CN106568870B - A kind of detection method irradiating aquatic products dried product - Google Patents
A kind of detection method irradiating aquatic products dried product Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 56
- 230000001678 irradiating effect Effects 0.000 title claims abstract description 10
- 238000010811 Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Methods 0.000 claims abstract description 19
- 238000002203 pretreatment Methods 0.000 claims abstract description 7
- 239000000523 sample Substances 0.000 claims description 82
- 239000000047 product Substances 0.000 claims description 80
- JZKXXXDKRQWDET-QMMMGPOBSA-N L-m-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC(O)=C1 JZKXXXDKRQWDET-QMMMGPOBSA-N 0.000 claims description 74
- JZKXXXDKRQWDET-UHFFFAOYSA-N meta-tyrosine Natural products OC(=O)C(N)CC1=CC=CC(O)=C1 JZKXXXDKRQWDET-UHFFFAOYSA-N 0.000 claims description 73
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims description 55
- WRFPVMFCRNYQNR-UHFFFAOYSA-N 2-hydroxyphenylalanine Chemical compound OC(=O)C(N)CC1=CC=CC=C1O WRFPVMFCRNYQNR-UHFFFAOYSA-N 0.000 claims description 44
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims description 44
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims description 35
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 claims description 34
- 239000007789 gas Substances 0.000 claims description 28
- 238000000034 method Methods 0.000 claims description 27
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
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- 238000011282 treatment Methods 0.000 claims description 12
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
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- DYUQAZSOFZSPHD-UHFFFAOYSA-N Phenylpropyl alcohol Natural products CCC(O)C1=CC=CC=C1 DYUQAZSOFZSPHD-UHFFFAOYSA-N 0.000 claims description 2
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- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 13
- 241000238366 Cephalopoda Species 0.000 description 12
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical class OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 9
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 8
- 235000001014 amino acid Nutrition 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 6
- 238000012544 monitoring process Methods 0.000 description 6
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- 150000001875 compounds Chemical class 0.000 description 5
- 238000009930 food irradiation Methods 0.000 description 5
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 235000004279 alanine Nutrition 0.000 description 4
- 229910021529 ammonia Inorganic materials 0.000 description 4
- 238000000904 thermoluminescence Methods 0.000 description 4
- KUNFMZSTKSLIEY-GRHHLOCNSA-N (2s)-2-azanyl-3-phenyl-propanoic acid Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1.OC(=O)[C@@H](N)CC1=CC=CC=C1 KUNFMZSTKSLIEY-GRHHLOCNSA-N 0.000 description 3
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- -1 hydroxyl radical free radical Chemical class 0.000 description 2
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- 238000007689 inspection Methods 0.000 description 2
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 2
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- 238000004885 tandem mass spectrometry Methods 0.000 description 2
- VVKRFZDUZMLMQM-UHFFFAOYSA-N 2-Dodecylcyclobutanone Chemical compound CCCCCCCCCCCCC1CCC1=O VVKRFZDUZMLMQM-UHFFFAOYSA-N 0.000 description 1
- COLNVLDHVKWLRT-MRVPVSSYSA-N D-phenylalanine Chemical compound OC(=O)[C@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-MRVPVSSYSA-N 0.000 description 1
- 229930182832 D-phenylalanine Natural products 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 241000237852 Mollusca Species 0.000 description 1
- 231100000678 Mycotoxin Toxicity 0.000 description 1
- BPQQTUXANYXVAA-UHFFFAOYSA-N Orthosilicate Chemical compound [O-][Si]([O-])([O-])[O-] BPQQTUXANYXVAA-UHFFFAOYSA-N 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
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- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 235000013332 fish product Nutrition 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 241000411851 herbal medicine Species 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- GPRLSGONYQIRFK-UHFFFAOYSA-N hydron Chemical compound [H+] GPRLSGONYQIRFK-UHFFFAOYSA-N 0.000 description 1
- OUUQCZGPVNCOIJ-UHFFFAOYSA-N hydroperoxyl Chemical compound O[O] OUUQCZGPVNCOIJ-UHFFFAOYSA-N 0.000 description 1
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- SYSQUGFVNFXIIT-UHFFFAOYSA-N n-[4-(1,3-benzoxazol-2-yl)phenyl]-4-nitrobenzenesulfonamide Chemical class C1=CC([N+](=O)[O-])=CC=C1S(=O)(=O)NC1=CC=C(C=2OC3=CC=CC=C3N=2)C=C1 SYSQUGFVNFXIIT-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- G—PHYSICS
- 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
- G01N30/02—Column chromatography
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a kind of detection methods for irradiating aquatic products dried product, including sample pre-treatments, QTRAP-UPLC-MS/MS are tested and analyzed and irradiation dose quantitative detection step.The present invention can separate a variety of amino acid and its isomer simultaneously, and it is carried out qualitative, quantitative and secondary qualitative, can judge whether aquatic products dried product by irradiation and quantitative to irradiation dose, as a result reliably, detection limit is low, quickly detects suitable for irradiation aquatic products dried product.
Description
Technical field
The present invention relates to aquatic products detection technique field, in particular to a kind of detection method for irradiating aquatic products dried product.
