Kit and method for simultaneously determining contents of 11 benzoate, metabolites thereof and chlorine-containing antibacterial agent, and application of composition

Abstract

The invention provides a kit and a method for simultaneously determining the contents of 11 benzoate, metabolites thereof and chlorine-containing antibacterial agents, and application of a composition. According to the method, acetonitrile and a zinc sulfate (containing an antioxidant VC) solution are adopted for protein precipitation, pretreatment is carried out in a liquid-liquid extraction mode by using ethyl acetate containing formic acid, an eluent is collected, blow-dried and redissolved, then liquid chromatography-tandem mass spectrometry detection can be carried out, and the quantification lower limit is reduced through a trap column. The method provided by the invention realizes detection of two water-soluble benzoic acids which are difficult to detect, and has the advantages of high extraction efficiency of detection index compounds, high recovery rate, low detection quantification lower limit, convenience in operation, better effect and lower cost.

Description

[0001] This application is a divisional application of parent case 202310690833.4, with application date June 12, 2023. Technical Field

[0002] The invention belongs to the technical field of chemical analysis and relates to a kit, a method and a composition for simultaneously determining the contents of 11 benzoic acid esters and their metabolites and chlorine-containing antibacterial agents. Background Art

[0003] Parabens are preservatives, triclosan and triclocarban are antibacterial agents, and are often used in various foods, medicines, and cosmetics. They are therefore ubiquitous in the daily environment and can easily enter the human body through skin contact and daily diet. The main metabolite of parabens in the body is parahydroxybenzoic acid, which will further undergo oxidative metabolism and introduce a hydroxyl group to become dihydroxybenzoic acid. This type of compound is an endocrine disruptor, has estrogenic activity, and has a certain effect on reproductive function. Therefore, in recent years, research on the correlation between the concentrations of this type of compound in urine and blood and various hormone abnormalities has become a hot topic.

[0004] At present, such substances are commonly detected by solid phase extraction or multiple liquid-liquid extraction methods. For example, in the literature Qiong Luo, Hongna Zhang, Zongwei Cai, et al. Simultaneous determination of triclosan, triclocarban, triclocarban metabolites and byproducts in urine and serum by ultra-high-performance liquid chromatography electrospray ionization tandemmass spectrometry [J]. Rapid Commun Mass Spectrom, 2021, 35(14): 136390, the sampling volume was 500 μL, and Waters Oasis HLB plate was used for SPE solid phase extraction. This method has a large sampling volume and requires the use of SPE plates, which is costly and has relatively complicated pretreatment. Another example is patent CN113671093A, in which the sample type is urine. Hydroxybenzoic acid ester compounds in urine will combine with glucuronic acid. After enzymatic hydrolysis, liquid-liquid extraction is used, and a total of three extractions are required. The content of hydroxybenzoic acid ester compounds in urine is higher than that in serum, so the detection difficulty of urine samples is lower than that of serum samples. However, the recovery rate of parahydroxybenzoic acid (4-HB) provided in the patent is only about 60%.

[0005] In addition, this type of compound is widely used as a preservative and antibacterial agent in daily life, so there is often background in plastic tubes, solvents and instrument pipelines during the detection process. According to the literature report, the lower limit of quantification of triclosan (TCS) is 0.9 ng / mL, which causes great interference to trace detection.

[0006] Therefore, there is an urgent need for a new technology for detecting benzoic acid esters and their metabolites and chlorine-containing antimicrobial agents, which can improve the efficiency of extracting such compounds from samples, the recovery rate of detection indicators, and reduce the quantitative limit to increase sensitivity. Summary of the invention

[0007] To solve the above problems, the purpose of the present invention is to provide a kit, method and use of a composition for simultaneously determining the contents of 11 benzoic acid esters and their metabolites, and chlorine-containing antibacterial agents; and to simultaneously determine 11 benzoic acid esters and their metabolites, and chlorine-containing antibacterial agents in human blood by liquid chromatography tandem mass spectrometry.

[0008] The 11 benzoic acid esters and their metabolites, and chlorine-containing antibacterial agents are: butyl parahydroxybenzoate (BuP), methyl parahydroxybenzoate (MeP), ethyl parahydroxybenzoate (EtP), ethyl 3,4-dihydroxybenzoate (OH-EtP), 3,4-dihydroxybenzoic acid (3,4-DHB), benzyl parahydroxybenzoate (BzP), heptyl parahydroxybenzoate (HeP), parahydroxybenzoic acid (4-HB) and methyl 3,4-dihydroxybenzoate (OH-MeP), triclocarban (TCC), and triclosan (TCS).

[0009] First, the invention mainly uses ZnSO4 solution and acetonitrile to coprecipitate proteins in serum, and the reagent used for liquid-liquid extraction is ethyl acetate (containing 1% formic acid (FA)), which improves the extraction recovery rate of 3,4-dihydroxybenzoic acid (3,4-DHB) and p-hydroxybenzoic acid (4-HB). Among these 11 detection indicators, two benzoic acids have good water solubility, and the remaining 9 compounds have good fat solubility and large differences in properties. Therefore, in most literatures, the methods for extracting these compounds mostly use SPE, and liquid-liquid extraction cannot guarantee the extraction recovery rate of the two benzoic acids with good water solubility.

