CN103675118B - The detection method of a kind of aquatic products nitrite, nitrate and polyphosphate Simultaneously test - Google Patents
The detection method of a kind of aquatic products nitrite, nitrate and polyphosphate Simultaneously test Download PDFInfo
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- 229910002651 NO3 Inorganic materials 0.000 title claims abstract description 46
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 title claims abstract description 44
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 title claims abstract description 42
- 229920000388 Polyphosphate Polymers 0.000 title claims abstract description 37
- 239000001205 polyphosphate Substances 0.000 title claims abstract description 37
- 235000011176 polyphosphates Nutrition 0.000 title claims abstract description 37
- 238000001514 detection method Methods 0.000 title claims abstract description 27
- 238000012360 testing method Methods 0.000 title claims abstract description 19
- 150000002500 ions Chemical class 0.000 claims abstract description 27
- 238000002604 ultrasonography Methods 0.000 claims abstract description 13
- 239000000047 product Substances 0.000 claims description 49
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 29
- 238000000034 method Methods 0.000 claims description 23
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 22
- 235000019832 sodium triphosphate Nutrition 0.000 claims description 17
- UNXRWKVEANCORM-UHFFFAOYSA-I triphosphate(5-) Chemical compound [O-]P([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O UNXRWKVEANCORM-UHFFFAOYSA-I 0.000 claims description 17
- AZSFNUJOCKMOGB-UHFFFAOYSA-K cyclotriphosphate(3-) Chemical compound [O-]P1(=O)OP([O-])(=O)OP([O-])(=O)O1 AZSFNUJOCKMOGB-UHFFFAOYSA-K 0.000 claims description 16
- 235000011180 diphosphates Nutrition 0.000 claims description 16
- 238000002414 normal-phase solid-phase extraction Methods 0.000 claims description 13
- 238000002203 pretreatment Methods 0.000 claims description 13
- 239000000706 filtrate Substances 0.000 claims description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- 238000002137 ultrasound extraction Methods 0.000 claims description 9
- 230000006920 protein precipitation Effects 0.000 claims description 8
- 239000012498 ultrapure water Substances 0.000 claims description 8
- 238000001914 filtration Methods 0.000 claims description 7
- 239000000284 extract Substances 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 150000001450 anions Chemical class 0.000 claims description 5
- 238000004255 ion exchange chromatography Methods 0.000 claims description 5
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 5
- 239000004677 Nylon Substances 0.000 claims description 4
- 238000010828 elution Methods 0.000 claims description 4
- 239000011521 glass Substances 0.000 claims description 4
- 238000002386 leaching Methods 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 229920001778 nylon Polymers 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 4
- 230000001629 suppression Effects 0.000 claims description 4
- 230000003213 activating effect Effects 0.000 claims description 3
- 230000010355 oscillation Effects 0.000 claims description 3
- 230000000717 retained effect Effects 0.000 claims description 3
- 238000011068 loading method Methods 0.000 claims description 2
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 claims 4
- 238000004587 chromatography analysis Methods 0.000 abstract description 16
- 238000011084 recovery Methods 0.000 abstract description 15
- IOVCWXUNBOPUCH-UHFFFAOYSA-N Nitrous acid Chemical compound ON=O IOVCWXUNBOPUCH-UHFFFAOYSA-N 0.000 abstract description 7
- 230000008901 benefit Effects 0.000 abstract description 6
- 239000000463 material Substances 0.000 abstract description 6
- 239000007790 solid phase Substances 0.000 abstract description 6
- 235000013305 food Nutrition 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 238000012544 monitoring process Methods 0.000 abstract description 4
- 238000004458 analytical method Methods 0.000 abstract description 2
- 238000005868 electrolysis reaction Methods 0.000 abstract description 2
- 239000003344 environmental pollutant Substances 0.000 abstract description 2
- 231100000719 pollutant Toxicity 0.000 abstract description 2
- 239000000654 additive Substances 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 39
- XPPKVPWEQAFLFU-UHFFFAOYSA-N diphosphoric acid Chemical compound OP(O)(=O)OP(O)(O)=O XPPKVPWEQAFLFU-UHFFFAOYSA-N 0.000 description 14
- 229940048084 pyrophosphate Drugs 0.000 description 14
- 229910019142 PO4 Inorganic materials 0.000 description 9
- 239000010452 phosphate Substances 0.000 description 9
- 239000000243 solution Substances 0.000 description 8
