A method for simultaneously determining 9 fungicides in tobacco products

Abstract

The present application belongs to the technical field of analysis and detection, and particularly relates to a method for simultaneously determining nine fungicides in tobacco products. The method for simultaneously determining the nine fungicides in tobacco products comprises the following steps: preparing a standard solution; processing a standard working solution; processing a sample to be measured; placing the sample to be measured into a sample bottle, adding a first internal standard and a second internal standard, adjusting pH, and extracting and enriching; and determining the sample to be measured. After the standard working solution and the extraction phase of the sample to be measured are on-line derivatized at a GC inlet, the mass spectrometry internal standard method is used to qualitatively and quantitatively analyze the components to be measured in the sample to be measured. Compared with three-phase hollow liquid-liquid microextraction, the two-phase mode can further improve the enrichment multiple of hydroxybenzoic acid ester compounds, and the sensitivity of the method is significantly improved.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection technology, specifically relating to a method for simultaneously determining nine bactericides in tobacco products. Background Technology

[0002] Reconstituted tobacco, also known as tobacco sheet or reconstituted tobacco, is a product of tobacco waste resource recycling. It primarily utilizes tobacco by-products and waste generated during tobacco harvesting, curing, re-drying, storage, and tobacco product processing, such as tobacco stems, dust, fragments, shredded tobacco, and even lower-grade tobacco leaves. These are processed according to specific formulas and techniques to create a reconstituted tobacco product resembling kraft paper. Because it is made from tobacco and its physicochemical properties are similar to or even superior to natural tobacco leaves, it is commonly used in tobacco products as a leaf blend formulation ingredient or filler, and is also one of the main tobacco segment materials in heated tobacco products (HTPs). Research has found that tobacco is rich in glucose, fructose, proline, and protein, which are carbon and nitrogen sources necessary for microbial growth and reproduction. Putrefactive microorganisms readily grow and multiply in the pulp, causing it to deteriorate and affecting the physical properties of the paper base, thus further impacting the overall quality of reconstituted tobacco. Therefore, in order to prevent spoilage caused by microbial growth and improve the shelf life of reconstituted tobacco during production, transportation and storage, it is often necessary to add some chemical substances to inhibit the growth of microorganisms or kill these microorganisms.

[0003] Benzoic acid and sorbic acid are widely used as food additives and preservatives to prevent product spoilage and extend shelf life. Parabens, on the other hand, act as preservatives by disrupting cell membranes and intracellular proteins and altering the activity of microbial enzymes; they are commonly used preservatives in the food and cosmetic industries. While these preservatives are generally considered safe, excessive use can pose risks. For example, excessive intake of benzoic acid and sorbic acid may lead to a range of adverse reactions, including metabolic acidosis, cramps, and allergies. Furthermore, studies have shown that parabens possess estrogenic activity, potentially interfering with the human endocrine system. Their estrogenic properties increase with chain length; therefore, since 2014, the use of long-chain parabens has been increasingly restricted by the laws of various countries.

[0004] Unlike daily chemical products, preservatives in reconstituted tobacco can directly enter the human body through smoking or oral inhalation, potentially posing a threat to consumer health. Currently, domestic industry standards for tobacco preservatives (YC / T423-2011) and preservative testing in grassroots laboratories mostly use liquid-liquid extraction as sample pretreatment, which has limitations in sensitivity and selectivity, and does not include the detection of long-chain parabens. Therefore, establishing simple, rapid, and sensitive sample pretreatment and analytical methods is of great significance for effectively regulating the use of preservatives in reconstituted tobacco and other tobacco auxiliary materials.

[0005] Hollow fiber liquid-phase microextraction (HF-LPME) is a relatively novel sample pretreatment technique proposed by Pedersen-Bjergaard and Rasmussen in 1999. HF-LPME uses porous hollow fibers as a carrier for organic solvents, essentially a liquid-liquid membrane extraction method. Its principle is similar to membrane-based liquid-phase extraction, avoiding contamination of the analyte or extractant by the sample matrix, thus acting as microfiltration and sample purification. Compared to traditional methods, HF-LPME features lower solvent consumption and higher enrichment rates. Because the extract phase is protected by the fiber membrane, HF-LPME exhibits superior selectivity and stability compared to general liquid-phase microextraction (LPME). The fiber membrane is also low-cost, suitable for batch testing, and has broad application prospects in the field of trace analysis of complex matrices, effectively compensating for the shortcomings of existing standard methods.

