CN114755336A - Detection method capable of simultaneously detecting multiple illegal additives in wheat product - Google Patents
Detection method capable of simultaneously detecting multiple illegal additives in wheat product Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 86
- 241000209140 Triticum Species 0.000 title claims abstract description 47
- 235000021307 Triticum Nutrition 0.000 title claims abstract description 47
- 239000000654 additive Substances 0.000 title claims abstract description 14
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 84
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 60
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 16
- 238000000605 extraction Methods 0.000 claims abstract description 16
- 239000000047 product Substances 0.000 claims abstract description 14
- 239000000203 mixture Substances 0.000 claims abstract description 11
- ROBLTDOHDSGGDT-UHFFFAOYSA-M sodium;pentane-1-sulfonate Chemical compound [Na+].CCCCCS([O-])(=O)=O ROBLTDOHDSGGDT-UHFFFAOYSA-M 0.000 claims abstract description 9
- 238000001914 filtration Methods 0.000 claims abstract description 8
- 239000006228 supernatant Substances 0.000 claims abstract description 7
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000000741 silica gel Substances 0.000 claims abstract description 6
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 6
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical group [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 claims abstract description 5
- 229910000077 silane Inorganic materials 0.000 claims abstract description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 27
- 238000010438 heat treatment Methods 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 15
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 14
- 239000007864 aqueous solution Substances 0.000 claims description 7
- 239000012528 membrane Substances 0.000 claims description 6
- 238000009210 therapy by ultrasound Methods 0.000 claims description 6
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- 238000005119 centrifugation Methods 0.000 claims description 2
- GHMLBKRAJCXXBS-UHFFFAOYSA-N resorcinol Chemical compound OC1=CC=CC(O)=C1 GHMLBKRAJCXXBS-UHFFFAOYSA-N 0.000 abstract description 72
- VDEUYMSGMPQMIK-UHFFFAOYSA-N benzhydroxamic acid Chemical compound ONC(=O)C1=CC=CC=C1 VDEUYMSGMPQMIK-UHFFFAOYSA-N 0.000 abstract description 38
- BEJNERDRQOWKJM-UHFFFAOYSA-N kojic acid Chemical compound OCC1=CC(=O)C(O)=CO1 BEJNERDRQOWKJM-UHFFFAOYSA-N 0.000 abstract description 36
- 229960004705 kojic acid Drugs 0.000 abstract description 36
- WZNJWVWKTVETCG-UHFFFAOYSA-N kojic acid Natural products OC(=O)C(N)CN1C=CC(=O)C(O)=C1 WZNJWVWKTVETCG-UHFFFAOYSA-N 0.000 abstract description 36
- 239000000126 substance Substances 0.000 abstract description 23
- NYVOYAFUSJONHU-UHFFFAOYSA-K trisodium;1,3,5-triazine-2,4,6-trithiolate Chemical compound [Na+].[Na+].[Na+].[S-]C1=NC([S-])=NC([S-])=N1 NYVOYAFUSJONHU-UHFFFAOYSA-K 0.000 abstract description 10
- 235000013305 food Nutrition 0.000 abstract description 6
- BYGYBSHPLSVNGL-UHFFFAOYSA-K trisodium trithiocyanate Chemical compound [Na+].[Na+].[Na+].[S-]C#N.[S-]C#N.[S-]C#N BYGYBSHPLSVNGL-UHFFFAOYSA-K 0.000 description 26
- 230000000052 comparative effect Effects 0.000 description 18
- 230000000694 effects Effects 0.000 description 14
- 238000000926 separation method Methods 0.000 description 12
- 238000002347 injection Methods 0.000 description 8
- 239000007924 injection Substances 0.000 description 8
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 6
- 230000014759 maintenance of location Effects 0.000 description 6
- 238000004090 dissolution Methods 0.000 description 5
- 235000013312 flour Nutrition 0.000 description 5
- 239000002244 precipitate Substances 0.000 description 5
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- 230000005284 excitation Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 238000000703 high-speed centrifugation Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- UMGDCJDMYOKAJW-UHFFFAOYSA-N thiourea Chemical compound NC(N)=S UMGDCJDMYOKAJW-UHFFFAOYSA-N 0.000 description 2
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 2
- NEAQRZUHTPSBBM-UHFFFAOYSA-N 2-hydroxy-3,3-dimethyl-7-nitro-4h-isoquinolin-1-one Chemical compound C1=C([N+]([O-])=O)C=C2C(=O)N(O)C(C)(C)CC2=C1 NEAQRZUHTPSBBM-UHFFFAOYSA-N 0.000 description 1
