A method for screening of sweeteners and sex hormones in food

Abstract

The present application relates to the field of food detection and analysis, and particularly relates to a screening method of sweeteners and sex hormones in food. The screening method comprises the following steps: mixing a detection sample with an extraction solvent to obtain a target extraction liquid; refrigerating, low-temperature centrifuging and filtering the target extraction liquid to obtain a target detection liquid; and analyzing and detecting the target detection liquid by using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry; wherein the sugar content of the detection sample is more than 20%, and the extraction solvent is a mixed extraction solvent containing 10% methanol, acetonitrile solution, water and n-hexane. The screening method provided by the present application has the characteristics of wide detection range, low detection limit and short detection time, can quickly identify sweeteners and sex hormones in food, and breaks through the technical barrier that the current national standard detection is mainly for a small class of substances with the same chemical property, and can simultaneously detect food additives and drug residues with a large span of chemical properties.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of food detection and analysis, in particular to a screening method of sweeteners and sex hormones in food. BACKGROUND

[0002] Sweeteners are substances that impart sweetness to food, and are an important food additive. Sweeteners are divided into artificial and natural. Appropriate addition of sweeteners in food can improve the flavor of food, but long-term and large intake of artificial sweeteners may cause metabolic system diseases such as hyperglycemia, glucose intolerance, and obesity. Sex hormones refer to steroid hormones synthesized by living organisms, which are divided into three categories of androgens, estrogens, and progestogens, and are used to regulate various physiological functions. Unreasonable intake of sex hormones can cause endocrine disorders, metabolic disorders, irregular menstruation in women, and other diseases. Some sex hormones are carcinogenic.

[0003] At present, the national standard detection of food additives and drug residues is mainly for a small class of substances with the same chemical properties, and there is no synchronous rapid detection method for simultaneous detection of food additives and drug residues. SUMMARY

[0004] The purpose of the present application is to analyze food safety risk factors in the food variety list and establish a method for simultaneous rapid preliminary screening and qualitative analysis of sweeteners and sex hormones in food safety detection.

[0005] In order to solve the above technical problems, the technical scheme provided by the present application is as follows:

[0006] A screening method of sweeteners and sex hormones in food, the sweeteners including alitame, aspartame, rebaudioside A, sucralose, neotame, glycyrrhetinic acid and stevioside;

[0007] The sex hormones include testosterone, methyltestosterone, 17β-estradiol, estrone, estriol, progesterone and neohesperidin dihydrochalcone, and the screening method comprises the following steps:

[0008] Mixing the detection sample with an extraction solvent to obtain a target extraction liquid by extraction;

[0009] Refrigerating, low-temperature centrifuging and filtering the target extraction liquid to obtain a target detection liquid;

[0010] Analyzing and detecting the target detection liquid by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry;

[0011] The sugar content of the detection sample is more than 20%, and the extraction solvent is a mixed extraction solvent containing 10% methanol, acetonitrile solution, water and n-hexane.

[0012] In a more preferable embodiment, the refrigeration is refrigerating the target extract at 4℃ for 20 minutes.

[0013] In a more preferable embodiment, the sample is a solid sample, and the extraction to obtain the target extract is as follows:

[0014] Obtaining a sample;

[0015] Mixing the sample with water and n-hexane, and separating n-hexane solution and residual material by low-temperature centrifugation;

[0016] Blowing dry the n-hexane solution with nitrogen at 40℃, and then mixing with acetonitrile solution containing 10% methanol to obtain a re-dissolved solution;

[0017] Letting the residual material stand, and separating water-soluble extract and sample residue by low-temperature centrifugation;

[0018] Mixing the re-dissolved solution with the sample residue, ultrasonicating, and low-temperature centrifugation, and then mixing with the water-soluble extract to obtain the target extract.

[0019] In a more preferable embodiment, the volume ratio of the methanol acetonitrile solution, water and n-hexane is (4-5):(2.5-3.5):(3.5-4.5).