[0006] The patent "A Method for Simultaneous Determination of Benzoic Acid, Sorbic Acid, and Parabens in Tobacco Flavorings and Fragrances" establishes a three-phase hollow fiber liquid-liquid microextraction-high performance liquid chromatography (HPLC) method, achieving simultaneous determination of sorbic acid, benzoic acid, and seven parabens in tobacco flavorings and fragrances, with good practical application results. However, using three-phase hollow fiber microextraction technology as sample pretreatment also has certain limitations. Parabens have the following characteristics: ① stronger lipophilicity; ② lower degree of dissociation in aqueous solution, resulting in a lower enrichment factor than sorbic acid and benzoic acid. Since current regulations have lower limits for parabens than for sorbic acid and benzoic acid, improving the detection sensitivity of parabens is of practical significance.

[0007] Therefore, this patent selects sorbic acid, benzoic acid and seven parabens as target compounds in reconstituted tobacco leaves to explore two-phase hollow fiber liquid-phase microextraction in the hope of obtaining better extraction and enrichment effects. Summary of the Invention

[0008] This invention addresses the limitations and shortcomings of existing technologies by developing a sample pretreatment method based on two-phase hollow fiber liquid-liquid microextraction for the determination of bactericides in reconstituted tobacco. This method integrates extraction, enrichment, and purification. After hollow fiber extraction / enrichment, the analyte is added with a derivatization reagent, derivatized online via a GC inlet, and then quantified using internal standard mass spectrometry. This method features a low detection limit and sensitivity exceeding that of tandem mass spectrometry.

[0009] A method for simultaneously determining nine bactericides in tobacco products, characterized by comprising the following steps:

[0010] 1) Preparation of standard solutions: Weigh out the standards of 9 fungicides, make up to volume to obtain a mixed standard stock solution of 9 fungicides. Weigh out the first internal standard and the second internal standard, make up to volume to obtain a mixed solution of the first internal standard and the second internal standard.

[0011] 2) Preparation of standard working solutions: Weigh reconstituted tobacco leaves that do not contain the analyte, and add a series of different volumes of the mixed standard stock solution and the first internal standard mixed solution and the second internal standard mixed solution obtained in step 1) to prepare a series of standard working solutions of different concentrations and adjust the pH.

[0012] 3) Sample preparation: Place the sample to be tested into a sample vial, add the first internal standard and the second internal standard, adjust the pH, and obtain the sample to be tested;

[0013] 4) Extraction and enrichment: Hollow fibers are placed into a series of standard working solutions of different concentrations obtained in step 2) and the test sample obtained in step 3), and the extraction phase is injected into the inner cavity of the hollow fibers for extraction and enrichment.

[0014] 5) Determination of the test sample: After the standard working solution and the extract phase of the test sample from step 4) are derivatized online at the GC inlet, the analyte in the test sample is qualitatively and quantitatively analyzed by the internal standard method of mass spectrometry.

[0015] Preferably, in step 1), the bactericide is selected from one or more of sorbic acid, benzoic acid, methylparaben, ethylparaben, isopropylparaben, propylparaben, isobutylparaben, butylparaben, or benzylparaben; the relevant information of the test compound is as follows:

[0016] Table 1. Relevant information of the test compounds

[0017]

[0018] Preferably, in step 1), the first internal standard is selected from deuterated benzoic acid;

[0019] Preferably, in step 1), the second internal standard is selected from propyl deuterated p-hydroxybenzoate;

[0020] Preferably, in step 1), the reagent used for volume adjustment is methanol.

[0021] Preferably, in step 2), the pH is 2 to 8; for example, it can be pH 2 to 3, pH 3 to 4, pH 4 to 5, pH 5 to 6, pH 6 to 7, pH 7 to 8, etc.

[0022] Preferably, in step 2), 22), the pH-adjusting reagent is an aqueous hydrochloric acid solution, and the molar concentration of the aqueous hydrochloric acid solution in the standard working solution can be 0–10 mmol / L, for example, 0–0.001 mmol / L, 0.001–0.01 mmol / L, 0.01–0.1 mmol / L, 0.1–1 mmol / L, 1–10 mmol / L, etc. Preferably, in step 3), 31), when the sample to be tested is a paste-like viscous or solid sample, it is homogenized;

[0023] Preferably, in step 3), pH = 2 to 8, for example, pH = 2 to 3, pH = 3 to 4, pH = 4 to 5, pH = 5 to 6, pH = 6 to 7, pH = 7 to 8, etc.

