CN103163239A - Method of rapid detection of histamine in aquatic products - Google Patents
Method of rapid detection of histamine in aquatic products Download PDFInfo
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- CN103163239A CN103163239A CN2013100596833A CN201310059683A CN103163239A CN 103163239 A CN103163239 A CN 103163239A CN 2013100596833 A CN2013100596833 A CN 2013100596833A CN 201310059683 A CN201310059683 A CN 201310059683A CN 103163239 A CN103163239 A CN 103163239A
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Abstract
The invention discloses a method of rapid detection of histamine in aquatic products. The method of rapid detection of the histamine in the aquatic products is characterized by comprising enabling full homogenate by a tissue pounding machine to be carried out on a fish sample under the condition of ice bath, adding the fish sample into a reagent bottle with a plug, adding distilled water and sodium chloride, and adjusting potential of hydrogen (pH) value to be in a range of 8.0-10.0, wherein the weight of the distilled water and the weight of the sodium chloride are respectively five times of the weight of fishes; adding a magnetic stirring adsorption extraction rod into the reagent bottle, and carrying out extraction in an electromagnetic stirrer for 0.5h-3h at the temperature of 45DEG C-60DEG C; washing the magnetic stirring adsorption extraction rod clean by using the distilled water, absorbing water, and directly carrying out quantitative analysis on the histamine in the aquatic products by using online heat analysis sample handing and a gas chromatography (GC) separation detection technology; and finally, calculating to obtain the content of the histamine in the fish sample according to a standard curve of a quantitative relationship between the content of the histamine and a peak area of the histamine. The method of rapid detection of the histamine in the aquatic products has the advantages of being simple and rapid, free from a complex pre-processing process and a cumbersome derivatization process.
Description
Technical field
The present invention relates to a kind of detection method of histamine, especially relate to the method for quick of histamine in a kind of aquatic products.
Background technology
Histamine is a kind of bioactive low molecule organic compounds containing nitrogen that has, and is prevalent in varieties of food items such as fermented product, dairy products, drinks, aquatic products and goods thereof, and especially aquatic products are the most outstanding.Histamine is as a kind of chemical transmitter matter in human body, by with cell membrane on two receptoroids (H1 and H2) effect bring into play toxicity, as eat excessive histamine, the lighter causes allergic reaction, severe one is with crisis life.The pelagic fishes that some protein contents are higher such as tuna, mackerel, saury etc. if after death rationally do not control at it, very easily produce histamine, and the histamine poisoning event of report mostly occurs in this type of fish both at home and abroad.Simultaneously, easily produce the pelagic fishes of histamine as the product of raw material take chub mackerel Scad, mackerel etc. in aquatic products, the event of the outlet that is under an embargo because histamine content exceeds standard happens occasionally in recent years, brings very large impact for the product water outlet trade.Therefore, for guaranteeing the aquatic food edible safety, promote the aquatic products foreign trade, need quick, accurate, a reliable histamine detection method badly.
At present, in aquatic products, the detection method of histamine mainly comprises HPLC method, capillary electrophoresis, colourimetry, biochemical sensor method, euzymelinked immunosorbent assay (ELISA), thin-layered chromatography etc.These methods generally all need to carry out acid extraction, and the pretreatment process that organic solvent extraction etc. are more loaded down with trivial details and derivatization process make these analytical approachs complicated, time-consuming; Although thin-layer chromatography does not need expensive device, have advantages of simple to operate, quick, amount of analysis is large and with low cost, this method is semi-quantitative detection method, can only be used for monitoring and the screening in early stage of histamine.
Summary of the invention
Technical matters to be solved by this invention is to provide a kind of complicated pretreatment process and loaded down with trivial details derivatization process of not needing, and can carry out the method for quick of histamine in the aquatic products of Simple fast of qualitative and quantitative analysis.