[0020] In a more preferable embodiment, the sample is a liquid sample, and the extraction to obtain the target extract is as follows:

[0021] Obtaining a sample;

[0022] Mixing the sample with water and n-hexane, and separating n-hexane solution and residual material by low-temperature centrifugation;

[0023] Blowing dry the n-hexane solution with nitrogen at 40℃, and then mixing with acetonitrile solution containing 10% methanol to obtain a re-dissolved solution;

[0024] Letting the residual material stand, and mixing with the re-dissolved solution, and ultrasonicating to obtain the target extract.

[0025] In a more preferable embodiment, the volume ratio of the methanol acetonitrile solution, mixed solvent and n-hexane is (4-5):(2.5-3.5):(3.5-4.5).

[0026] The total volume of the mixed solvent is equal to the sum of the volume of water and the liquid sample.

[0027] In a more preferable embodiment, the low-temperature centrifugation is carried out at 8000r / min for 5 minutes in a centrifuge at 4℃.

[0028] In a more preferable embodiment, the chromatographic conditions of the ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry are as follows:

[0029] Chromatographic column: Thermo scientific Hypersil GOLDTM, length 100 mm, inner diameter 2.1 mm, particle size 2.7 μm;

[0030] Mobile phase: water and acetonitrile;

[0031] Column temperature: 40℃; flow rate: 0.3 mL / min; injection volume: 10 μL.

[0032] In a more preferable embodiment, the gradient elution program of the mobile phase is as follows: 0.0-2.0 min (10% acetonitrile), 2.0-9.0 min (10%-100% acetonitrile), 9.0-16.0 min (100% acetonitrile), 16.0-18.0 min (100%-10% acetonitrile), 18.0-21.0 min (10% acetonitrile).

[0033] In a more preferable embodiment, the mass spectrometric conditions of the ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry are as follows: using an electrospray ion source (ESI), positive and negative ion mode detection;

[0034] Ion source temperature: 350℃; ion transmission temperature: 325℃; sheath gas pressure: 4 MPa; auxiliary gas pressure: 1 MPa; capillary voltage: 3.2 kV; scan mode: full scan; acquisition range: 70-900 m / z; first-order mass spectrometric resolution: 70000 FWHM.

[0035] The present application provides a method for synchronous rapid qualitative detection of sweeteners and sex hormones in food safety detection, which has the characteristics of wide detection range, low detection limit, and short detection time, and can quickly identify sweeteners and sex hormones in food. On the one hand, it saves a lot of manpower, material resources and time, and solves the difficulty of measuring ingredients not marked on food labels; on the other hand, it breaks through the technical barrier limitation of the current national standard detection of a small class of substances with the same chemical properties, and can simultaneously detect food additives and drug residues with a large span of chemical properties.

[0036] Other features and advantages of the present application will be set forth in the following description, and in part will become apparent to those skilled in the art from the description, or can be learned by practice of the present application. The objects and other advantages of the present application can be realized and achieved by the structures specifically pointed out in the specification, claims and drawings. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1Total ion current chromatogram for ACE Excel 1.7 SuperC18 in negative scan mode;

[0038] Figure 2 Total ion current chromatogram for ACQUITY Total ion current chromatogram for BEH C18 in negative scan mode;

[0039] Figure 3 Total ion current chromatogram for Thermo scientific Hypersil GOLD™ in negative scan mode;

[0040] Figure 4 Total ion current chromatogram for 1000 ng / mL standard solution initial elution procedure in positive scan mode;

[0041] Figure 5 Total ion current chromatogram for 1000 ng / mL standard solution initial elution procedure in negative scan mode;

[0042] Figure 6 Total ion current chromatogram for 1000 ng / mL standard solution optimal elution procedure in positive scan mode;

[0043] Figure 7 Total ion current chromatogram for 1000 ng / mL standard solution optimal elution procedure in negative scan mode;

[0044] Figure 8 The response values of hesperetin dihydrochalcone in different matrices are shown, and the response value ratio of the same concentration of the standard solution prepared using 10% methanol acetonitrile as a solvent is shown. DETAILED DESCRIPTION