[0024] Preferably, in step 3), the reagent for adjusting pH is an aqueous solution of hydrochloric acid.

[0025] Preferably, in feature 31), the homogenizing reagent is selected from one or more of water or methanol; the water is deionized water that has been boiled to remove CO2.

[0026] Preferably, in feature 33), the molar concentration of the hydrochloric acid aqueous solution in the sample to be tested is 0–10 mmol / L, for example, it can be 0–0.001 mmol / L, 0.001–0.01 mmol / L, 0.01–0.1 mmol / L, or 0.1–1 mmol / L.

[0027] 1–10 mmol / L, etc.

[0028] Preferably, the volume ratio of the sample homogenate to the sample to be tested is (1:15) to (2:5); for example, it can be (1:15) to (2:5).

[0029] 15)~(1:10), (1:10)~(2:15), (2:15)~(1:5), (1:5)~(2:5), etc.

[0030] Preferably, in step 4), the hollow fiber is first immersed in a reagent to remove impurities and then dried before use;

[0031] 42) In step 4), NaCl is further added to the sample to be tested;

[0032] 43) In step 4), the solvent of the extraction phase is selected from one or more of toluene, xylene, chloroform or ethyl acetate;

[0033] 44) In step 4), the mass-volume ratio of the component to be tested in the standard working solution to the extraction phase is (1:200) to (1:10000);

[0034] 45) In step 4), the volume ratio of the sample to be tested to the extraction phase is (1:15) to (2:5); for example, it can be (1:15) to (1:10), (1:10) to (2:15), (2:15) to (1:5), (1:5) to (2:5), etc.

[0035] 46) In step 4), the extraction time is 10 to 60 min; for example, it can be 10 to 20 min, 20 to 30 min, 30 to 40 min, 40 to 50 min, 50 to 60 min, etc.

[0036] 47) In step 4), during the extraction, the standard working solution and / or the sample to be tested is stirred.

[0037] Preferably, in 411), in feature 41), the reagent is selected from one or more of acetone, isopropanol or methanol, and more preferably acetone.

[0038] 412) In feature 41), the impurity removal time is 2 to 10 min; for example, it can be 2 to 3 min, 3 to 4 min, 4 to 5 min, 5 to 6 min, 6 to 7 min, 7 to 8 min, 8 to 9 min, 9 to 10 min, etc.

[0039] 413) In feature 41), the impurity removal method is ultrasonic;

[0040] 421) In feature 42), the mass percentage concentration of NaCl is X, 0 < X ≤ 30%, for example, it can be 0 < X ≤ 5%, 5% ≤ X ≤ 10%, 15% ≤ X ≤ 20%, 20% ≤ X ≤ 25%, 25% ≤ X ≤ 30%, etc.

[0041] 471) In feature 47), the stirring device is a magnetic stirrer;

[0042] 472) In feature 47), the stirring rate is 400 to 1200 rpm, for example, it can be 400 to 500 rpm, 500 to 600 rpm, 600 to 700 rpm, 700 to 800 rpm, 800 to 900 rpm, 900 to 1000 rpm, 1000 to 1100 rpm, 1100 to 1200 rpm, etc.

[0043] Preferably, in step 5), the derived reagent is selected from one or more of BSA, BSTFA, MTBSTFA or MSTFA.

[0044] Preferably, in step 5), the amount of the derivatizing reagent is 0.5 to 4 μL, for example, it can be 0.5 to 4 μL, 0.5 to 1 μL, 1 to 1.5 μL, 1.5 to 2 μL, 2 to 2.5 μL, 2.5 to 3 μL, 3.5 to 4 μL, etc.

[0045] Preferably, in step 5) of 53), the GC chromatographic conditions are:

[0046] Chromatographic column: DB-5MS capillary column (0.25μm×30m×250μm film thickness);

[0047] Carrier gas: selected from one or more of He, N2 and H2, with a purity of 99%; preferably He.

[0048] Inlet temperature: 220~300℃; for example, it can be 220~230℃, 230~240℃, 240~250℃, 250~260℃, 260~270℃, 270~280℃, 280~290℃, 290~300℃, etc.

[0049] Injection volume: 1–3.5 μL, for example, 1–1.5 μL, 1.5–2 μL, 2–2.5 μL, 2.5–3 μL, 3–3.5 μL, etc., splitless injection.