Background technique
Food irradiation be using ionising radiation (60Co or137What ray, the electron accelerator of Cs radioactive source generation generated is lower than
10MeV electron beam or X-ray) food is processed, the radiation chemistry and spoke generated in food using ionising radiation
Biological effects are penetrated, to reach control food-borne causal agent, reduce load of microorganisms and insect pest, inhibit physiology course and extend goods
The purpose of frame phase.Because it has the characteristics that cold work, without secondary pollution and chemical residual, low energy consumption, in packaging for foodstuff material
The fields such as the desinsections such as material, grain, fruit, sterilizing have a wide range of applications.According to another studies have shown that irradiation technique to mycotoxin,
The food contaminants such as pesticide, fishing medicine also have certain inhibition and degradation.Corruption, storage hardly possible are easy since aquatic products have
The disadvantages of, in recent years, irradiation technique is widely used in the sterilization fresh-keeping during processing of aquatic products, transport and storage, becomes
The main radiation treatment food other than spice, rhizome plants in the world.
The potential safety issue of irradiated food always exists dispute, and improperly irradiation can color to food, taste, battalion
It supports and generates some side effects, for example decolourize, have stink, destroy the nutritional ingredients such as protein, fat.Currently, countries in the world are to spoke
The problem of holding quite stringent and careful attitude according to food, being identified especially in irradiated food management obtained EU countries and
The attention of the states such as Japan and Korea S..Currently, there is more detailed requirement in the country such as the U.S., Britain, Germany to food irradiation, for food
Type, irradiation dose, irradiation facility, food irradiation mark have specific regulation.Instruction of the European Union in relation to food irradiation be
The skeleton instruction 1999/2/EC regulation of the food of processing " ion exposure ", all irradiated food or containing irradiated food at
That divides must indicate on food labelling；1999/3/EC is executed instruction to provide to allow the food irradiated only to allow at present in European Union
One substance of radiation treatment herbal medicine, fragrance and plant sauce.
Currently, irradiation detection method establishes the specific derivative analyte detection generated after being irradiated to variety classes food
On the basis of, the principle used by method itself is limited, and every kind of method is suitable for certain types of sample.It see the table below 1.
The different irradiation detection methods of table 1 compare
When fat-containing food is illuminated, fatty acid and acylglycerol in food can be decomposed to form accordingly with carbon atom number 2-
Alkyl cyclobutanone (2-alkylcyclobutanones, 2-ACBs).Since self-discovery, 2-ACBs is always to contain rouge irradiated food
The hot spot of research.As the significant compound of fat-containing food irradiation, 2-ACBs can be used to detect irradiated food containing rouge.But it is big
Part aquatic products fat content is lower, therefore determines whether not being suitable for spoke by irradiation by detecting 2- dodecyl cyclobutanone
According to the detection of aquatic products.Another common method thermoluminescence analytic approach (TL) of irradiation detection, which can be used to detect, can isolate mineral
Whether the food of matter passes through radiation treatment.But part aquatic product is because have passed through internal organ, decladding, steaming in its process
It boils, especially repeatedly rinsing, such as shredded squid, flesh of fish product, actually detected it is difficult to extract the minerals for arriving sufficient amount in the process
For detecting, thermoluminescence analytic approach has certain difficulty in the application whether such product detects by irradiation；In addition the method
There are problems that false negative；Its luminosity curve can not quantify irradiation dose.Therefore, it also lacks at present for irradiation aquatic products
The standardized method that product examine is surveyed, the especially method of irradiation dose quantitative detection.
Phenylalanine is the neutral amino acid (see structural formula 1) with aromatic rings, because of c-terminus band under the conditions of 7.0 pH
Negative electricity, aminoterminal is positively charged and deposit entire molecule can in a free form or by peptide linkage in cell in electroneutral
It is in protein.After water in food is irradiated, hydroxyl radical free radical, hydroperoxyl radical, hydrogen ion and aqueous electron can be generated,
O-, m-, the contraposition of middle hydroxyl radical free radical meeting phenylalanine of the attack containing aromatic ring, and then three kinds of corresponding hydroxy compounds are generated,
I.e. three kinds of isomers-of tyrosine are to tyrosine (p-Tyrosine, structural formula 2), adjacent tyrosine (o-Tyrosine, structural formula 4)
With m-Tyrosine (m-Tyrosine, structural formula 3).Wherein, relatively conventional to tyrosine, and have in matrix itself and largely deposit
?.And meta position tyrosine (m- tyrosine, 3- hydroxy phenylalanine or LM- tyrosine) and ortho position tyrosine (o- tyrosine or
2- hydroxy phenylalanine), though the two naturally occurring, be all it is rare, must be via being carried out under the oxidative stress condition of phenylalanine
Non- Antloxidation hydroxylation and generate.
1 Phenylalanine phenylalanine of structural formula
2 Tyrosine of structural formula is to tyrosine
3 m-Tyrosine m-Tyrosine of structural formula or 3- hydroxy phenylalanine
4 o-Tyrosine neighbour's tyrosine of structural formula or 2- hydroxy phenylalanine
Aquatic products include fish, shell-fish and enrich with mollusc water content, and protein content is in 15%-25%
Between, though wherein there is some difference in different aquatic products for the content of phenylalanine, all accounted in protein composition higher
Ratio.Aquatic products can generate o-Tyrosine and m-Tyrosine after being irradiated, therefore measure in aquatic products whether contain o-
Whether Tyrosine and m-Tyrosine, which can pass through irradiation to the product, determines.
Currently, the country has no based on QTRAP-UPLC-MS/MS technology, by by a variety of same points of tyrosine and phenylalanine
The content of isomer separation, measurement tyrosine, phenylalanine and 2- hydroxy phenylalanine and 3- hydroxy phenylalanine is to irradiation water
Product identifies and the method for quantitative detection.