[0045] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0046] The present application provides a screening method for sweeteners and sex hormones in food, wherein the sweeteners include alitame, aspartame, rebaudioside A, sucralose, neotame, glycyrrhetinic acid and stevioside;

[0047] The sex hormones include testosterone, methyltestosterone, 17β-estradiol, estrone, estriol, progesterone and hesperetin dihydrochalcone, and the screening method comprises the following steps:

[0048] Mix the detection sample with the extraction solvent to obtain a target extraction liquid;

[0049] Refrigerate, low-temperature centrifuge and filter the target extraction liquid to obtain a target detection liquid;

[0050] Analyze and detect the target detection liquid by using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry;

[0051] The sugar content of the detection sample is greater than 20%, and the extraction solvent is a mixed extraction solvent containing 10% methanol, acetonitrile solution, water and n-hexane.

[0052] The inventors consider that both sweeteners and sex hormones belong to different chemical substances, and the span of chemical detection is large, so the inventors select different purification methods for sample pretreatment, in order to test the purification effect of different types of purification methods and select the appropriate sample pretreatment method.

[0053] To this end, the inventors add 27 mu g / kg of standard in the negative sample, and use four kinds of extraction solvents in volume percentage, namely 60% methanol-40% water, 60% acetonitrile-40% water, 60% methanol-40% water combined with an appropriate amount of n-hexane, and 60% acetonitrile-40% water combined with an appropriate amount of n-hexane.

[0054] It is found that the use of acetonitrile-water as an extraction liquid cannot achieve the recovery of progesterone; methanol-water and methanol-water-n-hexane as extraction liquids can recover the target substances, but the recovery rate is not good; and acetonitrile-water-n-hexane as an extraction liquid can recover the target substances and has good recovery effect.

[0055] Moreover, the inventors find that when acetonitrile is used as an extraction solvent, in a low-temperature environment, acetonitrile has a good effect on removing fatty substances, so that the extraction liquid can be used for subsequent steps after centrifugation and filtration, and the solid-phase extraction purification step can be reduced.

[0056] However, it is found in the experiment that when the extraction methods of acetonitrile-water and acetonitrile-water-n-hexane are mixed with acetonitrile and water, after low-temperature refrigeration and low-temperature high-speed centrifugation, the sample with high sugar content has obvious stratification. The experiment of adding sucrose standard solution to acetonitrile-water mixed solution shows that when the sugar content of the sample is greater than 20%, after low-temperature centrifugation, 60% acetonitrile-40% water will have obvious stratification. Considering the matrix effect of the detection sample itself, the inventors creatively propose that using 10% methanol acetonitrile+water as an extraction solvent can eliminate the stratification in the low-temperature centrifugation process, ensure the uniform mixing of the machine liquid, and retain the extraction effect of acetonitrile on the target compound.

[0057] The pre-treatment process is simple and fast, and after simple sample pre-treatment, machine detection can be carried out, which makes up the technical blank of the current national standard detection which is mainly for a small class of substances with the same chemical property, and realizes the simultaneous detection of food additives and drug residues with large chemical property span.

[0058] The application provides a screening method for sweeteners and sex hormones in food.

[0059] (1) mixing the detection sample with an extraction solvent to obtain a target extraction solution.

[0060] The mixing method is not limited in the application, and is well known to those skilled in the art, for example, oscillation for 15-25 min, preferably vortex mixing for 45-75 s.

[0061] The extraction solvent is a mixed extraction solvent of 10% methanol acetonitrile solution, water and n-hexane; considering that the detection sample is a food sample, it can be a solid sample or a liquid sample, therefore, for a solid sample, the mass-volume ratio of the detection sample to the extraction solvent is 1: (10-13.5), preferably, the mass-volume ratio is 1:11.5; for a liquid sample, the volume ratio of the detection sample to the extraction solvent is 1: (10-13.5), preferably, the volume ratio is 1:10.5.