- KWSLGOVYXMQPPX-UHFFFAOYSA-N 5-[3-(trifluoromethyl)phenyl]-2h-tetrazole Chemical compound FC(F)(F)C1=CC=CC(C2=NNN=N2)=C1 KWSLGOVYXMQPPX-UHFFFAOYSA-N 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 239000004342 Benzoyl peroxide Substances 0.000 description 1
- OMPJBNCRMGITSC-UHFFFAOYSA-N Benzoylperoxide Chemical compound C=1C=CC=CC=1C(=O)OOC(=O)C1=CC=CC=C1 OMPJBNCRMGITSC-UHFFFAOYSA-N 0.000 description 1
- 206010012442 Dermatitis contact Diseases 0.000 description 1
- 238000004497 NIR spectroscopy Methods 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Natural products NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- LOPKSUUTTFZXSY-UHFFFAOYSA-N [Na].CCCCC Chemical compound [Na].CCCCC LOPKSUUTTFZXSY-UHFFFAOYSA-N 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 230000002421 anti-septic effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000019400 benzoyl peroxide Nutrition 0.000 description 1
- 229940105847 calamine Drugs 0.000 description 1
- 239000013064 chemical raw material Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 208000010247 contact dermatitis Diseases 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 239000000645 desinfectant Substances 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000000378 dietary effect Effects 0.000 description 1
- ZXOKVTWPEIAYAB-UHFFFAOYSA-N dioxido(oxo)tungsten Chemical compound [O-][W]([O-])=O ZXOKVTWPEIAYAB-UHFFFAOYSA-N 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000002848 electrochemical method Methods 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 238000005188 flotation Methods 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 229910052864 hemimorphite Inorganic materials 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 239000010842 industrial wastewater Substances 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000011056 performance test Methods 0.000 description 1
- JRKICGRDRMAZLK-UHFFFAOYSA-L peroxydisulfate Chemical compound [O-]S(=O)(=O)OOS([O-])(=O)=O JRKICGRDRMAZLK-UHFFFAOYSA-L 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 239000000718 radiation-protective agent Substances 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- -1 scheelite Chemical compound 0.000 description 1
- 229910001379 sodium hypophosphite Inorganic materials 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- VLLMWSRANPNYQX-UHFFFAOYSA-N thiadiazole Chemical compound C1=CSN=N1.C1=CSN=N1 VLLMWSRANPNYQX-UHFFFAOYSA-N 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 239000011787 zinc oxide Substances 0.000 description 1
- 235000014692 zinc oxide Nutrition 0.000 description 1
- CPYIZQLXMGRKSW-UHFFFAOYSA-N zinc;iron(3+);oxygen(2-) Chemical compound [O-2].[O-2].[O-2].[O-2].[Fe+3].[Fe+3].[Zn+2] CPYIZQLXMGRKSW-UHFFFAOYSA-N 0.000 description 1
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
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- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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Abstract
The application relates to the field of food detection, and particularly discloses a detection method capable of simultaneously detecting multiple illegal additives in a wheat product, which comprises the following steps: crushing a sample, adding a methanol solution for dissolving, carrying out vortex mixing, and then carrying out ultrasonic vacuum extraction process treatment to obtain a wheat dissolving solution; step two, carrying out centrifugal treatment on the wheat dissolved solution, filtering the supernatant and then carrying out sample detection; wherein the chromatographic column adopts a C18 silane bonded silica gel column, the mobile phase A is sodium pentanesulfonate water solution, and the mobile phase B is a mixture of acetonitrile and water-soluble alcohol. By adopting the detection method, the contents of kojic acid, benzohydroxamic acid, trithiocyanuric acid trisodium salt and resorcinol in the wheat sample can be simultaneously detected through one-time sample introduction, so that the detection time is saved, and the detection efficiency is obviously improved; meanwhile, the detection limit of each substance is reduced, and the accuracy of the detection result is improved.
