculate the spiked recovery rate and precision. As can be seen from Table 3, the average recovery rates of the 4 bacteriostatic agents at 3 spiked levels are between 94.2% and 108.2%, and the RSDs are all less than 10%, indicating that the recovery rate and precision of the method of the present invention are good, and it shows that this method is accurate, stable and reliable.

[0037] Table 3 Spiked Recovery Rate and Precision

[0038] Experimental Example 3 Anti-interference Experiment Take blank samples, add 1 μg / mL target substance and each interfering substance (the concentration of the interfering substance is the commonly used amount in actual products) respectively, perform pretreatment process and UHPLC-MS / MS analysis, and calculate the recovery rate.

[0039] As can be seen from Table 4, the recovery rates of the interfering substances in the 4 bacteriostatic agents are 92.5% - 106.2%, indicating that the method of the present invention is not easily interfered by other components in the hand sanitizer and has good selectivity.

[0040] Table 4 Anti-interference Results

[0041] Experimental Example 5 Sample Detection 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 sanitizer to be detected (K1, K2) according to the standard curve: 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 detected, calculate the concentrations (μg / mL, n = 6) of TCC, PCMC-ac, DCP-ac, and BCP-ac in the filtrate, and record them as C TCC 、C PCMC-ac 、C DCP-ac 、C BCP-ac ; Further calculate the concentration in the original hand sanitizer sample according to the concentration of each bacteriostatic agent in the filtrate: DF = (V 净化 / V 提取 ) × (V 复溶 / V 洗脱) = (2 mL / 15 mL) × (0.1 mL / 1.5 mL) = 1 / 112.5; For TCC: C TCC = C TCC-滤液 × (1 / DF) = C TCC-滤液 × 112.5 (μg / mL); For PCMC: 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); For DCP: 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); For BCP: 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); Accordingly, calculate the contents of TCC, PCMC, DCP, and BCP in the hand sanitizer sample to be tested, and the results are shown in Table 5.

[0042] Table 5 Contents of 4 bacteriostatic agents in the sample to be tested

[0043] The "Technical Specifications for Cosmetics Safety" 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 benzyl chlorophenol in rinse-off products is 0.1% (1000 μg / mL). Based on this, the contents of the 4 bacteriostatic agents in the 2 hand sanitizer samples tested in this example are all within the specified limits and comply with the safety specifications.

[0044] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A detection method for simultaneously and rapidly detecting 4 kinds of bacteriostatic agents in hand sanitizer, characterized in that: Specifically, it includes the following steps: S1: MMIP synthesis: including the preparation of Fe3O4@SiO2 magnetic carriers and the construction of the molecular imprinting layer; S2: Pretreatment: including sample extraction, MMIP enrichment and purification, and in-situ derivatization; S3: Plot the standard curve; S4: Detection: Perform UHPLC-MS / MS analysis, and calculate the contents of 4 bacteriostatic agents in the hand sanitizer sample to be detected according to the standard curve; The 4 bacteriostatic agents are trichlocarban, 4-chloro-3-methylphenol, dichlorophenol, and benzyl chlorophenol.

2. The detection method for simultaneously and rapidly detecting 4 kinds of bacteriostatic agents in hand sanitizer according to claim 1, wherein: The synthesis of MMIP in step S1 specifically includes the following steps: S11: Preparation of Fe3O4@SiO2 magnetic carriers: Preparation of Fe3O4 nanoparticles: Dissolve FeCl3·6H2O and FeSO4·7H2O in ultrapure water, deoxygenate by passing nitrogen for 30 min to obtain an iron salt solution; Under N2 protection, heat the iron salt solution to 80 °C, mechanically stir at 500 rpm, add 25% NH4OH dropwise until pH = 11 ± 0.2, react at 80 °C for 30 min, wash the precipitate by magnetic separation with ultrapure water 3 times, then wash with 0.02M NaCl once, and vacuum dry at 40 °C for 6 h to obtain; SiO2 coating: Take Fe3O4 and disperse it in an ethanol-water mixed solvent, dropwise add TEOS at 0.5 mL / min, add 28% NH4OH dropwise until pH = 9.5 ± 0.1, react at 25 °C with mechanical stirring at 300 rpm for 6 h, collect Fe3O4@SiO2 by magnetic separation, wash with ethanol 3 times, and vacuum dry at 60 °C for 12 h; S12: Construction of the molecular imprinting layer: Pre-assembly solution: Dissolve the template dichlorophenol standard and the monomer methacrylic acid in acetonitrile, ultrasonically treat at 25 °C for 30 min, and let stand at 4 °C for 12 h to complete pre-polymerization; Polymerization reaction: Add the cross-linking agent EGDMA, the initiator AIBN, and Fe3O4@SiO2, bubble with nitrogen for 15 min to remove oxygen, perform water bath at 60 °C, and magnetically stir at 200 rpm under nitrogen protection for 12 h; Template elution: Collect the polymer by magnetic separation, perform Soxhlet extraction with the eluent for 48 h, change the eluent every 12 h until no template residue is detected by HPLC, vacuum dry at 40 °C for 12 h, and sieve to obtain MMIP.