[0062] In particular, the application provides the following preferred steps for obtaining the target extraction solution for a solid sample:

[0063] Step 1, obtaining a detection sample;

[0064] Step 2, mixing the detection sample with water and n-hexane, and separating n-hexane solution and residual material by low-temperature centrifugation;

[0065] In step 2, the detection sample is placed in a container, and water and n-hexane are added to the container for mixing and oscillation, which can make water-soluble substances in the detection sample and substances dissolved in n-hexane be extracted respectively, in particular, before mixing, the detection sample is crushed uniformly to increase the contact area of the detection sample with water and n-hexane and improve the extraction probability; preferably, the low-temperature centrifugation is carried out at 8000 r / min for 5 min in a 4℃ centrifuge; after oscillation centrifugation, the reagents in the container are stratified, and therefore, the n-hexane solution in the upper layer and the residual material can be separated;

[0066] Step 3, blowing dry the n-hexane solution by using 40℃ nitrogen, and then adding 10% methanol acetonitrile solution to mix to obtain a redissolution solution;

[0067] In step 3, the separated n-hexane solution is transferred into a containing vessel, and the n-hexane solution is blown to near dryness using nitrogen at 40℃, and then a 10% methanol acetonitrile solution is added and vortexed to obtain a re-dissolved solution; nitrogen at 40℃ is blown to the surface of the solution through a nozzle to rapidly volatilize the volatile substances (n-hexane) on the surface of the solution, so that the remaining sample is in a dry state.

[0068] In step 4, the remaining material is allowed to stand, and water-soluble extract and sample residue are separated by low-temperature centrifugation.

[0069] In step 4, the remaining material is allowed to stand, and water-soluble extract and sample residue are separated by low-temperature centrifugation.

[0070] In step 5, the re-dissolved solution is mixed with the sample residue, ultrasonicated, and low-temperature centrifuged, and then mixed with the water-soluble extract to obtain the target extract.

[0071] In step 5, the re-dissolved solution obtained in step 3 is poured into a vessel containing the sample residue, vortexed and ultrasonicated, and then centrifuged at 8000r / min for 5min in a 4℃ centrifuge to further remove fatty substances in the material in the vessel, and finally the obtained extract is mixed with the water-soluble extract to obtain the target extract.

[0072] The ultrasonic time in the present application can be 15-25min, preferably 20min.

[0073] In addition, the present application provides the following preferred extraction steps for obtaining the target extract from a liquid sample:

[0074] Obtain a detection sample;

[0075] Mix the detection sample with water and n-hexane, and separate the n-hexane solution and the remaining material by low-temperature centrifugation;

[0076] The n-hexane solution is blown dry using nitrogen at 40℃, and then mixed with a 10% methanol acetonitrile solution to obtain a re-dissolved solution;

[0077] The remaining material is allowed to stand, mixed with the re-dissolved solution, and ultrasonicated to obtain the target extract.

[0078] Since the extraction steps of the liquid sample are similar to those of the solid sample, those skilled in the art can know the steps based on the above-mentioned extraction steps of the solid sample and the above-mentioned steps, and therefore no further description is given here.

[0079] (2) refrigerating, centrifuging at low temperature, and filtering the target extract to obtain a target test solution;

[0080] The target extract was placed in a 4°C refrigerator for 20 minutes, centrifuged at 8000 rpm for 5 minutes at 4°C, and the final extract was filtered through a 0.45 μm filter membrane and used as the target detection solution for subsequent analysis.

