A method for rapid identification of sulfur-smoked Fritillaria

Abstract

The present invention discloses a method for quickly identifying sulfur-fumed Fritillaria. The method comprises the following steps: S1, crushing the sample to be tested and dissolving it in a methanol solution, mixing it and then centrifuging it, and filtering it to obtain a filtrate; S2, subjecting the filtrate to UPLC‑Q‑TOF‑MS analysis to obtain a total ion current chromatogram. When the total ion current chromatogram includes a molecular ion peak of 508.27±0.0003, that is, it contains sulfonated Fritillaria thunbergii, then the sample to be tested is sulfur-fumed Fritillaria. The present invention uses UPLC‑Q‑TOF‑MS in combination with metabolomics statistical methods. On the basis of chemical research on the sulfur-fumigated Fritillaria system, a common sulfur-containing marker is found in the sulfur-fumigated Fritillaria for rapid identification of sulfur-fumigated Fritillaria. The method of the present invention can also quickly detect samples with a sulfur dioxide residue of only 35 mg / kg. It is fast, simple, and highly specific, and has great promotional significance.

Description

Technical Field

[0001] The present invention relates to a method for rapidly identifying sulfur-fumigated Fritillaria thunbergii, and belongs to the field of analytical technology. Background Art

[0002] Fritillaria buds are derived from the dried bulbs of the genus Fritillaria in the Liliaceae family. The 2020 edition of the Pharmacopoeia of the People's Republic of China (Volume 1) includes five species of Fritillaria buds, in addition to Anhui Fritillaria buds. Fritillaria buds are a popular cough suppressant, with their main medicinal ingredients being alkaloids and saponins, which have antitussive, expectorant, anti-inflammatory, antibacterial, and anti-tumor properties. They are widely used in Traditional Chinese Medicine (TCM) to relieve cough and expectoration, and as a raw material for throat-clearing and soothing health foods, possessing excellent medicinal and nutritional value.

[0003] Sulfur fumigation (hereinafter referred to as "sulfur fumigation") is a commonly used primary processing method for Chinese medicinal materials. It is often used to dry Chinese medicinal materials with high starch polysaccharide content. However, due to its simple operation and low cost, it has been abused in recent years. At present, the phenomenon of sulfur fumigation of Fritillaria thunbergii still exists in the market, especially Fritillaria thunbergii. Studies have shown that the chemical composition and pharmacological activity of Fritillaria thunbergii will undergo corresponding changes after sulfur fumigation, but the effect is not significant within the appropriate sulfur fumigation range. At present, a large number of studies have focused on the analysis of the differential components of a single variety of Fritillaria thunbergii before and after sulfur fumigation, and there is a lack of research on the common differential components of Fritillaria thunbergii of the same genus before and after sulfur fumigation.

[0004] Numerous studies have shown that sulfur fumigation affects the quality of Chinese medicinal materials. The sulfurous acid produced during sulfur fumigation can react with compounds containing different types of functional groups to generate corresponding sulfur fumigation markers. Chinese medicinal materials with the same functional groups are prone to undergo the same or similar transformation processes during sulfur fumigation. For example, aldehydes and ketone compounds can undergo addition reactions with sodium bisulfite to generate corresponding hydroxysulfonates. Alkaloids, the main active ingredients in Fritillaria medicinal materials, contain ketone groups in their structures. The transformation mechanism during sulfur fumigation is still unclear, and research on their sulfur fumigation markers is still lacking. Therefore, exploring the common sulfur-containing compounds in different varieties of Fritillaria before and after sulfur fumigation is of great significance to explaining how sulfur fumigation affects the quality of Fritillaria. Summary of the Invention

[0005] The present invention aims to provide a method for rapidly identifying sulfur-fumigated Fritillaria. The present invention utilizes UPLC-Q-TOF-MS combined with a metabolomics statistical method to separate common sulfur-fumigation characteristic components from the sulfur-fumigated Fritillaria, and the method is used for rapidly identifying the sulfur-fumigated Fritillaria. The method has the characteristics of being fast, simple, and highly specific.

[0006] The present invention separates the sulfonated Fritillaria cirrhosae shown in formula I from sulfur-fumigated Fritillaria cirrhosae.

