SU1364987A1 - Method of determining nonvolatile biogenic amines in food products - Google Patents

Abstract

FIELD OF THE INVENTION The invention relates to the food industry and can be used to determine the trace amounts of unwanted toxicides, nts in food products and biological objects. The purpose of the invention is to increase the reliability of the determination. The method consists in alkalizing to a pH of 9 to 14 solutions containing biogenic amines and volatile amines. Ammonia and volatile amines such as melamine, dimethylamine, ethylamine, diethylamine, propylamine, isopropylamine, butyl amine, isobutylamine, piperidine and others are removed during alkalization, while nonvolatile biogenic amines remain in the residue. 1 tab. WITH/)

Description

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This invention relates to the food industry and can be used to determine trace amounts of unwanted toxicants in food products and biological objects.

The purpose of the invention is to increase the reliability of the determination.

The method consists in the fact that when alkalizing the evaporated extract, ammonia and volatile amines are removed - methylamine, dimethylamine, ethylamine, diethylamine, propylamine, isopropyl13

Biogenic amines identify

amine, butylamine, isobutylamine, piperidine and others, and nonvolatile biogenic fluorescent centers on UV fluorescence compared

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lysing the object. For example, 20 mg each of histamine, phenethylamine, putrescin, cadaverine is dissolved in 100 ml of 0.02 n. hydrochloric acid. Aliquots of coli 1: Standard solutions, for example, 20 and 100 μl, are converted to the same: derivatives as in the experimental sample.

10 preparations of derivatives of 5-10 µl of benzene solutions of the experimental and standard samples are applied to one plate and chromatographed.

Biogenic amines identify

u on fluorescence in UV light

amines remain in the residue. Further processing with reagents to obtain derivatives, for example, dansyl chloride, in the sample, only derivatives of the analyzed biogenic amines are formed, which are easily separated and determined by TLC. In this case, interference caused by the presence of ammonia derivatives and volatile amines in the sample is absent, which facilitates the interpretation of chromatograms and significantly increases the reliability of the determination in comparison with the known methods.

The method is carried out as follows.

A sample of the food product is homogenized with an aqueous acid solution, for example, 0.02 n. hydrochloric acid, or with a mixture of an aqueous solution of acid and acetone, centrifuged or filtered and the aliquot part is evaporated to dryness. The residue is thoroughly mixed with 50% methanol or ethanol. Using 0.01 n. alcohol solution of alkali set the pH of the sample 9-14, mostly 9-11, and evaporated at 90-100 ° C to dryness. In this case, ammonia and volatile amines are removed from the extract. The nonvolatile b-gene amines in the residue are converted to the corresponding derivatives, for example, dancylamides. To do this, distilled water and acetyl solution of dansyl chloride are added to the residue, kept for 14-16 hours at room temperature or 1.5-2 hours at 50 ° C, then water and benzene are added, shaken vigorously and the organic layer is taken off. At the same time, the same derivatives are prepared from a mixture of chemically pure biogenic amines, the set of which is determined by the expected nomenclature of biogenic amines in analogy in experimental and standard samples. In this case, you can visually assess the content of biogenic amines. To quantify, the corresponding zones are eluted with the same amount of benzene, centrifuged or filtered, and the fluorescence intensity is measured on a fluorometer, using a comparison sample as a sample.

standard sample, the closest to the experimental content of the analyzed substances.

The calculation of the concentration (mg / kg or mg / l) of biogenic amines is made

on the fermule

C Klfi-ii-E, le-V-M

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lo.Ic

0

five

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where K is a coefficient taking into account an aliquot taken for analysis; fluorescence intensity of eluates from experimental and standard samples, relative units; V is the volume of the experimental sample deposited on the chromatogram of the test sample, μL;

V is the volume of the standard sample applied to the chromatogram, μl;