The present invention solves the problems of the technologies described above the technical scheme that adopts: the method for quick of histamine in a kind of aquatic products specifically comprises the following steps:
(1) will oppress sample under ice bath with after the abundant homogenate of tissue mashing machine, to oppress sample joins in band plug headspace sample bottle, then add the distilled water of 5 times of flesh of fish weight, be 8.0-10.0 with NaOH solution adjusting pH value, and add solid sodium chloride to make its content reach 30wt%;
(2) magnetic agitation sorption extraction rod is added in the sample bottle of step (1), cover bottle cap, in 30-45 ℃ of extraction 0.5-3h, wherein electromagnetic agitation speed is 1000-1500r/Min on magnetic stirrer;
(3) extraction is complete, and magnetic agitation sorption extraction rod is clean with distilled water flushing, and is after sucking moisture on magnetic agitation sorption extraction rod with dustless thieving paper, stand-by;
(4) compartment analysis of histamine
Analytical conditions for gas chromatography:
Resolve the gas chromatograph Agilent6890 gas chromatograph of sampling system with direct heat;
Chromatographic column: possess temperature range and be the bonded stationary phase capillary column more than 280 ℃;
Sampling system one-level pyrolysis eutectoid temperature is analysed in pyrolysis: 280 ℃, time 5min captures temperature-20--30 ℃, 280 ℃ of secondary pyrolysis eutectoid temperatures, time 3-5min, 300 ℃ of hydrogen flameionization (FID) detector temperatures;
Chromatographic column column temperature: 150 ℃, keep 2min; Then the speed with 15 ℃/min is warming up to 230 ℃, keeps 1min, then is warming up to 290 ℃ with the speed of 50 ℃/min, keeps 5min;
(5) typical curve is set up
Adopt internal standard method, the histamine standard items and the interior mark compound 1 that add variable concentrations in sample, the 9-nonanediol, under the analytical conditions for gas chromatography of step (4), measure the histamine peak area corresponding to histamine standard items of a series of variable concentrations, and according to after interior mark compound calibration peak area, determine the quantitative relationship of content and the histamine peak area of histamine, the Criterion curve;
(6) sample determination
Obtain the histamine peak area of testing sample according to above-mentioned steps (1)-(4), the quantitative relationship according to content and the histamine peak area of histamine calculates the histamine content in testing sample.
Described magnetic agitation sorption extraction rod surface is provided with olefin(e) acid ester coating (PA:Polyacrylate) or ethylene glycol silicone (EG-Silicone: Ethylene Glycol (EG)-Silicone) coating.Because these two coatings are to have certain polarity, be more suitable for the extraction of this polar compound of histamine.
Described chromatographic column adopting OV-1701, its length * internal diameter * film thickness=30m * 0.53mm * mm * 1.5mm.
Compared with prior art, the invention has the advantages that: the invention discloses the method for quick of histamine in a kind of aquatic products, adopt solvent-free magnetic stick sorption extraction technology to analyse sample introduction in conjunction with online pyrolysis and separate detection technique with GC and directly the histamine in aquatic products is carried out quantitative test.due to the boiling point of histamine up to 209.5 ℃, generally can not directly carry out GC analyzes, need to carry out separation and purification, derivatization just can be analyzed again, this method utilizes the characteristics of magnetic stick sorption extraction technology to realize the direct extraction of the histamine in complicated aquatic products, and by using resistant to elevated temperatures Bonded Phase capillary gas chromatographic column can realize the separation of higher-boiling compound, can be in the situation that not by the histamine in complicated pretreatment process and loaded down with trivial details derivatization process Direct Analysis sample, detection limit reaches 50 μ g/kg, it is a kind of Simple fast, solvent-free histamine extraction easy and simple to handle detects with separating without the derivatization histamine histamine method for qualitative and quantitative detection that combines.
Description of drawings
Fig. 1 is the content of histamine in the mackerel sample and the quantitative relationship canonical plotting of histamine peak area.
Embodiment
Embodiment is described in further detail the present invention below in conjunction with accompanying drawing.
Specific embodiment 1
The method for quick of histamine in a kind of aquatic products of the present invention specifically comprises the following steps:
(1) will oppress sample under ice bath with after the abundant homogenate of tissue mashing machine, to oppress sample joins in band plug headspace sampling bottle, then the distilled water that adds 5 times of flesh of fish weight, regulating pH value with NaOH solution is 8.0-10.0, and to add the NaCl solid to make its content reach the massfraction of 30wt%(NaCl in oppressing sample solution be 30%);
(2) be that the Twister magnetic agitation sorption extraction rod of 1cm adds in the sample bottle of step (1) with length, cover bottle cap, in 30-45 ℃ of extraction 0.5-3h, electromagnetic agitation speed is 1000-1500r/Min on magnetic stirrer;
(3) extraction is complete, and magnetic agitation sorption extraction rod is clean with distilled water flushing, sucks moisture on magnetic agitation sorption extraction rod with dustless thieving paper, and magnetic agitation sorption extraction rod is put into memory ready;
(4) compartment analysis of histamine
Analytical conditions for gas chromatography:
Resolve the gas chromatograph Agilent6890 gas chromatograph of sampling system with direct heat
Chromatographic column: OV-1701(length * internal diameter * film thickness=30m * 0.53mm * mm * 1.5mm), or other high-temperature stability chromatographic columns of the same type.Non-polar column, low-pole column, Semi-polarity post, temperature limit are that the bonded stationary phase capillary column more than 280 ℃ all can use, and wherein Semi-polarity post effect is best.