Summary of the invention
The purpose of the present invention is to provide a kind of detection methods for irradiating aquatic products dried product, can separate a variety of amino acid simultaneously
And its isomer, and it is carried out it is qualitative, quantitative and secondary qualitative, can judge aquatic products dried product whether by irradiation and it is right
Irradiation dose is quantitative, as a result reliably, detection limit is low, quickly detects suitable for irradiation aquatic products dried product.
The technical solution adopted by the present invention to solve the technical problems is:
A kind of detection method irradiating aquatic products dried product, steps are as follows:
One, sample pre-treatments
(1) sample treatment: after sample to be tested is crushed be packed into container in, seal, refrigerate it is spare；
(2) acidolysis: weighing sample 0.2g and be added in 20mL acidolysis glass tube, addition 10mL 6moL/L hydrochloric acid, after inflated with nitrogen
Spiral cover is covered tightly, acid hydrolysis solution is stayed overnight to obtain in acidolysis at 110 DEG C；
(3) it purifies: after taking acid hydrolysis solution to be filtered with filter paper, being placed in glass centrifuge tube, be centrifuged at 4 DEG C, take supernatant 5mL,
It is dried with nitrogen at 35 DEG C, then freeze dryer is lyophilized, and dissolves lyophilized products, 0.22 μm of water phase nylon with 0.1% aqueous formic acid of 1mL
Membrane filtration, filtrate are used for the detection of 2- hydroxy phenylalanine and 3- hydroxy phenylalanine as sample liquid；Meanwhile taking 100 μ L
Sample liquid after filtrate with 0.1% aqueous formic acid is settled to 1mL and must dilute 10 times is detected for tyrosine and phenylalanine；
Two, QTRAP-UPLC-MS/MS is tested and analyzed
It is mark with tyrosine, phenylalanine and irradiation specific product 2- hydroxy phenylalanine and 3- hydroxy phenylalanine
Quasi- product, using irradiation mass spectrum response peak area as abscissa, to tyrosine, phenylalanine, 2- hydroxy phenylalanine and 3- hydroxyl
Base concentration of phenylalanine draws standard curve as ordinate；With MRM-IDA-EPI mode, by the second level of four kinds of standard substances
Map deposit spectrum library, so that sample compares therewith；
By the upper machine testing of the sample liquid after 10 times of sample liquid and dilution, by the correspondence ion pair and standard drawing of test substance
Spectrum is matched, and using Purity value as judgment basis, is considered same substance greater than 70%, and quantify to it；
Sample to be tested hydroxy phenylalanine containing 2- and the representative of 3- hydroxy phenylalanine received radiation treatment, and according to standard
Curve calculates the content of contained 2- hydroxy phenylalanine and 3- hydroxy phenylalanine；
Three, irradiation dose quantitative detection
Using irradiation dose as abscissa, 3- hydroxy phenylalanine and 2- hydroxy phenylalanine content are that ordinate draws standard
The content of 3- hydroxy phenylalanine and 2- hydroxy phenylalanine in sample to be tested is substituted into standard curve to determine to test sample by curve
The irradiation dose that product received.
Because tyrosine contained by aquatic products and phenylalanine content are higher, therefore with machine on the sample after 10 times of dilution to this
Two kinds of substances carry out quantitative detection and qualitative confirmation.Although detection irradiation specific product 2- hydroxy phenylalanine and 3- hydroxy benzenes
Alanine can judge whether further quantitative by irradiating and carrying out.But tyrosine and phenylalanine are the third ammonia of 2- hydroxy benzenes
The isomer of acid and 3- hydroxy phenylalanine, if not detecting the two and compareing, so directly for 2- hydroxy benzenes third
Propylhomoserin and the measurement of 3- hydroxy phenylalanine will appear false positive, be difficult in detection different to both same point of tyrosine and phenylalanine
Structure body distinguishes.Therefore, while tyrosine and phenylalanine being detected as comparing, the accuracy of detection can be improved.
Since free 3- hydroxy phenylalanine and 2- hydroxy phenylalanine contained in aquatic products dried product are less, this hair
It is bright to design specific extracting mode for aquatic products dried product, substantially increase tyrosine, phenylalanine, 3- hydroxy phenylalanine and
The extraction efficiency of 2- hydroxy phenylalanine, and low-temperature and high-speed centrifugation is combined to carry out purification and freeze drying technology, improve practical inspection
The sensitivity of survey；And QTRAP-UPLC-MS/MS is with stronger separating capacity, the qualitative analysis is reliable, detection limit is low quickly
The advantages that accurate, specific irradiation product in the aquatic products of QTRAP-UPLC-MS/MS technology energy Accurate Determining high protein content
The content of 3- hydroxy phenylalanine and 2- hydroxy phenylalanine simultaneously carries out secondary qualitative, avoids isomer and other miscellaneous peaks
Interference, be compared by the specific product content of the aquatic products after different known dose irradiation, determine sample to be tested
The irradiation dose received.The present invention is suitble to irradiate the qualitative and quantitative detection and food safety monitoring of aquatic products.
Preferably, the chromatographic parameter setting that QTRAP-UPLC-MS/MS is tested and analyzed are as follows:
Chromatographic separation condition: Waters Acquity UPLC BEH C18Column, flow velocity 0.4mL/min；Mobile phase A: methanol,
Mobile phase B: 0.1% aqueous formic acid；Sample volume: 2 μ L；Column temperature: 35 DEG C；
The gradient elution program of mobile phase are as follows: 0-5min, Mobile phase B ratio are adjusted to 30% from 5%, later 5.1min-
Mobile phase B ratio is adjusted to 5% from 30% in 13-13.5min by 8min.