[0010] The present invention ensures the extraction recovery rate of each compound through the improvement of pre-treatment, especially the extraction recovery rate of two water-soluble benzoic acids. Protein precipitation is first performed and then liquid-liquid extraction is performed. 1% formic acid is added to the extraction solvent ethyl acetate, so that the two water-soluble benzoic acids are in a molecular state in the solution, which improves the extraction efficiency. The added formic acid will make the acetonitrile precipitation of protein incomplete, resulting in reduced extraction efficiency. Therefore, in the protein precipitation step, we use ZnSO4 solution and acetonitrile for co-precipitation, which can improve the extraction efficiency after protein precipitation, thereby improving the recovery rate. Compared with the SPE pre-treatment in the literature, this pre-treatment method has higher extraction efficiency, better effect and lower cost.

[0011] Secondly, the present invention connects a Trap column between a mobile phase mixer and a six-way valve, thereby avoiding interference of background peaks in the mobile phase and the front-end pipeline of the instrument on triclosan (TCS), delaying the peak time of the background interference and staggering the peak of the compound in the sample, significantly reducing the background peak area by 35 times, and further reducing the quantitative lower limit of triclosan (TCS).

[0012] On the one hand, the present invention provides a kit for simultaneously determining the contents of 11 benzoic acid esters and their metabolites and chlorine-containing antibacterial agents in human blood by liquid chromatography tandem mass spectrometry, the kit comprising an internal standard working solution, a zinc sulfate solution and an extractant; the internal standard working solution comprises acetonitrile, and the extractant comprises ethyl acetate.

[0013] The 11 benzoic acid esters and their metabolites and chlorine-containing antibacterial agents are: butyl parahydroxybenzoate, methyl parahydroxybenzoate, ethyl parahydroxybenzoate, ethyl 3,4-dihydroxybenzoate, 3,4-dihydroxybenzoic acid, benzyl parahydroxybenzoate, heptyl parahydroxybenzoate, parahydroxybenzoic acid, methyl 3,4-dihydroxybenzoate, triclocarban, and triclosan.

[0014] Furthermore, the concentration of the zinc sulfate solution is 30-89 mg / ml, and the volume ratio of the internal standard working solution to the zinc sulfate solution is 9:1-1:1.

[0015] Furthermore, the extractant also contains formic acid.

[0016] In some embodiments, the extractant is ethyl acetate containing 1% formic acid.

[0017] In principle, adding 1% formic acid to the extraction solvent ethyl acetate can make the two benzoic acids present in a molecular state in the solution and improve the extraction efficiency. However, the added formic acid will cause incomplete precipitation of the protein with acetonitrile, resulting in reduced extraction efficiency. Therefore, in the protein precipitation step, we use ZnSO4 solution and acetonitrile for co-precipitation, which can improve the extraction efficiency after protein precipitation, thereby improving the recovery rate.

[0018] Furthermore, the extractant is used for liquid-liquid extraction.

[0019] Furthermore, the volume ratio of the internal standard working solution to the zinc sulfate solution is 9:1-1:1.

[0020] Furthermore, the zinc sulfate solution has a concentration of 89 mg / ml and also contains vitamin C.

[0021] In some embodiments, the mass ratio of zinc sulfate to vitamin C is 89:1, and the concentration of vitamin C is 1 mg / ml.

[0022] In principle, the two detection indicators of 3,4-dihydroxybenzoic acid (3,4-DHB) and p-hydroxybenzoic acid (4-HB) in the present invention are easily oxidized during the extraction process. Adding antioxidant vitamin C can effectively avoid their oxidation and increase the extraction efficiency and recovery rate.

[0023] On the other hand, the present invention provides a method for simultaneously determining the contents of 11 benzoic acid esters and their metabolites and chlorine-containing antibacterial agents in human blood by liquid chromatography-tandem mass spectrometry, wherein the method uses the above-mentioned kit for sample pretreatment.

[0024] The 11 benzoic acid esters and their metabolites and chlorine-containing antibacterial agents are: butyl parahydroxybenzoate, methyl parahydroxybenzoate, ethyl parahydroxybenzoate, ethyl 3,4-dihydroxybenzoate, 3,4-dihydroxybenzoic acid, benzyl parahydroxybenzoate, heptyl parahydroxybenzoate, parahydroxybenzoic acid, methyl 3,4-dihydroxybenzoate, triclocarban, and triclosan.

[0025] Furthermore, the method comprises the following steps:

[0026] (1) adding an internal standard working solution and a zinc sulfate solution to the sample, wherein the internal standard working solution contains acetonitrile;

[0027] (2) adding an extractant to perform liquid-liquid extraction, wherein the extractant comprises ethyl acetate;

[0028] (3) Blow dry and re-dissolve;

[0029] (4) Liquid chromatography tandem mass spectrometry detection.

[0030] Furthermore, the volume ratio of the internal standard working solution to the zinc sulfate solution in step (1) is 9:1-1:1.

[0031] Preferably, the volume ratio of the internal standard working solution to the zinc sulfate solution in step (1) is 9:1.

[0032] Furthermore, the zinc sulfate solution in step (1) further contains vitamin C (VC), and the amount of vitamin C added is

[0033] 0.2~1mg / mL.

[0034] Furthermore, the extractant in step (2) also contains formic acid, and the formic acid content is 0.5% to 2%.

[0035] This method mainly adopts liquid-liquid extraction, which is more convenient and less expensive than SPE, and at the same time improves the extraction rate of two benzoic acid compounds (4-HB and 3,4-DHB).