[0050] The non-splitting time is 1–3.5 min, for example, it can be 1–1.5 min, 1.5–2 min, 2–2.5 min, 2.5–3 min, 3–3.5 min, etc.

[0051] The heating process is as follows:

[0052] The initial temperature is 75-85℃, preferably 80℃; the temperature is increased to 255-265℃, preferably 260℃, at a rate of 3-10℃ / min.

[0053] 54) In step 5), the mass spectrometry conditions are:

[0054] Ion source: ESI source;

[0055] Scanning methods: SACN (full scan) and SIM (selective ion monitoring);

[0056] Scan range: 35-300 amu; for example, it can be 35-50 amu, 50-100 amu, 100-150 amu, 150-200 amu, 200-250 amu, 250-00 amu, etc.

[0057] Ion source temperature: 220~280℃; for example, it can be 220~230℃, 230~280℃, 220~240℃, 240~250℃, 250~260℃, 260~270℃, 270~280℃, etc.

[0058] Quadrupole temperature: 130~180℃; for example, it can be 130~140℃, 140~150℃, 150~160℃, 160~170℃, 170~180℃, etc.

[0059] Solvent delay: 5-10 min, for example, 5-6 min, 6-7 min, 7-8 min, 8-9 min, 9-10 min, etc.

[0060] As described above, the method for simultaneously determining benzoic acid, sorbic acid, and parabens in tobacco flavorings and fragrances provided by the present invention has the following beneficial effects:

[0061] 1) Hollow fiber liquid phase microextraction technology has the advantages of small solvent consumption, high extraction efficiency and large enrichment factor. In addition, the micropores on the hollow fiber wall can block macromolecules and particles in the donor phase solution from entering the receiver phase, which plays a micro-filtration role on the sample and can obtain a cleaner extract that can be directly used for subsequent analysis.

[0062] 2) This invention is the first to apply two-phase hollow fiber liquid-liquid microextraction technology to the extraction, purification, and enrichment of bactericides in reconstituted tobacco, tobacco flavorings, and tobacco extracts. After derivatization via the GC inlet, the samples are separated by chromatography, and quantified using mass spectrometry with a deuterated internal standard. Compared to three-phase hollow fiber liquid-liquid microextraction, the two-phase mode can further enhance the enrichment factor of parabens, and the method sensitivity is significantly improved. This method is simple to operate, environmentally friendly, and highly sensitive, and can be used for rapid screening of sorbic acid, benzoic acid, and parabens in matrices such as reconstituted tobacco, tobacco flavorings, and tobacco extracts. Attached Figure Description

[0063] Figure 1 Schematic diagram of a two-phase hollow fiber liquid-liquid microextraction device;

[0064] Figure 2 This shows the effect of inlet temperature on derivatization efficiency;

[0065] Figure 3 This is shown as the impact of non-splitting time on derivation efficiency;

[0066] Figure 4 The effect of BSTFA reagent dosage on derivatization efficiency is shown.

[0067] Figure 5 This shows the effect of different extraction solvents on the extraction effect;

[0068] Figure 6 This shows the effect of sample phase pH on extraction efficiency;

[0069] Figure 7 The effect of salt concentration on extraction efficiency is shown.

[0070] Figure 8 The effect of sample phase volume on extraction efficiency is shown.

[0071] Figure 9 This shows the effect of extraction time on the extraction effect;

[0072] Figure 10 The effect of stirring rate on extraction efficiency is shown.

[0073] Figure 11 The image shows a selected ion scan (SIM) plot comparing the microextraction of the mixed standard solution before and after the extraction.

[0074] Figure 12 The image shown is a spiked (100 ppb) selected ion scan (SIM) image of a reconstituted tobacco sample. Detailed Implementation

[0075] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the process equipment or apparatus not specifically specified in the following embodiments are all conventional equipment or apparatus in the art.

[0076] Example 1

[0077] 1) Hollow fiber treatment

[0078] Take hollow fibers, cut them to a length of 10 cm, immerse them in analytical grade acetone, sonicate for 5 minutes to remove impurities, and then air dry for later use.

[0079] 2) Prepare standard solutions

[0080] Take 100 mg of sorbic acid, benzoic acid, methylparaben, ethylparaben, isopropylparaben, propylparaben, isobutylparaben, butylparaben, and 25 mg of paraben into a 100 mL volumetric flask, add methanol to make up to volume, and store in a refrigerator at 4 °C for later use.