Preferably, the Mass Spectrometry Conditions that QTRAP-UPLC-MS/MS is tested and analyzed are as follows: ion source Turbo V ionizes mode
ESI+, acquisition mode MRM mode, 550 DEG C of ionization temperature；Spray voltage 5500V, atomization gas 55psi assist gas 60psi, gas
Curtain gas 30psi, collision gas High；Entrance potential 10v, collision cell exit potential 13v.
Preferably, MRM-IDA-EPI mode parameter is arranged are as follows: IDA trigger signal intensity threshold 5000；EPI condition: it sweeps
Retouch rate 10000Da/s；Scanning range: 50-200Da；The same MRM of Souce/Gas condition；DP voltage 80v；Collision energy 35v, is touched
Hitting energy spectrum width is 15.
Preferably, in step 2, when drawing standard curve, the concentration gradient setting of standard items are as follows: 5 μ g/L, 10 μ g/L,
50 μ g/L, 100 μ g/L, 500 μ g/L contain tyrosine, phenylpropyl alcohol ammonia according to corresponding prepare of concentration gradient setting of standard items simultaneously
The mixed standard solution of acid, 2- hydroxy phenylalanine and 3- hydroxy phenylalanine.Contain tyrosine, phenylalanine, 2- hydroxyl simultaneously
In mixed standard solution, that is, mixed standard solution of base phenylalanine and 3- hydroxy phenylalanine simultaneously tyrosine, phenylalanine,
The concentration of 2- hydroxy phenylalanine and 3- hydroxy phenylalanine is all the same and is normal concentration design value, such as 5 μ g/L or 10 μ
g/L。
Preferably, in step 3, the method for drawing standard curve specifically: take not irradiated control sample, use X-ray
Irradiation instrument carries out the irradiation of 0kGy, 1kGy, 2kGy, 5kGy, 10kGy, 20kGy dosage respectively, according to step 1 sample pre-treatments
After method is handled, the 3- in the control sample after various dose irradiates is detected respectively with machine in MRM-IDA-EPI mode
The content of hydroxy phenylalanine and 2- hydroxy phenylalanine and secondary qualitative, by irradiation dose as abscissa, 3- hydroxy benzenes
Alanine and 2- hydroxy phenylalanine content find out regression equation and related coefficient respectively and draw standard song as ordinate
Line.
Preferably, 12000r/min is centrifuged 10min at 4 DEG C in step 1.
Measurement is all made of blank and positive control sample every time.Blank control is the same type that negative sample is not irradiated
For aquatic products as blank control, positive sample is measuring samples same type aquatic products, is used if measuring samples are single-freeze peeled shrimp
Irradiated peeled shrimp does not carry out the irradiation of 2kGy dosage in X-ray irradiation instrument, and the sample after this is irradiated is made as positive sample
For the means of Quality Control.
The beneficial effects of the present invention are:
1, have a wide range of application.The present invention can reinforce the monitoring of aquatic products irradiation, be conducive to Distribution of Aquatic Products link and disengaging
Monitoring in mouth trade, the international trade dispute caused by reducing because of aquatic products irradiation, reduces the economic loss of import and export enterprise.
2, the present invention can separate a variety of amino acid and its isomer simultaneously, and carry out to it qualitative, quantitative and secondary
It is qualitative.Solve the problems, such as the separation of amino acid isomer difficulty, difficult measurement.
3, the present invention is solved the problems, such as currently without suitable for the identification of irradiation aquatic products dried product and detection method.Utilize water
Product high protein content, and specific irradiation product 3- hydroxy phenylalanine and 2- hydroxy phenylalanine are generated after irradiation
The characteristics of, the method for being suitable for irradiating aquatic products dried product identification and detection is established, solves irradiation aquatic products dried product detection nothing
Method can according to the problem of.
4, it present invention employs UPLC-MS/MS technology, can be rapidly completed in 5min to amino acid and isomer
Separation and quantitative detection.Due to isomer molecular weight having the same, and it can generate identical ion pair, it is necessary to pass through
The mode that ion pair is combined with retention time is come qualitative and quantitative.UPLC-MS/MS technology quickly and effectively solves different with dividing
The problem of structure body difficulty separates.
5, present invention employs MRM-IDA-EPI, that is, MRM to trigger enhancer ion scan technology, while obtaining MRM chromatography
Figure and MS2Mass spectrogram carries out a variety of isomers secondary qualitative true by the matching degree with standard substance second level spectrogram
Card.Remain series connection quadrupole mass spectrometer is selectively good, sensitivity is high MRM, at the same enhance secondary fragment compose entirely it is qualitative, effectively
It solves the problems, such as the qualitative of isomer and is interfered vulnerable to other substances.
6, the present invention passes through the preparation of first time standard curve, irradiates specific product 2- hydroxy phenylalanine and 3- hydroxyl
Phenylalanine whether detect can fast qualitative judge whether sample to be tested passes through radiation treatment.As contained 2- hydroxyl in measuring samples
Base phenylalanine and 3- hydroxy phenylalanine, that is, can determine that and receive irradiation.Method is simple and easy, can treat in a short time
Whether sample, which passes through irradiation, is determined.
7, the present invention using measurement irradiate through various dose sample generation specific product content and its be subjected to
Irradiation dose relationship as standard curve, pass through 3- hydroxy phenylalanine and 2- hydroxy phenylalanine in detection sample to be tested
Content determine its receive irradiation dose.The quantitative detection of irradiated food is realized for the first time.
8,2- hydroxy phenylalanine and 3- hydroxy phenylalanine has been selected to carry out in the present invention as irradiation specific product
As the foundation determined whether by irradiation, both material stabilities are preferable for measurement, and irradiated treated sample is when long
Between save after redeterminate and can still detect both substances.Therefore the method high reliablity, especially frozen fish have long-term
The characteristics of freezing, the present disclosure applies equally to the irradiation detections of the sample after long-term frozen or storage.
