CN102650628B - Method for quickly detecting biogenic amine - Google Patents
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- CN102650628B CN102650628B CN201210156963.1A CN201210156963A CN102650628B CN 102650628 B CN102650628 B CN 102650628B CN 201210156963 A CN201210156963 A CN 201210156963A CN 102650628 B CN102650628 B CN 102650628B
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Abstract
The invention provides a method for quickly detecting biogenic amine, which comprises the following steps: performing derivatization on a biogenic amine standard sample and a to-be-tested sample by derivating agent and then providing a chromophoric group, applying the sample on a chromatography material polyamide thin film, unfolding by using a developer, stopping chromatography when the front edge of the developer is 0.5 to 1 cm far away from the top of a thin film, developing, and observing the developing results. According to the invention, the polyamide thin film is used as chromatography detecting material for biogenic amine, the material is easy to obtain, the unfolding is quick, an unfolded thin layer plate can be preserved for a long time for recheck, and the quantitative estimation can be performed by using a thin layer scanner; through utilizing the developer with appropriate proportion, a target object and other components can be separated completely. Therefore, the method provided by the invention can be used for quick determination and semiquantitative detection of biogenic amine, and by using the method, the results can be obtained in the whole-process of 1 hour, and the instrument analysis requires longer time.
Description
Technical field
The invention belongs to stratographic analysis field, be specially a kind of method of fast detecting biogenic amine.
Background technology
Biogenic amine is the nitrogenous aliphatics of a class or heterocyclic low molecular compound, finds more in the Food & Drink that contain albumen or free amino acid.It is extensively present among various vegeto-animal tissues, has important physiologically active.The biogenic amine of low concentration has obvious diastole or contraction to blood vessel and muscle, can suppress the outbreak of epilepsy, and cerebration and cerebral cortex are had to important regulating action, and heart is had to positive inotropic and Positive Chronotropic Action in various degree.But, when the excessive biogenic amine of absorption of human body, may cause headache, the allergic reactions such as disordered breathing, palpitaition blood pressure change.
The toxic action of biogenic amine can cause the safety problem of food.Secondly wherein healthy have the greatest impact of histamine to the mankind be tyrasamine.U.S. FDA is by the damaging effect level of determining histamine of the mass data of outburst histamine poisoning being: 500mg/kg (food).The requirement of limiting the quantity of has been done to histamine content in Partial Food by America and Europe and China: U.S. FDA requires import aquatic products histamine must not surpass 50mg/kg; In regulation mackerel section of European Union fish, histamine content must not surpass 100mg/kg; In other food, histamine must not surpass 1000mg/kg; Tyrasamine must not surpass 100～800mg/kg; In China regulation mackerel, histamine must not surpass 1000mg/kg; Other fishs must not surpass 300mg/kg.
Based on biogenic amine, in food, extensively exist and potential safety issue, biogenic amine is detected significant.Biogenic amine itself does not have the color development group of fluorescence and ultraviolet, therefore all will could under chromatographic apparatus, detect through derivatization.Dansyl Cl as liquid-phase chromatographic column before derivative reagent have that derivation operation is simple, derivant good stability, can quantitatively complete sulfonylation, have stronger fluorescence and uv absorption, highly sensitive, reaction range is wide, collective disturbs the advantages such as not obvious, so become biogenic amine high performance liquid chromatography detect in maximum derivative reagents.
At present, research comprises enzyme biologic sensor method, thin-layered chromatography, vapor-phase chromatography, high performance liquid chromatography, electrophoresis etc. to the detection method of biogenic amine both at home and abroad.Although the qualitative and quantitative analysis method of biogenic amine can the progress along with analytical instrument reach certain level at aspects such as accuracy, precision, instrument analytical method length consuming time, complicated operation, cost are high.Therefore study quick, easy, cheap analytical approach very necessary.This research provides the sxemiquantitative technology of biogenic amine aspect food quality monitoring, for differentiating its security, provides rapid analysis.
Summary of the invention
For the weak point of existing detection of biological amine technology, the object of the invention is to propose a kind of method of fast detecting biogenic amine.