[0007]

[0008] The sulfonated Fritillaria cirrhosa shown in formula I is used as a sulfur-fumigation marker. Only sulfur-fumigated Fritillaria cirrhosa contains this component, which can be used to distinguish sulfur-fumigated Fritillaria cirrhosa from sulfur-free Fritillaria cirrhosa.

[0009] The experiments of the present invention confirmed that when the sulfonated Fritillaria cirrhosae compound shown in formula I was used as a sulfur fumigation marker for identification, the compound (peak of 508.27) could also be detected in the Fritillaria cirrhosae sample with the lowest sulfur dioxide residue of 35.1 mg / kg, demonstrating the high sensitivity of the method.

[0010] The sulfonated fritillary cinnamate shown in formula I can be prepared according to the following method:

[0011] The beimuxin was fully reacted with a supersaturated aqueous sodium metabisulfite solution, and the supernatant was passed through a 0.22 μm microporous filter membrane. After filtration, HPLC preparative separation was performed using a YMC-Pack ODS-A (250×10.0 mml.DS-5 μm, 12 nm) column with water-acetonitrile as the mobile phase at a flow rate of 2.5 mL / min and an ultraviolet detection wavelength of approximately 200 nm (terminal absorption) as the detection wavelength, as follows:

[0012] First, individual product fractions were collected and analyzed by UPLC-QTOF-MS to identify the target product fraction and its purity. Retention time, molecular weight, secondary fragments, and other information confirmed that the fraction was the target product and had high purity. Based on the peak area, the purity was estimated to be greater than 95%. Secondly, after collecting the target fractions on a large scale, the collected solution was concentrated to approximately 1 mL using a rotary evaporator. The concentrated liquid was then freeze-dried using a freeze dryer.

[0013] Liquid phase detection conditions for the UPLC-QTOF-MS detection:

[0014] Mobile phase: 0.1% formic acid in water as phase A, 0.1% formic acid in acetonitrile as phase B;

[0015] The gradient elution program was as follows: 0-3.0 min, 2%-12% phase B; 3.0-11.0 min, 12%-50% phase B; 11.0-17.0 min, 50%-90% phase B; 17.0-20.0 min, 90%-98% phase B; 20.0-23.0 min, 98% phase B; 23.0-23.05 min, 98%-2% phase B; 23.05-26.0 min, 2% phase B.

[0016] Mass spectrometry detection conditions for the UPLC-QTOF-MS detection:

[0017] The data were collected using an ESI ion source and negative ion mode;

[0018] Ion source parameters: detection data format: continuum; ion source temperature: 100°C; desolvation gas: nitrogen; cone voltage: 40 V; collision energy: 30-60 eV; capillary voltage: 2 kV; cone gas flow rate: 50 L / Hr; desolvation gas flow rate: 650 L / Hr; data acquisition range: 50-1500 Da.

[0019] The identification of sulfur-fumigated Fritillaria cirrhosa by using the sulfonated Fritillaria cirrhosa succinate represented by formula I as a sulfur-fumigation marker comprises the following steps:

[0020] S1. Crush the sample to be tested and dissolve it in methanol solution. Mix well and then centrifuge to obtain filtrate.

[0021] S2. The filtrate is subjected to UPLC-Q-TOF-MS analysis to obtain a total ion current chromatogram. When the total ion current chromatogram includes a 508.27±0.01 molecular ion peak, the sample to be tested is sulfur-fumigated Fritillaria.

[0022] The Fritillaria thunbergii involved in the present invention can be Fritillaria thunbergii, Fritillaria cirrhosa, Fritillaria hupehensis, Fritillaria irifolium and Fritillaria cirrhosa.

[0023] In the above method, in step S1, the particle size of the sample to be tested after crushing is 250±9.9 μm;

[0024] The ratio of the sample to be tested to the methanol solution is: 0.1 g / 1.5-2 mL;

[0025] The volume content of the methanol solution is 70-85%, preferably 80%;

[0026] After ultrasonic treatment, centrifugation was performed.

[0027] In the above method, in step S2, the liquid phase detection conditions of the UPLC-Q-TOF-MS analysis are as follows:

[0028] Mobile phase: 0.1% formic acid in water as phase A, 0.1% formic acid in acetonitrile as phase B;

[0029] The gradient elution program was as follows: 0-3.0 min, 2%-12% phase B; 3.0-11.0 min, 12%-50% phase B; 11.0-17.0 min, 50%-90% phase B; 17.0-20.0 min, 90%-98% phase B; 20.0-23.0 min, 98% phase B; 23.0-23.05 min, 98%-2% phase B; 23.05-26.0 min, 2% phase B;

[0030] Injection volume: 2 μL, flow rate: 0.5 mL / min, column temperature: 40°C.

