AU2020102328A4

AU2020102328A4 - Method for determination of heterocyclic aromatic amine in oil

Info

Publication number: AU2020102328A4
Application number: AU2020102328A
Authority: AU
Inventors: Yuxiang MA; Zhao QIN; Xuede WANG; Jun Xi; Chenxia ZHANG; Tianpei ZHAO
Original assignee: Henan University of Technology
Current assignee: Henan University of Technology
Priority date: 2019-10-29
Filing date: 2020-09-18
Publication date: 2020-10-29
Anticipated expiration: 2028-09-18
Also published as: CN110618217A

Abstract

The present invention relates to the technical field of heterocyclic aromatic amines (HAAs) detection, and in particular to a method for the determination of HAAs in oils. The method for detecting HAAs in oils provided by the present invention includes the following steps: extracting and purifying HAAs from oil samples; and identifying and quantitating HAAs in oil samples by ultra-high performance liquid chromatography - triple quadrupole mass spectrometry (UPLC MS/MS); the extraction and purification includes alkaline organic solvent extraction and solid phase extraction (SPE). The method provided by the present invention is effective, sensitive, accurate, easy to operate, and suitable for the determination and confirmation of HAAs in oils. 1 /2 1400000 1200000- 8 1000000 800000 600000 1 400000 200000 0 0 1 2 3 4 5 Time (min) FIG. 1 120 | e iAcetonitrile Alkaline acetonitrile 100 80 o 60 40 20 0 --- FIG. 2

Description

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120 | e iAcetonitrile Alkaline acetonitrile 100

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METHOD FOR DETERMINATION OF HETEROCYCLIC AROMATIC AMINE IN OIL

TECHNICAL FIELD The present invention relates to the technical field of heterocyclic aromatic amines (HAAs) detection, and in particular to a method for the determination of HAAs in oils. BACKGROUND With the rapid development of economy and the incessant improvement of people's living standard in China, food safety problems have received more and more attention. Oils are important sources of daily energy and essential fatty acids, and safety problems thereof have gained more and more attention. However, oils are susceptible to contamination of heterocyclic aromatic amines (HAAs) during oil processing, and these contaminants are carcinogenic and mutagenic (Sanz-Alaejos, M., & Afonso, A. M. (2011). Factors that affect the content of heterocyclic aromatic amines in foods. Comprehensive Reviews in Food Science and Food Safety, 10, 52-108. De Meester, C. (1995). Genotoxic potential of beta-carbolines: A review. Mutation Research, 339, 139-153.). The presence of HAAs leads to increased risk factors for oils, and the oil safety problems are doubted by people because these problems are going against their increasing food safety awareness. Currently, most published reports focus on the detection of HAAs in meat products, coffee products, and alcoholic beverages, and there has not been a method used in detecting HAAs in oils yet. SUMMARY An objective of the present invention is to provide a method for the determination of HAAs in oils. To achieve the above purpose, the present invention provides the following technical solutions. The present invention provides a method for the determination of HAAs in oils includes the following steps: extracting and purifying HAAs from oil samples; and identifying and quantitating HAAs in oil samples by ultra-high performance liquid chromatography (UPLC) and triple quadrupole mass spectrometry (MS/MS); where the extraction and purification includes solvent extraction, defatting, and solid phase extraction (SPE); the solvent extraction is organic solvent extraction or alkaline organic solvent extraction. Preferably, the solvent extraction includes the following steps:

1) mixing the oil sample and an internal standard with an extractant, and centrifuging to obtain a first supernatant and residues; 2) repeating operations of step 1) for the residues to obtain a second supernatant; and 3) mixing the first and second supernatants to obtain a solution for defatting. Preferably, the extractant is an organic solvent or an alkaline organic solvent; the organic solvent is acetonitrile, and the alkaline organic solvent is1% aqueous ammonia acetonitrile solution; a ratio of the extractant to the oil sample is 10 mL: (0.5-3) g; the 1% aqueous ammonia-acetonitrile solution includes aqueous ammonia and acetonitrile; a volume ratio of the aqueous ammonia to the acetonitrile is 1:99. Preferably, a defatting solvent for the defatting is an n-hexane solution saturated with acetonitrile; a volume ratio of the solution for defatting after mixing the first and second supernatants to the n-hexane solution saturated with acetonitrile is 2:1. Preferably, the solid phase extraction includes the following steps: adding a defatting solution into solid phase extraction (SPE) cartridges, rinsing, suctioning dry, and eluting sequentially to obtain an eluent; and blow-drying the eluent, dissolving in methanol, and filtering to obtain a pretreated oil. Preferably, a defatting solution to be purified has a sample-loading rate of1 mL/min; the rinsing is conducted by using an acid solution at pH1 and methanol successively; in the eluting, the eluent is a methanol-aqueous ammonia solution, and a volume ratio of the methanol to the aqueous ammonia in the methanol-aqueous ammonia solution is (90 95):(10-5). Preferably, the blow-drying is conducted by using a water bath nitrogen blower; during the blow-drying, a water bath temperature is 40-60°C. Preferably, a C18 column is used as a chromatographic column in the UPLC; the chromatographic column has a column temperature of 20-40°C; a mobile phase system of the UPLC has a flow rate of 0.10-0.50 m/min. Preferably, the mobile phase system includes acetonitrile-water phase or methanol-water phase; the water phase is aqueous formic acid solution, aqueous ammonium formate solution, aqueous formic acid/ammonium formate solution, aqueous acetic acid solution, aqueous ammonium acetate solution, or aqueous acetic acid/ ammonium acetate solution. Preferably, a positive electrospray ionization (ESI) mode is used as an ionization source in the MS/MS; in the MS/MS, capillary voltage is 0.5-0.8 kV, source temperature is 100-150°C, desolvation temperature is 650-700°C, desolvation gas flow is 44-55 L/h, argon is used as a collision gas, and multi-reaction monitoring (MRM) mode is used as a scan mode. The present invention provides a method for the determination of HAAs in oils, including the following steps: extracting and purifying HAAs from oil samples; and identifying and quantitating HAAs in oil samples by UPLC-MS/MS successively; the extraction and purification includes alkaline organic solvent extraction and SPE. The method provided by the present invention is effective, sensitive, accurate, easy to operate, and suitable for the determination and confirmation of HAAs in oils. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is an ultra-high performance liquid chromatogram of HAA standards; FIG. 2 illustrates a comparison of extraction effects of HAAs in oils of Examples 1 and 2; FIG. 3 is an ultra-high performance liquid chromatogram of HAAs in a soybean oil sample of Example 1; FIG. 4 is an ultra-high performance liquid chromatogram of HAAs in a soybean oil sample of Example 2; FIG. 5 is an ultra-high performance liquid chromatogram of HAAs in a sesame oil sample of Example 3; where: 1. IQ; 2. MeIQ; 3. MeIQx; 4.4,8-DiMeIQx; 5.4,7,8-TriMeIQx; 6. PhIP; 7. Harman; 8. Norharman. DETAILED DESCRIPTION The present invention provides a method for the determination of HAAs in oils, including the following steps: extracting and purifying HAAs from oil samples to obtain a pretreated oil sample; and identifying and quantitating HAAs in oil samples by UPLC-MS/MS; where the extraction and purification includes solvent extraction, defatting, and SPE; the solvent extraction is organic solvent extraction or alkaline organic solvent extraction. In the present invention, unless otherwise specified, all raw materials are commercially available products conventional in the art. In the present invention, HAAs are extracted and purified from oil samples; the extraction and purification includes solvent extraction, defatting, and SPE; the solvent extraction is organic solvent extraction or alkaline organic solvent extraction. In the present invention, the oil sample to be detected is preferably an edible oil, and the edible oil is preferably an edible vegetable oil; the edible vegetable oil is not particularly limited in the present invention, as long as the edible vegetable oil used is well known to those skilled in the art. In particular embodiments of the present invention, soybean oil and sesame oil are used as test samples to verify the detection method of the present invention. In the present invention, the solvent extraction preferably includes