[0036] Furthermore, in the step (4) of liquid chromatography tandem mass spectrometry, a trap column needs to be connected between the mobile phase mixer and the six-way valve of the injector.

[0037] In principle, a Trap column is connected between the mobile phase mixer and the six-way valve to avoid the interference of background peaks in the mobile phase and the front-end pipeline of the instrument on triclosan (TCS), delay the elution time of the background interference peak, and stagger the elution time of the compound in the sample, significantly reducing the background peak area by 35 times, thereby reducing the quantitative limit of TCS.

[0038] In some embodiments, in the liquid chromatography tandem mass spectrometry of step (4), the mobile phases are: mobile phase A: water (containing 0.05% HAc), mobile phase B: acetonitrile (0.05% HAc).

[0039] Adding a small amount of acetic acid to the mobile phase can optimize the peak shape of 3,4-DHB and solve the problem of carboxylic acid tailing; under the above mobile phase, the extraction peaks of 11 detection index compounds have high separation and can all be detected simultaneously.

[0040] In another aspect, the present invention provides a use of a composition for preparing a preparation for improving the extraction recovery rate of 3,4-dihydroxybenzoic acid (3,4-DHB) and p-hydroxybenzoic acid (4-HB), wherein the composition comprises acetonitrile and zinc sulfate.

[0041] In some embodiments, the volume ratio of acetonitrile to zinc sulfate solution is 9:1.

[0042] In some embodiments, the zinc sulfate solution also contains vitamin C to combat oxidation by 3,4-dihydroxybenzoic acid (3,4-DHB) and p-hydroxybenzoic acid (4-HB).

[0043] The beneficial effects of the present invention are:

[0044] 1. The method for detecting the contents of 11 benzoic acid esters and their metabolites and chlorine-containing antibacterial agents in human blood provided by the present invention can realize the simultaneous detection of the contents of 11 benzoic acid esters and their metabolites and chlorine-containing antibacterial agents in human blood.

[0045] 2. Through the improvement and innovative combination of protein precipitants and extractants, the recovery rates of 11 detection index compounds were significantly improved, and the extraction peak areas of 11 detection index compounds were significantly increased. In particular, the problem of two water-soluble benzoic acid compounds (4-HB and 3,4-DHB) being difficult to extract and detect by liquid-liquid extraction was solved.

[0046] 3. This method mainly adopts liquid-liquid extraction, which is more convenient to operate than SPE, replaces the extraction technology using SPE plates, reduces costs, and at the same time improves the extraction rate of two benzoic acid compounds (4-HB and 3,4-DHB).

[0047] 4. Add a trap column to the liquid phase-mass spectrometry system, increase the connection of the trap column between the mobile phase mixer and the six-way valve of the injector, stagger the retention time of the background peak in the mobile phase and the front-end pipeline of the instrument and the triclosan (TCS) in the sample, and significantly reduce the impact of the interfering peak in the blank on the quantitative results. It can effectively reduce the interference of the background in the mobile phase and pipeline on TCS, reduce the quantitative lower limit of TCS to 0.05ng / mL, and realize trace detection of detection indicators. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Chromatogram without trap column + blank solvent 50% CH3OH

[0049] Figure 2 Chromatogram using trap column + blank solvent 50% CH3OH

[0050] Figure 3 Chromatogram of C1 without trap column + TCS calibrator

[0051] Figure 4 Chromatogram using trap column + TCS calibrator C1

[0052] Figure 5 Chromatogram of MeP detected by liquid chromatography-tandem mass spectrometry

[0053] Figure 6 Chromatogram of TCC detected by liquid chromatography-tandem mass spectrometry

[0054] Figure 7 Chromatogram of TCS detected by liquid chromatography-tandem mass spectrometry

[0055] Figure 8 Chromatogram of EtP detected by liquid chromatography-tandem mass spectrometry

[0056] Fig. 9 Chromatogram of BuP detected by liquid chromatography-tandem mass spectrometry

[0057] Fig.10Chromatogram of HeP detected by liquid chromatography-tandem mass spectrometry

[0058] Fig.11 Chromatogram of BzP detected by liquid chromatography-tandem mass spectrometry

[0059] Fig.12 Chromatogram of OH-MeP detected by liquid chromatography-tandem mass spectrometry

[0060] Fig.13 Chromatogram of OH-EtP detected by liquid chromatography-tandem mass spectrometry

[0061] Fig.14 Chromatogram of 3,4-DHB detected by liquid chromatography-tandem mass spectrometry

[0062] Fig.15 Chromatogram of 4-HB detected by liquid chromatography-tandem mass spectrometry DETAILED DESCRIPTION

[0063] The preferred embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings. It should be pointed out that the following embodiments are intended to facilitate the understanding of the present invention and do not have any limiting effect on it. All features disclosed in the embodiments of the present invention, or all steps in the methods or processes disclosed, except for mutually exclusive features and / or steps, can be combined in any way.