[0031] In the above method, in step S2, the mass spectrometry detection conditions of the UPLC-Q-TOF-MS analysis are as follows:

[0032] The data were collected using an ESI ion source and negative ion mode;

[0033] Ion source parameters: detection data format: continuum; ion source temperature: 100°C; desolvation gas: nitrogen; cone voltage: 40 V; collision energy: 30-60 eV; capillary voltage: 2 kV; cone gas flow rate: 50 L / Hr; desolvation gas flow rate: 650 L / Hr; data acquisition range: 50-1500 Da; the calibration solution (lock mass) was leucine enkephalin, and the accurate molecular mass was set to 554.2620 in negative ion mode.

[0034] Fritillaria medicinal materials have a wide range of sources and varieties. Currently, most studies focus on the differential component analysis of a single variety of Fritillaria medicinal materials before and after sulfur fumigation. There is no simple and effective method for detecting multiple varieties of sulfur-fumigated Fritillaria. The present invention uses UPLC-Q-TOF-MS combined with metabolomics statistical methods. Based on the chemical research of the sulfur-fumigated Fritillaria system, a common sulfur-containing marker is discovered in sulfur-fumigated Fritillaria for rapid identification of sulfur-fumigated Fritillaria. The method of the present invention can also quickly detect samples with a sulfur dioxide residue of only 35 mg / kg. It is fast, simple, and highly specific, and has great promotional significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The mass spectrometric fragmentation pathway of the sulfonated fritillary cin shown in formula I is shown in FIG.

[0036] Figure 2 The sulfonated benzyl alcohol of formula I 1 H NMR (400 MHz, CD3OD-d4) spectrum.

[0037] Figure 3 The sulfonated benzyl alcohol of formula I 13 C NMR (100 MHz, CD3OD-d4) spectrum.

[0038] Figure 4 This is the DEPT spectrum of the sulfonated fritillary cin shown in formula I.

[0039] Figure 5 This is the HMBC spectrum of the sulfonated benzyl alcohol represented by formula I.

[0040] Figure 6 HSQC spectrum of the sulfonated benzyl alcohol represented by formula I

[0041] Figure 7The key HMBC and 1 H, 1 H-COSY correlation.

[0042] Figure 8 The peak containing 508.27 is extracted from Fritillaria with different degrees of sulfur fumigation. DETAILED DESCRIPTION

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0044] The instruments and reagents used in the following examples were: ACQUITY I-Class ultra-high performance liquid chromatograph-Waters Xevo-G2-S QTof MS mass spectrometer system (Waters, USA), ACQUITY UPLC™ I-Class system with an electrospray ionization source, Waters ACQUITY UPLC-CSH-C18 chromatographic column (2.1 mm × 100 mm, 1.7 μm, Waters, USA), Mettler Toledo New classic MF type 100,000 analytical balance Mettler Toledo (Mettler Toledo, Switzerland), BSA224S type 10,000 analytical balance (Sartorius, Germany), Centrifuge 5415D centrifuge (Eppendorf, Germany), SB-800-DTD type ultrasonic cleaner, ultrasonic power 500 W (Ningbo Xinzhi Biotechnology Co., Ltd.), and Pacific T-II type ultrapure water instrument (Thermo Fisher Scientific, USA). 0.22 μm hydrophobic PTFE microporous filter (Millipor, USA). Water was ultrapure water, methanol, and acetonitrile were chromatographic grade (Fisher Scientific, USA). All other reagents were analytical grade. HPLC (Agilent, Santa Clara, CA, USA)

[0045] The experimental materials used in the following examples are as follows: the thunbergii sample was collected in Pan'an County, Jinhua City, Zhejiang Province on April 30, 2021, the flat Fritillaria sample was collected in Xinbin Manchu Autonomous County, Fushun City, Liaoning Province on June 5, 2021, the Hubei Fritillaria and Ili Fritillaria samples were purchased from Bozhou Medicinal Materials Market on August 21, 2021, and the Sichuan Fritillaria sample was purchased from Beijing Tong Ren Tang (Group) Co., Ltd. on August 26, 2021. All samples used were identified by the Center for Chinese Materia Medica Resources of the China Academy of Chinese Medical Sciences as fresh bulbs of Fritillaria plants of the genus Fritillaria F.thunbergii Miq., flat Fritillaria F.ussuriensis Maxim., Hubei Fritillaria F.hupehensis Hsiao etK.C.Hsia, Ili Fritillaria F.pallidiflora Schrenk, and Sichuan Fritillaria F.cirrhosa D.Don.

