Extraction method of spices containing ethylene oxide and 2-chloroethanol

Abstract

The invention relates to a method for extracting spices containing ethylene oxide and 2-chloroethanol, and particularly relates to a method for extracting spices containing ethylene oxide and 2-chloroethanol, which comprises the following steps of: adding an inorganic salt solution into spices by using an acetonitrile solution as an extraction solvent to obtain two aqueous phases to promote the transfer of ethylene oxide and 2-chloroethanol to an organic phase. Meanwhile, the extraction efficiency of the compound is improved by utilizing a diluent swing effect generated by the eutectic solvent, and the efficient extraction of ethylene oxide and 2-chloroethanol in the spices is successfully realized.

Description

Technical Field

[0001] The invention relates to an extraction method for spices containing ethylene oxide and 2-chloroethanol, which specifically adopts an acetonitrile solution added with a deep eutectic solvent combined with a two-aqueous phase extraction technology. Background Art

[0002] Food microbial contamination has become a food safety issue of great concern worldwide, especially Salmonella and E. coli, which cause huge economic losses every year. In order to deal with food microbial contamination, countries such as India or Turkey usually use fumigants to fumigate spices to remove pathogenic microorganisms and bacteria in food. Usually, ethylene oxide is used as a fumigant. Ethylene oxide (EO) has a boiling point of 10.3°C. Due to its small size, high diffusivity and strong penetrability, it is very effective in deinsectizing or disinfecting dry food products. Ethylene oxide is an excellent fumigant and has been used as a fumigant in the food industry for more than 40 years. Due to the highly toxic nature of ethylene oxide, it has been stopped in most parts of the world. The International Agency for Research on Cancer (IARC) classifies it as a Class I carcinogen (carcinogenic to humans). In 2024, my country has carried out relevant risk assessments on spices originating in India, Turkey and other countries containing ethylene oxide and 2-chloroethanol. As of May 18, 2025, the EU Rapid Alert System for Food and Feed RSAFF notification system has received more than 950 reports involving incidents of excessive ethylene oxide, including 596 originating in India and Turkey. The highest ethylene oxide content in their spices is 186mg / kg and 20.4mg / kg. The EU limit for ethylene oxide and 2-chloroethanol in spices is 0.05mg / kg.

[0003] Ethylene oxide (EO) is a highly unstable molecule whose ring structure enables it to react with halogen atoms and water present in the surrounding environment or in food. This results in the formation of less volatile and more stable metabolites such as 2-chloroethanol (2-CE), 2-bromoethanol, and ethylene glycol. Although 2-CE is less hazardous than EO, it still needs to be monitored alongside EO, as Regulation (EU) No. 396 / 2005 defines EO residues as the sum of EO and 2-CE. Therefore, strict monitoring measures for the residues of these contaminants in food are essential to ensure food safety and facilitate international trade.

[0004] As a new generation of ionic liquids, the concept of DES was first proposed by Abbot in 2003. DES are typically composed of low-melting-point mixtures of hydrogen bond acceptors (HBAs) such as quaternary ammonium salts and quaternary salts, and hydrogen bond donors (HBDs) such as amides, carboxylic acids, and alcohols, formed through hydrogen bonding in a specific ratio. DES not only possess the advantages of ionic liquids but also possess unique properties such as low cost, ease of preparation, biodegradability, and good biocompatibility. These properties enable DES to demonstrate excellent performance in gas absorption, extraction and separation, chemical reactions, and electrochemistry. In the field of extraction and separation, DES, in addition to its environmentally friendly properties, also offers a significant advantage in designability compared to traditional organic extractants. By varying the type and ratio of HBAs and HBDs in a DES, its physical properties can be tailored to suit the extraction operating window. Its hydrogen bond-forming capacity and strength with solute components can also be manipulated to improve solute recognition, laying the theoretical foundation for its application in extraction and separation processes.