[0081] 3) Prepare internal standard mixed solution

[0082] Weigh 0.1 g of deuterated benzoic acid into a 100 mL volumetric flask, dilute to volume with methanol, and prepare a 1 g / L deuterated benzoic acid internal standard stock solution. Weigh 0.1 g of deuterated benzoic acid into a 100 mL volumetric flask, dilute to volume with methanol, and prepare a 1 g / L deuterated propylparaben internal standard stock solution. Take 1 mL of each of the above stock solutions and place them into 100 mL volumetric flasks, dilute to volume with methanol, and prepare a 10 μg / mL internal standard mixture. Store at 4°C for later use.

[0083] 4) Treatment of standard working solutions

[0084] Matrix spiking was used. 0.2g of reconstituted tobacco leaves without the analyte was weighed and mixed with a series of different volumes of the standard solution obtained in step 3). 15mL of 0.1mmol / L hydrochloric acid aqueous solution was added, and 15μL of the internal standard mixture from step 3) was added to each standard solution. The pH was then adjusted to 2.

[0085] 5) Extraction and enrichment of standard working solutions

[0086] Place the hollow fiber from step 1) into 15 mL of standard working solutions of different concentrations from step 4). The hollow fiber is U-shaped, with both ends of the hollow fiber coated with organic solvent facing the mouth of the sample bottle. See Appendix for details. Figure 1 Approximately 30 μL of butyl acetate was injected into the inner cavity of a hollow fiber containing organic solvent. The resulting extraction apparatus was then placed on a magnetic stirrer and stirred at 800 rpm. After 30 min of extraction, 30 μL of the extract phase was drawn from the hollow fiber cavity using a microsyringe needle. The volume was accurately recorded and transferred to the liner of a chromatographic vial. 2 μL of BSTF was added, and derivatization was performed by setting the injection port temperature (220–300 °C, 20 °C intervals) and the splitless time (1–3.5 min, 0.5 min intervals).

[0087] 6) Determination of standard samples

[0088] The derivatized samples from step 5) were subjected to gas chromatography-mass spectrometry (GC-MS) for detection, with three sets of samples per sample. The GC conditions were as follows:

[0089] Chromatographic conditions:

[0090] Chromatographic column: DB-5MS capillary column (0.25μm×30m×250μm film thickness)

[0091] Carrier gas: He, 99% purity; Injector temperature: 260℃; Injection volume: 1μL, splitless injection, splitless injection time 2.5min; Temperature program:

[0092]

[0093] Mass spectrometry conditions:

[0094] Scanning methods: SACN (full scan) and SIM (selected ion monitoring); scan range: 35-300 amu; ion source temperature: 230℃; quadrupole temperature: 150℃; solvent delay: 8 min. Retention times, quantitative and qualitative ions for each analyte are detailed in Table 1.

[0095] Table 1. Elution order, retention time, and characteristic ions of each analyte compound under selected ion monitoring mode in gas chromatography-mass spectrometry.

[0096]

[0097]

[0098] 7) Regress the peak area response ratio of the target compound and the internal standard selected ion (Y-axis) against the concentration ratio (X-axis) to obtain the linear regression equation for the standard working curve. Spikes were performed at two concentration levels, 5 and 50 ng / mL, with five replicates per sample. The enrichment factor (EF) at each spiked concentration was calculated using the formula... Where C0 is the initial concentration of the target analyte in the sample phase, C f This refers to the concentration of the target analyte in the acceptor phase within the hollow fiber cavity. The limits of detection for each analyte are between 0.05 and 0.18 ng / mL. -1 Between these values, the enrichment factor EF reached a maximum of 620.5, indicating that the two-phase hollow fiber microextraction mode significantly improved the enrichment effect of p-hydroxybenzoic acid esters, which have strong lipophilicity, in the test compounds, as detailed in Table 2.

[0099] Table 2. Analytical capability of two-phase hollow fiber liquid-liquid microextraction method for bactericides.