[0046] Example 1. Preparation and structural identification of sulfonated fritillary cinnamate of formula I

[0047] 1. Preparation of sulfonated fritillary cinnamate shown in formula I

[0048] Beimuxin was fully reacted with a supersaturated aqueous sodium metabisulfite solution. The supernatant was filtered through a 0.22 μm microporous filter membrane and then eluted using a YMC-Pack ODS-A (250×10.0 mm L.DS-5 μm, 12 nm) column with a water (A)-acetonitrile (B) mobile phase. The gradient elution program was as follows: 0-30.0 min, 10%-20% B; 30.0-35.0 min, 20%-90% B; 35.0-40.0 min, 90%-10% B; 40.0-50.0 min, 10% B. The flow rate was 2.5 mL / min, and preparative separation was performed by HPLC using an ultraviolet detection wavelength of approximately 200 nm (terminal absorption).

[0049] First, each product fraction was collected for UPLC-QTOF-MS analysis to identify the target product fraction and its purity. The retention time, molecular weight, secondary fragments, and other information confirmed that the fraction was the target product and had a high purity. Based on the peak area, the purity was determined to be greater than 95%. Second, after collecting the target fractions on a large scale, the collected solution was concentrated to approximately 1 mL using a rotary evaporator. The concentrated liquid was then freeze-dried using a freeze dryer to obtain the sulfonated fritillary cinnamate of Formula I.

[0050] UPLC-QTOF-MS detection conditions:

[0051] Liquid phase detection conditions are as follows:

[0052] Mobile phase: 0.1% formic acid in water as phase A, 0.1% formic acid in acetonitrile as phase B;

[0053] The gradient elution program was as follows: 0-3.0 min, 2%-12% phase B; 3.0-11.0 min, 12%-50% phase B; 11.0-17.0 min, 50%-90% phase B; 17.0-20.0 min, 90%-98% phase B; 20.0-23.0 min, 98% phase B; 23.0-23.05 min, 98%-2% phase B; 23.05-26.0 min, 2% phase B;

[0054] Injection volume: 2 μL, flow rate: 0.5 mL / min, column temperature: 40°C.

[0055] Mass spectrometry detection conditions are as follows:

[0056] The data were collected using an ESI ion source and negative ion mode;

[0057] Ion source parameters: detection data format: continuum; ion source temperature: 100°C; desolvation gas: nitrogen; cone voltage: 40 V; collision energy: 30-60 eV; capillary voltage: 2 kV; cone gas flow rate: 50 L / Hr; desolvation gas flow rate: 650 L / Hr; data acquisition range: 50-1500 Da; the calibration solution (lock mass) was leucine enkephalin, and the accurate molecular mass was set to 554.2620 in negative ion mode.

[0058] 2. Structural Identification of the Sulfonated Beimuxin Represented by Formula I

[0059] PMS-S (4 mg, 0.010 mmol) was dissolved in 500 μL of pyridine-d5 in an NMR tube, (-)-MTPACl (4 μL, 0.021 mmol) was added, and the mixture was kept at room temperature under argon protection overnight.

[0060] The compound obtained in step 1 was a white amorphous solid, UV (acetonitrile-water): λmax (logε) 200.4 nm, ESI-MS: m / z 509.27 ([M+H] - , C 27 H 42 NO6S - Compared with Beimuxin, there is one more molecule of H2SO3 in the molecular formula. Therefore, it is speculated that this compound is produced by the addition reaction between Beimuxin and H2SO3. Its mass spectrometry fragmentation pathway is as follows Figure 1 shown.