[0064] Example 1: Sample preparation, pretreatment, detection, and analysis

[0065] 1. Sample preparation

[0066] 1. Preparation of Calibrators and Quality Control Samples

[0067] Triclocarban (TCC), triclosan (TCS), butyl parahydroxybenzoate (BuP), methyl parahydroxybenzoate (MeP), ethyl parahydroxybenzoate (EtP), ethyl 3,4-dihydroxybenzoate (OH-EtP), 3,4-dihydroxybenzoic acid (3,4-DHB), benzyl parahydroxybenzoate (BzP), heptyl parahydroxybenzoate (HeP), parahydroxybenzoic acid (4-HB) and methyl 3,4-dihydroxybenzoate (OH-MeP) standards were prepared into mixed solutions as the stock solutions of standard working solutions and quality control working solutions. The stock solutions of the standard working solutions were mixed with methanol at a volume ratio of 1:19, and the stock solutions of the quality control working solutions were mixed with mixed blank serum at a volume ratio of 1:99 to prepare calibration curves and quality control samples, respectively. The calibration curve was prepared with 8 concentration series (C1-C8), and the quality control samples were prepared with 3 concentration series (LQC, MQC and HQC), as shown in Tables 1 and 2, respectively.

[0068] Table 1: 8 series of concentrations in the calibration curve (C1 to C8)

[0069]

[0070] Table 2: Three series of spike concentrations in quality control samples (LQC, MQC and HQC)

[0071]

[0072] 2. Preparation of internal standard solution

[0073] (1) Preparation of internal standard stock solution

[0074] A mixed internal standard working solution was prepared. The concentrations of TCC-13C6, TCS-D3, BuP-D4, MeP-D4, EtP-D4, OH-EtP-13C, 3,4-DHB-D3, BzP-D7, HeP-D4, and 4-HB-D4 were 10 μg / mL, 40 μg / mL, 20 μg / mL, 20 μg / mL, 16 μg / mL, 10 μg / mL, 40 μg / mL, 20 μg / mL, 20 μg / mL, and 80 μg / mL, respectively.

[0075] (2) Preparation of internal standard secondary stock solution

[0076] Pipette 15 μL of the internal standard stock solution and add 1485 μL of methanol. The concentrations of the internal standards TCC-13C6, TCS-D3, BuP-D4, MeP-D4, EtP-D4, OH-EtP-13C, 3,4-DHB-D3, BzP-D7, HeP-D4, and 4-HB-D4 are 100 ng / mL, 400 ng / mL, 200 ng / mL, 200 ng / mL, 160 ng / mL, 100 ng / mL, 400 ng / mL, 200 ng / mL, 200 ng / mL, and 800 ng / mL, respectively.

[0077] (3) Preparation of internal standard working solution

[0078] Pipette 0.5 mL of the internal standard stock solution and add 99.5 mL of acetonitrile thereto to obtain the final internal standard working solution. The final concentrations of the internal standards TCC-13C6, TCS-D3, BuP-D4, MeP-D4, EtP-D4, OH-EtP-13C, 3,4-DHB-D3, BzP-D7, HeP-D4, and 4-HB-D4 were 0.5 ng / mL, 2 ng / mL, 1 ng / mL, 1 ng / mL, 0.8 ng / mL, 0.5 ng / mL, 2 ng / mL, 1 ng / mL, 1 ng / mL, and 4 ng / mL, respectively.

[0079] 3. Preparation of ZnSO4 solution

[0080] Weigh 890 mg ZnSO4 and 10 mg VC on a balance, add 10 mL water to dissolve and prepare 89 mg / mL ZnSO4 solution (containing 1 mg / mL VC).

[0081] 2. Sample pretreatment

[0082] The human serum samples, calibrators and quality control products were pre-treated using the following method.

[0083] (1) 200 μL of double blank (methanol), single blank (methanol), standard curve and serum (including quality control) were pipetted and added to 96-well plates respectively; single blank is the solvent with internal standard added only without standard substance, double blank is the solvent with neither standard substance nor internal standard added, both single blank and double blank are pre-treated, double blank is used to measure the background of solvent after pre-treatment, and single blank is used to measure the background of solvent after adding internal standard;

[0084] (2) Add 60 μL of ZnSO4 solution (89 mg / mL) to all samples, add 540 μL of acetonitrile to the double blank, and add 540 μL of internal standard solution to the remaining samples, and vortex for 5 min;

[0085] (3) Add 800 μL of ethyl acetate (containing 1% FA) and vortex for 15 min;

[0086] (4) Centrifuge at 4000 rpm for 10 min and take 900 μL of the upper organic phase;

[0087] (5) Add 900 μL of ethyl acetate (containing 1% FA) to the residual solution again and vortex for 15 min;

[0088] (6) Centrifuge at 4000 rpm for 10 min, take 900 μL of the upper organic phase, and combine the two organic phases;

[0089] (7) Dry with nitrogen gas, add 150 μL of reconstitution solution (80% methanol) to the residue for reconstitution;

[0090] (8) Centrifuge at 4000 rpm for 5 min and inject 5 μL of the sample for detection.

[0091] 3. Sample testing

[0092] Liquid chromatography tandem mass spectrometry system: AB Sciex LC-MS / MS system Triple Quad 6500 system

[0093] Column: Luna @ Omega Polar C18(3μm,3.0×50mm,Phenomenex)

[0094] Mobile phase A: water (containing 0.05% HAc), mobile phase B: acetonitrile (0.05% HAc)

[0095] Flow rate: 0.5 mL / min, column temperature: 40°C

[0096] Injector temperature: 15°C

[0097] Injection volume: 5 μL

[0098] The gradient elution conditions are shown in Table 3 below.

[0099] Table 3: Gradient elution conditions

[0100]

[0101] Mass spectrometry detection conditions: the ion source is an electrospray ion source (ESI), negative ion mode, ion source parameters are: ion source heating temperature is 500 ° C, curtain gas (CUR) is 30 psi, collision gas (CAD) is 9 psi, desolvation gas (GS1) is 55 psi, auxiliary heating gas (GS2) is 45 psi. The scanning mode is multiple reaction monitoring (MRM) mode.