Description
Technical Field
The application relates to the field of food detection, in particular to a detection method capable of simultaneously detecting multiple illegal additives in a wheat product.
Background
Wheat flour and wheat products are important components in the traditional dietary structure of China, and are second to rice as the second major food crops. In order to guarantee the food safety of common people, the national food and drug administration of 11 months in 2017 issues an announcement on further strengthening the safety supervision of wheat flour quality (No. 132 in 2017) that 'manufacturers are strictly prohibited to add non-food raw materials such as benzoyl peroxide, sodium hypophosphite, thiourea, resorcinol, persulfate, thiadiazole, kojic acid and the like into wheat flour'. However, in order to improve the appearance and quality of wheat flour, there are illegal companies who add kojic acid, benzohydroxamic acid, resorcinol, trisodium trithiocyanate and other compounds to wheat flour and products thereof.
Benzohydroxamic acid is a hydroximic acid substance commonly used in industry, is mainly used for flotation of refractory minerals such as wolframite, scheelite, calamine and the like, belongs to organic matters which are difficult to degrade, and can generate certain physiological toxicity when added into food. Resorcinol is a disinfectant, antiseptic and chemical raw material, and can cause adverse reactions such as contact dermatitis and the like. Trisodium trithiocyanate is a treating agent commonly used for treating heavy metal industrial wastewater. Kojic acid is an organic compound, is a colorless prismatic crystal, and can be used as an antioxidant and an antiradiation agent.
At present, the detection of benzohydroxamic acid, resorcinol, trithiocyanuric acid trisodium salt and kojic acid mainly comprises high performance liquid chromatography, high performance liquid chromatography-tandem mass spectrometry, near infrared spectroscopy and an electrochemical method, but the inventor finds that the detection of each substance needs to be matched with a corresponding detection method for sample injection detection independently in the detection process, and the detection efficiency is low.
Disclosure of Invention
The application provides a method for detecting multiple illegal additives in a wheat product simultaneously, and by improving the pretreatment conditions, the liquid chromatography conditions and other aspects of a wheat sample, the detection time of the multiple illegal additives is greatly shortened by once sample introduction through the content of the benzohydroxamic acid, the resorcinol, the trisodium trithiocyanate and the kojic acid, the detection efficiency is obviously improved, and meanwhile, the accuracy of the detection result can be effectively improved.
The detection method capable of simultaneously detecting various illegal additives in the wheat product adopts the following technical scheme:
the detection method capable of simultaneously detecting various illegal additives in the wheat product comprises the following steps:
crushing a sample, adding a methanol solution for dissolving, carrying out vortex mixing, and then carrying out ultrasonic vacuum extraction process treatment to obtain a wheat dissolving solution;
step two, carrying out centrifugal treatment on the wheat dissolved solution, filtering the supernatant and then carrying out sample detection; wherein the chromatographic column adopts a C18 silane bonded silica gel column, the mobile phase A is sodium pentanesulfonate water solution, and the mobile phase B is a mixture of acetonitrile and water-soluble alcohol.
In the second step, the kojic acid, the benzohydroxamic acid and the trisodium trithiocyanate adopt DAD detectors, the detection wavelength of the benzoic acid and the benzohydroxamic acid is 228nm, and the detection wavelength of the trisodium trithiocyanate is 296 nm; resorcinol adopts a fluorescence detector, and the excitation wavelength is 273 nm; the emission wavelength was 304 nm.
By adopting the detection method, the contents of kojic acid, benzohydroxamic acid, trisodium trithiocyanurate and resorcinol in the wheat sample can be simultaneously detected by one-time sample injection, and the detection is performed by one-time sample injection without aiming at a detection substance, so that the detection time is greatly saved, and the detection efficiency is obviously improved. Meanwhile, the detection limits of kojic acid, benzohydroxamic acid, trithiocyanuric acid trisodium salt and resorcinol can be effectively reduced, and the accuracy of the detection result is improved.