the following steps: 1) mixing the oil sample and an internal standard with an extractant, and centrifuging to obtain a first supernatant and residues; 2) repeating operations of step 1) for the residues to obtain a second supernatant; and 3) mixing the first and second supernatants to obtain a solution to be defatted. In the present invention, an oil sample, an internal standard with an extractant, and centrifuged to obtain a first supernatant and residues; in the present invention, the internal standard is preferably 500 ng/mL 2-amino-3,4,7,8-tetramethyl-3H-imidazo[4,5-F]quinoxaline (4,7,8-TriMeIQx) solution. In the present invention, the extractant is preferably an organic solvent or an alkaline organic solvent; the organic solvent is preferably acetonitrile, and the alkaline organic solvent is preferably 1% aqueous ammonia-acetonitrile solution; the 1% aqueous ammonia-acetonitrile solution includes aqueous ammonia (the aqueous ammonia preferably has a mass concentration of 25.0-28.0%) and acetonitrile; a volume ratio of the aqueous ammonia to the acetonitrile is 1:99. In the present invention, the extractant has high extraction efficiency for HAAs; meanwhile, the extractant is easily separated from oil matrix during extraction, which is more conducive to subsequent SPE process. In the present invention, a ratio of the extractant to the oil sample to be detected is preferably 10 mL: (0.5-3) g, more preferably 10 mL: (1.0-2.0) g, and most preferably 10 mL: (1.2-1.8) g. In the present invention, a ratio of the oil sample to the internal standard is preferably (0.5 3) g: 20 g, more preferably (1.0-2.0) g: 20 g, and most preferably (1.2-1.8) g: 20 g; an amount of the internal standard is on a 4,7,8-TriMeIQx basis. In the present invention, the mixing is preferably conducted by adding the internal standard and the extractant in the oil sample successively. In the present invention, the mixing is preferably conducted by sonication in a vortexer. Ultrasonic conditions and rotational speed of the vortexer are not particularly limited in the present invention, as long as the ultrasonic conditions and the rotational speed used are well known to those skilled in the art. In the present invention, the centrifugation is preferably low speed centrifugation, high speed centrifugation, or refrigerated centrifugation; the low speed centrifugation is preferably conducted at 3,500-4,000 r/min; the high speed centrifugation is preferably conducted at 10,000 r/min; the refrigerated centrifugation is preferably conducted at 10,000 r/min and 4°C. After the first supernatant and the residues are obtained, the extractant is added to the residues, and operations of step 1) are repeated to obtain a second supernatant; in the present invention, target residues incompletely extracted in step 1) are further extracted by the procedure to further achieve a better extraction effect. In the present invention, after the second supernatant is obtained, the first and second supernatants are mixed and defatted to obtain an oil sample to be purified. The mixing is not particularly limited in the present invention, as long as the process used is well known to those skilled in the art. In the present invention, a degreaser for the defatting is preferably a saturated acetonitrile-n-hexane solution; a volume ratio of the solution to be defatted after mixing the first and second supernatants to the saturated acetonitrile-n-hexane solution is preferably 2:1. In the present invention, because the oil matrix is relatively complex, the extractant obtained by the above extraction is mixed up with a large proportion of triglycerides of oils; the defatting process further removes the above residual impurities from the supernatant, thereby reducing the burden during subsequent SPE and effects on chromatographic columns, as well as the influence of oily components on detection results. In the present invention, extraction cartridges in the SPE are preferably mixed weak cation exchange SPE cartridges, and particularly PCX SPE cartridges; the SPE preferably includes the following steps: adding a defatted extraction solution into SPE cartridges, rinsing, suctioning dry, and eluting to obtain an eluent; and blow-drying the eluent, dissolving in methanol, and filtering to obtain a pretreated oil. In the present invention, a defatted extraction solution was added into SPE cartridges, rinsed, suctioned dry, and eluted to obtain an eluent; in the present invention, the SPE cartridges are preferably PCX SPE cartridges; the PCX SPE cartridges are preferably activated before extraction. In the present invention, during the activation, an activator is preferably methanol and ultrapure water. In the present invention, the oil to be purified preferably has a sample loading rate of 1 mL/min; the rinsing is preferably conducted by using an acid solution at pH 1 and methanol successively; in the present invention, the acid solution is preferably hydrochloric, sulfuric or phosphoric acid, and more preferably hydrochloric acid. The suctioning-dry is not particularly limited in the present invention, as long as the suctioning-dry process