[0102] The mass spectrometry parameters of the compounds are shown in Table 4:

[0103] Table 4: Mass spectrometry parameters of the compounds

[0104]

[0105]

[0106] 4. Data Processing and Analysis

[0107] Plotting a standard curve

[0108] The standard curve uses the concentration of each compound calibrator as the horizontal axis and the ratio of the peak area of ​​each compound to its corresponding internal standard peak area as the vertical axis to perform linear fitting to obtain the linear regression equation. The linear equation and correlation coefficient r are shown in Table 5. The linearity is good, and the correlation coefficient r is greater than 0.9900.

[0109] Table 5: Linear equation and correlation coefficient r

[0110]

[0111] 5. Accuracy, precision and spike recovery

[0112] 1. Accuracy and precision

[0113] The accuracy and precision of the 11 detection index compounds in the present invention are obtained by analyzing the quality control products at three concentration points of low, medium and high distributed in the linear range. The acceptance standard is that the accuracy between the mean of the measured value and the theoretical value is between 85% and 115%, and the precision is represented by the coefficient of variation (CV%). The precision (CV) ≤ 15% meets the requirements. Five batches of precision tests show that the accuracy of the three concentrations of low, medium and high is between 91.8% and 110.7%, and the batch-to-batch precision (CV) is between 2.14% and 5.56%, which meets the requirements.

[0114] 2. Spike recovery rate

[0115] The stock solution of the mixed standard working solution was used to prepare serum quality control samples of three concentrations: low, medium and high. The samples were pre-treated according to the method of the best embodiment. The spiked recovery and precision data of each compound are shown in Table 6.

[0116] Table 6: Statistical results of the recovery rate of each compound

[0117]

[0118]

[0119] Test results: The recovery rates of the spiked compounds were all in the range of 85%-115%, and the precision CV was less than 15%, meeting the quantitative requirements of biological sample analysis.

[0120] Example 2: Effects of different pre-treatment schemes on test results

[0121] Different pre-treatment schemes have a huge impact on the test results. In this embodiment, we comprehensively consider the pre-treatment schemes to find a most convenient, practical, economical, fast and effective pre-treatment scheme.

[0122] We set up seven schemes as shown in Table 7, selected the quality control product LQC described in Example 1 as the sample to be tested, and used the pretreatment schemes 1 to 7 described in Table 7 for pretreatment. Since the 11 compounds to be tested are mainly divided into two categories according to their physical properties: fat-soluble and water-soluble. The fat-soluble compounds are TCS, TCC, MeP, EtP, BuP, HeP, BzP, OH-EtP, and OH-MeP. The detection results of the above nine compounds show the same trend, and we selected TCS as the representative; the water-soluble compounds are 3,4-DHB and 4-HB, which are difficult to detect in other technical solutions. They are the objects we focus on solving, and we consider them separately; the detection results are shown in Table 7.

[0123] Table 7: Effects of different pre-treatment schemes on test results

[0124]

[0125]

[0126] N / A means no chromatographic peak was detected.

[0127] The results showed that among the above pretreatment schemes, acetonitrile, zinc sulfate (containing VC) protein precipitation + ethyl acetate (containing formic acid) liquid-liquid extraction was the optimal pretreatment scheme.

[0128] By comparing different solutions, it is not difficult to see that:

[0129] (1) Comparison between Scheme 1 and Scheme 3: The peak area of ​​the detected index compound was higher when acetonitrile was used for protein precipitation than when methanol was used.

[0130] (2) Comparison between Scheme 1 and Scheme 5: The additional addition of zinc sulfate solution during protein precipitation can significantly increase the protein precipitation efficiency, making compound extraction easier and resulting in a higher extraction peak area for the detection index compound.

[0131] (3) Comparison between Scheme 1 and Scheme 4: When VC was added, the peak area of ​​the detected index compounds was higher than that when VC was not added, especially for the two water-soluble compounds 3, 4-DHB and 4-HB. The reason is that the addition of VC can resist the oxidation of 3, 4-DHB and 4-HB.

[0132] (4) Comparison between Scheme 1 and Scheme 2: Adding formic acid to the liquid phase extractant ethyl acetate can significantly increase the extraction peak area of ​​the detection index compound. The reason is that the addition of formic acid can make the two benzoic acids present in a molecular state in the solution, thereby improving the extraction efficiency.

[0133] (5) Comparison of Scheme 1 with Scheme 6 and Scheme 7: When the two steps of protein precipitation and liquid-liquid extraction are missing, the detection results are poor and the peak area is very small. In order to achieve the detection of the 11 detection indicators, the two steps of protein precipitation and liquid-liquid extraction are necessary.

[0134] It can be seen that Scheme 1 is preferred. The selection of any compound in this scheme has been carefully screened and none of them can be missing. In this combination alone, the 11 detection index compounds we selected have the highest extraction efficiency, the highest recovery rate, and the largest detection peak area, especially for the two water-soluble compounds 3,4-DHB and 4-HB, the extraction efficiency, recovery rate and detection peak area are greatly improved. They are difficult to detect under the existing technology, but can be conveniently and efficiently detected by the method provided by the present invention.