[0100]

[0101] a 5ng·mL -1

[0102] b 50 ng·mL -1

[0103] Example 2

[0104] Three representative samples were selected from reconstituted tobacco: reconstituted tobacco leaves, tobacco flavorings (6#), and tobacco ethanol extract. Solid samples (1#-5#) or water-insoluble samples (7#) were prepared into homogenized solutions according to the following steps: 10g of reconstituted tobacco leaves or tobacco ethanol extract samples were weighed into stoppered centrifuge tubes, and approximately 10mL of 30% methanol / water solution was added. After ultrasonic extraction for 5min, the mixture was transferred to a 100mL volumetric flask, washed repeatedly three times, and the supernatant was collected each time. The solution was then diluted to the mark to prepare a 100g / L homogenized solution and stored at 4℃. An appropriate amount of the above samples (0.2g of water-soluble sample and 1mL of homogenized solution) was placed in a sample bottle and dissolved in deionized water that had been boiled to remove CO2. 15μL of an internal standard mixture with a mass concentration of 10μg / mL was added, and the pH was adjusted to 2 with 1mol / L hydrochloric acid solution, maintaining the sample solution volume at approximately 15mL. Then, extraction and enrichment were performed under the same conditions as in step 5) of Example 1, and determination was performed under the same conditions as in step 6). The results are shown in Table 3.

[0105] Table 3. Measurement results of sample 2 in Example 2

[0106]

[0107] a Reconstituted tobacco

[0108] b Tobacco flavorings

[0109] c Tobacco ethanol extract

[0110] Example 3

[0111] The 1# sample from Example 2) was subjected to three levels of spiked recovery experiments. The recoveries and relative standard deviations (n=5) are shown in Table 4. The recoveries of each analyte ranged from 91.1% to 115.6%, and the precision, except for benzyl benzoate, was <10%. This method can meet the needs of practical analysis. A comparison of the chromatograms of the 1# spiked sample before and after microextraction is shown in [Table 4]. Figure 11 The enrichment effect of the method described in this patent is obvious.

[0112] Table 4 Spike Recovery and Precision (1#)

[0113]

[0114]

[0115] a 2ng·mL -1(of which benzyl benzoate is 4.5 ng / mL) -1 )

[0116] b 20 ng·mL -1 (of which benzyl benzoate was 45 ng·mL) -1 )

[0117] c 100 ng·mL -1 (of which benzyl benzoate is 225 ng·mL) -1 )

[0118] Example 4

[0119] Spiked sample #1 was selected and microextraction experiments were performed under different modes to compare the differences in enrichment effects of different extraction modes on the analytes. See Table 5 for details.

[0120] Table 5 Comparison of enrichment folds of analytes under two-phase and three-phase modes (sample #1 spiked)

[0121]

[0122] *Spike concentration 50 ng / mL -1 (benzyl p-hydroxybenzoate 112.5 ng·mL) -1 )

[0123] The results show that 3p-HF-LPME has a better enrichment effect on substances with strong hydrophilicity and high polarity, such as sorbic acid and benzoic acid. In the two-phase mode, since the acceptor phase is organic, diffusion and mass transfer between the two phases rely solely on the analyte partition coefficient, thus significantly improving the enrichment effect of hydroxybenzoic esters, with an enrichment factor EF much greater than that obtained in the three-phase mode. This indicates that for substances with low dissociation in aqueous solution and Log K... ow For compounds with high levels of parabens, a two-phase method usually yields better enrichment results.

[0124] Example 5

[0125] In Example 2), the effect of injection port temperature on derivatization efficiency was determined using spiked sample #1. Other conditions were the same as those for sample #1 in Example 2, except that the injection port temperatures were 220℃, 240℃, 260℃, 280℃, and 300℃, respectively. The results are shown in [Figure number missing]. Figure 2 .Depend on Figure 2 It can be seen that the derivatization efficiency is highest when the sample inlet temperature is 260℃.

[0126] Example 6

[0127] The effect of splitless time on derivatization efficiency was determined using spiked sample #1 in Example 2. Other conditions were the same as those for sample #1 in Example 2, except that the splitless time was 0.5 min, 1 min, 1.5 min, 2 min, 2.5 min, 3 min, and 3.5 min, respectively. The results are shown in [Figure Number]. Figure 3 .Depend on Figure 3 It can be seen that the derivatization efficiency is highest when the non-splitting time is 2.5 minutes.

[0128] Example 7

[0129] The effect of BSTFA reagent dosage on derivatization efficiency was determined using sample #1 spiked in Example 2. Other conditions were the same as those for sample #1 in Example 2, except that the BSTFA reagent dosages were 0.5 μL, 1 μL, 2 μL, 3 μL, and 0.4 μL, respectively. The results are shown in [Figure Number]. Figure 4 .Depend on Figure 4 It can be seen that using 1 μL of BSTFA reagent results in good derivatization efficiency.