used is well known to those skilled in the art. In the present invention, in the eluting, the eluent is preferably a methanol-aqueous ammonia solution, and a volume ratio of the methanol to the aqueous ammonia in the methanol-aqueous ammonia solution is preferably (90-95):(10-5), and more preferably 95:5. In the present invention, after the eluent is obtained, the eluent is blow-dried, dissolved with methanol, and filtered to obtain a pretreated oil. In the present invention, the blow-drying is preferably conducted by using a water bath nitrogen blower; during the blow-drying, a water bath temperature preferably is 40-60°C, more preferably 45-55°C, and most preferably 45°C. In the present invention, redissolution is preferably conducted by using methanol; the methanol and the oil sample to be detected are preferably used in a ratio of (0.2-1.50) mL: (0.5-3) g, and more preferably 0.5 mL: (0.5-3) g. In the present invention, a 0.22 m organic filter membrane is preferably used in the filtration; material for the organic filter membrane is not particularly limited in the present invention, as long as the material used is well known to those skilled in the art. In the present invention, the SPE achieves enrichment in an extract and further improves detection limit and sensitivity. In the present invention, after the pretreated oil is obtained, the sample is separated by UPLC and subjected to the MS/MS for identification and quantitation In the present invention, a C18 column is preferably used as a chromatographic column in the UPLC; the chromatographic column is further preferably a BEH C18 Column; the chromatographic column preferably has a column temperature of 20-40°C, and more preferably 30°C; a mobile phase system of the UPLC preferably has a flow rate of 0.10-0.50 mL/min, and more preferably 0.3 mLmin. In the present invention, the mobile phase system preferably includes acetonitrile water phase or methanol-water phase, and more preferably acetonitrile-water phase; the water phase is preferably aqueous formic acid solution, aqueous ammonium formate solution, aqueous formic acid/ammonium formate solution, aqueous acetic acid solution, aqueous ammonium acetate solution, or aqueous acetic acid/ ammonium acetate solution, and more preferably aqueous formic acid solution, aqueous acetic acid solution, or aqueous ammonium formate solution. In the present invention, a mass concentration of the formic acid in the water phase is preferably 0.05-0.1%, and more preferably 0.1%; a mass concentration of the acetic acid is preferably 0.5-1%, and more preferably 1%; a concentration of the ammonium formate is preferably 10-15 mmol/L, and more preferably 10 mmol/L. In the present invention, the linear gradient program is as follows preferably (i.e., gradient elution is used): at 0-0.2 min, 10% acetonitrile and 90% water phase; at 0.2-1.0 min, 10-30% acetonitrile and 90-70% water phase; at 1.0-3.0 min, 30-60% acetonitrile and 70-40% water phase; at 3.0-3.5 min, 60-90% acetonitrile and 40-10% water phase; and at 3.5-5.0 min, 10% acetonitrile and 90% water phase. In the present invention, a positive electrospray ionization (ESI) mode is preferably used as an ionization source in the MS/MS; in the MS/MS, capillary voltage is preferably 0.5-0.8 kV, and more preferably 0.5 kV; source temperature is preferably 100-150°C, and more preferably 110°C; desolvation temperature is preferably 650-700°C, and more preferably 650°C; desolvation gas (N2, 99.9% pure) flow is preferably 44-55 L/h, and more preferably 50 L/h; argon (99.9% pure) is used as a collision gas, and multi-reaction monitoring (MRM) mode is used as a scan mode. In the present invention, settings of the above conditions and parameters can reduce matrix effect and increase sensitivity. The method for the determination of HAAs in oils provided by the present invention will be described in detail below in conjunction with examples, but should not be construed as limiting the scope of the invention. Example 1 Approximately 2 g of soybean oil was weighed into a Teflon-type centrifuge tube, mixed with 40 L of internal standard working solution (4,7,8-TriMeIQx solution, 1.0 g/mL) and 10 mL of 1% aqueous ammonia-acetonitrile solution successively. The mixture was vortexed for 5 min, sonicated for 10 min and centrifuged for 5 min at 3,500 r/min to obtain a first supernatant and residues. The residues were re-extracted according to the above step, to obtain a second supernatant. The first supernatant was mixed with the second supernatant, and then defatted with 10 mL of n-hexane saturated with acetonitrile to obtain a defatted extraction solution (10 mL). The defatted extraction solution was loaded (at a sample-loading rate of 1 mL/min) onto Cleanert PCX cartridges (3 mL/60 mg), which had been activated with 3 mL of methanol and 3 mL of ultrapure water; next, the cartridges were rinsed with 3 mL of 0.1 mol/L hydrochloric acid and 3 mL of methanol, suctioned dry after rinsing, and eluted with 3 mL of methanol aqueous ammonia solution (95:5 v/v) to obtain an eluent. The eluent was blow-dried by using a water bath nitrogen blower (water bath