P is the amount of detectable amine in the standard sample selected for comparison, mg; M is the mass of the food sample (4g) or volume (l). Example 1. 10 ml of model extract, which is a solution containing 20 mg / l of ammonia, 10 mg / l each of methylamine, dimethylamine, diethylamine, propylamine, isopropyl amine (a, butylamine and isobutylamine, as well as 0.5 mg / l putrescine, cadavering1, phenethylamine in 0.02 N. HCl, evaporated to dryness. The precipitate is dissolved in 1 ml of 50% methanol, added

0.01 n. KOH solution in 50% methanol to pH 9 and evaporated at 100 ° C to dryness. The residue is mixed with 0.5 ml of distilled water, and 1.5 ml of an acetone solution of dansyl chloride (3 mg / ml) is added. pH is adjusted to 10 and allowed to stand at room temperature for 16 hours. Then 2 ml of water and 1 ml of benzene were introduced into the sample, mixed thoroughly, and the benzene layer was removed. Similarly, 20 and 100 µl of the standard mixture are given, representing solutions (200 µg / ml each) of non-volatile biogenic amines (phenethylamine, 15 na. Petna with the corresponding R

putrescine, cadaverine) 0.01 n.NS1.

5 µl of the experimental and both standard samples are applied onto the Siloufla plate and chromatographed in the benzene-triethylamine system (5: 1). When viewed in UV light (360 nm) of the chromatogram obtained, only dansyl derivatives of biogenic amines — putrescine, cadaverine, and phenethylamine (respectively, 0.4A, 0.56, and 0.63) were found in the standard mixture.

The samples of dansyl derivatives of volatile amines and ammonia are absent in the experimental sample.

The spots with the same R in the test and standard samples were eluted with 1 ml of benzene, filtered and the fluorescence intensity was measured at 360/490 nm.

The calculation carried out by the formula showed that the model extract contains, mg / l: putrescine 0.48; cadaverine 0.49; phenethylamine 0.46.

PRI mme R 2. 0.01 kg (10 g) of crushed Swiss cheese is homogenized with 60 ml of a mixture of 0.02 NN and acetone (3: 1) and centrifuged. The liquid phase is separated, 6 ml are taken and evaporated to dryness. The dry residue is thoroughly mixed with 0.5 ml of 50% methanol, 0.01 n is added. KOH solution in 50% methanol to pH 10 and evaporated at 90 ° C to dryness. To the residue, 0.5 ml of distilled water and 1.5 ml of an acetone solution of dansyl chloride with a concentration of 3 mg / ml are added, the pH is adjusted to 9 and held for 2 hours at 50 ° C. Then the sample is cooled and 2 ml of distilled

Experimental and most interesting standard samples for elution with 1 ml of benzene and measuring fluorescence (360, 490 nm) per spectrofluorometer

20 The calculation carried out according to the formula showed that the Swiss cheese contains, µ / kg: histamine 10.7; pu rescine 17.6; cadaverine 11.2.

Froze South (0.01 kg)

25 cod fresh-frozen homogenizer with 50 ml of 0.02 n. HC1 and filtered. Take 5 ml of the filtrate and evaporate to dryness. The dry residue is thoroughly mixed with 1 ml of 50% ethanol,

30 alkalinized 0.01 n. KOH to pH 11 and evaporated at 100 ° C to dryness. To the residue, 0.5 ml of distilled water and 1.5 ml of acetone solution of dansyl chloride are added to a concentration of 4 mg.

2 ml, adjusted to pH 9.5 and left overnight (16 hours) at room temperature. Then 2 ml of distilled water is added to the sample and 1 ml of benzene is vigorously shaken

4Q manic layer. In parallel, 30 and 150 µl of a standard solution of chemically pure histamine, putrescine are introduced into two tubes containing 0.5 ml of distilled water.

g cadaverine, spermine and spermidine 0.02 n. HC1 (200 µg / ml each and each dansylate like a test sample and chromatographed in chloroform - triethylamine - benzene 15: 3: 10.

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Consider the plate in the UV light (360 nm). In the test sample, spots with R equal to R spot are observed.