[0135] Example 3: Effect of protein precipitant on test results

[0136] This example adopts the pretreatment method provided in Example 1 to discuss the effects of different protein precipitants on the test results. We selected the quality control product LQC described in Example 1 as the sample to be tested, and used four groups of protein precipitants (MeOH+ZnSO4, MeOH, ACN+ZnSO4, ACN) for protein precipitation, wherein the ratio of ZnSO4 to the organic solvent was 1:9, and then the treatment and detection process was the same as in Example 1, and the organic solvent was optimized according to the peak area of ​​compound extraction. The peak areas of the eleven compounds after extraction with different protein precipitants are shown in Table 8.

[0137] Table 8: Effects of different protein precipitants on test results

[0138] <![CDATA[MeOH+ZnSO4]]> MeOH <![CDATA[ACN+ZnSO4]]> ACN TCS 4.04E+04 3.78E+04 6.81E+04 6.04E+04 TCC 2.67E+05 2.38E+05 1.03E+06 8.86E+05 BnP 7.78E+04 7.63E+04 9.48E+04 9.75E+04 MeP 5.83E+04 5.51E+05 1.95E+05 1.62E+05 EtP 2.14E+05 2.07E+05 2.99E+05 3.11E+05 OH-EtP 6.73E+04 6.31E+04 2.16E+05 2.07E+05 BZ 7.14E+04 6.89E+04 1.97E+05 1.87E+05 HkD 1.68E+04 1.76E+04 2.21E+05 1.93E+05 OH-MeP 3.06E+04 2.46E+04 1.05E+05 8.72E+04 3,4-DHB 8.33E+03 7.76E+03 1.45E+05 1.08E+04 4-HB 5.89E+05 5.81E+05 7.02E+06 8.80E+05

[0139] The results showed that when the extraction peak areas of the 11 compounds were comprehensively compared, the extraction peak area when acetonitrile was used as a protein precipitant was much larger than that when methanol was used as a protein precipitant; when ZnSO4 and ACN were used to precipitate proteins together, the extraction rates of 3,4-dihydroxybenzoic acid (3,4-DHB) and p-hydroxybenzoic acid (4-HB) were significantly improved; among them, the peak area of ​​3,4-dihydroxybenzoic acid (3,4-DHB) increased by 12 times compared with that when only ACN was used to precipitate proteins, and the peak area of ​​p-hydroxybenzoic acid (4-HB) increased by 8 times compared with that when only ACN was used to precipitate proteins.

[0140] When acetonitrile and zinc sulfate were used as protein precipitants, the extraction efficiency of 9 fat-soluble compounds among the 11 detection indicators was improved, and the peak areas of some of them, such as EtP and BuP, were slightly reduced. However, the extraction efficiency of 2 water-soluble compounds, 3, 4-DHB and 4-HB, was increased several times. For this reason, we chose acetonitrile and zinc sulfate as protein precipitants. It is this protein precipitant that enables this method to detect these 11 types at the same time, and the detection effect is good.

[0141] Example 4: Effect of the ratio of zinc sulfate and acetonitrile in the protein precipitant on the test results

[0142] In Example 3, we determined that adding zinc sulfate to the protein precipitant can significantly improve the extraction efficiency of the two hydroxybenzoic acids. In this example, we further considered the effects of different ratios of zinc sulfate and acetonitrile on the test results. The sample to be tested was the quality control product LQC described in Example 1. The results are shown in Table 9.

[0143] Table 9: Effects of different ratios of zinc sulfate solution and acetonitrile as protein precipitant on compound extraction peak area

[0144] <![CDATA[ZnSO4:ACN(1:9)]]> <![CDATA[ZnSO4:ACN(3:7)]]> <![CDATA[ZnSO4:ACN(5:5)]]> <![CDATA[ZnSO4:ACN(7:3)]]> TCS 6.81E+04 4.38E+04 5.68E+04 2.29E+04 TCC 1.03E+06 6.56E+05 7.85E+05 9.36E+04 BnP 9.48E+04 6.27E+04 8.76E+04 5.48E+04 MeP 1.95E+05 1.94E+05 1.37E+05 1.40E+05 EtP 2.99E+05 2.04E+05 2.59E+05 2.22E+05 OH-EtP 2.16E+05 1.47E+05 1.26E+05 6.75E+04 3,4-DHB 1.45E+05 2.21E+05 2.23E+05 2.48E+05 BZ 1.97E+05 1.27E+05 1.83E+05 1.22E+05 4-HB 7.02E+06 6.65E+06 6.18E+06 5.82E+06 HkD 2.21E+05 1.37E+05 1.85E+05 3.88E+04 OH-MeP 1.05E+05 5.23E+04 2.21E+04 2.67E+04

[0145] A comprehensive comparison shows that the protein precipitant with a formula of ZnSO4 and ACN has a better overall extraction efficiency for each compound than the protein precipitant with a formula of ZnSO4 and MeOH; a comprehensive comparison of protein precipitants with different ratios shows that the protein precipitant with a formula of ZnSO4:ACN=1:9 has the best extraction efficiency for each compound, and the detection effect is best at this time.

[0146] Example 5: Effect of extraction conditions on test results

[0147] Among the 11 detection indicators of the present invention, two benzoic acids have good water solubility, and the remaining 9 compounds have good fat solubility and large differences in properties. Therefore, in most literatures, the methods for extracting these compounds mostly use solid phase extraction (SPE), and liquid-liquid extraction (LLE) cannot guarantee the extraction recovery rate of the two benzoic acids with good water solubility. The present invention increases the extraction peak area of ​​each compound by improving the pretreatment.