Pyrolysis analysis system one-level pyrolysis eutectoid temperature: 280 ℃, 5min captures temperature-20--30 ℃, 280 ℃ of secondary pyrolysis eutectoid temperatures, 3-5min, 300 ℃ of hydrogen flameionization (FID) detector temperatures; Column temperature: 150 ℃, 2min; Be warming up to 230 ℃ with 15 ℃/min, keep 1min, then be warming up to 290 ℃ with 50 ℃/min;
(5) histamine is qualitative, carry with sample analysis the same terms under carry out the analysis of histamine standard items, the chromatographic peak of comparative sample and standard items carries out qualitative, and confirms by GC-MS;
(6) qualitative, quantitative of histamine: adopt internal standard method, the histamine standard items and the interior mark compound 1 that add variable concentrations in sample, the 9-nonanediol, under the same conditions, measure interpolation, do not add the middle histamine peak area of standardized sample, and according to after interior mark compound calibration peak area, make the content of histamine and the typical curve of peak area, according to the content of typical curve calculating with histamine in testing sample under condition.
Specific embodiment 2
In a kind of aquatic products of the present invention, the method for quick of histamine is with embodiment 1, and its difference is:
In step (1), flesh of fish sample is got commercially available mackerel sample, and regulating the pH value with NaOH solution is 9.0;
In step (2) on magnetic stirrer in 38 ℃ the extraction 1.5h, electromagnetic agitation speed is 1250r/Min;
The compartment analysis of histamine in step (4):
The GC conditions analysis condition:
Pyrolysis analysis system one-level pyrolysis eutectoid temperature: 280 ℃, 5min captures temperature-25 ℃, 280 ℃ of secondary pyrolysis eutectoid temperatures, 4min, 300 ℃ of hydrogen flameionization (FID) detector temperatures; Column temperature: 150 ℃, 2min; Be warming up to 230 ℃ with 15 ℃/min, keep 1min, then be warming up to 290 ℃ with 50 ℃/min,
The content of the histamine that the method by embodiment 2 obtains and the typical curve of peak area, as shown in Figure 1.
Specific embodiment 3
The mensuration of detection limit
Method according to above-described embodiment 1, do not add sample in step 1, but add the histamine standard solution of dilution gradually, and analyze according to 2-6 step in above-described embodiment 1 successively, setting the fid detector scope is 1, decays to 1, signal to noise ratio (S/N ratio) is 3, until be diluted to the signal peak that can't detect histamine under this condition, the concentration that the corresponding histamine standard items of histamine signal peak can be detected is the detection limit of the method, and the detection limit of measuring according to the method is about 50 μ g/kg.
Specific embodiment 4
Recovery test
get commercially available mackerel sample 4.0g, after processing according to above-mentioned steps 1, add respectively 20, 41, 80, 119, 159 μ g histamine standard solutions are according to the method for above-described embodiment 2, the test set amine content, the mackerel histamine content that does not add standard items is 47.5 ± 2.3 μ g, the sample sets amine content that adds the histamine standard items is respectively 69.2 ± 2.0(coefficient of variation CV 2.4%) μ g, ± 3.1 90.4 (CV 3.4%) μ g, ± 11.2 126.3 (CV8.9%) μ g, ± 9.2 167.6 (CV 5.5%) μ g, ± 8.9 201.3 (CV 4.4%) μ g, the result recovery that obtains the method is respectively 108.5% accordingly, 104.6%, 97.3%, 100.9%, 96.7%.Illustrate that this detection method has the higher recovery.
Specific embodiment 5
The impact of test parameters salt content on analytical effect
Get commercially available mackerel sample 4.0g, according to above-described embodiment 2 methods, wherein step 1 adopts respectively that to add massfraction be 30% NaCl and do not add the NaCl dual mode, measures histamine content, and each experiment is carried out 5 times and repeated.Experimental result is that the sample sets amine content that does not add NaCl is 273 ± 33(CV 12.1%) μ g/kg, the testing result that adds NaCl is 352 ± 16(CV 4.5%) μ g/kg.The percentage extraction that can improve sample that adds of NaCl is described, significantly improves the accuracy of testing result.
Specific embodiment 6
The mensuration of histamine content in the aquatic products sample
Get chilled mackerel, will place 24h, commercially available poor mackerel, commercially available shrimp alive, shrimp room temperature placement 24h in this sample chamber warm air, get respectively above-mentioned 5 samples and analyze its histamine content according to the detection method of above-described embodiment 1, result is that the histamine content of placing the shrimp of the mackerel of 24h, commercially available poor mackerel, commercially available shrimp alive, room temperature placement 24h in chilled mackerel, air at room temperature is respectively: 31.3mg/kg, 152.5mg/kg, 253.2 mg/kg, 101 μ g/kg, 146 μ g/kg.
Certainly, above-mentioned explanation is not limitation of the present invention, and the present invention also is not limited to above-mentioned giving an example.Those skilled in the art are in essential scope of the present invention, and the variation of making, remodeling, interpolation or replacement also should belong to protection scope of the present invention.