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 241000251468 Actinopterygii Species 0.000 description 5
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 229910017604 nitric acid Inorganic materials 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- 235000011007 phosphoric acid Nutrition 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- YWEUIGNSBFLMFL-UHFFFAOYSA-N diphosphonate Chemical compound O=P(=O)OP(=O)=O YWEUIGNSBFLMFL-UHFFFAOYSA-N 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 125000004437 phosphorous atom Chemical group 0.000 description 2
- DLYUQMMRRRQYAE-UHFFFAOYSA-N phosphorus pentoxide Inorganic materials O1P(O2)(=O)OP3(=O)OP1(=O)OP2(=O)O3 DLYUQMMRRRQYAE-UHFFFAOYSA-N 0.000 description 2
- 231100000572 poisoning Toxicity 0.000 description 2
- 230000000607 poisoning effect Effects 0.000 description 2
- 238000003969 polarography Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 229940005657 pyrophosphoric acid Drugs 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000002798 spectrophotometry method Methods 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 235000010149 Brassica rapa subsp chinensis Nutrition 0.000 description 1
- 235000000536 Brassica rapa subsp pekinensis Nutrition 0.000 description 1
- 241000499436 Brassica rapa subsp. pekinensis Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 101710178133 Exotoxin type C Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 230000004308 accommodation Effects 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- -1 after filtration Substances 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 150000001449 anionic compounds Chemical class 0.000 description 1
- 230000002528 anti-freeze Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 239000012496 blank sample Substances 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000003185 calcium uptake Effects 0.000 description 1
- MWKXCSMICWVRGW-UHFFFAOYSA-N calcium;phosphane Chemical compound P.[Ca] MWKXCSMICWVRGW-UHFFFAOYSA-N 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
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- 238000009833 condensation Methods 0.000 description 1
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- 210000002249 digestive system Anatomy 0.000 description 1
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- 230000007613 environmental effect Effects 0.000 description 1
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- 235000021393 food security Nutrition 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 229910001412 inorganic anion Inorganic materials 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005360 mashing Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 150000001455 metallic ions Chemical class 0.000 description 1
- 125000005341 metaphosphate group Chemical group 0.000 description 1
- 208000005135 methemoglobinemia Diseases 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- XKLJHFLUAHKGGU-UHFFFAOYSA-N nitrous amide Chemical compound ON=N XKLJHFLUAHKGGU-UHFFFAOYSA-N 0.000 description 1
- 238000001225 nuclear magnetic resonance method Methods 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 230000007096 poisonous effect Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920000137 polyphosphoric acid Polymers 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
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- 238000003860 storage Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 description 1
- 229940048102 triphosphoric acid Drugs 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to the detection method of multiple additives and pollutant, particularly the detection method of a kind of aquatic products nitrite, nitrate and polyphosphate Simultaneously test.The present invention adopts quick ultrasound on extracting-Solid-Phase Extraction-chromatography of ions (UAE-SPE-IC) to detect nitrate in aquatic products simultaneously, nitrous acid and polyphosphate, by quick ultrasound on extracting, substantially increase nitrate, the recovery of nitrite and polyphosphate, and in conjunction with C18 pillar, effectively eliminate the interference of other materials, improve the sensitivity of actual detection, and the chromatography of ions (IC) has stronger separating power to the larger material of electrolysis coefficient especially negative ion, different valence state analysis can be carried out, the advantages such as detectability is low, the present invention is applicable to nitrate in aquatic products, the quantitative detection of nitrite and polyphosphate and food safety monitoring.
Description
Technical field
The present invention relates to the detection method of plurality of inorganic salt, particularly the method that simultaneously detects of aquatic products nitrite, nitrate and polyphosphate.