[0005] The concept of an aqueous two-phase system was first proposed by Roger's team in 2003. Since then, it has been widely applied in the biology, pharmaceutical, and food industries. As a new green separation and purification technology, aqueous two-phase extraction is simple, time-saving, highly efficient, and pollution-free. It overcomes the shortcomings of traditional organic solvent extraction and meets the development requirements of green biochemical separation technologies.

[0006] A deep eutectic solvent aqueous two-phase system refers to a novel extraction system that combines a deep eutectic solvent with small molecules or inorganic salts. This system leverages the excellent solubility, non-volatility, and biodegradability of deep eutectic solvents, resulting in additional extraction advantages, including high efficiency, rapidity, and environmental friendliness. Summary of the Invention

[0007] This patent, leveraging the low boiling point of ethylene oxide and combined with eutectic aqueous two-phase extraction technology, establishes a mild sample pretreatment method. This method successfully achieves efficient extraction of ethylene oxide and 2-chloroethanol from spice samples, ensuring compliance with the EURL-SRM ethylene oxide standard. A method for extracting ethylene oxide and 2-chloroethanol from spices, characterized by using acetonitrile as the extraction solvent and adding an inorganic salt solution to the spices to produce an aqueous two-phase solution, which promotes the transfer of ethylene oxide and 2-chloroethanol to the organic phase.

[0008] The specific preparation steps are:

[0009] 1) Preparation of raw materials: Prepare spices treated with fumigants; 2) Preparation of deep eutectic solvent: Accurately weigh choline bicarbonate (molecular weight 165.19) and geranic acid (molecular weight 168.23) in a molar ratio of 1:0.5-4, and mix at 80-100°C under vacuum (150-200 rpm) for 10 hours;

[0010] 3) preparing the extractant: mixing acetonitrile and the deep eutectic solvent prepared in step 2) at a volume ratio of 1% to 20%;

[0011] 4) Extraction process: After adding the extractant and inorganic salt solution of step 3) to the spices obtained in step 2), a biphasic aqueous phase is obtained to promote the transfer of ethylene oxide and 2-chloroethanol to the organic phase.

[0012] The spices treated by the fumigant method are one or more of cumin, chili, turmeric, oregano containing ethylene oxide and 2-chloroethanol. The spices are purchased from India and Turkey. The fumigant uses ethylene oxide,

[0013] The spice is one or more than two of cumin, pepper, turmeric and oregano.

[0014] A deep eutectic solvent is added to the acetonitrile solution, wherein the hydrogen bond donor of the deep eutectic solvent is choline chloride bicarbonate, the hydrogen bond acceptor is geranic acid, and the molar ratio of the deep eutectic solvent to the spice is 1:0.5-4, wherein the molecular weight of choline chloride bicarbonate is 165.19, and the molecular weight of geranic acid is 168.23.

[0015] The spice contains one or more of cumin, pepper, turmeric and oregano, and contains ethylene oxide and 2-chloroethanol. The spice is purchased from India and Türkiye.

[0016] The volume fraction of the deep eutectic solvent in the extractant acetonitrile is 1 to 20%.

[0017] The inorganic salt forming the aqueous two phase is one or more of ammonium sulfate, potassium sulfate and potassium chloride.

[0018] The mass fraction of the inorganic salt is 10 to 30%.

[0019] The mass fraction of the spice in the reaction system is 20 to 60%.

[0020] The advantages of the present invention are: the present invention combines the characteristics of a deep eutectic solvent and a two-phase aqueous extraction, establishes an acetonitrile and inorganic salt construction system, the extraction system has mild conditions and is particularly suitable for the efficient extraction of low-boiling-point compound ethylene oxide. The Quechers method used in the conventional method generates a large amount of heat during the extraction process, which easily causes the loss of low-boiling-point compounds. The present invention uses a mild two-phase aqueous extraction condition and utilizes the diluent swing effect generated by the deep eutectic solvent to improve the extraction efficiency of the compound. The invention successfully achieves the efficient extraction of ethylene oxide and 2-chloroethanol in spices. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 These are the test results for ethylene oxide and 2-chloroethanol in cumin samples.