[0061] The compound 1 H-NMR and 13 C-NMR data are shown in Table 1. 1H-NMR ( Figure 2 ), 13 C-NMR ( Figure 3 )、DEPT( Figure 4 )HMBC( Figure 5 ) and HSQC( Figure 6 ) spectrum, combined with the NMR spectrum data of the compound's precursor, Beimuxin

[17] , it can be judged that the compound has 4 methyl groups [δH 0.75 (3H, s, H-19), 1.05 (3H, d, J = 6.5 Hz, H-27), 1.49 (3H, d, J = 7.5 Hz, H-21), 2.03 (3H, s, H-18); δC 12.45 (C-19), 16.87 (C-21), 17.66 (C-18), 18.44 (C-27)]; 9 methylene groups [δC 30.60 (C-4, 14), 31.28 (C-2, 15), 34.54 (C-11), 38.14 (C-1), 43.07 (C-24), 46.84 (C-7), 52.04 (C-26), 30.60 (C-4)] and 9 methine groups [δC 71.35 (C-3), 70.89 (C-23), 65.43 (C-22), 57.30 (C-5), 53.60 (C-9), 49.29 (C-13), 47.38 (C-8), 29.24 (C-20, 25)] and 5 quaternary carbons [δC 213.36 (C-6), 142.02 (C-17), 132.18 (C-16), 74.18 (C-12), 39.20 (C-10)], and the carbonyl group [δC 213.36 (C-6)] in the precursor benzyl cinnamate still exists after the reaction. 1 H, 1 The H-COSY spectrum shows the following correlations: H-1 / H-2, H-3, H-4, H-8 / H-9, H-7 / H-8, H-9, H-11, H-13, H-14, H-15, H-20 / H-22, H-23, H-24, H-25, H-26 ( Figure 7 ); HMBC spectrum shows the following correlations: H-1 / C-3, C-5, C-9, CH3-19; H-5 / C-3, C-7, C-9; CH3-19 / C-1, C-9, C-5; H-11 / C-8, C-13, C-17; CH3-18 / C-12, C-16; H-15 / C-13, C-17, C-20; H-20 / C-15, C-17, C-23; CH3-27 / C-24, C-26( Figure 7 ); Combined with the above 1 H, 1The correlation between H-COSY and HMBC spectra can be used to construct the entire skeleton structure of the compound, which is a sulfonated fritillary cin. Compared with fritillary cin, the ether bond between C-16 and C-23 of this compound is broken, and the double bond between C-12 and C-17 migrates to between C-16 and C-17; combined with 1 D and 2 The D-NMR information indicated that the sulfonic acid group was attached to the C-12 position of the compound.

[0062] Table 1 Compounds of formula I 1 H-NMR (400 MHz) and 13 C-NMR (100 MHz) data

[0063]

[0064] Example 2, identification of sulfur-fumigated Fritillaria

[0065] 1. Preparation of sulfur-smoked Fritillaria samples

[0066] Five fresh Fritillaria herbs were washed and air-dried. They were divided into a non-sulfur group and a sulfur-fumigated group. The sulfur-fumigated group used a sulfur-to-sample ratio of 1:10 (three samples per group, approximately 200g each). The divided samples were placed in two plastic boxes and ignited with 12ml of alcohol per group. After 24 hours, the samples were removed, freeze-dried, powdered, and stored at 4°C.

[0067] 2. Preparation of test solution

[0068] Approximately 0.1 g of each sample powder (particle size 250 ± 9.9 μm) was added to 1.5 ml of 80% methanol solution, mixed thoroughly, sonicated for 1 hour, weighed differentially, and centrifuged at 12 rpm for 10 minutes. The samples were filtered through a 0.22 μm filter to obtain the sample solutions. 50 μL of each sample solution was then mixed to obtain the mixed sample solution (serving as a quality control). UPLC-Q-TOF-MS analysis was then performed.

[0069] 3. UPLC-Q-TOF-MS analysis

[0070] 1. Liquid phase conditions

[0071] The elution solvents were 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The gradient elution program was as follows: 0-3.0 min, 2%-12% B; 3.0-11.0 min, 12%-50% B; 11.0-17.0 min, 50%-90% B; 17.0-20.0 min, 90%-98% B; 20.0-23.0 min, 98% B; 23.0-23.05 min, 98%-2% B; 23.05-26.0 min, 2% B. Injection volume: 2 μL, flow rate: 0.5 mL / min, column temperature: 40°C.