Background technology
Nitrate in marine product can be reduced into nitrite under the effect of bacterium and enzyme.Some storage times are longer, have putrid and deteriorated tendency and the higher marine product of protein content nitrous acid content contained by it is higher.Nitrite can make the oxygen carrying capability of blood decline, and causes methemoglobinemia.It is combined with amine can form nitrosamine, brings out the canceration of digestive system.About nitrite poisoning event also time have report.For controlling the intake of human body to nitric acid and nitrous acid, nitric acid and nitrous acid are listed in the important indicator in food hygienic standard, and China proposes clear and definite limit standard to nitric acid in food and nitrite.At present, in aquatic products, the assay method of NO3-N and NO2-N mainly contains the chromatography of ions, spectrophotometric method, oscilloscopic polarography etc., and spectrophotometric method, oscilloscopic polarography need substep to measure nitric acid and nitrous acid, and complex operation, sensitivity is low.The chromatography of ions can detect NO3-N and NO2-N simultaneously.
Phosphate can be divided into orthophosphate and condensed phosphate: orthophosphate is made a comment or criticism phosphoric acid (H
3pO
4) various salt.The condensation of orthophosphate thermal dehydration forms condensed phosphate, and its general formula is M
n+2p
no
3n+1, in formula, M is monovalent metallic ion, and n is phosphorus atoms number, and when n increases, phosphate-stabilized property is deteriorated.The various salt of pyrophosphoric acid are called pyrophosphate, M
4p
2o
7; The various salt of triphosphoric acid are called tripolyphosphate, M
5p
3o
10; The condensed phosphate that molecule contains the phosphorus atoms of more than 3 is referred to as polyphosphate, and metaphosphate molecular formula is (MPO
3) n.At present, in aquatic products processing, polyphosphate mainly refers to pyrophosphate, tripolyphosphate, trimetaphosphate.Polyphosphate is water-loss reducer and the antifreeze of a class excellent performance, by many aquatic product enterprises in a large number in the marine product particularly processing of freezing marine product and preservation process, be used for improving effective moisture holding capacity of marine product, and effectively extend the shelf life of marine product, reduce the drip loss after thawing.Meanwhile, there are some researches prove that the growth of microorganism in polyphosphoric acids salt pair aquatic products has inhibiting effect.In the last few years, have some illegal retailers to make use of polyphosphate and can strengthen aquatic products water absorptivity, suppress the features such as growth of microorganism, in commodity, people was water filling, adulterated, many to fill less.But human body is as taken in phosphate too much, then can reduce calcium uptake and cause human body calcium phosphorus unbalance, then diseases induced, therefore various countries have done clear and definite limitation requirement to content of polyphosphate in aquatic products.At present, domestic national standard and the industry standard also not detecting content of polyphosphate in aquatic products.Phosphatic polymer is converted into orthophosphate by the multiplex hydrogen peroxide of method of traditional detection polyphosphate and sulfuric acid, then detects by the chromatography of ions.But inherently there is in aquatic products the orthophosphate of certain content, therefore, the content of polyphosphate can not be measured in this way accurately.Also there are some researchers once to attempt different pre-treating methods, rear nuclear magnetic resonance method direct-detection polyphosphate is extracted to marine product, but due to apparatus expensive, effect are also not satisfactory.The people such as Yang Min adopt nitrate, nitrite, phosphate and total phosphorus in ion-chromatographic determination Chinese cabbage, total nitrogen.But can not carry out quantitatively different phosphate.Because biosome itself is containing certain phosphoric acid, therefore the method still can not Simultaneously test nitrate, nitrite and polyphosphate.The mensuration of the chromatography of ions to pollutant in aquatic products and adjuvant is current study hotspot, but not yet sets up the detection method that a kind of quick ultrasound assisted extraction-Solid-Phase Extraction-chromatography of ions (UAE-SPE-IC) detects nitrate, nitrite and polyphosphate in aquatic products simultaneously so far.Domestic have a kind of method detecting sour salted vegetables nitrite, nitrate disclosed in application number 201210014139.2, applying date 2012-01-18 in the relevant patent of invention of declaring, but sensitivity is not high during the actual detection of these methods, application difficulty is detected fast for the nitrate detected in aquatic products, nitrite and polyphosphate simultaneously larger.