The technical scheme that realizes the object of the invention is:
A kind of method of detection of biological amine, comprise step: by biogenic amine standard items and detected sample with being with upper chromophoric group after derivating agent derivatization, point sample on chromatographic material polyamide film, with developping agent, launch, at solvent front, stop chromatography during apart from film top 0.5～1cm, colour developing, observes colour developing result.
Wherein, described biogenic amine is a kind of in phenyl ethylamine, putrescine, histamine, tyrasamine, tryptamines, cadaverine, spermine, spermidine.
Wherein, the pH value of described biogenic amine standard items and detected sample is adjusted to 9.4～10.0.
Wherein, described derivating agent is dansyl Cl.
Wherein, the condition of described derivatization be biogenic amine standard items and detected sample respectively with equivalent derivating agent vibration after mixing, at 58～65 ℃, react 25～35min.
Wherein, described point sample is point sample 5～12 μ L on chromatographic material.
Wherein, described developping agent is the dimethylbenzene of volume ratio proportioning: glacial acetic acid=5～20: 1 potpourri, deployment step is to carry out in this developping agent.
Preferably, described developping agent is the dimethylbenzene of volume ratio proportioning: glacial acetic acid=10: 1 potpourri.
Wherein, described colour developing be biogenic amine standard items after chromatography and detected sample under 250～280nm ultraviolet ray is irradiated, there is yellow green spot.
Wherein, the observation of described colour developing result, comprise step: visual inspection under ultraviolet ray, if observe the yellowish green spot that testing sample is identical with standard items Rf value position between Rf value 0.5～0.7 simultaneously, think in testing sample and contain this kind of biogenic amine, then chromatography spot and standard items are relatively carried out to semi-quantitative analysis; If do not observe the yellow green spot that testing sample is identical with standard items Rf value position between Rf value 0.5～0.7, think in testing sample and do not contain this kind of biogenic amine.
Beneficial effect of the present invention is:
The present invention proposes to use polyamide film as the measurement in chromatography material of biogenic amine first, and this material is conveniently easy to get, and launches rapidly, and the thin layer plate after expansion can be preserved for a long time in order to check, and can apply thin-layer chromatogram scanner and carry out quantitative measurement; Utilize the developping agent of suitable proportioning, separate targets thing and other components completely, so this method can realize fast qualitative, half-quantitative detection to biogenic amine, the method overall process is about 1h can obtain result, and that instrumental analysis takes is longer.The biogenic amine that in food, wine, content is higher has phenyl ethylamine, putrescine, histamine, tyrasamine etc., by polyamide film chromatography, it is carried out to half-quantitative detection fast and easy.
Accompanying drawing explanation
Fig. 1 is the photo of colour developing result.In figure, the spot of light color is from left to right followed successively by the colour developing result that the derivative grape wine sample of the mixed mark of biogenic amine, DNS-Cl (dansyl Cl), 1/2 the derivative grape wine of DNS-Cl (dansyl Cl) mix 1/2 putrescine (100mg/L).
Embodiment
Now with following most preferred embodiment, the present invention is described, but is not used for limiting the scope of the invention.
Embodiment 1:
With 0.1M hydrochloric acid, be mixed with the putrescine solution that mass concentration is 5mg/L, 10mg/L, 15mg/L, 20mg/L, 25mg/L; get standard solution and each 1mL of grape wine sample to be detected; with 2M NaOH, regulate pH value to 9.4～10.0; add 1mL dansyl Cl derivating agent (5mg/L acetone), vibration mixes.Be placed in and at 60 ℃, react 30min (during vibrate 3～8 times).