[0072] 2. Mass spectrometry conditions

[0073] Data were acquired using an ESI ion source in negative ion mode using a MassLynx 4.1 acquisition system. Source parameters included: Survey Data Format: Continuous; Source Temperature: 100°C; Desolvation Gas: Nitrogen; Sample Cone: 40 V; Ramp High Energy: 30–60 eV; Capillary Voltage: 2 kV; Cone Gas Flow: 50 L / Hr; Desolvation Gas Flow: 650 L / Hr; Acquisition Mass Range: 50–1500 Da; Leucine enkephalin (LEK) as the calibration fluid, with an exact molecular mass of 554.2620 in negative ion mode.

[0074] 4. Identification of sulfur-smoked Fritillaria samples

[0075] The sulfonated Fritillaria cirrhosa shown in formula I is used as a specific component to identify sulfur-smoked Fritillaria cirrhosa and sulfur-free Fritillaria cirrhosa.

[0076] like Figure 1 As shown, the characteristic ion of the compound is 508.27. 508.27 was extracted from the total ion chromatogram of Fritillaria with different sulfur fumigation degrees. The results are as follows Figure 8 shown.

[0077] Figure 8 The results showed that the peak at 508.27 had a high response in all sulfur-fumigated Fritillaria, while no response was found in the non-sulfur Fritillaria, indicating the feasibility of the method. Among all sulfur-fumigated Fritillaria, the peak at 508.27 was also detected in the Fritillaria sample (Chuan Fritillaria-Sulfur-Fumigated-2) with the lowest sulfur dioxide residue of 35 mg / kg, indicating the high sensitivity of the method.

[0078] The method of extracting the peak of 508.27 in the total ion current graph using high-resolution mass spectrometry technology can distinguish between sulfur-fumigated and sulfur-free Fritillaria, which has the advantages of being simple, rapid, highly specific and highly sensitive.

Claims

1. Sulfonated fritillary cinnamate shown in formula I, 2. Use of the sulfonated Fritillaria cirrhosae represented by formula I in claim 1 in identifying sulfur-fumigated Fritillaria cirrhosae.

3. The use according to claim 2, characterized in that: The sulfonated fritillary cin shown in formula I is used as a sulfur fumigation marker.

4. The use according to claim 2 or 3, characterized in that: The sulfur dioxide residue in the sulfur-smoked Fritillaria is as low as 35 mg / kg.

5. A method for identifying sulfur-smoked Fritillaria thunbergii, comprising the steps of: S1. Crush the sample to be tested and dissolve it in methanol solution. Mix well and then centrifuge to obtain filtrate. S2, described filtrate is carried out UPLC-Q-TOF-MS analysis, obtain total ion current chromatogram, when described total ion current chromatogram includes 508.27 ± 0.01 molecular ion peak, then testing sample is sulfur-smoked Fritillaria; In step S2, the liquid phase detection conditions of the UPLC-Q-TOF-MS analysis are as follows: Mobile phase: 0.1% formic acid in water as phase A, 0.1% formic acid in acetonitrile as phase B; The gradient elution program was as follows: 0-3.0 min, 2%-12% phase B; 3.0-11.0 min, 12%-50% phase B; 11.0-17.0 min, 50%-90% phase B; 17.0-20.0 min, 90%-98% phase B; 20.0-23.0 min, 98% phase B; 23.0-23.05 min, 98%-2% phase B; 23.05-26.0 min, 2% phase B; The mass spectrometry detection conditions for the UPLC-Q-TOF-MS analysis are as follows: The data were collected using an ESI ion source and negative ion mode; Ion source parameters: detection data format: continuum; ion source temperature: 100°C; desolvation gas: nitrogen; cone voltage: 40 V; collision energy: 30-60 eV; capillary voltage: 2 kV; cone gas flow rate: 50 L / Hr; desolvation gas flow rate: 650 L / Hr; data acquisition range: 50-1500 Da.

6. The method according to claim 5, characterized in that: In step S1, the particle size of the sample to be tested after crushing is 250±9.9 μm; The ratio of the sample to be tested to the methanol solution is: 0.1 g / 1.5-2 mL; The volume content of the methanol solution is 70% to 85%; After ultrasonic treatment, centrifugation was performed.

7. The method according to claim 5 or 6, characterized in that: The residual amount of sulfur dioxide in the sample to be tested is as low as 35 mg / kg.

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