[0081] (3) analyzing and detecting the target test solution using ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry;

[0082] Based on the wide range of target compounds screened by the present invention, the present invention selects a chromatographic column in order to obtain a more suitable chromatographic column. The specific process is as follows:

[0083] By comparing ACE Excel 1.7 SuperC18 (2.1mm×100mm, 1.7μm), ACQUITY The ion currents of BEH C18 (2.1mm×50mm, 1.7μm) and Thermo scientific Hypersil GOLDTM (2.1mm×100mm, 1.9μm) revealed that:

[0084] like Figure 1 As shown, when the chromatographic column is ACE Excel 1.7 SuperC18, the target compound has a late peak time, obvious noise, and unstable baseline;

[0085] like Figure 2 As shown, the chromatographic column is ACQUITY When BEH C18 was used, the target compound eluted early and the baseline was stable, but the number of retained target compounds was small and the separation effect was poor;

[0086] like Figure 3 As shown, when the chromatographic column is Thermo scientific Hypersil GOLDTM, the target compound has a moderate peak time, a stable baseline, a large number of target compounds are retained, and the separation effect is good;

[0087] This shows that Thermo scientific Hypersil GOLDTM has better results, so the chromatographic column finally selected for this experiment is Thermo scientific Hypersil GOLDTM.

[0088] The mobile phase of the present application is water and acetonitrile, and the initial elution program is set as: 0.0-0.5 min (60% acetonitrile), 0.5-8.0 min (60%-10% acetonitrile), 8.0-10.0 min (10% acetonitrile), 10.0-12.0 min (10%-100% acetonitrile), 12.0-17.0 min (100% acetonitrile), 17.0-19.0 min (100%-60% acetonitrile), 19.0-20.0 min (60% acetonitrile).

[0089] It can be seen from the ion flow diagram ( Figure 4 、 5 ) that the target compounds are mainly concentrated in the early detection time, and the compound separation effect is poor.

[0090] Therefore, the mobile phase elution program is optimized, the target peak time is mainly concentrated in the middle of the whole detection by reducing the change rate of acetonitrile, and a better compound separation effect ( Figure 6 、 7 ) is obtained, and the optimal elution program is: 0.0-2.0 min (10% acetonitrile), 2.0-9.0 min (10%-100% acetonitrile), 9.0-16.0 min (100% acetonitrile), 16.0-18.0 min (100%-10% acetonitrile), 18.0-21.0 min (10% acetonitrile).

[0091] Based on the total ion flow diagram ( Figure 6 、 7 ) analysis, the sample is detected in positive and negative ion modes by using UPLC-Q Orbitrap HRMS in full scan mode. Due to the high resolution of UPLC-Q Orbitrap HRMS, the mass-to-charge ratio of the extracted ion can be accurate to 5 decimal places, and by comparing with the theoretical mass-to-charge ratio of each standard, it is determined that 8 sweeteners and 7 sex hormones can be detected in positive and negative ion scanning modes. The information is shown in the following table.

[0092] Table 1 Information of UPLC-Q Orbitrap HRMS for detecting food additives and drug residues in food

[0093]

[0094] Note: a is a sweetener, a total of 8; b is a sex hormone, a total of 7.

[0095] Further, by using the matrix solution as the solvent to prepare the standard solution and using the acetonitrile solution containing 10% methanol as the solvent to prepare the standard solution at the same concentration, SPSS significance analysis is performed, and it is found that the items in the negative ion scanning mode: sucralose, neohesperidin dihydrochalcone, rebaudioside A, stevioside, aspartame, and the Friedman test asymptotic significance is less than 0.05, and there is a significant matrix effect (for example, the response value ratio of neohesperidin dihydrochalcone in different matrices to the response value of the standard solution prepared by using 10% methanol acetonitrile as the solvent at the same concentration is shown in Figure 8 ), which may be not conducive to the determination of experimental results. In order to ensure the accuracy of the experimental results, the matrix curve is prepared, and the results are qualitatively analyzed.

[0096] For this purpose, the matrix curve is used for qualitative analysis, and in the range of 50-500 ng / mL, the linear regression equation and the correlation coefficient of the target item are shown in the following table. The results show that the linearity of the detection method is good, R 2 ≥ 0.99, which meets the requirements of GB / T 27404-2008.