[0022] Figure 2 is the reaction equation for a deep eutectic solvent. DETAILED DESCRIPTION

[0023] Preparation Example

[0024] Preparation of raw materials in the present invention

[0025] 1. Preparation of Raw Materials: Spices were prepared by fumigation at 30°C for 120 hours using a fumigant (ethylene oxide). The spice was cumin, purchased from India, and tested using the EURL-SRM method to determine the presence of ethylene oxide and 2-chloroethanol. The following examples all used spices obtained using this method.

[0026] 2. Preparation of deep eutectic solvent: Accurately weigh choline chloride bicarbonate and geranic acid in a molar ratio of 1:0.5-4 (1:2 used in the following examples), and mix at 80°C under vacuum (100 rpm) for 10 h.

[0027] 3. Extractant preparation: Accurately pipette 100 ml of acetonitrile, with the volume ratio of acetonitrile to deep eutectic solvent being 1% to 20% (10 ml / time is used in the present invention).

[0028] 4. Preparation of inorganic salt solution: Weigh one or more of ammonium sulfate, potassium sulfate, and potassium chloride to prepare an aqueous solution with a mass fraction of 10-30%.

[0029] Example 1

[0030] Taking cumin as an example, the temperature of cumin was set to 30°C on the fumigation machine and fumigated for 120 hours. The obtained spices were tested by the EURL-SRM method, and cumin containing ethylene oxide and 2-chloroethanol was obtained. The test results showed that ethylene oxide was 0.05 mg / kg and 2-chloroethanol was 0.50 mg / kg. 2 g of the obtained cumin sample was weighed in a 50 mL centrifuge tube, 5 mL of inorganic salt solution (15% by mass of potassium chloride) was added, 50 μg / mL of ethylene oxide and 2-chloroethanol isotope internal standard mixed solution (wherein the concentration ratio of D4-ethylene oxide to D4-2-chloroethanol was 1:2) was added, 10 mL of acetonitrile (containing a low eutectic solvent volume ratio of 1%) was added, and the mixture was tumbled and mixed for 10 minutes at a speed of 70 r / min and centrifuged at 10000 r / min for 10 minutes. 1 mL of the supernatant was taken and solid phase extraction (waters PRIME HLB 3cc) was performed. 60mg) was purified, the first 0.1mL of the effluent was discarded, and the remaining filtrate was collected. The results of chromatography-mass spectrometry detection showed that the content of ethylene oxide was 0.035mg / kg and that of 2-chloroethanol was 0.039mg / kg. The results are shown in the attached Figure 1 , the method of Example 1 is lower than the standard values ​​of ethylene oxide and 2-chloroethanol.

[0031] Example 2:

[0032] Weigh 2 g of the cumin sample of the preparation example into a 50 mL centrifuge tube, add 5 mL of an inorganic salt solution (10% by mass of potassium sulfate), add 50 μg / mL of a mixed solution of ethylene oxide and 2-chloroethanol isotope internal standard (wherein the concentration ratio of D4-ethylene oxide to D4-2-chloroethanol is 1:2), add 10 mL of acetonitrile (containing a 5% volume ratio of a deep eutectic solvent), turn and mix for 10 minutes at a speed of 70 r / min, centrifuge at 10000 r / min for 10 minutes, take 1 mL of the supernatant, and purify it by solid phase extraction (watersPRIME HLB 3cc 60 mg), discard the first 0.1 mL of the effluent, and receive the remaining filtrate. The results of chromatography-mass spectrometry detection were 0.032 mg / kg for ethylene oxide and 0.035 mg / kg for 2-chloroethanol. See the attached results. Figure 1 , the method of Example 2 is lower than the standard values ​​of ethylene oxide and 2-chloroethanol.