With micro-sampling device point sample 10 μ L on the chromatographic material polyamide film at 10cm * 10cm, by having put, the polyamide film shiny surface that sample dries up is outside, polyamide is towards interior, with rubber band, tie, vertically put into airtight, saturated developping agent (dimethylbenzene: glacial acetic acid=10: expansion cylinder ascending development 1), the balance solvent in expansion cylinder is developping agent and mixes standing obtaining in airtight expansion cylinder is housed.At solvent front, stop chromatography during apart from film top 0.5～1cm, volatilize solvent, can realize target compound and separated from impurities, in 280nm ultraviolet, detect under lamp naked eyes after colour developing and directly observe.Development distance is about 4.5cm, observes the yellow-green fluorescence spot that testing sample is identical with standard items Rf value position between Rf value 0.5～0.7 simultaneously.With quantitative point in the putrescine standard items comparison of same thin laminate, can observe in this wine sample putrescine fluorescent brightness higher than 5mg/L lower than 15mg/L putrescine standard items (as shown in Figure 1), in preliminary this wine sample of judgement, putrescine content is close to 10mg/L, far below domestic and international limiting the quantity of to biogenic amine in food.
Embodiment 2:
With 0.1M hydrochloric acid, be mixed with the histamine solution that mass concentration is 5mg/L, 10mg/L, 15mg/L, 20mg/L, 25mg/L; get standard solution and each 1mL of beer sample to be detected; with 2M NaOH, regulate pH value to 9.4, add 1mL dansyl Cl derivating agent (5mg/L acetone), vibration mixes.Be placed in and at 65 ℃, react 25min (during vibrate 5 times).
With micro-sampling device point sample 5 μ L on the chromatographic material polyamide film at 10cm * 10cm, by having put, the polyamide film shiny surface that sample dries up is outside, polyamide is towards interior, with rubber band, tie, vertically put into airtight, saturated developping agent (dimethylbenzene: glacial acetic acid=10: expansion cylinder ascending development 1) is housed, at solvent front, stop chromatography during apart from film top 0.8cm, volatilize solvent, in 254nm ultraviolet, detect under lamp and develop the color.Development distance is about 4.5cm, observes the yellow green spot that testing sample is identical with standard items Rf value position between Rf value 0.5～0.7 simultaneously.In the putrescine standard items comparison of same thin laminate, can observe that in this wine sample, histamine spot intensity is lower than 5mg/L with quantitative point, sxemiquantitative judges in this wine sample that histamine content is close to 5mg/L.
Embodiment 3:
With 0.1M hydrochloric acid, be mixed with the tyrasamine solution that mass concentration is 5mg/L, 10mg/L, 15mg/L, 20mg/L, 25mg/L; get standard solution and each 1mL of Wine Sample to be detected; with 2M NaOH, regulate pH value to 10.0; add 1mL dansyl Cl derivating agent (5mg/L acetone), vibration mixes.Be placed in and at 58 ℃, react 35min (during vibrate 3 times).
With micro-sampling device point sample 12 μ L on the chromatographic material polyamide film at 10cm * 10cm, by having put, the polyamide film shiny surface that sample dries up is outside, polyamide is towards interior, with rubber band, tie, vertically put into airtight, saturated developping agent (dimethylbenzene: glacial acetic acid=10: expansion cylinder ascending development 1) is housed, at solvent front, stop chromatography during apart from film top 1cm, volatilize solvent, in 280nm ultraviolet, detect under lamp and develop the color.Development distance is about 4.5cm, observes the yellow-green fluorescence spot that testing sample is identical with standard items Rf value position between Rf value 0.5～0.7 simultaneously.In the tyrasamine standard items comparison of same thin laminate, can observe that in this wine sample, tyrasamine fluorescent brightness is lower than 5mg/L with quantitative point, sxemiquantitative judges in this wine sample that tyramine content is close to 5mg/L.
Embodiment 4:
Testing sample is beans sauce 10g, pulverizes and grinds, and is dissolved in 0.1M hydrochloric acid solution, is settled to 100ml.
With 0.1M hydrochloric acid, be mixed with the cadaverine solution that mass concentration is 5mg/L, 10mg/L, 15mg/L, 20mg/L, 25mg/L; get standard solution and each 1mL of beans sauce sample to be detected; with 2M NaOH, regulate pH value to 9.8, add 1mL dansyl Cl derivating agent (5mg/L acetone), vibration mixes.Be placed in and at 60 ℃, react 30min (during vibrate 5 times).