[0097] Table 2 Linear regression equation and correlation coefficient of target item

[0098]

[0099] Based on the above experiments, the chromatographic conditions of the ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry method are as follows:

[0100] The chromatographic column is Thermo scientific Hypersil GOLDTM, the length is 100 mm, the inner diameter is 2.1 mm, and the particle size is 2.7 μm;

[0101] The mobile phase is water and acetonitrile;

[0102] The column temperature is 40℃, the flow rate is 0.3 mL / min, and the injection volume is 10 μL.

[0103] In a more preferred embodiment, the gradient elution program of the mobile phase is as follows: 0.0-2.0 min (10% acetonitrile), 2.0-9.0 min (10%-100% acetonitrile), 9.0-16.0 min (100% acetonitrile), 16.0-18.0 min (100%-10% acetonitrile), and 18.0-21.0 min (10% acetonitrile).

[0104] The mass spectrometry conditions of the ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry method are as follows: an electrospray ion source (ESI) is used, and positive and negative ion modes are detected.

[0105] Ion source temperature: 350℃; ion transmission temperature: 325℃; sheath gas pressure: 4 MPa; auxiliary gas pressure: 1 MPa; capillary voltage: 3.2 kV; scan mode: full scan; acquisition range: 70-900 m / z; first mass spectrum resolution: 70000 FWHM.

[0106] The technical solutions of the present application are further described and explained below through specific examples. However, the protection scope of the present application is not limited thereto.

[0107] Examples

[0108] The reagents or instruments used in the screening method of sweeteners and sex hormones in food according to the present application can be purchased from the market.

[0109] 1.1 Main instruments and devices:

[0110] Dione UltiMate 3000 high-pressure liquid chromatography system, quadrupole / electrostatic field orbitrap mass spectrometer: product of Thermo Fisher Company, USA; AB X500R high-performance liquid chromatography-quadrupole-time-of-flight mass spectrometer: BioSystems Company, USA.

[0111] 1.2 Main materials and reagents:

[0112] Standard substances: alitame (purity 99.3%), glycyrrhetinic acid (purity 99.1%), rebaudioside A (purity 98.0%), stevioside (purity 99.7%), all products of Tanmo Quality Inspection Technology Co., Ltd.; testosterone (purity 99.2%), methyltestosterone (purity 98.3), 17β-estradiol (purity 99.6%), estrone (purity 99.6%), estriol (purity 96.4%), sucralose (purity 99.7%), all products of Beijing Zhenxiang Technology Co., Ltd.; neotame (purity 95.1%) is a product of Shanghai Anpusuierui Standard Technology Service Co., Ltd.; neohesperidin dihydrochalcone (purity 100.0%) is a product of Zhuhai GeTe Biological Technology Co., Ltd.; aspartame (purity 99.7%) and progesterone (purity 99.5%) are products of Aladdin Reagent (Shanghai) Co., Ltd.

[0113] Acetonitrile, n-hexane: chromatographically pure, product of Meker Company, Germany.

[0114] 1.3 Sample preparation

[0115] 1.3.1 Preparation of standard solution

[0116] Take appropriate amount of the above standard, respectively, to prepare 1000 mg / L of single standard solution stock solution (alitame, aspartame, rebaudioside A, sucralose, neotame, neohesperidin dihydrochalcone with water as solvent; glycyrrhizic acid, stevioside, testosterone, methyltestosterone, 17β-estradiol, estrone, estriol, progesterone with acetonitrile as solvent).

[0117] Take appropriate amount of the standard stock solution, respectively, to dilute the methanol acetonitrile solution to obtain 200 μg / L, 500 μg / L of mixed standard solution, now use now.

[0118] 1.3.2 Sample extraction

[0119] Liquid sample:

[0120] Take 1.00 mL of mixed uniform sample in a 15 mL plastic centrifuge tube with a plug, add 2 mL of water, 4 mL of n-hexane, shake for 20 min, then put it in a 4℃ centrifuge at 8000 r / min for 5 min, transfer the upper n-hexane to a 15 mL test tube, blow it to near dryness at 40℃ under nitrogen, add 4.5 mL of methanol acetonitrile solution and vortex to obtain a re-dissolved solution;

[0121] After the lower aqueous solution is placed for 30 min to make the n-hexane completely evaporate, add the methanol acetonitrile re-dissolved solution, vortex for 1 min, and then ultrasonic for 20 min, and then put it in a 4℃ refrigerator for 20 min, and then put it in a 4℃ centrifuge at 8000 r / min for 5 min.