Summary of the invention
The invention provides the detection method of a kind of aquatic products nitrite, nitrate and polyphosphate Simultaneously test, the method quantitative analysis results is reliable, detectability is low, be applicable to nitrate in Simultaneously test aquatic products, nitrite and polyphosphate (comprising orthophosphate, pyrophosphate, tripolyphosphate and trimetaphosphate) and detect fast.
The technical solution adopted for the present invention to solve the technical problems is:
A detection method for aquatic products nitrite, nitrate and polyphosphate Simultaneously test, the method comprises the steps:
A, sample pre-treatments: aquatic products sample to be measured is fully mixed and blends, load in container, seal, refrigerate for subsequent use;
B, fast ultrasound assisted extraction: the aquatic products sample accurately taken through pre-treatment is put in glass container, adds appropriate ultrapure water and makes homogeneous system, after capping, ultrasound wave extracts;
C, protein precipitation: the sample after being extracted by ultrasound wave is poured in centrifuge tube, and centrifugal filtration, gets filtrate and mix with trifluoroacetic acid solution, and the volume ratio of filtrate and trifluoroacetic acid solution is 20:3-4,0-4 DEG C and preserves;
D, Solid-Phase Extraction: by centrifugal for the sample after protein precipitation, adopt C18 post to carry out Solid-Phase Extraction to filtrate after filtration, crosses post liquid and be collected in 5mL automatic sampler sample bottle after nylon leaching film filters, measure for ion chromatograph;
E, ion chromatography;
The preparation of f, typical curve, get nitrate, nitrite, orthophosphate, pyrophosphate, tripolyphosphate and trimetaphosphate and be mixed with series concentration, obtain regression equation and related coefficient, according to typical curve to determine the content of nitrate, nitrite, orthophosphate, pyrophosphate, tripolyphosphate and trimetaphosphate in detected sample.
The present invention adopts quick ultrasound assisted extraction-Solid-Phase Extraction-chromatography of ions (UAE-SPE-IC) to detect nitrate in aquatic products simultaneously, nitrous acid and polyphosphate, by quick ultrasound assisted extraction, substantially increase nitrate, the recovery of nitrite and polyphosphate, and in conjunction with C18 pillar, effectively eliminate the interference of other materials, improve the sensitivity of actual detection, and the chromatography of ions (IC) has stronger separating power to the larger material of electrolysis coefficient especially negative ion, different valence state analysis can be carried out, the advantages such as detectability is low, the present invention is applicable to nitrate in aquatic products, the quantitative detection of nitrite and polyphosphate and food safety monitoring.
As preferably, experimental water used in pre-treatment is ultrapure water, resistivity 18.2M Ω cm.
As preferably, the condition of quick ultrasound assisted extraction is: sample is after homogeneous, and supersonic oscillations extract 30min, and every 5min jolting once, disperses completely to keep solid phase.
As preferably, the volumetric concentration of trifluoroacetic acid solution is 20%.In step c, precipitation agent adopts trifluoroacetic acid, and usually adopt the concentration of 20%, sedimentation effect is better, if testing sample protein content is less, can suitably reduce trifluoroacetic acid concentration.
As preferably, SPE Solid-Phase Extraction C
18before post loading, carry out activating rear 15mL high purity water with 5mL methyl alcohol and clean, flow velocity is less than 3mL/min, and when post crossed by sample, pre-treatment post keeps vertical.
As preferably, in step e, chromatographic condition is: chromatographic column: separating column: IonPac AS11-HC; Guard column IonPac AG11-HC; Leacheate: KOH solution, flow velocity 1.0mL/min; The automatic suppression mode of AMMS-ICE300 (4mm) rejector, suppresses electric current 200mA, and suppressed conductivity detector detects; Column temperature: 30.0 DEG C; Sample size: 30.0 μ L; External aqueous mode; Qualitative with retention time, peak area quantification.