With micro-sampling device point sample 10 μ L on the chromatographic material polyamide film at 10cm * 10cm, by having put, the polyamide film shiny surface that sample dries up is outside, polyamide is towards interior, with rubber band, tie, vertically put into expansion cylinder ascending development airtight, that saturated developping agent is housed, at solvent front, stop chromatography during apart from film top 1cm, volatilize solvent, in 280nm ultraviolet, detect under lamp and develop the color.Development distance is about 4.5cm, observes the yellow-green fluorescence spot that testing sample is identical with standard items Rf value position between Rf value 0.5～0.7 simultaneously.With quantitative point in the cadaverine standard items comparison of same thin laminate, can observe the brightness that cadaverine fluorescent brightness in this sample is approximately equivalent to 5mg/L standard items, sxemiquantitative judges that in this sample, cadaverine content is close to 5mg/L, and in beans sauce, cadaverine content is about 0.05mg/g.
Above embodiment is described the preferred embodiment of the present invention; not scope of the present invention is limited; design under the prerequisite of spirit not departing from the present invention; various modification and improvement that the common engineering technical personnel in this area make technical scheme of the present invention, all should fall in the definite protection domain of claims of the present invention.
Claims (1)
1. a method for detection of biological amine, comprises step:
With 0.1M hydrochloric acid, be mixed with the putrescine solution that mass concentration is 5mg/L, 10mg/L, 15mg/L, 20mg/L, 25mg/L; get standard solution and each 1mL of grape wine sample to be detected; with 2M NaOH, regulate pH value to 9.4～10.0; add and be dissolved in the dansyl Cl derivating agent 1mL that acetone, concentration are 5mg/L; vibrate and mix; be placed at 60 ℃ and react 30min, during vibrate 3～8 times;
With micro-sampling device point sample 10 μ L on the chromatographic material polyamide film of 10cm * 10cm, by having put, the polyamide film shiny surface that sample dries up is outside, polyamide is towards interior, with rubber band, tie, vertically put into expansion cylinder ascending development airtight, that saturated developping agent is housed, saturated developping agent is dimethylbenzene: glacial acetic acid=10:1; Balance solvent in expansion cylinder is developping agent and in airtight expansion cylinder, mixes standing obtaining; At solvent front, stop chromatography during apart from film top 0.5～1cm, volatilize solvent, can realize target compound and separated from impurities, in 280nm ultraviolet, detect under lamp naked eyes after colour developing and directly observe; Development distance is about 4.5cm, observes the yellow-green fluorescence spot that testing sample is identical with standard items Rf value position between Rf value 0.5～0.7 simultaneously; With quantitative point in the putrescine standard items comparison of same thin laminate, can observe in this wine sample putrescine fluorescent brightness higher than 5mg/L lower than 15mg/L putrescine standard items, in preliminary this wine sample of judgement, putrescine content is close to 10mg/L, far below domestic and international limiting the quantity of to biogenic amine in food.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103149317A (en) * | 2012-12-21 | 2013-06-12 | 江南大学 | Method for promptly detecting biogenic amine in food |
| CN103983733A (en) * | 2014-05-21 | 2014-08-13 | 江南大学 | Method for detecting biogenic amine in yellow wine by using chromatographic sheet |
| CN104597199B (en) * | 2015-02-03 | 2016-04-13 | 江南大学 | An a kind of step thin-layer chromatography detects the method for biogenic amine fast |
| CN109358151B (en) * | 2018-12-19 | 2021-01-05 | 广东盛泰华生物制药有限公司 | Thin-layer chromatography detection method for 1, 4-butanediamine impurity in L-2-amino-5-guanidino valeric acid raw material |
| CN110208426A (en) * | 2019-07-03 | 2019-09-06 | 仲恺农业工程学院 | A kind of pork freshness evaluation method based on biogenic amine |
| CN112304929B (en) * | 2019-07-24 | 2023-10-27 | 高雄大学 | Method and device for detecting biogenic amine in food material |
| CN110794060B (en) * | 2019-11-12 | 2022-11-18 | 华润三九（雅安）药业有限公司 | Method for determining spermidine content in safflower medicinal material and method for enriching spermidine |
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