[0122] Filter the extract through a 0.45 μm filter membrane to obtain the target extract for machine analysis.

[0123] Solid sample:

[0124] Take 1.0000 g (accurate to 0.0001 g) of crushed uniform sample in a 15 mL plastic centrifuge tube with a plug, add 3 mL of water, 4 mL of n-hexane, shake for 20 min, then put it in a 4℃ centrifuge at 8000 r / min for 5 min, transfer the upper n-hexane to a 15 mL test tube, blow it to near dryness at 40℃ under nitrogen, add 4.5 mL of methanol acetonitrile solution and vortex to obtain a re-dissolved solution;

[0125] After the lower aqueous solution is placed for 30 min to make the n-hexane completely evaporate, put it in a 4℃ centrifuge at 8000 r / min for 5 min, and then transfer the aqueous solution to a 15 mL centrifuge tube with a plug;

[0126] The methanol-acetonitrile solution was poured into the sample residue, vortexed for 1 min, ultrasonicated for 20 min, centrifuged at 8000 r / min in a 4℃ centrifuge for 5 min, the two extraction solutions were combined, and after oscillation for 20 min, the mixture was placed in a 4℃ refrigerator for 20 min, and then centrifuged at 8000 r / min in a 4℃ centrifuge for 5 min. The final extraction solution was filtered through a 0.45 μm filter membrane and used as the target extraction solution for machine analysis.

[0127] The chromatographic conditions of the ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry method are as follows:

[0128] The chromatographic column is Thermo scientific Hypersil GOLDTM, with a length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 2.7 μm.

[0129] The mobile phase is water and acetonitrile.

[0130] The column temperature is 40℃, the flow rate is 0.3 mL / min, and the injection volume is 10 μL.

[0131] In a more preferred embodiment, the gradient elution program of the mobile phase is as follows: 0.0-2.0 min (10% acetonitrile), 2.0-9.0 min (10%-100% acetonitrile), 9.0-16.0 min (100% acetonitrile), 16.0-18.0 min (100%-10% acetonitrile), and 18.0-21.0 min (10% acetonitrile).

[0132] The mass spectrometry conditions of the ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry method are as follows: an electrospray ion source (ESI) is used, and positive and negative ion modes are detected.

[0133] The ion source temperature is 350℃, the ion transmission temperature is 325℃, the sheath gas pressure is 4 MPa, the auxiliary gas pressure is 1 MPa, the capillary voltage is 3.2 kV, the scan mode is full scan, the acquisition range is 70-900 m / z, and the first mass spectrometry resolution is 70000 FWHM.

[0134] A blank sample was taken, and a six-parallel standard addition recovery experiment was performed on the solid sample at 27 μg / kg and on the liquid sample at 40 μg / L. The recovery rate and relative standard deviation of the detection limit were calculated, as shown in the following table.

[0135]

[0136]

[0137] The recovery rate of the solid sample at a 27 μg / kg standard addition level is 62.4% to 98.8%, and the RSD value is 3.2 to 13.7; the recovery rate of the liquid sample at a 40 μg / L standard addition level is 72.6% to 99.4%, and the RSD value is 3.2 to 9.7, which all meet the requirements of GB / T 27404-2008.

[0138] In summary, the application provides a screening method for simultaneously and rapidly identifying eight kinds of sweeteners and seven kinds of sex hormones which are prone to exceed the standard in food, which has the characteristics of wide detection range, low detection limit and short detection time, and can quickly identify sweeteners and sex hormones in food. On the one hand, it saves a lot of manpower, material resources and time, and facilitates the supervision of unknown risk factors outside food labels, and is suitable for wide promotion and use in food quality supervision. On the other hand, it breaks through the technical barriers of the current national standard detection which is mainly for a small class of substances with the same chemical properties, and can simultaneously detect food additives and drug residues with a large span of chemical properties.