Claims (3)
1. the method for quick of histamine in aquatic products is characterized in that specifically comprising the following steps:
(1) will oppress sample under ice bath with after the abundant homogenate of tissue mashing machine, to oppress sample joins in band plug headspace sample bottle, then add the distilled water of 5 times of flesh of fish weight, be 8.0-10.0 with NaOH solution adjusting pH value, and add solid sodium chloride to make its content reach 30wt%;
(2) magnetic agitation sorption extraction rod is added in the sample bottle of step (1), cover bottle cap, in 30-45 ℃ of extraction 0.5-3h, wherein electromagnetic agitation speed is 1000-1500r/Min on magnetic stirrer;
(3) extraction is complete, and magnetic agitation sorption extraction rod is clean with distilled water flushing, and is after sucking moisture on magnetic agitation sorption extraction rod with dustless thieving paper, stand-by;
(4) compartment analysis of histamine
Analytical conditions for gas chromatography:
Resolve the gas chromatograph Agilent6890 gas chromatograph of sampling system with direct heat;
Chromatographic column: possess temperature range and be the bonded stationary phase capillary column more than 280 ℃;
Sampling system one-level pyrolysis eutectoid temperature is analysed in pyrolysis: 280 ℃, time 5min captures temperature-20--30 ℃, 280 ℃ of secondary pyrolysis eutectoid temperatures, time 3-5min, 300 ℃ of hydrogen flameionization (FID) detector temperatures;
Chromatographic column column temperature: 150 ℃, keep 2min; Then the speed with 15 ℃/min is warming up to 230 ℃, keeps 1min, then is warming up to 290 ℃ with the speed of 50 ℃/min, keeps 5min;
(5) typical curve is set up
Adopt internal standard method, the histamine standard items and the interior mark compound 1 that add variable concentrations in sample, the 9-nonanediol, under the analytical conditions for gas chromatography of step (4), measure the histamine peak area corresponding to histamine standard items of a series of variable concentrations, and according to after interior mark compound calibration peak area, determine the quantitative relationship of content and the histamine peak area of histamine, the Criterion curve;
(6) sample determination
Obtain the histamine peak area of testing sample according to above-mentioned steps (1)-(4), the quantitative relationship according to content and the histamine peak area of histamine calculates the histamine content in testing sample.
2. the method for quick of histamine in a kind of aquatic products according to claim 1 is characterized in that: described magnetic agitation sorption extraction rod surface is provided with olefin(e) acid ester coating or ethylene glycol silicone coating.
3. the method for quick of histamine in a kind of aquatic products according to claim 1, is characterized in that: described chromatographic column adopting OV-1701, its length * internal diameter * film thickness=30m * 0.53mm * mm * 1.5mm.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106770716A (en) * | 2016-11-28 | 2017-05-31 | 无锡艾科瑞思产品设计与研究有限公司 | Histamine detection method and kit in a kind of animal tissue |
| CN107176614A (en) * | 2017-05-16 | 2017-09-19 | 中国天辰工程有限公司 | A kind for the treatment of fluid for washing sial phosphorus molecular sieve and the method for detecting amine component and content in the treatment fluid |
| CN107345911A (en) * | 2017-06-06 | 2017-11-14 | 浙江大学 | A kind of method of histamine in SERS qualitative and quantitative analysis rice fish tissue |
-
2013
- 2013-02-26 CN CN201310059683.3A patent/CN103163239B/en not_active Expired - Fee Related
Non-Patent Citations (5)
| Title |
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| BAO-SHYUNG HWANG等: "A rapid gas chromatographic method for the determination of histamine in fish and fish products", 《FOOD CHEMISTRY》 * |
| FRANCIS R.ANTOINE等: "Gas Chromatographic Analysis of Histamine in Mahi-mahi (Coryphaena hippurus)", 《J.AGRIC.FOOD CHEM.》 * |
| 张阳等: "超高效液相色谱-柱前衍生法同时测定水产品中8种生物胺", 《食品科学》 * |
| 戴煌等: "GC-MS结合模式识别评价鱼新鲜度的研究", 《食品安全质量检测学报》 * |
| 禹春鹤等: "搅拌棒吸附萃取研究进展", 《分析化学》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106770716A (en) * | 2016-11-28 | 2017-05-31 | 无锡艾科瑞思产品设计与研究有限公司 | Histamine detection method and kit in a kind of animal tissue |
| CN107176614A (en) * | 2017-05-16 | 2017-09-19 | 中国天辰工程有限公司 | A kind for the treatment of fluid for washing sial phosphorus molecular sieve and the method for detecting amine component and content in the treatment fluid |
| CN107345911A (en) * | 2017-06-06 | 2017-11-14 | 浙江大学 | A kind of method of histamine in SERS qualitative and quantitative analysis rice fish tissue |
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