9, present invention employs QTRAP-UPLC-MS/MS methods detects irradiation aquatic products, improves actually detected
Sensitivity, and QTRAP-UPLC-MS/MS has that stronger separating capacity, the qualitative analysis are reliable, detection limit is low, secondary
The advantages that qualitative, has better utility value compared with traditional detection method.
10, present invention employs QTRAP-UPLC-MS/MS methods detects irradiation aquatic products dried product, liquid chromatogram-
Tandem mass spectrometer is the more general and common detection device in current laboratory, in analysis testing field and food safety control neck
Domain has a wide range of applications and Research Prospects.The special instrument that its application in irradiation detection can avoid purchasing irradiation detection is set
Standby such as thermoluminescence detector, electron spin resonanceapparatus, light release light measurement system, such equipment is removed particular kind of for identifying
Whether sample is outer by irradiation, if thermoluminescence detector is served only for may separate out the qualitative detection of the irradiated food of silicate,
Remaining field using less, and such equipment acquisition cost is higher, and selectable instrument brand is few and expensive, greatly
Increase the testing cost and operating cost in laboratory.Application of the liquid chromatography-tandem mass spectrometry instrument in irradiation aquatic products detection,
The dedicated irradiation detection device that can avoid laboratory acquisition price valuableness, improves the utilization efficiency of instrument, greatly reduces
Testing cost.
11, the present invention can once handle gross sample, be suitble to the quick detection of batch samples.
Detailed description of the invention
Fig. 1 is the MRM figure of four kinds of amino acid；
In figure: 1 tyrosine；2. m-Tyrosine (m-tyrosine, 3- hydroxy phenylalanine)；3. adjacent tyrosine (o-
Tyrosine, 2- hydroxy phenylalanine)；4. phenylalanine.
Fig. 2 is the EPI figure of four kinds of amino acid；
In figure: A tyrosine (p-tyrosine)；B m-Tyrosine (m-tyrosine, 3- hydroxy phenylalanine)；C neighbour's junket ammonia
Sour (o-tyrosine, 2- hydroxy phenylalanine)；D phenylalanine (Phenylalanine).
Fig. 3 is that sample MRM schemes after being irradiated, in figure: 1 tyrosine；2. m-Tyrosine (m-tyrosine, 3- hydroxy benzenes third
Propylhomoserin)；3. adjacent tyrosine (o-tyrosine, 2- hydroxy phenylalanine)；4. phenylalanine.
Specific embodiment
Below by specific embodiment, technical scheme of the present invention will be further explained in detail.
In the present invention, if not refering in particular to, used raw material and equipment etc. are commercially available or commonly used in the art.
Method in following embodiments is unless otherwise instructed the conventional method of this field.
Embodiment:
A kind of detection method irradiating aquatic products dried product, steps are as follows:
One, sample pre-treatments
(1) sample treatment: after sample to be tested is crushed be packed into container in, seal, refrigerate it is spare；
(2) acidolysis: weighing sample 0.2g and be added in 20mL acidolysis glass tube, addition 10mL 6moL/L hydrochloric acid, after inflated with nitrogen
Spiral cover is covered tightly, acid hydrolysis solution is stayed overnight to obtain in acidolysis at 110 DEG C；
(3) it purifies: after taking acid hydrolysis solution to be filtered with filter paper, being placed in glass centrifuge tube, 12000r/min is centrifuged at 4 DEG C
10min takes supernatant 5mL, is dried with nitrogen at 35 DEG C, and then freeze dryer is lyophilized, and is dissolved and is lyophilized with 0.1% aqueous formic acid of 1mL
Object, the filtering of 0.22 μm of water phase nylon leaching film, filtrate are used for 2- hydroxy phenylalanine and 3- hydroxy phenylalanine as sample liquid
Detection；Meanwhile the sample liquid after taking 100 μ L filtrates with 0.1% aqueous formic acid to be settled to 1mL and must dilute 10 times is for tyrosine
It is detected with phenylalanine.
Two, QTRAP-UPLC-MS/MS is tested and analyzed
It is mark with tyrosine, phenylalanine and irradiation specific product 2- hydroxy phenylalanine and 3- hydroxy phenylalanine
Quasi- product, using irradiation mass spectrum response peak area as abscissa, to tyrosine, phenylalanine, 2- hydroxy phenylalanine and 3- hydroxyl
Base concentration of phenylalanine draws standard curve as ordinate, when drawing standard curve, the concentration gradient setting of standard items are as follows: 5
μ g/L, 10 μ g/L, 50 μ g/L, 100 μ g/L, 500 μ g/L contain junket according to corresponding prepare of concentration gradient setting of standard items simultaneously
Propylhomoserin, phenylalanine, 2- hydroxy phenylalanine and 3- hydroxy phenylalanine mixed standard solution.
With MRM-IDA-EPI mode, library is composed into the second order spectrum deposit of four kinds of standard substances, so that sample compares therewith；
By the upper machine testing of the sample liquid after 10 times of sample liquid and dilution, by the correspondence ion pair and standard drawing of test substance
Spectrum is matched, and using Purity value as judgment basis, is considered same substance greater than 70%, and quantify to it；
Sample to be tested hydroxy phenylalanine containing 2- and the representative of 3- hydroxy phenylalanine received radiation treatment, and according to standard
Curve calculates the content of contained 2- hydroxy phenylalanine and 3- hydroxy phenylalanine.