As preferably, the leacheate Gradient program in step e is specially: in 0-10min, and concentration is 6mM/L, afterwards concentration be reduced to 2mM/L and stablize 1 minute, keep concentration 2mM/L in 11-20min, then in 1min, concentration adjusted and be stabilized in 25mM/L, keep 5min, between 26min-28min, concentration is continued to be increased to 60mM/L, and be retained to 31min, afterwards concentration is reduced to 25mM/L, chromatographic column is rinsed, and when 45min, terminate whole testing process.
As preferably, described step also comprises recovery experiment.
As preferably, the method concrete steps are as follows:
A, sample pre-treatments: aquatic products sample to be measured is fully mixed and blends, load in container, seal, refrigerate for subsequent use;
B, fast ultrasound assisted extraction: the aquatic products sample accurately taken through pre-treatment is put in glass container, adds 50mL ultrapure water and makes homogeneous system, after capping, ultrasound wave extracts 30min;
C, protein precipitation: the sample after being extracted by ultrasound wave is poured in centrifuge tube, the centrifugal 10min of 7000r/min filters, gets filtrate 20mL, adds the trifluoroacetic acid solution mixing that 3mL volumetric concentration is 20%, preserves 30 minutes for 0-4 DEG C;
D, Solid-Phase Extraction: by centrifugal for the sample 8000rpm after protein precipitation 10 minutes, by C18 post good for filtrate overactivation after filtration, remove and just cross post liquid 3mL, post liquid is crossed in access, be collected in 5mL automatic sampler sample bottle after 0.22 μm of nylon leaching film filters, measure for ISC-3000 ion chromatograph;
E, ion chromatography: adopt anion chromatographic column, external aqueous mode, KOH automatic generating apparatus, gradient elution, suppressive Conductivity detection;
The preparation of f, typical curve, get nitrate, nitrite, orthophosphate, pyrophosphate, tripolyphosphate and trimetaphosphate and be mixed with series concentration, obtain regression equation and related coefficient, according to typical curve to determine the content of nitrate, nitrite, orthophosphate, pyrophosphate, tripolyphosphate and trimetaphosphate in detected sample.
The present invention has the following advantages:
1, applied range.The present invention can strengthen aquatic products nitrite, the monitoring of nitrate and polyphosphate, and the monitoring be conducive in Distribution of Aquatic Products link reduces aquatic products nitrite and polyphosphate poisoning, ensures food safety.
2, present invention employs quick ultrasound assisted extraction (UAE) to extract, utilize its physicochemical property such as cavitation effect, enhanced dispersion to extract target product, compared with traditional extraction process, have fast, normal temperature, energy consumption are low, extraction ratio advantages of higher.Improve extraction efficiency, and economic environmental protection.
3, present invention employs the chromatography of ions to measure nitrous acid, nitrate and polyphosphate, this method is the prefered method measuring inorganic anion at present, and can be separated the ion of different valence state.Using water as mobile phase in this method, without the need to special reagent, deleterious effect is not produced to environment and personnel health.
4, present invention employs KOH and leacheate device occurs automatically, the leacheate of actual concentrations can be produced at short notice, be suitable for carrying out gradient, nitrate, nitrite, phosphate and three kinds of polyphosphates effectively can be separated, and remove the interference of Common Anions in aquatic products.
5, present invention employs quick ultrasound assisted extraction-Solid-Phase Extraction-chromatography of ions (UAE-SPE-IC) and detect nitrate, nitrite, phosphate, pyrophosphate, tripolyphosphate and trimetaphosphate in aquatic products, substantially increase the recovery, and in conjunction with C
18post, effectively eliminates the interference of other materials, improves the sensitivity of actual detection, and has the advantages such as stronger separating power, detectability be low by gradient elution, compared with traditional detection method, has better value.
6, instrument auto injection, is applicable to the detection of biological sample in enormous quantities.
7, The inventive method achieves in aquatic products and detect while multiple poisonous and harmful substance, effectively reduce testing cost.