Examine the plate in UV light (360 nm). In the test sample, spots with R equal to R spot are observed.

water and 1 ml of benzene, carefully re-gg histamine, putrescine, cadaverine

mix and select organic and spermine. Spot with the same R

layer. In parallel in two test tubes, in an experimental sample and in the most bizkom

containing 0.5 mp distilled according to the intensity standard eluted

water, 20 and 100 µl of 0.5 ml of benzene are injected and the intensity is measured

Dart solution of histamine, phenethyl-amine, putrescine and cadavarin in 0.02 n. HC1 (200 µg / ml each) and dansiliruyut similar to the experimental sample. On a chromatographic plate with KSK silica gel, 5 µl of both standards and 10 µl of the experimental sample are applied and chloroform-triethylamine-benzene is developed in the system 15: 3: 10.

Visually in UV light (360 nm), the plate is examined. In the test sample, spots with R, equal to R of histamine, putrescine and cadavariant and closest in intensity to standard samples were observed to elute with 1 ml of benzene and fluorescence (360, 490 nm) was measured on a spectrofluorimeter.

The calculation carried out according to the formula showed that Swiss cheese contains, microns / kg: histamine 10.7; putrescine 17.6; cadaverine 11.2.

Froze South (0.01 kg)

cod fresh-frozen homogenized with 50 ml of 0.02 n. HC1 and filtered. Take 5 ml of the filtrate and evaporate to dryness. The dry residue is thoroughly mixed with 1 ml of 50% ethanol,

alkalinized 0.01 n. KOH to pH 11 and evaporated at 100 ° C to dryness. 0.5 ml of distilled water and 1.5 ml of 4 mg acetone solution of dansyl chloride are added to the residue.

ml, adjusted to pH 9.5 and left overnight (16 hours) at room temperature. Then 2 ml of distilled water and 1 ml of benzene are added to the sample, shaken vigorously and the organic layer is taken off. In parallel, 30 and 150 µl of a standard solution of chemically pure histamine, putrescine, are introduced into two tubes containing 0.5 ml of distilled water.

cadaverine, spermine and spermidine 0.02 n. HC1 (200 µg / ml each) and dansilyrut similarly to the experimental sample and chromatographic in the system chloroform - triethylamine - benzene 15: 3: 10.

Examine the plate in UV light (360 nm). In the test sample, spots with R equal to R spot are observed.

histamine, putrescine, cadaverine

fluorescence (360/490 nm) in the experience of the relative standard.

The calculation by the formula showed that in the cod sample freshly frozen, there are mg / kg: histamine 7.4; putrescine 12.3; cadaverine 18.8; spemen 2.7.

The effect of pH on the complete removal of volatile amines during evaporation of solutions containing various amines with a concentration of 100 mg / ml is shown in the table.

From the data in the table it can be seen that alkalinization of the solution to a pH above 8 ensures complete removal of volatile amines. The introduction of an excess of alkali is impractical, therefore, it is preferable to bring the pH to 9-11.

Reliable identification and determination of nonvolatile biogenic amines are achieved by the proposed method with their content of 10-30 ng in the spot. Spo364987

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tested on various types of food products. The proposed method for identifying and determining non-volatile biogenic amines eliminates the interfering effect on the determination of biogenic amines ammonia and volatile amines contained in extracts from food products, greatly facilitates the decoding of chromatograms and the identification of biogenic amines, determining the accuracy of the determination by reducing the likelihood getting false results.

Due to its high sensitivity, simplicity, speed and convenience of implementation, the method can be used both in research institutions and in laboratories of hygienic and biochemical profiles, such as sanitary and epidemiological devices, for the analysis of biogenic amines in food products and biological objects.

Claims (1)

Invention Formula Method for determination of non-volatile bio-. genes amines in food products, which involves extracting them from the sample, removing the solvent, chromatographic separation and quantifying them, characterized in that, in order to increase the accuracy of the determination, the resulting extract separates the volatile amines by adjusting the pH of the extract to 9 - 14.