[0148] After the pretreatment described in Example 1, the sample to be tested is the quality control product LQC described in Example 1. We first extract the pretreated sample using solid phase extraction and liquid-liquid extraction. Among them, two 96-well plates with ion exchange mechanisms are selected for solid phase extraction, namely PAX (anion exchange 96-well plate) and PWAX (weak anion exchange 96-well plate).

[0149] The solid phase extraction method is as follows:

[0150] (1) Sample preparation: Add the substance to be tested to the sample, then add the internal standard substance and mix well.

[0151] (2) Preparation of 96-well solid phase extraction plate: Place the 96-well solid phase extraction plate in a holder and activate it with methanol and purified water, respectively.

[0152] (3) Sample processing: The pretreated samples were loaded into a solid phase extraction 96-well plate and eluted with solvents containing different ratios.

[0153] (4) Elution: Collect the eluate and then conduct further analysis

[0154] The method of liquid-liquid extraction used in the present invention is as follows:

[0155] (1) Pipette 200 μL of double blank (methanol), single blank (methanol), standard curve and serum (including quality control) into a 96-well plate respectively;

[0156] (2) Add 60 μL of ZnSO4 solution (89 mg / mL) to all samples, add 540 μL of acetonitrile to the double blank, and add 540 μL of internal standard solution to the remaining samples, and vortex for 5 min;

[0157] (3) Add 800 μL of ethyl acetate (containing 1% FA) and vortex for 15 min;

[0158] (4) Centrifuge at 4000 rpm for 10 min and take 900 μL of the upper organic phase;

[0159] (5) Add 900 μL of ethyl acetate (containing 1% FA) to the residual solution again and vortex for 15 min;

[0160] (6) Centrifuge at 4000 rpm for 10 min, take 900 μL of the upper organic phase, and combine the two organic phases;

[0161] (7) Dry with nitrogen gas, add 150 μL of reconstitution solution (80% methanol) to the residue for reconstitution;

[0162] (8) Centrifuge at 4000 rpm for 5 min and inject 5 μL of the sample for detection.

[0163] The extraction was carried out by solid phase extraction and liquid phase extraction. The other steps were the same as in Example 1. The peak area of ​​the compound after extraction was measured. The results are shown in Table 10.

[0164] Table 10: Extraction peak areas of index compounds detected by solid phase extraction and liquid-liquid extraction

[0165]

[0166]

[0167] The results show that the extraction peak areas of TCC, BuP, MeP, 3,4-DHB, BzP, and 4-HB by solid phase extraction are all lower than those of the liquid-liquid extraction in the present invention. Taking all factors into consideration, the liquid-liquid extraction method selected in the present invention has better effect, can increase the extraction peak areas of 3,4-DHB and 4-HB, which are difficult to detect in the prior art, has strong operation convenience, and has lower cost.

[0168] According to the above test results, after the pretreatment method described in Example 1, the extraction should preferably be carried out by liquid-liquid extraction. Therefore, we then carried out a screening test of liquid phase extractants in liquid-liquid extraction. We used methyl tert-butyl ether, ethyl acetate and isooctane: methyl tert-butyl ether = 1:1 for liquid-liquid extraction, and the results of the extraction peak area of ​​the index compound detected in 11 are shown in Table 11.

[0169] Table 11: Effects of different extractants on the extraction peak area of ​​the detection index compounds

[0170]

[0171]

[0172] N / A means no chromatographic peak was detected.

[0173] The results showed that when methyl tert-butyl ether was used as the extractant, the extraction peak area was small; when isooctane: methyl tert-butyl ether = 1:1 was used as the extractant, the chromatographic peak of 3,4-DHB could not be detected, the detection of all detection indicators could not be completed, and the extraction peak area was small; when ethyl acetate was used as the extractant, the extraction peak area of ​​the 11 detection indicator compounds was the largest overall, which was the preferred choice.

[0174] Example 6: Effect of Antioxidants on Test Results

[0175] Among the 11 detection indicators selected by the present invention, two water-soluble benzoic acids are easily oxidized during the extraction process. This embodiment adopts the pretreatment method described in Example 1 to adjust the antioxidant, study the influence of the antioxidant on the detection results, and then screen out the antioxidant that is most suitable for detecting the 11 detection indicators selected by the present invention.

[0176] In this example, 2,6-di-tert-butyl-p-cresol (BHT) and vitamin C (VC) were selected as antioxidants, and were prepared into 1 mg / ml respectively and added into zinc sulfate solution. The sample to be tested was the quality control product LQC described in Example 1, and was treated according to the pretreatment method in Example 1. A blank control group was additionally added, and the recovery rates of the index compounds detected under different antioxidants were measured respectively. The results are shown in Table 12.

[0177] Table 12: Effect of antioxidants on the recovery of detected index compounds

[0178]

[0179]

[0180] The results showed that adding antioxidants to zinc sulfate solution could increase the recovery rate of the detected index compounds, and significantly increased the recovery rates of two water-soluble benzoic acids, 3,4-DHB and 4-HB; among different antioxidants, VC was the preferred one and could increase the recovery rate to the greatest extent.