The wheat sample is dissolved by adopting a methanol solution after being crushed, and the dissolution effect of kojic acid, benzohydroxamic acid, trisodium trithiocyanate and resorcinol can be effectively improved by matching with an ultrasonic vacuum extraction process, so that the separation effect is improved. The method is characterized in that a sodium pentanesulfonate aqueous solution is used as a specific mobile phase A, a mixture of acetonitrile and water-soluble alcohol is used as a specific mobile phase B, and a corresponding chromatographic column is matched, so that kojic acid, benzohydroxamic acid, trisodium trithiocyanate and resorcinol are driven by the mobile phase to enter the chromatographic column, sufficient retention time is provided for separation, corresponding peak areas are detected, the separation degree of kojic acid, benzohydroxamic acid, trisodium trithiocyanate and resorcinol is good, the detection limit of each substance is low, and the accuracy of a detection result is improved.
Preferably, in the first step, the ultrasonic vacuum extraction process includes the following steps:
wherein the step of increasing the temperature comprises: in the first stage, the temperature is kept between 10 ℃ below zero and 2 ℃ below zero for 3 to 5 min; in the second stage, the temperature is kept between 2 ℃ below zero and 10 ℃ for 5 to 10 min; the temperature of the third section is 10-20 deg.C and is maintained for 8-13min, and the temperature of the third section is 20-35 deg.C and is maintained for 4-8 min.
By adopting the technical scheme, the dissolution effect of the components can be further improved by carrying out ultrasonic treatment at low temperature, so that the separation effect of each substance under the matching of a mobile phase and a chromatographic column is better, and the adverse effect caused by the temperature rise of the wheat dissolution liquid due to ultrasonic operation can be reduced. The precooling can also gather the centrifugal precipitate, so that the centrifugal precipitate is not easy to disperse, and the subsequent operators can conveniently absorb the supernatant and discard the centrifugal precipitate. And the dissolution effect of kojic acid, benzohydroxamic acid, trisodium trithiocyanate and resorcinol can be further effectively improved by heating in sections, so that the separation effect is improved. By matching with subsequent mobile phase and chromatographic column, the contents of kojic acid, benzohydroxamic acid, trithiocyanuric acid trisodium salt and resorcinol in the wheat sample can be detected simultaneously through one-time sample introduction, so that the detection time is saved, and the detection efficiency is obviously improved; meanwhile, the detection limit of each substance is reduced, and the accuracy of the detection result is improved.
Preferably, in the second step, the water-soluble alcohol is methanol, ethylene glycol or propanol.
Preferably, in the second step, the water-soluble alcohol is methanol, and the mass ratio of the acetonitrile to the methanol is (2-4.5): (1-1.7).
By adopting the technical scheme, the selection of the types of the water-soluble alcohol is optimized, the dosage relation of the water-soluble alcohol and the acetonitrile is optimized, and the corresponding chromatographic column is matched, so that the kojic acid, the benzohydroxamic acid, the trisodium trithiocyanate and the resorcinol are driven by the mobile phase to enter the chromatographic column and have enough retention time to be separated, the corresponding peak areas are detected under the corresponding detection conditions, the separation degrees of the kojic acid, the benzohydroxamic acid, the trisodium trithiocyanate and the resorcinol are good, the detection limit of each substance is low, and the accuracy of the detection result is improved.
Preferably, in the second step, the mass fraction of the sodium pentane sulfonate aqueous solution is 0.5-5 per mill.
By adopting the technical scheme, the retention time of kojic acid, benzohydroxamic acid, trisodium trithiocyanate and resorcinol after being driven by the mobile phase to enter the chromatographic column can be further improved by adopting 50-65% of pentane sodium sulfonate aqueous solution by mass fraction, and the separation effect among all substances is improved, so that the accuracy of the detection result is improved.
Preferably, in the first step, a methanol solution with a mass fraction of 40-60% is adopted.