Chromatographic parameter setting are as follows:
Chromatographic separation condition: Waters Acquity UPLC BEH C18Column (100mm*2.1mm, 1.7 μm), flow velocity
0.4mL/min；Mobile phase A: methanol, Mobile phase B: 0.1% aqueous formic acid；Sample volume: 2 μ L；Column temperature: 35 DEG C.
The gradient elution program of mobile phase are as follows: 0-5min, Mobile phase B ratio are adjusted to 30% from 5%, later 5.1min-
Mobile phase B ratio is adjusted to 5% from 30% in 13-13.5min by 8min.
Mass Spectrometry Conditions are as follows: ion source Turbo V ionizes mode ESI+, acquisition mode MRM mode, ionization temperature (TEM)
550℃；Spray voltage 5500V, atomization gas (GS1) 55psi assist gas (GS2) 60psi, gas curtain gas (CUR) 30psi, collision gas
It (CAD) is High；Entrance potential (EP) 10v, collision cell exit potential (CXP) 13v.The specific acquisition parameter of each compound is shown in Table
2。
The Mass Spectrometry Conditions parameter that table 2 optimizes
Using MRM-IDA-EPI mode monitoring.MRM triggers enhancer ion scan IDA-EPI condition: IDA trigger signal
Intensity threshold 5000；EPI condition: sweep speed (Scan rate) 10000Da/s；Scanning range: 50-200Da；Souce/Gas
The same MRM of condition；DP voltage 80v；Collision energy (CE) 35v, collision energy spectrum width (CES) are 15.
Three, irradiation dose quantitative detection
Using irradiation dose as abscissa, 3- hydroxy phenylalanine and 2- hydroxy phenylalanine content are that ordinate draws standard
Curve, the method for drawing standard curve specifically: take not irradiated control sample, carried out respectively with X-ray irradiation instrument 0kGy,
The irradiation of 1kGy, 2kGy, 5kGy, 10kGy, 20kGy dosage after being handled according to step 1 sample-pretreating method, is used
Machine detects 3- hydroxy phenylalanine and 2- hydroxyl in the control sample after various dose irradiates respectively in MRM-IDA-EPI mode
The content of base phenylalanine and secondary qualitative, by irradiation dose as abscissa, 3- hydroxy phenylalanine and 2- hydroxy benzenes third
Histidine content finds out regression equation and related coefficient respectively and draws standard curve as ordinate.By 3- hydroxyl in sample to be tested
The content of base phenylalanine and 2- hydroxy phenylalanine substitutes into standard curve to determine irradiation dose that sample to be tested received.
Actual sample detection:
Whether shredded squid (dried product) is by irradiation and the quantitative detection of irradiation dose.
1, sample treatment: mixing well shredded squid and blend, be packed into container in, seal, refrigerate it is spare；
2, acidolysis: weighing sample 0.2g (being accurate to 0.01g), and in 20mL acidolysis glass tube, 10mL 6moL/L salt is added
Acid covers tightly spiral cover after inflated with nitrogen, and acidolysis is stayed overnight at 110 DEG C.
3, it purifies: after taking acid hydrolysis solution to be filtered with filter paper, being placed in glass centrifuge tube, at a temperature of 4 DEG C, 12000r/min centrifugation
10min。
4, supernatant 5mL is taken, is dried with nitrogen at 35 DEG C, then freeze dryer is lyophilized, water-soluble with 1mL 0.1% (v/v) formic acid
Liquid dissolves lyophilized products, is filtered with 0.22 μm of water phase nylon leaching film.100 μ L filtrates are taken to be settled to 0.1% (v/v) aqueous formic acid
1mL dilutes 10 times and detects for tyrosine and phenylalanine, and residual filtrate is (undiluted) to be used as sample liquid to be used for 2- hydroxy benzenes
The detection of alanine and 3- hydroxy phenylalanine.The upper machine of the sample of two concentration is to be measured.
5, it by step 4 treated sample liquid QTRAP-UPLC-MS/MS condition analysis, is supervised using MRM-IDA-EPI mode
It surveys.
Chromatographic separation condition: Waters Acquity UPLC BEH C18 column (100mm*2.1mm, 1.7 μm), flow velocity
0.4mL/min；Mobile phase A: methanol, Mobile phase B: 0.1% (v/v) aqueous formic acid；Sample volume: 2 μ L；Column temperature: 35 DEG C.
The gradient elution program of liquid phase mobile phase are as follows: 0-5min, Mobile phase B ratio are adjusted to 30%, Zhi Houcong from 5%
Mobile phase B ratio is adjusted to 5% from 30% in 13-13.5min between 5.1min-8min, chromatographic column is rinsed, is terminated
Entire testing process.
Using Mass Spectrometry Conditions: ion source Turbo V ionizes mode ESI+, acquisition mode MRM mode, ionization temperature
(TEM)550℃；Spray voltage 5500V, atomization gas (GS1) 55psi assist gas (GS2) 60psi, gas curtain gas (CUR) 30psi,
Collision gas (CAD) is High；Entrance potential (EP) 10v, collision cell exit potential (CXP) 13v.The specific acquisition of each compound is joined
Number is shown in table 2 above.
Using MRM-IDA-EPI mode monitoring.MRM triggers enhancer ion scan IDA-EPI condition:
A.IDA trigger signal intensity threshold 5000；
B.EPI condition: sweep speed (Scan rate) 10000Da/s；Scanning range: 50-200Da；Souce/Gas item
The same MRM of part；DP voltage 80v；Collision energy (CE) 35v, collision energy spectrum width (CES) are 15.