Accompanying drawing explanation
Fig. 1 is the standard colors spectrogram of nitrite, nitrate, orthophosphate, pyrophosphate, tripolyphosphate and trimetaphosphate, wherein 1-nitrite, 2-nitrate, 3-orthophosphate, 4-pyrophosphate, 5-tripolyphosphate, 6-trimetaphosphate;
Fig. 2 is nitrite, nitrate, orthophosphate, pyrophosphate, tripolyphosphate and trimetaphosphate and Common Anions chromatogram, wherein 1-F-, 2-Cl-, 3-NO
2-, 4-Br
-, 5-NO
3-, 6-SO
4 2-, 7-PO
4 3-, 8-P
2o
7 4-, 9-P
3o
10 5-, 10-P
3o
9 3-;
Fig. 3 is embodiment 1 empty sample ions chromatogram, wherein 1-nitrite anions, 2-orthophosphoric acid root;
Fig. 4 is sample mark-on chromatography of ions figure, wherein 1-nitrite, 2-nitrate, 3-orthophosphate, 4-pyrophosphate, 5-tripolyphosphate, 6-trimetaphosphate in embodiment 1.
Embodiment
Below by specific embodiment, technical scheme of the present invention is described in further detail.Should be appreciated that enforcement of the present invention is not limited to the following examples, any pro forma accommodation make the present invention and/or change all will fall into scope.
In the present invention, if not refer in particular to, all parts, number percent are unit of weight, and all equipment and raw material etc. all can be buied from market or the industry is conventional.
The embodiment 1 Simultaneous Determination flesh of fish nitrite, nitrate and polyphosphate
1, sample pre-treatments:
Flesh of fish sample to be measured is fully mixed and blended, loads in container, sealing, refrigeration (-18 DEG C) are for subsequent use;
2, quick ultrasound assisted extraction (UAE):
Take the sample 5g(after tissue mashing and be accurate to 0.1g) in 150mL conical flask, add 50mL deionized water, ultrasonic 30min in homogeneous rearmounted ultrasonic oscillation device, every 5min jolting once, disperses completely to keep solid phase.Then with the centrifugal 10min of 7000r/min, filter, get filtrate 20mL, add the mixing of 3mL20% trifluoroacetic acid solution, put into 0-4 DEG C of refrigerator and preserve 30 minutes, then in the hydro-extractor of 8000rpm centrifugal 10 minutes.
3, Solid-Phase Extraction (SPE):
Get SPE Solid-Phase Extraction C
18post, first carry out activating rear 15mL high purity water with 5mL methyl alcohol and clean, flow velocity is less than 3mL/min.Get centrifugal in step 2 after sample, after filtration, filtrate is crossed the vertical SPE-C placed with the flow velocity being less than 3mL/min
18post, discards front 3mL efflux, gets 2mL efflux.Get 0.5mL deionized water and be settled to 5mL, in automatic sampler sample injection bottle, upper machine is to be measured.
4, ion chromatography:
Chromatography of ions condition: separating column: IonPac AS11-HC; Guard column IonPac AG11-HC; Leacheate: KOH solution gradient elution, flow velocity 1.0mL/min; Rejector: AMMS-ICE300 (4mm) rejector, the automatic suppression mode of automatic suppression mode, suppresses electric current 200mA; Detecting device: suppressed conductivity detector; Sample size: 30 μ L; Column temperature: 30 DEG C (sample is the aqueous solution that step 3 obtains); Qualitative with retention time, peak area quantification.
Leacheate Gradient program: KOH leacheate occurs automatically, external aqueous mode.In 0-10min, concentration is 6mM/L, afterwards concentration is reduced to 2mM/L and stablizes 1 minute, keeps concentration 2mM/L in 11-20min, in 1min, concentration adjusted again and be stabilized in 25mM/L, keep between 5min, 26min-28min, concentration being continued to be increased to 60mM/L, and be retained to 31min, afterwards concentration is reduced to 25mM/L, chromatographic column is rinsed, and when 45min, terminates whole testing process.Leacheate Gradient program is specifically in table 1.