[0139] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the application, and not to limit them; although the application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the application.

Claims

1. A method for screening sweeteners and sex hormones in food, wherein the sweeteners include alitame, aspartame, rebaudioside A, sucralose, neotame, glycyrrhetinic acid, and stevioside; The sex hormones include testosterone, methyltestosterone, 17β-estradiol, estrone, estriol, progesterone and neohesperidin dihydrochalcone, characterized in that: The screening method comprises the following steps: Mixing the test sample with the extraction solvent to obtain the target extract; refrigerating the target extract, centrifuging at low temperature, and filtering to obtain a target detection solution; The target test solution was analyzed and detected by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry. The mass spectrometry conditions of the ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry were as follows: electrospray ionization (ESI) source, positive and negative ion mode detection; ion source temperature of 350°C; ion transmission temperature of 325°C; sheath gas pressure of 4 MPa; auxiliary gas pressure of 1 MPa; capillary voltage of 3.2 kV; scan mode: full scan; acquisition range of 70-900 m / z; primary mass spectrometry resolution of 70,000 FWHM; The test sample is a solid sample, and the steps of extracting to obtain the target extract are as follows: The method comprises the following steps: obtaining a test sample; mixing the test sample with water and n-hexane, and separating the n-hexane solution and the remaining material by low-temperature centrifugation; drying the n-hexane solution with nitrogen at 40° C., adding an acetonitrile solution containing 10% methanol, and mixing to obtain a reconstituted solution; allowing the remaining material to stand, and separating the water-soluble extract and the sample residue by low-temperature centrifugation; mixing the reconstituted solution with the sample residue, ultrasonicating, and centrifuging at low temperature, and then mixing with the water-soluble extract to obtain a target extract.

2. The method for screening sweeteners and sex hormones in food according to claim 1, characterized in that: The refrigeration is as follows: placing the target extract in a 4° C. environment and refrigerating for 20 minutes.

3. The method for screening sweeteners and sex hormones in food according to claim 1, characterized in that: The volume ratio of acetonitrile solution containing 10% methanol, water and n-hexane is (4-5):(2.5-3.5):(3.5-4.5).

4. The method for screening sweeteners and sex hormones in food according to claim 1, characterized in that: The test sample may also be a liquid sample, and the steps of extracting to obtain the target extract are as follows: Obtain test samples; The test sample is mixed with water and n-hexane, and centrifuged at low temperature to separate the n-hexane solution and the remaining material; The n-hexane solution was dried using nitrogen at 40° C., and then an acetonitrile solution containing 10% methanol was added and mixed to obtain a reconstituted solution; The remaining material was allowed to stand, mixed with the reconstituted solution, and ultrasonicated to obtain the target extract.

5. The method for screening sweeteners and sex hormones in food according to any one of claims 3 or 4, characterized in that: The low-temperature centrifugation is as follows: centrifuging at 8000 r / min in a centrifuge at 4°C for 5 minutes.

6. The method for screening sweeteners and sex hormones in food according to claim 1, characterized in that: The chromatographic conditions of the ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry are: Chromatographic column: Thermo scientific Hypersil GOLD™, length 100 mm, inner diameter 2.1 mm, particle size 2.7 μm; Mobile phase: water and acetonitrile; Column temperature: 40°C; flow rate: 0.3 mL / min; injection volume: 10 μL.

7. The method for screening sweeteners and sex hormones in food according to claim 6, characterized in that: The gradient elution program of the mobile phase is: 10% acetonitrile from 0.0 to 2.0 min, 10% to 100% acetonitrile from 2.0 to 9.0 min, 100% to 16.0 min, 100% to 10% acetonitrile from 16.0 to 18.0 min, and 10% to 10% acetonitrile from 18.0 to 21.0 min.

Images