[0033] Example 3:

[0034] Weigh 2 g of the cumin sample of the preparation example into a 50 mL centrifuge tube, add 5 mL of an inorganic salt solution (20% by mass of ammonium sulfate), add 50 μg / mL of a mixed solution of ethylene oxide and 2-chloroethanol isotope internal standard (wherein the concentration ratio of D4-ethylene oxide to D4-2-chloroethanol is 1:2), add 10 mL of acetonitrile (containing a low eutectic solvent volume ratio of 20%), turn and mix for 10 minutes at a speed of 70 r / min, centrifuge at 10000 r / min for 10 minutes, take 1 mL of the supernatant, and purify it by solid phase extraction (watersPRIME HLB 3cc 60 mg), discard the first 0.1 mL of the effluent, and receive the remaining filtrate. The results of chromatography-mass spectrometry detection were 0.047 mg / kg for ethylene oxide and 0.049 mg / kg for 2-chloroethanol. See the attached results. Figure 1 , the standard values ​​of ethylene oxide and 2-chloroethanol are consistent using the method of Example 3.

[0035] Example 4:

[0036] Weigh 2 g of the cumin sample of the preparation example into a 50 mL centrifuge tube, add 5 mL of ultrapure water, add 50 μg / mL of a mixed solution of ethylene oxide and 2-chloroethanol isotope internal standard (wherein the concentration ratio of D4-ethylene oxide to D4-2-chloroethanol is 1:2), add 10 mL of acetonitrile (containing a low eutectic solvent volume ratio of 10%), turn and mix for 10 minutes at a speed of 70 r / min, centrifuge at 10000 r / min for 10 minutes, take 1 mL of the supernatant, and purify it by solid phase extraction (waters PRIME HLB 3cc 60 mg), discard the first 0.1 mL of the effluent, and receive the remaining filtrate. The results of chromatography-mass spectrometry detection were 0.023 mg / kg for ethylene oxide and 0.026 mg / kg for 2-chloroethanol. See the attached results. Figure 1 , the method of Example 4 is lower than the standard values ​​of ethylene oxide and 2-chloroethanol.

[0037] Example 5

[0038] Weigh 2 g of the cumin sample of the preparation example into a 50 mL centrifuge tube, add 5 mL of ultrapure water, add 50 μg / mL of a mixed solution of ethylene oxide and 2-chloroethanol isotope internal standard (wherein the concentration ratio of D4-ethylene oxide to D4-2-chloroethanol is 1:2), add 10 mL of acetonitrile, flip and mix for 10 min at a speed of 70 r / min, centrifuge at 10000 r / min for 10 min, take 1 mL of the supernatant, and purify it by solid phase extraction (waters PRIME HLB 3cc 60 mg), discard the first 0.1 mL of the effluent, and collect the remaining filtrate. The results of chromatography-mass spectrometry detection were 0.018 mg / kg for ethylene oxide and 0.024 mg / kg for 2-chloroethanol. See the attached results. Figure 1, the method of Example 5 is lower than the standard values ​​of ethylene oxide and 2-chloroethanol.

[0039] Example 6:

[0040] Weigh 2 g of the cumin sample of the preparation example into a 50 mL centrifuge tube, add 5 mL of an inorganic salt solution (20% potassium chloride), add 50 μg / mL of a mixed solution of ethylene oxide and 2-chloroethanol isotope internal standard (wherein the concentration ratio of D4-ethylene oxide to D4-2-chloroethanol is 1:2), add 10 mL of acetonitrile, flip and mix for 10 min at a speed of 70 r / min, centrifuge at 10000 r / min for 10 min, take 1 mL of the supernatant, and purify it by solid phase extraction (waters PRIME HLB 3cc 60 mg), discard the first 0.1 mL of the effluent, and receive the remaining filtrate. The result of chromatography-mass spectrometry detection is that the ethylene oxide content is 0.019 mg / kg.

[0041] 2-Chloroethanol is 0.025mg / kg. See attached for the results. Figure 1 , the method of Example 6 is lower than the standard values ​​of ethylene oxide and 2-chloroethanol.