Through adopting above-mentioned technical scheme, the methanol solution is volatile, chooses for use suitable methanol solution, can enough guarantee corresponding dissolving effect, cooperates ultrasonic vacuum extraction technology to exert effect, can save the cost of enterprises again.
Preferably, in the second step, filtration is performed by using an organic microporous filter membrane with the diameter of 0.2-0.45 μm.
By adopting the technical scheme, the filtering condition is optimized, the influence of impurities on the detection result can be effectively reduced, and the detection accuracy is improved.
Preferably, in the second step, the centrifugation is carried out for 4-8min under the conditions of 8000-9000 r/min.
Through adopting above-mentioned technical scheme, during high-speed centrifugation, utilize the rotatory strong centrifugal force that produces at a high speed, the material that subsides the coefficient difference takes place to subside in the suspension or the emulsion that make in the centrifuging tube to make it separate, concentrate and purify, high-speed centrifugation not only can centrifuge out the large granule precipitate, can also centrifuge out the insoluble substance of nanometer level, makes its deposit, so that follow-up operating personnel absorbs the supernatant, abandons centrifugal precipitate.
In summary, the present application has the following beneficial effects:
1. by adopting the detection method, the contents of kojic acid, benzohydroxamic acid, trithiocyanuric acid trisodium salt and resorcinol in the wheat sample can be simultaneously detected through one-time sample introduction, one-time sample introduction for one detection substance is not needed, the detection time is saved, and the detection efficiency is obviously improved.
2. The wheat sample is dissolved by adopting a methanol solution after being crushed, and the ultrasonic vacuum extraction process is matched, so that the dissolving effect of kojic acid, benzohydroxamic acid, trisodium trithiocyanate and resorcinol can be effectively improved, and the separation effect is improved. The specific mobile phase is adopted and matched with a corresponding chromatographic column, so that the kojic acid, the benzohydroxamic acid, the trisodium trithiocyanate and the resorcinol are driven by the mobile phase to enter the chromatographic column and have enough retention time to be separated, the corresponding peak areas are detected, the separation degree of the kojic acid, the benzohydroxamic acid, the trisodium trithiocyanate and the resorcinol is good, the detection limit of each substance is low, and the accuracy of the detection result is improved.
Drawings
FIG. 1 is a liquid chromatogram of example 9;
FIG. 2 is a graph of the linear relationship of kojic acid;
FIG. 3 is a graph of the linear relationship of resorcinol;
FIG. 4 is a graph of the linear relationship of benzohydroxamic acid;
figure 5 is a linear plot of trisodium trithiocyanate.
Detailed Description
The present application will be described in further detail with reference to examples.
The raw materials used in the application are all common commercially available raw materials, wherein the microporous filter membrane is purchased from Jinteng experimental equipment Limited company in Tianjin; c18 column specification: the length is 250mm, the inner diameter is 4.6mm, and the silica gel particle size is 5 μm.
Examples
Example 1
The detection method capable of simultaneously detecting various illegal additives in the wheat product comprises the following steps:
crushing a wheat sample, adding a methanol solution with the mass fraction of 40% for dissolving, uniformly mixing in a vortex manner, and then processing by adopting an ultrasonic vacuum extraction process to prepare a wheat dissolving solution;
the ultrasonic vacuum extraction process comprises the following steps:
wherein the step of raising the temperature comprises: in the first stage, heating to-10 deg.C, and maintaining for 3 min; in the second stage, heating to-2 deg.C, and maintaining for 10 min; the third stage is heating to 10 deg.C for 13min, and the third stage is heating to 20 deg.C for 8 min;
centrifuging the wheat dissolved solution for 8min under the condition of 8000r/min, filtering the supernatant by a 0.45 mu m organic microporous filter membrane, and introducing a sample of 0.5 mu g/mL for detection; wherein the chromatographic column adopts a C18 silane bonded silica gel column, the mobile phase A is a sodium pentanesulfonate aqueous solution with the mass fraction of 0.5 per mill, and the mobile phase B is a mixture of acetonitrile and propanol, wherein the mass ratio of the acetonitrile to the propanol is 2: 1.7;
wherein, the kojic acid, the benzohydroxamic acid and the trisodium trithiocyanate adopt DAD detectors, the detection wavelength of the benzoic acid and the benzohydroxamic acid is 228nm, and the detection wavelength of the trisodium trithiocyanate is 296 nm; resorcinol adopts a fluorescence detector, and the excitation wavelength is 273 nm; the emission wavelength was 304 nm.