3. The detection method for simultaneously and rapidly detecting 4 bacteriostatic agents in hand sanitizer according to claim 1, characterized in that: The pretreatment in step S2 specifically includes the following steps: S21: Sample extraction: Take the hand sanitizer sample into a centrifuge tube, add a methanol-water mixed solvent, immediately vortex for 2 min, perform ultrasonic-assisted extraction at 40 °C with 300 W for 10 min, centrifuge at 8000 rpm for 5 min, and transfer the supernatant to a new centrifuge tube; Add the methanol-water mixed solvent to the residue again, repeat the vortexing, ultrasonic, and centrifugation steps, combine the two supernatants to obtain a sample extract; Slowly blow to near dry with nitrogen in a 40 °C water bath, add acetonitrile to redissolve the residue, vortex for 1 min, and filter through a 0.22 μm organic filter membrane to obtain a sample extract for purification; S22: MMIP enrichment and purification: S221: Activate MMIP: Place MMIP in a centrifuge tube, add methanol, vortex for 1 min, magnetically separate and discard the supernatant, and repeat once; then equilibrate 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 at 1200 rpm for 5 min, magnetically separate and discard the supernatant; add an acetonitrile - water mixed solvent, vortex for 2 min, magnetically separate and discard the supernatant, and repeat 2 times; S223: Elution of Target Substance: Add a methanol - acetic acid eluent, vortex for 3 min, magnetically separate, and collect the eluate; add the eluent again, combine the two eluates, and set aside for use; S23: In - situ Derivatization: S231: Pretreatment: Blow - dry the eluate to near - dryness under nitrogen at 40 °C, redissolve the residue with 2 volumes of acetonitrile, and transfer it to a glass derivatization bottle; S232: Derivatization Reaction: Sequentially add acetic anhydride and pyridine, immediately vortex and shake for 10 s to mix evenly, react in a 70 °C water bath in the dark for 15 min; S233: Terminate the Reaction: Take out the derivatization bottle, immediately cool it on ice for 2 min, blow - dry to near - dryness under nitrogen at 40 °C; add a 0.1% formic acid water - acetonitrile mixed solvent, vortex for 1 min, filter through a 0.22 - μm nylon filter membrane, and the filtrate is directly used for UHPLC - MS / MS analysis.

4. The detection method for simultaneously and rapidly detecting 4 bacteriostatic agents in hand sanitizer according to claim 2, wherein: In the said step S11: Prepare Fe3O4 nanoparticles: The molar ratio of FeCl3·6H2O to FeSO4·7H2O is 2:1; SiO2 Coating: The solid - solution ratio of Fe3O4, the mixed solvent to TEOS is 100 mg:100 mL:1 mL; the volume ratio of ethanol to water in the mixed solvent is 4:

1.

5. The detection method for simultaneously and rapidly detecting 4 kinds of bacteriostatic agents in hand sanitizer according to claim 2, wherein: In the said step S12: Pre - assembled solution: The dosage ratio of the template, monomer to acetonitrile is 0.1 mmol:0.4 mmol:20 mL; Polymerization reaction: The dosage ratio of the template, cross - linker, initiator to Fe3O4@SiO2 is 0.1 mmol:1 mmol:10 mg:50 mg; Template elution: The volume ratio of methanol to acetic acid in the eluent is 9:

1.

6. The detection method for simultaneously and rapidly detecting 4 kinds of bacteriostatic agents in hand sanitizer according to claim 3, wherein: In the said step S21, the dosage ratio of the hand sanitizer sample, the mixed solvent to acetonitrile is 1.0 g:15 mL:2 mL; the volume ratio of methanol to water in the mixed solvent is 8:

2.

7. The detection method for simultaneously and rapidly detecting 4 bacteriostatic agents in hand sanitizer according to claim 3, characterized in that: In the said step S22: The mass ratio of MMIP to the hand sanitizer sample is 1:100; the dosage ratio of MMIP, methanol, acetonitrile, the mixed solvent, and the eluent is: 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.

8. The detection method for simultaneously and rapidly detecting 4 bacteriostatic agents in hand sanitizer according to claim 3, wherein: In the said step S23: The volume ratio of acetonitrile, acetic anhydride, pyridine, and the mixed solvent is 10:5:2:20; the volume ratio of 0.1% formic acid water to acetonitrile in the mixed solvent is 95:

5.

9. The detection method for simultaneously and rapidly detecting 4 kinds of bacteriostatic agents in hand sanitizer according to claim 1, wherein: In the said 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 in water, and Phase B is acetonitrile; Elution mode: Gradient elution. The elution program is as follows: at 0 min, 5% B; from 0 to 2.0 min, Phase B linearly changes from 5% to 95%; from 2 to 5 min, 95% B; from 5 to 7 min, Phase B linearly changes from 95% to 5%; from 7 to 9 min, 5% B.

10. The detection method for simultaneously and rapidly detecting 4 kinds of bacteriostatic agents in hand sanitizer according to claim 1, characterized in that: In the said step S4, the mass spectrometry conditions are: Ion source: Electrospray ionization source (ESI-); Scanning mode: Negative ion scanning; Spray voltage: -4500 V; Ion source temperature: 500 °C; Nebulizing 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; Qualitative 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; Qualitative ion pair: m / z 231.0→119.0, Collision energy: 25 eV.

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