temperature: 45°C), and redissolved with 500 L of methanol, and filtered through a 0.22 m organic filter membrane for further analysis. It was separated by UPLC. Chromatographic conditions were as follows: chromatographic column: BEH C18 Column; column temperature: 30°C; injection volume: 1 L; mobile phase system: 10 mmol/L ammonium formate solution (pH=6.80)-acetonitrile; flow rate: 0.3 mL/min. The linear gradient program was as follows (i.e., gradient elution was used): at 0-0.2 min, 10% acetonitrile and 90% ammonium formate solution; at 0.2-1.0 min, 10-30% acetonitrile and 90-70% ammonium formate solution; at 1.0-3.0 min, 30-60% acetonitrile and 70-40% ammonium formate solution; at 3.0-3.5 min, 60-90% acetonitrile and 40-10% ammonium formate solution; and at 3.5-5.0 min, 10% acetonitrile and 90% ammonium formate solution. The above test sample was separated by UPLC and subjected to the MS/MS for identification and quantitation, and the corresponding retention time and signal response were obtained. MS conditions were as follows: a positive ESI mode was used as an ionization source; capillary voltage of a detector was 0.5 kV; source temperature was 110°C; desolvation temperature was 650°C; desolvation gas (N2, 99.9% pure) flow was 1,000 L/h; cone gas (N2, 99.9% pure) flow was 50 L/h; argon (99.9% pure) was used as a collision gas. The results are shown in FIG. 3. From FIG. 3, the technical example is suitable for the determination of HAAs in the soybean oil. Example 2 Approximately 2 g of soybean oil was weighed into a Teflon-type centrifuge tube, mixed with 40 L of internal standard working solution (4,7,8-TriMeIQx solution, 1.0 g/mL) and 10 mL of acetonitrile successively. The mixture was vortexed for 5 min, sonicated for 10 min and centrifuged for 5 min at 3,500 r/min to obtain a first supernatant and residues. The residues were re-extracted according to the above step, to obtain a second supernatant. The first supernatant was mixed with the second supernatant, and then defatted with 10 mL of n-hexane saturated with acetonitrile to obtain a defatted extraction solution (10 mL). The defatted extraction solution was loaded (at a sample-loading rate of 1 mL/min) onto Cleanert PCX cartridges (3 mL/60 mg), which had been activated with 3 mL of methanol and 3 mL of ultrapure water; next, the cartridges were rinsed with 3 mL of 0.1 mol/L hydrochloric acid and 3 mL of methanol, suctioned dry after rinsing, and eluted with 3 mL of methanol aqueous ammonia solution (95:5 v/v) to obtain an eluent. The eluent was blow-dried by using a water bath nitrogen blower (water bath temperature: 45°C), and redissolved with 500 L of methanol, and filtered through a 0.22 m organic filter membrane for further analysis. It was separated by UPLC. Chromatographic conditions were as follows: chromatographic column: BEH C18 Column; column temperature: 30°C; injection volume: 1 L; mobile phase system: 10 mmol/L ammonium formate solution (pH=6.80)-acetonitrile; flow rate: 0.3 mL/min. The linear gradient program was as follows (i.e., gradient elution was used): at 0-0.2 min, 10% acetonitrile and 90% ammonium formate solution; at 0.2-1.0 min, 10-30% acetonitrile and 90-70% ammonium formate solution; at 1.0-3.0 min, 30-60% acetonitrile and 70-40% ammonium formate solution; at 3.0-3.5 min, 60-90% acetonitrile and 40-10% ammonium formate solution; and at 3.5-5.0 min, 10% acetonitrile and 90% ammonium formate solution. The above test sample was separated by UPLC and subjected to the MS/MS for identification and quantitation, and the corresponding retention time and signal response were obtained. MS conditions were as follows: a positive ESI mode was used as an ionization source; capillary voltage of a detector was 0.5 kV; source temperature was 110°C; desolvation temperature was 650°C; desolvation gas (N2, 99.9% pure) flow was 1,000 L/h; cone gas (N2, 99.9% pure) flow was 50 L/h; argon (99.9% pure) was used as a collision gas. The results are shown in FIG. 4. From FIG. 4, the technical example is suitable for the determination of HAAs in the soybean oil. Example 3 Approximately 1 g of sesame oil was weighed into a Teflon-type centrifuge tube, mixed with 80 L of internal standard working solution (4,7,8-TriMeIQx solution, 500 ng/mL) and 10 mL of 1% aqueous ammonia-acetonitrile solution successively, vortexed for 5 min, sonicated for 10 min, and centrifuged for 5 min at 3,500 r/min to obtain a first supernatant and residues. The residues were re-extracted according to the above step, to obtain a second supernatant. The first supernatant was mixed with the second supernatant, and then defatted with 10 mL of n-hexane saturated with acetonitrile to obtain a defatted extraction solution (10 mL). The defatted extraction solution was loaded (at a sample-loading rate of 1 mL/min) onto Cleanert PCX cartridges (3 mL/60 mg), which had been activated with 3 mL of methanol and 3 mL of ultrapure water; next, the cartridges were rinsed with 3 mL of 0.1 mol/L hydrochloric acid and 3 mL of methanol, suctioned dry after rinsing, and eluted with 3 mL of methanol aqueous ammonia solution (95:5 v/v) to obtain an eluent.