Example 2
The detection method capable of simultaneously detecting various illegal additives in the wheat product comprises the following steps:
crushing a wheat sample, adding a methanol solution with the mass fraction of 60% for dissolving, uniformly mixing in a vortex manner, and then processing by adopting an ultrasonic vacuum extraction process to prepare a wheat dissolving solution;
the ultrasonic vacuum extraction process comprises the following steps:
wherein the step of raising the temperature comprises: in the first stage, heating to-2 deg.C, and maintaining for 5 min; in the second stage, heating to 10 ℃ and keeping for 10 min; heating to 20 deg.C for 8min in the third stage, and heating to 35 deg.C for 4min in the third stage;
step two, centrifuging the wheat dissolved solution for 4min under the condition of 9000r/min, taking supernate, filtering the supernate by using a 0.45 mu m organic microporous filter membrane, and then injecting a sample of 0.5 mu g/mL for detection; wherein the chromatographic column adopts a C18 silane bonded silica gel column, the mobile phase A is a sodium pentanesulfonate aqueous solution with the mass fraction of 5 per mill, the mobile phase B is a mixture of acetonitrile and glycol, and the mass ratio of the acetonitrile to the methanol is 4.5: 1.7;
adopting DAD detectors for kojic acid, benzohydroxamic acid and trisodium trithiocyanate, wherein the detection wavelength of benzoic acid and benzohydroxamic acid is 228nm, and the detection wavelength of trisodium trithiocyanate is 296 nm; resorcinol adopts a fluorescence detector, and the excitation wavelength is 273 nm; the emission wavelength was 304 nm.
Example 3
The difference from the example 1 is that in the first step, a methanol solution with a mass fraction of 50% is used, and the rest is the same as the example 1.
Example 4
The difference from the embodiment 3 is that in the first step, the ultrasonic vacuum extraction process comprises the following steps:
wherein the step of raising the temperature comprises: in the first stage, heating to-5 deg.C, and maintaining for 3 min; in the second stage, heating to 6 deg.C, and maintaining for 8 min; heating to 15 deg.C for 10min in the third stage, and heating to 25 deg.C for 6min in the third stage; the rest is the same as in example 3.
Example 5
The difference from the embodiment 4 is that in the second step, the wheat dissolving solution is centrifuged for 7min under the condition of 8800r/min, and the supernatant is filtered by a 0.22 μm organic microporous filter membrane and then is injected with 0.5 μ g/mL for detection; the rest is the same as in example 4.
Example 6
The difference from the embodiment 5 is that in the second step, the mobile phase a is a sodium pentanesulfonate aqueous solution with the mass fraction of 2.5 per mill; the rest is the same as example 5.
Example 7
The difference from the example 6 is that in the second step, the mobile phase B is a mixture of acetonitrile and ethylene glycol, and the mass ratio of the acetonitrile to the ethylene glycol is 2: 1; the rest is the same as example 6.
Example 8
The difference from the example 7 is that the mobile phase B is a mixture of acetonitrile and methanol, and the mass ratio of the acetonitrile to the methanol is 2: 1; the rest is the same as in example 7.
Example 9
The difference from the example 7 is that the mobile phase B is a mixture of acetonitrile and methanol, and the mass ratio of the acetonitrile to the methanol is 3.1: 1.4; the rest was the same as in example 7.
Example 10
The difference from the embodiment 7 is that the mobile phase B is a mixture of acetonitrile and methanol, and the mass ratio of the acetonitrile to the methanol is 1.5: 3; the rest is the same as in example 7.