Table 1 gradient elution program
5, the preparation of typical curve, configure the nitrate of a series of concentration, nitrite and polyphosphate mixed standard solution, production standard curve under optimum experimental condition, with target compound concentration for horizontal ordinate, standard items peak area response is ordinate, drawing standard curve.Measure according to 4. chromatography of ions condition, obtain equation of linear regression and the related coefficient (see table 2) of test substance.In formula, X represents Ion response value peak area, and Y represents corresponding material concentration.Standard solution and anion chromatographic figure are shown in Fig. 1 and Fig. 2.
The linear relationship of table 2 nitrate, nitrite and polyphosphate, detection limit, working curve equation
Note: polyphosphate is in phosphorus pentoxide
6, recovery experiment: precision takes flesh of fish sample 5.0g, quantitatively adds variable concentrations standard items, measures the recovery of standard addition of varying level.The recovery=sample measured concentration/sample theoretical concentration × 100%.Result shows, average recovery rate at 77.4% ~ 93.7%(in table 3), relative standard deviation (n=6), below 9.6%, meets the requirement of quantitative test.Blank sample chromatogram and mark-on sample chromatogram figure are shown in Fig. 3 and Fig. 4 respectively.Detection limit calculates (see table 2) with 3 times of signal to noise ratio (S/N ratio)s (S/N), can meet the detection needs of food security.
Table 3 difference adds the recovery (%) under scalar
Above-mentioned the results show, this method is for the detection of nitrate, nitrite and polyphosphate, and good in corresponding concentration range internal linear, related coefficient, all more than 0.99, adds target average recovery rate more than 77.4%.This method has the good recovery and extraction efficiency, and can detect many kinds of substance simultaneously, can reduce testing cost.
Embodiment 2 Simultaneous Determination dried product nitrite, nitrate and polyphosphate
Concrete scheme is with embodiment 1, and difference is: taking sample size is that 2.5g(is accurate to 0.1g).According to measurement result, average recovery of standard addition, more than 75.3%, meets testing requirement.
Embodiment 3 utilizes the chromatography of ions to monitor the content of aquatic products nitrite, nitrate and polyphosphate
Sample source is in aquatic product manufactory and circulation market stochastic buying, totally 10 lot samples, and instrument and reagent are as previously mentioned.
The preparation of sample-pretreating method, chromatography of ions parameter and typical curve is with embodiment 1.
Every batch sample all adopts same flesh of fish sample to control (only containing orthophosphate and nitrate) and sample interpolation recovery mensuration carries out quality control and guarantee.Precision takes flesh of fish sample 5.0g, adds 50mL deionized water, quantitatively adds standard items after homogeneous, sample pre-treatment simultaneously and upper machine after mixing.
Result shows: with negative Quality control testing result all only containing orthophosphate and nitrate, all the other several test substances are feminine gender, orthophosphoric acid, pyrophosphoric acid, tripolyphosphate, NO3-N and NO2-N recovery scope 83.5%-102.1%.The recovery of trimetaphosphate is between 74.1%-85.8%.Meet method requirement.The testing result of the 10 parts of all kinds of aquatic products deriving from aquatic product manufactory and circulation market stochastic buying is shown, all not containing nitrite in 10 increment product, wherein 3 increment product are only containing nitrate and orthophosphate, containing nitrate, orthophosphate, pyrophosphate, tripolyphosphate and trimetaphosphate in all the other 7 increment product, in one of them sample, the summation (in phosphorus pentoxide) of pyrophosphate, tripolyphosphate and trimetaphosphate content is more than 5g/kg.
More than show and describe ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and instructions just illustrates principle of the present invention; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications, and these improve and change all falls in the claimed scope of the invention.