6, quality control experiment.It takes respectively without drying shredded squid sample irradiated and after 7kGy is irradiated, and it is to be measured
Sample carries out pre-treatment together, measures according to QTRAP-UPLC-MS/MS condition in step 5, and according to as a result, the entire inspection of judgement
Experiment accuracy during survey, avoids the testing results such as false positive or false negative.
7, will irradiation mass spectrum response peak area as abscissa, to tyrosine, phenylalanine, 2- hydroxy phenylalanine and
3- hydroxy phenylalanine concentration draws standard curve as ordinate.Standard curve prepares same embodiment.In 10-500 μ g/L
Being respectively obtained in range to the equation of linear regression of tyrosine (p-tyrosine) is Y=1.10986e6x+5.2e3, r=
0.9987；Adjacent tyrosine (o-tyrosine, 2- hydroxy phenylalanine) Y=7.95756e6x+7.6e3, r=0.9995；With junket
Propylhomoserin (m-tyrosine, 3- hydroxy phenylalanine) Y=1.10049e6x+3.2e3；The line of phenylalanine (Phenylalanine)
Property regression equation be Y=7.77349e6x+2.5e4, r=0.9990；X indicates that corresponding compound concentration, Y indicate mass spectrum in formula
Response peak area.Mass spectrogram and the EPI figure of standard solution are shown in Fig. 1 and Fig. 2 respectively, and second order ms figure is stored in spectrum library.
8, whether the product passes through the judgement of irradiation: in the shredded squid sample and for finding spoke in the irradiation sample of Quality Control
According to specific product m-Tyrosine (m-tyrosine, 3- hydroxy phenylalanine) and adjacent tyrosine (o-tyrosine, 2- hydroxy benzenes
Alanine), shredded squid sample experiments result is shown in Fig. 3, and EPI second order ms figure result and spectrum library are compared.Similarity degree master
Same substance is regarded as greater than 70% with Purity for main judgment basis.M-Tyrosine (m- in shredded squid sample
Tyrosine) scheme and corresponding standard diagram similarity degree Purity in spectrum library with the EPI of adjacent tyrosine (o-tyrosine)
Value is respectively 93.253% and 92.825%.Therefore, it can determine that the shredded squid sample by radiation treatment.
9, the preparation of the standard curve of irradiation dose measurement.Not irradiated shredded squid sample is taken, is distinguished with X-ray irradiation instrument
Carry out the irradiation of 0kGy, 1kGy, 2kGy, 5kGy, 10kGy, 20kGy dosage.Sample pre-treatments are the same as step 1-4, QTRAP-UPLC-
It is analyzed under the conditions of MS/MS, using MRM-IDA-EPI mode monitoring with step 5.Upper machine testing and with the standard curve in step 7
Calculate the m-Tyrosine (m-tyrosine) and adjacent tyrosine (o- after various dose irradiates in shredded squid sample
Tyrosine content).According to the corresponding relationship of irradiation dose and content, irradiation agent is obtained in the range of 1-20kGy dosage
The polynomial fitting of amount and m-Tyrosine (m-tyrosine) content is Y=-0.0016+0.03112x-0.0002x2, r=
0.98；Irradiation dose and the equation of linear regression of adjacent tyrosine (o-tyrosine) content are Y=-0.0224+0.09718x-
0.0009x2, r=0.98；It is irradiation dose (kGy) by X, Y is respective compound content (mg/kg).
10, sample to be tested testing result.Shredded squid sample is measured according to the standard curve in step 7 containing to tyrosine
1.31g/kg, m-Tyrosine (m-tyrosine, 3- hydroxy phenylalanine) 0.022mg/kg and adjacent tyrosine (o-tyrosine,
2- hydroxy phenylalanine) 0.528mg/kg, phenylalanine (Phenylalanine) 1.704g/kg.According to m-Tyrosine (m-
Tyrosine) and the content of adjacent tyrosine (o-tyrosine), according to second standard curve in step 9, it may be determined that tested
Surveying the irradiation dose that shredded squid received is 6kGy.
11, the rate of recovery is tested: precision weighs sample 1.0g, is quantitatively adding standard items, and additive amount is made to be equivalent to 20,50,100
μ g/L m-Tyrosine (m-tyrosine) and adjacent tyrosine (o-tyrosine), measure recovery of standard addition.The rate of recovery=sample is real
Survey concentration/sample theoretical concentration × 100%.The result shows that average recovery rate is 81.3%~105.1%, relative standard deviation
(n=6) 8.45% hereinafter, meeting the requirement of quantitative analysis.Detection, which is calculated, with 3 times of signal-to-noise ratio (S/N) is limited to 3 μ g/L, junket
Propylhomoserin (m-tyrosine) and adjacent tyrosine (o-tyrosine) concentration are up to 5.0 μ g/L, 20 μ L loadings, with 10 times of signal-to-noise ratio (S/
N m-Tyrosine (m-tyrosine, 3- hydroxy phenylalanine) and adjacent tyrosine (the third ammonia of o-tyrosine, 2- hydroxy benzenes) are calculated
Acid) lower limit of quantitation be 10 μ g/kg, be able to satisfy the detection needs of food safety.
Above-mentioned the results show, this method is linear good within the scope of 5-500 μ g/L, related coefficient 0.99 or more,
Detection is limited to 3 μ g/L, is quantitatively limited to 10 μ g/L, the rate of recovery of matrix mark-on is 81.3% or more.Use QTRAP-UPLC-MS/MS
The irradiation specific product in aquatic products dried product is measured, there is the preferable rate of recovery and extraction efficiency.