Comparative example
Comparative example 1
The difference from the example 9 is that in the step one, the ultrasonic vacuum extraction process is not adopted, and the rest is the same as the example 9.
Comparative example 2
The difference from the example 9 is that the first step is specifically: crushing a wheat sample, dissolving the wheat sample by adopting an ethanol solution with the mass fraction of 50%, uniformly mixing the wheat sample by vortex, and extracting the wheat sample for 30min under the condition that the vacuum degree is 15 Pa; the rest was the same as in example 9.
Comparative example 3
The difference from the example 9 is that in the second step, the mobile phase B is ethanol solution, and the rest is the same as the example 9.
Comparative example 4
The difference from the example 9 is that in the second step, the mobile phase is water, and the rest is the same as the example 9.
Performance test
The detection limits of kojic acid, benzohydroxamic acid, trisodium trithiocyanate and resorcinol were determined in examples 1 to 10 and comparative examples 1 to 4 according to GB/T27417-.
TABLE 1
The liquid chromatogram obtained in example 9 and comparative examples 1 to 4 were analyzed to calculate the retention times of kojic acid, benzohydroxamic acid, trisodium trithiocyanate and resorcinol, and the results are shown in Table 2.
TABLE 2
As can be seen from examples 1 to 10 in combination with table 1, the content of kojic acid, benzohydroxamic acid, trithiocyanuric acid trisodium salt and resorcinol in a wheat sample can be simultaneously detected by the detection method of the present application through one-time sample injection, and one-time sample injection is not required for one detection substance, so that the detection time is saved, and the detection efficiency is significantly improved. Meanwhile, the detection limits of kojic acid, benzohydroxamic acid, trithiocyanuric acid trisodium salt and resorcinol can be effectively reduced, and the accuracy of the detection result is improved.
As can be seen from example 9 in combination with fig. 1 and tables 1 and 2, the detection limit of each substance in example 9 is low, and from both fig. 1 and table 2, it can be seen that the peak appearance time interval between the substances in example 9 is long, the peak areas of the substances do not overlap, and the detection accuracy is good.
As can be seen from comparative examples 1 to 4 in combination with tables 1 and 2, comparative example 1 and comparative example 2 do not employ the ultrasonic vacuum extraction process of the present application in combination with a methanol solution as a solvent in step one, and comparative example 1 and comparative example 2 employ a pretreatment method different from example 9, so that comparative example 1 and comparative example 2 cannot simultaneously detect a plurality of substances by one sample injection, the detection efficiency is significantly reduced, and the detection limit of the detectable substances is significantly increased, but the peak time interval of the detected substances corresponding to comparative example 1 and comparative example 2 is not much different from that of example 9. In the comparative example 3, an ethanol solution is used as the mobile phase B, and in the comparative example 4, water is used as the mobile phase A, although kojic acid, benzohydroxamic acid, trisodium trithiocyanate and resorcinol can be detected at the same time, the detection limit of each substance is obviously increased, the accuracy of the detection result is poor, the peak emergence time of the four substances is close, the detected peak areas are overlapped, and the accuracy of the detection result is obviously reduced. The wheat sample is dissolved by adopting a methanol solution after being crushed, and the dissolution effect of kojic acid, benzohydroxamic acid, trisodium trithiocyanate and resorcinol can be effectively improved by matching with an ultrasonic vacuum extraction process, so that the separation effect is improved. Meanwhile, a mixture of acetonitrile and methanol is used as a specific mobile phase under a certain proportion condition, and a corresponding chromatographic column is matched, so that the kojic acid, the benzohydroxamic acid, the trithiocyanic acid trisodium salt and the resorcinol are driven by the mobile phase to enter the chromatographic column, sufficient retention time is provided for separation, corresponding peak areas are detected, the separation degree of the kojic acid, the benzohydroxamic acid, the trithiocyanic acid trisodium salt and the resorcinol is good, the detection limit of each substance is low, and the accuracy of detection results is improved.