Claims (3)
1. a detection method for aquatic products nitrite, nitrate and polyphosphate Simultaneously test, is characterized in that the method comprises the steps:
A, sample pre-treatments: aquatic products sample to be measured is fully mixed and blends, load in container, seal, refrigerate for subsequent use;
B, fast ultrasound assisted extraction: the aquatic products sample accurately taken through pre-treatment is put in glass container, adds appropriate ultrapure water and makes homogeneous system, after capping, ultrasound wave extracts; The condition of quick ultrasound assisted extraction is: sample is after homogeneous, and supersonic oscillations extract 30min, and every 5min jolting once;
C, protein precipitation: the sample after being extracted by ultrasound wave is poured in centrifuge tube, and centrifugal filtration, gets filtrate and mix with trifluoroacetic acid solution, and the volume ratio of filtrate and trifluoroacetic acid solution is 20:3-4,0-4 DEG C and preserves; The volumetric concentration of trifluoroacetic acid solution is 20%;
D, Solid-Phase Extraction: by centrifugal for the sample after protein precipitation, adopt C18 post to carry out Solid-Phase Extraction to filtrate after filtration, crosses post liquid and be collected in 5mL automatic sampler sample bottle after nylon leaching film filters, measure for ion chromatograph;
E, ion chromatography; Chromatographic condition is: chromatographic column: separating column: IonPac AS11-HC; Guard column IonPac AG11-HC; Leacheate: KOH solution, flow velocity 1.0 mL/min; The automatic suppression mode of AMMS-ICE 300 thickness 4mm rejector, suppresses electric current 200 mA, and suppressed conductivity detector detects; Column temperature: 30.0 DEG C; Sample size: 30.0 μ L; External aqueous mode;
Leacheate Gradient program is specially: in 0-10min, concentration is 6mM/L, afterwards concentration is reduced to 2mM/L and stablizes 1 minute, keeps concentration 2mM/L in 11-20min, in 1min, concentration adjusted again and be stabilized in 25mM/L, keep between 5min, 26min-28min, concentration being continued to be increased to 60 mM/L, and be retained to 31min, afterwards concentration is reduced to 25 mM/L, chromatographic column is rinsed, and when 45min, terminates whole testing process;
The preparation of f, typical curve, get nitrate, nitrite, orthophosphate, pyrophosphate, tripolyphosphate and trimetaphosphate and be mixed with series concentration, obtain regression equation and related coefficient, according to typical curve to determine the content of nitrate, nitrite, orthophosphate, pyrophosphate, tripolyphosphate and trimetaphosphate in detected sample.
2. detection method according to claim 1, is characterized in that: Solid-Phase Extraction C
18before post loading, carry out activating rear 15mL high purity water with 5mL methyl alcohol and clean, flow velocity is less than 3mL/min, and when post crossed by sample, pre-treatment post keeps vertical.
3. detection method according to claim 1, is characterized in that the method comprises the steps:
A, sample pre-treatments: aquatic products sample to be measured is fully mixed and blends, load in container, seal, refrigerate for subsequent use;
B, fast ultrasound assisted extraction: the aquatic products sample accurately taken through pre-treatment is put in glass container, adds 50mL ultrapure water and makes homogeneous system, after capping, ultrasound wave extracts 30min;
C, protein precipitation: the sample after being extracted by ultrasound wave is poured in centrifuge tube, centrifugal 10 min of 7000r/min filter, get filtrate 20mL, add the trifluoroacetic acid solution mixing of 3 mL volumetric concentrations 20%, preserve 30 minutes for 0-4 DEG C;
D, Solid-Phase Extraction: by centrifugal for the sample 8000rpm after protein precipitation 10 minutes, by C18 post good for filtrate overactivation after filtration, remove and just cross post liquid 3 mL, post liquid is crossed in access, be collected in 5mL automatic sampler sample bottle after 0.22 μm of nylon leaching film filters, measure for ISC-3000 ion chromatograph;
E, ion chromatography: adopt anion chromatographic column, external aqueous mode, KOH automatic generating apparatus, gradient elution, suppressive Conductivity detection;
The preparation of f, typical curve, get nitrate, nitrite, orthophosphate, pyrophosphate, tripolyphosphate and trimetaphosphate and be mixed with series concentration, obtain regression equation and related coefficient, according to typical curve to determine the content of nitrate, nitrite, orthophosphate, pyrophosphate, tripolyphosphate and trimetaphosphate in detected sample.
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