The eluent was blow-dried by using a water bath nitrogen blower (water bath temperature: 45°C), redissolved with 500 L of methanol, and filtered through a 0.22 m organic filter membrane for further analysis. It was separated by UPLC. Chromatographic conditions were as follows: chromatographic column: BEH C18 Column; column temperature: 30°C; injection volume: 1 L; mobile phase system: 10 mmol/L ammonium formate solution (pH=6.80)-acetonitrile; flow rate: 0.3 mL/min. The linear gradient program was as follows (i.e., gradient elution was used): at 0-0.2 min, 10% acetonitrile and 90% ammonium formate solution; at 0.2-1.0 min, 10-30% acetonitrile and 90-70% ammonium formate solution; at 1.0-3.0 min, 30-60% acetonitrile and 70-40% ammonium formate solution; at 3.0-3.5 min, 60-90% acetonitrile and 40-10% ammonium formate solution; and at 3.5-5.0 min, 10% acetonitrile and 90% ammonium formate solution. The above test sample was separated by HPLC and subjected to MS/MS for identification and quantitation, and the corresponding retention time and signal response were obtained. MS conditions were as follows: a positive ESI mode was used as an ionization source; capillary voltage of a detector was 0.5kV; source temperature was110°C; desolvation temperature was 650°C; desolvation gas (N2, 99.9% pure) flow was 1,000 L/h; cone gas (N2, 99.9% pure) flow was 50 L/h; argon (99.9% pure) was used as a collision gas. The results are shown in FIG. 5. From FIG. 5, the technical example is suitable for the determination of HAAs in the sesame oil. Specifically, retention times (Table 1), ion pairs of second order MS (Table 1), and linear equations and correlation coefficients (Table 2) of reference solutions of HAAs (related test conditions are the same as those in Example 1) are shown in Tables 1 and 2:

Table 1 Retention times and ion pairs of second order MS of reference solutions of HAAs

Parent ion Cone voltage Collison energy HAA Time Product ion pair (m/z) (m/z) (V) (eV)

IQ 1.98 199.09 58 199.09/184.03*, 26,30 199.09/156.99 MeIQ 2.11 213.11 58 213.11/198.05*, 24,40 213.11/144.04 MeIQx 2.25 214.12 54 214.12/199.04*, 214,12/130.99 24,36

4,8-DiMeIQx 2.31 228.13 40 228.13/213.10*, 25,34 228.13/212.99 4,7,8-TriMeIQx 242.16/227.00* (internal 2.46 242.16 62 242.16/145.03 28,40 standard) PhIP 2.89 225.11 54 225.11/210.04*, 225,11/114.75 42,50

Harman 2.93 183.01 56 183.01/114.97*, 183,01/142.03 40,26

Norharman 3.08 169.03 56 169.03/1141' 34,24

Table 2 Linear equations and correlation coefficients of reference solutions HAA Linear range (ng/mL) Linear equation R2

IQ 0.5 -20 y =0.0955 x - 0.0161 0.9995 MeIQ 0.5- 20 y =0.0932 x - 0.0073 0.9999 MeIQx 0.5- 20 y =0.0429 x + 0.0006 0.9993 4,8-DiMeIQx 0.5 -20 y =0.0512 x - 0.0135 0.9994 PhIP 1- 100 y =0.0179 x + 0.0026 0.9991 Harman 1- 100 y =0.0967 x - 0.0108 0.9996 Norharman 1- 100 y =0.1233 x + 0.0287 0.9999

Herein, FIG. 1 is an ultra-high performance liquid chromatogram of HAA standards; from FIG. 1, all target compounds is separated by UPLC-MS/MS excellently and accurately, and chromatographic peaks obtained are sharp and symmetrical, which is suitable for the separation of HAAs. FIG. 2 illustrates a comparison of extraction effects of HAAs in oils of Examples 1 and 2; from FIG. 2, when 1% aqueous ammonia-acetonitrile solution and acetonitrile are used as extractants, there is an excellent recovery of HAAs; while, when 1% aqueous ammonia acetonitrile solution is used as an extractant, the recovery of HAAs increases as a whole compared with acetonitrile extraction, where recoveries of IQ, 4,8-DiMeIQx, PhIP, Harman, and Norharman increase, except that recoveries of MeIQ and MeIQx decrease. FIG. 3 is an ultra-high performance liquid chromatogram of HAAs in a soybean oil sample of Example 1; from FIG. 3, the technical example is suitable for the determination of HAAs in the soybean oil, and indicates that the determination of HAAs can use 1% aqueous ammonia acetonitrile solution as an extractant, and the extraction effect obtained in Example 1 is relatively better than that obtained in Example 2. FIG. 4 is an ultra-high performance liquid chromatogram of HAAs in a soybean oil sample of Example 2; from FIG. 4, the technical example is suitable for the determination of HAAs in the soybean oil, and indicates that the determination of HAAs can use acetonitrile as an extractant. FIG. 5 is an ultra-high performance liquid chromatogram of HAAs in a sesame oil sample of Example 3; from FIG. 5, the technical example is suitable for the determination of HAAs in the sesame oil, and indicates that color of oil has little influence on the determination provided by the technical solution. As can be seen from the above the examples, the method provided by the present invention is effective, sensitive, accurate, easy to operate, and suitable for the determination and confirmation of HAAs in oils. The foregoing descriptions is merely preferred examples of the present invention; it should be noted that various modifications and variations can be made by those skilled in the art without departing from the principles of the present invention and are within the scope of the invention. In the claims which follow, and in the preceding description, except where the context requires otherwise due to express language or necessary implication, the word "comprise" and variations such as "comprises" or "comprising" are used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the apparatus and method as disclosed herein.