The detection steps of example 9 were adopted for sample injection detection, and the linear relationship graphs of kojic acid, benzohydroxamic acid, trithiocyanuric acid trisodium salt and resorcinol were respectively plotted according to the results of kojic acid and benzohydroxamic acidThe corresponding standard detection methods of the hydroxamic acid, the trisodium trithiocyanate and the resorcinol are corrected, and the linear relation graph of the kojic acid, the benzohydroxamic acid, the trisodium trithiocyanate and the resorcinol and the correlation coefficient R of a marked line are recorded2Coefficient of correlation R2The results are reported in Table 3, R2The closer to 1, the more accurate the detection result.
TABLE 3
As can be seen from example 9 in combination with fig. 2 to 5 and table 3, with the detection method of the present application, the linear correlation coefficients are all greater than 0.999, the detected linear correlation lines of kojic acid, benzohydroxamic acid, trithiocyanuric acid trisodium salt and resorcinol have high goodness of fit with the standard line, the detection accuracy is high, multiple substances can be detected simultaneously by one-time sample injection, and the detection efficiency is good.
The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.
Claims (8)
1. The detection method capable of simultaneously detecting various illegal additives in the wheat product is characterized by comprising the following steps of:
crushing a wheat sample, adding a methanol solution for dissolving, performing vortex mixing, and performing ultrasonic vacuum extraction to obtain a wheat dissolved solution;
step two, carrying out centrifugal treatment on the wheat dissolved solution, filtering the supernatant and then carrying out sample detection; wherein the chromatographic column adopts a C18 silane bonded silica gel column, the mobile phase A is sodium pentanesulfonate water solution, and the mobile phase B is a mixture of acetonitrile and water-soluble alcohol.
2. The method of claim 1, wherein the method comprises the steps of: in the first step, the ultrasonic vacuum extraction process comprises the following steps:
step 1, ultrasonic precooling: adjusting the temperature to below-10 ℃, and carrying out ultrasonic treatment for 5-9 min;
step 2, heating in sections, wherein the vacuum degree is set to be 20 +/-10 Pa;
wherein the step of raising the temperature comprises: in the first stage, the temperature is kept between 10 ℃ below zero and 2 ℃ below zero for 3 to 5 min; in the second stage, the temperature is kept between-2 ℃ and 10 ℃ for 5-10 min; the temperature of the third section is 10-20 deg.C and is maintained for 8-13min, and the temperature of the third section is 20-35 deg.C and is maintained for 4-8 min.
3. The detection method for simultaneously detecting multiple illegal additives in a wheat product according to claim 1 or 2, characterized in that: in the second step, the water-soluble alcohol is methanol, ethylene glycol or propanol.
4. The method of claim 3, wherein the method comprises the steps of: in the second step, the water-soluble alcohol is methanol, and the mass ratio of the acetonitrile to the methanol is (2-4.5) to (1-1.7).
5. The method of claim 1, wherein the method comprises the steps of: in the second step, the mass fraction of the sodium pentane sulfonate aqueous solution is 0.5-5 per mill.
6. The method of claim 1 or 5 for simultaneously detecting multiple illegal additives in a wheat product, wherein the method comprises the steps of: in the first step, a methanol solution with the mass fraction of 40-60% is adopted.
7. The method of claim 1, wherein the method comprises the steps of: in the second step, an organic microporous filter membrane with the diameter of 0.2-0.45 μm is adopted for filtration.
8. The method of claim 7 for detecting multiple illegal additives in a wheat product simultaneously, wherein the method comprises: in the second step, the centrifugation is carried out for 4-8min under the conditions of 8000-9000 r/min.
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| CN111721863A (en) * | 2020-06-16 | 2020-09-29 | 山东省食品药品检验研究院 | Method for measuring non-edible substance trithiocyanuric acid trisodium salt in wheat flour and additives thereof by ultra-high performance liquid chromatography-tandem mass spectrometry |
| US20210289794A1 (en) * | 2018-07-25 | 2021-09-23 | Flagship Pioneering Innovations V, Inc. | Compositions and related methods for agriculture |
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| CN107167532A (en) * | 2017-05-18 | 2017-09-15 | 东莞市中鼎检测技术有限公司 | A kind of method of food additives in use high performance liquid chromatography test food |
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