[0130] Example 8

[0131] In Example 2), the effect of the solvent of the extract phase on the extraction efficiency was determined using sample #1 spiked. Other conditions were the same as those for sample #1 in Example 2, except that toluene, chloroform, dihexyl ether, dioctanone, or butyl acetate were used as the solvent for the extract phase. The results are shown in [Figure Number]. Figure 5 .Depend on Figure 5 It is known that n-octanol, dihexyl ether, toluene, dioctyl ketone, and chlorobenzene can all be used as organic solvents to treat hollow fibers, but dihexyl ether (DHE) has the best extraction effect.

[0132] Example 9

[0133] In Example 2), the effect of sample phase pH on extraction efficiency was determined using spiked sample #1. Other conditions were the same as those for sample #1 in Example 2, except that the sample phase pH was set to 2, 3, 4, 5, 6, 7, and 8. The results are shown in [Figure Number]. Figure 6 .

[0134] Example 10

[0135] According to the effect of salt concentration on extraction efficiency of sample phase determined by spiked sample #1 in Example 2, other conditions were the same as those for sample #1 in Example 2, except that NaCl was added to the sample phase at amounts of 5% (w / v), 10% (w / v), 15% (w / v), 20% (w / v), and 30% (w / v), respectively. The results are shown in [Figure number missing]. Figure 7 .Depend on Figure 7 It can be seen that adding NaCl is beneficial for extraction.

[0136] Example 11

[0137] According to the determination of the effect of sample phase volume on extraction efficiency by spiked sample #1 in Example 2, other conditions were the same as those for sample #1 in Example 2, but the sample phase volume was adjusted to 5 mL, 10 mL, 15 mL, 20 mL and 30 mL respectively. The results are shown in [Figure 1]. Figure 8 .Depend on Figure 8 It can be seen that a good extraction effect can be achieved when the volume of the sample phase is 15 mL.

[0138] Example 12

[0139] According to the effect of extraction time on extraction efficiency determined by spiked sample #1 in Example 2, other conditions were the same as those for sample #1 in Example 2, but the extraction time was controlled to be 10, 20, 30, 40 and 60 min respectively. The results are shown in [Figure 1]. Figure 9 .Depend on Figure 9 It can be seen that the enrichment and extraction of the sample can be achieved when the extraction time is 10-60 min, but the effect is best when the extraction time is 60 min.

[0140] Example 13

[0141] Based on the effect of magnetic stirrer speed on extraction efficiency determined in Example 2, other conditions were the same as those for sample #1 in Example 2, except that the magnetic stirrer speed was controlled at 400, 600, 800, 1000, and 1200 rpm respectively. The results are shown in [Figure 1]. Figure 10 .Depend on Figure 10 It can be seen that the enrichment and extraction of the sample can be achieved when the extraction time is 400-1200 rpm, but the effect is best when the extraction time is 600 rpm.

[0142] Furthermore, it should be understood that the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps, does not preclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps, unless otherwise stated. It should also be understood that the combined connection relationship between one or more devices / apparatus mentioned in this invention does not preclude the existence of other devices / apparatus before or after the combined devices / apparatus, or the insertion of other devices / apparatus between these explicitly mentioned devices / apparatus, unless otherwise stated. Moreover, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not for limiting the order of the method steps or limiting the scope of the invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the invention.