[0181] Example 7: Effect of adding a Trap column on the detection results

[0182] TCS exists in pipelines, and the TCS background peak is relatively high in the liquid phase system. In the present invention, a Trap column is connected between the mobile phase mixer and the six-way valve of the injector, so that the retention time of the background peak in the mobile phase and the front-end pipeline of the system is staggered with the retention time of the TCS chromatographic peak in the sample, thereby reducing the interference of the background on quantification and reducing the lower limit of quantification.

[0183] Based on the sample detection method described in Example 1, the sample to be tested is the quality control product LQC described in Example 1, and the following tests are performed: no trap column + blank solvent 50% CH3OH, using trap column + blank solvent 50% CH3OH, no trap column + calibrator C1, using trap column + calibrator C1, and the results are as follows: Figure 1 , Figure 2 , Figure 3 , Figure 4 shown.

[0184] As can be seen from the figure: adding a connection of a Trap column between the mobile phase mixer and the six-way valve can separate the background peak of triclosan (TCS) in the mobile phase and the front-end pipeline of the instrument from the target peak of triclosan (TCS) in the sample, significantly reducing the interference caused by the background peak, improving the quantitative accuracy, and reducing the quantitative lower limit. According to the literature, the quantitative lower limit of TCS is 0.9ng / mL, and the present invention can effectively reduce the interference of the background in the mobile phase and pipeline on TCS by adding a connection of a Trap column between the mobile phase mixer and the six-way valve of the injector, and reduce the quantitative lower limit of TCS to 0.05ng / mL.

[0185] Example 8: Effect of Liquid Chromatography Mobile Phase on Detection Results

[0186] In order to detect 11 detection indicators at the same time and ensure that the extraction peaks of the 11 detection indicator compounds do not overlap, we studied the liquid chromatography mobile phase and set up the following sets of mobile phases:

[0187] Group 1: Mobile phase A: water (containing 0.05% HAc), mobile phase B: acetonitrile (0.05% HAc)

[0188] Group 2: Mobile phase A: water, mobile phase B: acetonitrile

[0189] The sample to be tested is the quality control product LQC described in Example 1. The test was performed according to the detection method described in Example 1, and only the mobile phase was changed. We found that adding a small amount of acetic acid to the mobile phase can optimize the peak shape of 3,4-DHB and solve the problem of carboxylic acid tailing. The chromatogram is shown in FIG. Figure 5 to Figure 15 shown.

[0190] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A composition for preparing a preparation for improving the extraction recovery rate of 3,4-dihydroxybenzoic acid and p-hydroxybenzoic acid, characterized in that: The composition includes acetonitrile and zinc sulfate.

2. The use according to claim 1, characterized in that The extraction is liquid-liquid extraction using ethyl acetate and formic acid, and the sample is human blood.

3. A kit for simultaneously determining the contents of 11 benzoic acid esters and their metabolites and chlorine-containing antibacterial agents in human blood by liquid chromatography-tandem mass spectrometry, characterized in that: The kit contains an internal standard working solution, a zinc sulfate solution and an extractant; the internal standard working solution includes acetonitrile, and the extractant includes ethyl acetate and formic acid; the 11 benzoic acid esters and their metabolites and chlorine-containing antibacterial agents are: butyl p-hydroxybenzoate, methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, ethyl 3,4-dihydroxybenzoate, 3,4-dihydroxybenzoic acid, benzyl p-hydroxybenzoate, heptyl p-hydroxybenzoate, p-hydroxybenzoic acid, methyl 3,4-dihydroxybenzoate, triclocarban, and triclosan.

4. The kit according to claim 3, characterized in that The concentration of the zinc sulfate solution is 30-89 mg / ml, and the volume ratio of the internal standard working solution to the zinc sulfate solution is 9:1-1:

1.

5. The kit according to claim 4, characterized in that The zinc sulfate solution also contains an antioxidant.

6. A method for simultaneously determining the contents of 11 benzoic acid esters and their metabolites and chlorine-containing antibacterial agents in human blood by liquid chromatography-tandem mass spectrometry, characterized in that: The sample pretreatment is carried out using the kit as described in any one of claims 3 to 5; the 11 benzoic acid esters and their metabolites and chlorine-containing antibacterial agents are: butyl parahydroxybenzoate, methyl parahydroxybenzoate, ethyl parahydroxybenzoate, ethyl 3,4-dihydroxybenzoate, 3,4-dihydroxybenzoic acid, benzyl parahydroxybenzoate, heptyl parahydroxybenzoate, parahydroxybenzoic acid, methyl 3,4-dihydroxybenzoate, triclocarban, and triclosan.

7. The method according to claim 6, characterized in that The following steps are involved: (1) adding an internal standard working solution and a zinc sulfate solution to the sample, wherein the internal standard working solution contains acetonitrile; (2) adding an extractant to perform liquid-liquid extraction, wherein the extractant comprises ethyl acetate; (3) Blow dry and re-dissolve; (4) Liquid chromatography tandem mass spectrometry detection.

8. The method according to claim 7, characterized in that The volume ratio of the internal standard working solution to the zinc sulfate solution in step (1) is 9:1-1:1; and the extractant in step (2) further contains formic acid, with a formic acid content of 0.5% to 1%.

9. The method according to claim 8, characterized in that The zinc sulfate solution also contains vitamin C, and the added amount of vitamin C is 0.2-1 mg / mL.

10. The method according to claim 9, characterized in that In step (4), when the liquid chromatography-tandem mass spectrometry is performed, a trap column needs to be connected between the mobile phase mixer and the six-way valve.

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