Above-mentioned embodiment is only a preferred solution of the present invention, not the present invention is made in any form
Limitation, there are also other variations and modifications on the premise of not exceeding the technical scheme recorded in the claims.
Claims (3)
1. a kind of detection method for irradiating aquatic products dried product, which is characterized in that steps are as follows:
One, sample pre-treatments
(1) sample treatment: after sample to be tested is crushed be packed into container in, seal, refrigerate it is spare；
(2) acidolysis: weighing sample 0.2g and be added in 20mL acidolysis glass tube, and 10mL 6moL/L hydrochloric acid is added, covers tightly after inflated with nitrogen
Spiral cover, acid hydrolysis solution is stayed overnight to obtain in acidolysis at 110 DEG C；
(3) it purifies: after taking acid hydrolysis solution to be filtered with filter paper, being placed in glass centrifuge tube, 12000r/min is centrifuged 10min at 4 DEG C, takes
Supernatant 5mL is dried with nitrogen at 35 DEG C, and then freeze dryer is lyophilized, and dissolves lyophilized products, 0.22 μ with 0.1% aqueous formic acid of 1mL
The filtering of m water phase nylon leaching film, filtrate are used for the detection of 2- hydroxy phenylalanine and 3- hydroxy phenylalanine as sample liquid；Together
When, the sample liquid after taking 100 μ L filtrates with 0.1% aqueous formic acid to be settled to 1mL and must dilute 10 times is for tyrosine and phenylpropyl alcohol
Propylhomoserin detection；
Two, QTRAP-UPLC-MS/MS is tested and analyzed
Using tyrosine, phenylalanine and irradiation specific product 2- hydroxy phenylalanine and 3- hydroxy phenylalanine as standard
Product, using irradiation mass spectrum response peak area as abscissa, to tyrosine, phenylalanine, 2- hydroxy phenylalanine and 3- hydroxyl
Concentration of phenylalanine draws standard curve as ordinate；With MRM-IDA-EPI mode, by the second level figure of four kinds of standard substances
Spectrum deposit spectrum library, so that sample compares therewith；
By the upper machine testing of sample liquid after 10 times of sample liquid and dilution, by the correspondence ion pair of test substance and standard diagram into
Row matching, using Purity value as judgment basis, is considered same substance greater than 70%, and quantify to it；
Sample to be tested hydroxy phenylalanine containing 2- and the representative of 3- hydroxy phenylalanine received radiation treatment, and according to standard curve
Calculate the content of contained 2- hydroxy phenylalanine and 3- hydroxy phenylalanine；
Three, irradiation dose quantitative detection
Using irradiation dose as abscissa, 3- hydroxy phenylalanine and 2- hydroxy phenylalanine content are that ordinate draws standard song
The content of 3- hydroxy phenylalanine and 2- hydroxy phenylalanine in sample to be tested is substituted into standard curve to determine sample to be tested by line
The irradiation dose received；
Four, quality controls
Measurement is all made of blank and positive control sample every time；Blank control is negative sample, i.e., the same type water not being irradiated
For product as blank control, positive sample is the measuring samples same type aquatic products after the irradiation of known irradiation dose；Wherein
The chromatographic parameter setting that QTRAP-UPLC-MS/MS is tested and analyzed are as follows: chromatographic separation condition: Waters Acquity UPLC
BEH C18 column, flow velocity 0.4mL/min；Mobile phase A: methanol, Mobile phase B: 0.1% aqueous formic acid；Sample volume: 2 μ L；Column temperature:
35℃；The gradient elution program of mobile phase are as follows: 0-5min, Mobile phase B ratio are adjusted to 30% from 5%, later 5.1min-
Mobile phase B ratio is adjusted to 5% from 30% in 13-13.5min by 8min；
The Mass Spectrometry Conditions that QTRAP-UPLC-MS/MS is tested and analyzed are as follows: ion source Turbo V ionizes mode ESI+, acquisition mode
MRM mode, 550 DEG C of ionization temperature；Spray voltage 5500V, atomization gas 55psi assist gas 60psi, gas curtain gas 30psi to touch
Hitting gas is High；Entrance potential 10v, collision cell exit potential 13v；
MRM-IDA-EPI mode parameter setting are as follows: IDA trigger signal intensity threshold 5000；EPI condition: sweep speed
10000Da/s；Scanning range: 50-200Da；The same MRM of Souce/Gas condition；DP voltage 80v；Collision energy 35v, collision energy
Spectrum width is 15.
2. detection method according to claim 1, which is characterized in that in step 2, when drawing standard curve, standard items
Concentration gradient setting are as follows: 5 μ g/L, 10 μ g/L, 50 μ g/L, 100 μ g/L, 500 μ g/L, according to the concentration gradient setting pair of standard items
Should prepare simultaneously containing tyrosine, phenylalanine, 2- hydroxy phenylalanine and 3- hydroxy phenylalanine mixed standard solution.
3. detection method according to claim 1 or 2, which is characterized in that in step 3, draw the method tool of standard curve
Body are as follows: take not irradiated control sample, carry out 0kGy, 1kGy, 2kGy, 5kGy, 10kGy, 20kGy respectively with X-ray irradiation instrument
The irradiation of dosage after being handled according to step 1 sample-pretreating method, detects warp with machine in MRM-IDA-EPI mode respectively
3- hydroxy phenylalanine in control sample and the content of 2- hydroxy phenylalanine after various dose irradiation and secondary qualitative,
By irradiation dose as abscissa, 3- hydroxy phenylalanine and 2- hydroxy phenylalanine content are found out back respectively as ordinate
Return equation and related coefficient and draws standard curve.
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