Claims

1. A method for simultaneously determining nine bactericides in tobacco products, characterized in that, Includes the following steps: 1) Preparation of standard solutions: Weigh out the standards of 9 fungicides, dilute to volume to obtain a mixed standard stock solution of 9 fungicides, weigh out the first internal standard and the second internal standard, dilute to volume to obtain a mixed solution of the first internal standard and the second internal standard. 2) Preparation of standard working solutions: Weigh reconstituted tobacco leaves that do not contain the analyte, and add a series of different volumes of the mixed standard stock solution and the first internal standard mixed solution and the second internal standard mixed solution obtained in step 1) to prepare a series of standard working solutions of different concentrations and adjust the pH. pH = 2-5; 3) Sample preparation: Place the sample to be tested into a sample vial, add the first internal standard and the second internal standard, adjust the pH to obtain the sample to be tested; pH=2-5; 4) Extraction and enrichment: Hollow fibers are placed into a series of standard working solutions of different concentrations obtained in step 2) and the test sample obtained in step 3), respectively. The extractant phase is injected into the inner cavity of the hollow fiber for extraction and enrichment. The solvent of the extractant phase is selected from one or more of toluene, xylene, chloroform or ethyl acetate. The first internal standard is selected from deuterated benzoic acid. The second internal standard is selected from propyl deuterated p-hydroxybenzoate. 5) Determination of the test sample: After online derivatization of the standard working solution and the extract phase of the test sample in step 4) at the GC injection port, the analytes in the test sample are qualitatively and quantitatively analyzed by the internal standard method of mass spectrometry; the bactericide is selected from sorbic acid, benzoic acid, methylparaben, ethylparaben, isopropylparaben, propylparaben, isobutylparaben, butylparaben, and benzylparaben; the derivatization reagent is selected from one or more of BSA, BSTFA, MTBSTFA, or MSTFA; the GC chromatographic conditions are: column: DB-5MS capillary column; temperature program: 80℃ for 1 minute, then increase to 260℃ at a rate of 5℃ / minute, then increase to 280℃ at a rate of 10℃ / minute, and hold for 5 minutes.

2. The method for simultaneously determining nine bactericides in tobacco products according to claim 1, characterized in that, In step 1), the reagent used for volume adjustment is methanol.

3. The method for simultaneously determining nine bactericides in tobacco products according to claim 2, characterized in that, In step 2), the reagent for adjusting the pH is an aqueous solution of hydrochloric acid.

4. The method for simultaneously determining nine bactericides in tobacco products according to claim 1, characterized in that, Includes at least one of the following technical features: 31) In step 3), when the sample to be tested is a paste-like viscous sample or a solid sample, it is homogenized; 33) In step 3), the reagent for adjusting pH is an aqueous solution of hydrochloric acid.

5. The method for simultaneously determining nine bactericides in tobacco products according to claim 4, characterized in that, Includes at least one of the following technical features: 311) In feature 31), the homogenizing reagent is selected from one or more of water or methanol; In feature 312), the molar concentration of the hydrochloric acid aqueous solution in the sample to be tested is 0~10 mmol / L.

6. The method for simultaneously determining nine bactericides in tobacco products according to claim 5, characterized in that, The volume ratio of the homogenized sample liquid to the sample to be tested is (1:15) to (2:5).

7. The method for simultaneously determining nine bactericides in tobacco products according to claim 1, characterized in that, Includes at least one of the following technical features: 41) In step 4), when the hollow fiber is used, it is first immersed in a reagent to remove impurities and then dried. 42) In step 4), the sample to be tested also includes the addition of NaCl; 44) In step 4), the mass-to-volume ratio of the analyte in the standard working solution to the extract phase is (1:200) to (1:10000). 45) In step 4), the volume ratio of the sample to be tested to the extract phase is (1:15) to (2:5). 46) In step 4), the extraction time is 10~60 min; 47) In step 4), during the extraction, the standard working solution and / or the sample to be tested are stirred.

8. The method for simultaneously determining nine bactericides in tobacco products according to claim 7, characterized in that, Includes at least one of the following technical features: 411) In feature 41), the reagent is selected from one or more of acetone, isopropanol, or methanol. 412) In feature 41), the impurity removal time is 2~10 min; 413) In feature 41), the method of impurity removal is ultrasound; In feature 42), the mass percentage concentration of NaCl is X, 0 <X≤30%; 471) In feature 47), the stirring device is a magnetic stirrer; 472) In feature 47), the stirring rate is 400~1200 rpm.

9. The method for simultaneously determining nine bactericides in tobacco products according to claim 1, characterized in that, Includes at least one of the following technical features: 52) In step 5), the amount of derivatizing reagent used is 0.5~4µL; 53) In step 5), the GC chromatographic conditions are: Chromatographic column: DB-5MS capillary column, 0.25μm×30m×250μm film thickness; Carrier gas: selected from one or more of He, N2 and H2, with a purity of not less than 99%; Inlet temperature: 220~300℃; Injection volume: 1~3.5µL, splitless injection; Non-splitting time: 1~3.5min; 54) In step 5), the mass spectrometry conditions are: Ion source: ESI source; Scanning methods: SASN and SIM; Scan range: 35-300 amu; Ion source temperature: 220~280℃; Quadrupole temperature: 130~180℃; Solvent delay: 5~10 min.

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