Claims

What is claimed is: 1. A method for the determination of detecting heterocyclic aromatic amines (HAAs) in oils, comprising the following steps: extracting and purifying HAAs from oil samples; and identifying and quantitating HAAs in oil samples by ultra-high performance liquid chromatography (UPLC) and triple quadrupole mass spectrometry (MS/MS) successively; wherein the identification and quantification comprises solvent extraction, defatting, and solid phase extraction (SPE); the solvent extraction is organic solvent extraction or alkaline organic solvent extraction.
2. The method according to claim 1, wherein the solvent extraction comprises the following steps: 1) mixing the oil sample and an internal standard with an extractant, and centrifuging to obtain a first supernatant and residues;

2) repeating operations of step 1) for the residues to obtain a second supernatant; and 3) mixing the first and second supernatants to obtain a solution to be defatted; wherein the extractant is an organic solvent or an alkaline organic solvent;

the organic solvent is acetonitrile, and the alkaline organic solvent is1% aqueous ammonia acetonitrile solution; a ratio of the extractant to the oil sample is 10 mL: (0.5-3) g; the 1% aqueous ammonia-acetonitrile solution comprises aqueous ammonia and acetonitrile; a volume ratio of the aqueous ammonia to the acetonitrile is 1:99.

3. The method according to claim 1, wherein a degreaser for the defatting is a saturated acetonitrile-n-hexane solution; a volume ratio of the solution to be defatted after mixing the first and second supernatants to the saturated acetonitrile-n-hexane solution is 2:1.

4. The method according to claim 1, wherein the solid phase extraction comprises the following steps: adding a defatted extraction solution into solid phase extraction (SPE) cartridges, rinsing, suctioning dry, and eluting to obtain an eluent; and blow-drying the eluent, dissolving in methanol, and filtering to obtain a pretreated oil; wherein an oil to be purified has a sample-loading rate of1 mL/min; the rinsing is conducted by using an acid solution at pH1 and methanol successively; in the eluting, the eluent is a methanol-aqueous ammonia solution, and a volume ratio of the methanol to the aqueous ammonia in the methanol-aqueous ammonia solution is (90 95):(10-5) wherein the blow-drying is conducted by using a water bath nitrogen blower; during the blow-drying, a water bath temperature is 40-60°C.

5. The method according to claim 1, wherein a C18 column is used as a chromatographic column in the UPLC; the chromatographic column has a column temperature of 20-40°C; a mobile phase system of the UPLC has a flow rate of 0.10-0.50 mL/min; wherein the mobile phase system comprises acetonitrile-water phase or methanol-water phase; the water phase is aqueous formic acid solution, aqueous ammonium formate solution, aqueous formic acid/ammonium formate solution, aqueous acetic acid solution, aqueous ammonium acetate solution, or aqueous acetic acid/ ammonium acetate solution.

1 /2 18 Sep 2020

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FIG. 1

120 乙Acetonitrile Alkaline acetonitrile 碱性乙 100

80 Recovery/% 回收率/ %

60

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0 IP IQ

IQ

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an

an x IQ

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FIG. 2

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120000

Response 8 80000 响 40000 7

2 6 2020102328

0 0 1 2 3 4 5 t/min Time (t/min)

FIG. 3

160000 5 120000 8 Response

80000 7 响

40000 2 6 0 0 1 2 3 4 5 t / min Time (t/min)

FIG. 4

16000000

12000000 7 8 Response

8000000 响

4000000

0 0 1 2 3 4 5 Time (t/min) t / min

FIG. 5