[0042] Application Examples

[0043] Test method:

[0044] GC-MS / MS analysis conditions for ethylene oxide and 2-chloroethanol residues:

[0045] Chromatographic conditions:

[0046] Chromatographic column: highly inert quartz capillary column DB-624UI (6% cyanopropyl / phenyl, 94% polydimethylsiloxane), 60 m × 0.25 mm × 1.4 um; carrier gas: helium (99.999%); carrier gas flow rate: 1.0 mL / min; column temperature: initial temperature 35°C for 5 min, then increase to 220°C at 20°C / min and hold for 5 min; injection port temperature: 240°C; injection volume: 2 μL; injection mode: split injection, split ratio 10:1.

[0047] Mass spectrometry conditions:

[0048] Solvent delay: 4.5 min; ionization mode: electron impact (EI); ionization energy: 70 eV; ion source temperature: 250°C; quadrupole temperature: 150°C; transfer line temperature: 240°C; collision gas: high-purity argon (99.999%); detection mode: multiple reaction monitoring (MRM). Retention times and mass spectrometric parameters for EtO and 2-CE are shown in Table 1.

[0049] Table 1 Retention time and mass spectrometry parameters of ethylene oxide and 2-chloroethanol

[0050]

[0051] in conclusion:

[0052] The conditions of the application example were measured for Examples 1-6. It can be clearly seen from the comparison of specific examples 4 and 5 that when pure acetonitrile alone and acetonitrile containing a deep eutectic solvent are used as extraction agents, the detection results of ethylene oxide and 2-chloroethanol in Example 4 are 0.023 mg / kg for ethylene oxide and 0.026 mg / kg for 2-chloroethanol, 0.018 mg / kg for ethylene oxide and 0.024 mg / kg for 2-chloroethanol in Example 5, and it can be seen from the comparison of specific examples 6 and 5 that only the two-phase aqueous extraction method is used. The results of Example 5 are ethylene oxide. The test results of ethylene oxide and 2-chloroethanol in Specific Example 4, Specific Example 5, and Specific Example 6 were lower than those of the extraction method using a combination of low eutectic and double hydrate. At the same time, the results of the extraction system containing different deep eutectic solvents in acetonitrile were different. The result of Specific Example 3 was significantly higher than that of Specific Example 1 and Specific Example 2.

Claims

1. A method for extracting spices containing ethylene oxide and 2-chloroethanol, characterized in that: 1) Preparation of raw materials: Prepare spices treated with fumigants; 2) Preparation of deep eutectic solvent: Accurately weigh choline bicarbonate and geranic acid in a molar ratio of 1:0.5-4, and mix at 80-100°C under vacuum (150-200 rpm) for 10 hours; 3) preparing the extractant: mixing acetonitrile and the deep eutectic solvent prepared in step 2) at a volume ratio of 1% to 20%; 4) Extraction process: After adding the extractant and inorganic salt solution of step 3) to the spices obtained in step 2), a biphasic aqueous phase is obtained to promote the transfer of ethylene oxide and 2-chloroethanol to the organic phase.

2. The extraction method according to claim 1, wherein: The spices treated by the fumigant method are one or more of cumin, pepper, turmeric and oregano containing ethylene oxide and 2-chloroethanol, and the spices are purchased from India and Türkiye.

3. The extraction method according to claim 1, wherein: The hydrogen bond donor of the deep eutectic solvent is choline chloride bicarbonate, the hydrogen bond acceptor is geranic acid, and the molar ratio of the deep eutectic solvent to the spice is 1:0.5-4.

4. The extraction method according to claim 3, wherein: The volume fraction of the deep eutectic solvent in the extractant acetonitrile is 1 to 20%.

5. The extraction method according to claim 1, wherein: The inorganic salt forming the aqueous two phase is one or more of ammonium sulfate, potassium sulfate and potassium chloride.

6. The extraction method according to claim 5, wherein: The mass fraction of the inorganic salt is 10 to 30%.

7. The extraction method according to claim 1, wherein: The mass fraction of the spice in the reaction system is 20 to 60%.

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