Methods for the determination of mycotoxins. Laboratory methods for the diagnosis of mycotoxicoses. Mycotoxins and methods for their determination
After the birth of puppies, a responsible period begins for the bitch and her owners, when one should take care not only of the feeding and health of the bitch, but also of newborn babies. After the birth of puppies, the first one or two days the bitch may have a poor appetite, indigestion, especially if she ate a lot of afterbirth.
Remember that during this period you should not resort to antibiotics - this will adversely affect the health of babies. Try to get by with safer drugs (such as herbs, probiotics, gastric lavage, activated charcoal, and preferably lignitin). Very good results in this case are obtained by the use of absolutely harmless homeopathic preparations from Heel, and it is not necessary to look for veterinary ones, “human” ones work no worse. Try one or two subcutaneous injections of Echinacea compositum, Nux vomica, Berebe-ris-hommacord. Good results are obtained by using the drug in tablets of the same Diar-heel company. The drug is given 3 times a day, 1 tablet. The results are better if it is pre-dissolved in 1.5 tablespoons of boiled water and mixed thoroughly.
In the first three days after childbirth, care must be taken in feeding so as not to aggravate a possible disorder. Foreign authors during this period suggest giving the bitch boiled small chickens, ground together with bones and mixed with boiled rice.
After the first days after giving birth, the bitch noticeably improves her appetite, and this is quite natural - the needs of her body increase markedly. Colostrum begins to be actively produced first, and then milk. To a large extent, the quantity and quality of milk depend not only on the individual characteristics of the bitch, but also on her feeding.
In the first week, the amount of feed exceeds the usual diet by 1.5 times. But during this period, too much meat should not be given to the bitch, so as not to cause eclampsia. As a protein component at this time, you can give fish or cottage cheese. Feed the bitch should be frequent (every 4-5 hours), in small portions. After the normal function of the stomach is restored, it is necessary to resume giving mineral supplements and means to strengthen the coat.
- meat, fish and offal - 45%,
- cereals - 30%,
- milk and dairy products - 10%,
- vegetables - 15%.
In order for a lactating bitch to have more milk, she should include foods that promote lactation in her diet: raw carrots, meat, fish, broth, oatmeal, etc. As often as possible, offer the bitch to drink: milk (if it does not cause disorders), tea with milk, weak coffee with milk. As a means to increase the amount of milk, you can try the following:
- decoction of oregano;
- decoction of lemon balm: pour one teaspoon of grass with two cups of boiling water, let it brew, add sugar;
- place two teaspoons of tea in a thermos, pour two cups of boiling milk, let it brew for 3-4 hours, then add sugar and cool;
- anise decoction.
Apilac, which many owners give to bitches to increase lactation, did not give the desired results in my practice, although I tried to give it to different bitches.
If the dog flatly refuses to drink, you can, of course, try to force him to drink, but it’s better to first try to “deceive” him by putting a small piece of butter in a still warm enough drink. It may very well be that the smell of butter will seduce her.
In order to prevent exhaustion of the bitch, her feeding during this period should be complete and the food should contain a sufficient amount of calories, proteins, vitamins and minerals.
However, throughout the lactation period, the amount of food is not constant. As already mentioned, in the first week the diet should be increased by 1.5 times. As the amount of milk in the bitch increases, the ration should also increase: in the second week, the ration should be doubled, and from the third week until the end of lactation, it should be tripled: Comparison is made with the diet of a dog in its normal state. The amount of food a bitch needs depends on the size of her litter. Four puppies in a litter require a 2-fold increase in the diet, and eight puppies require at least a three-fold increase.
Usually the bitch feeds the puppies for 4-6 weeks. The amount of milk produced by a bitch is not constant throughout lactation. Until the 20-25th day, the amount of milk increases, and then decreases. Western experts offer a very easy way to calculate the calorie content of feed during this period. This method consists in weighing the entire litter at 3-4 days of age, adding 250 cal of additional energy to the diet of the bitch for each kilogram of puppies and, in accordance with this, increasing the amount and calorie content of food.
The duration of lactation depends on the individual characteristics and feeding of the dog. In each case, of course, the individual characteristics of the dog should be taken into account. There are bitches that puppies literally “suck out”, they produce milk for a long time, in large quantities, and they feed puppies for up to two months. Naturally, their diet should be larger and more complete than that of bitches, who have little milk even at the very “peak of milk production”, which coincides with the 14-17th day of lactation, and whose owner simply dreams that she will feed the children up to three weeks. To a large extent, the bitch's milk production is influenced by a complete protein diet rich in essential amino acids. The lack of amino acids primarily affects the quality of milk, as well as its quantity, and this causes a decrease in the growth rate of puppies and slows down their development.
Vitamins also play an important role in the nutrition of lactating bitches. They are needed not only for the bitches themselves, but also for the development of puppies. It is necessary to ensure that the bitch receives the required amount of vitamins during the entire lactation period. For example, the content of vitamin A in milk, which is so necessary for the growth of puppies, depends only on the presence of it in the mother's feed *. Therefore, the owner should monitor the constant presence of this vitamin in the diet of a nursing bitch. This also applies to vitamin D and B vitamins, which are excreted in large quantities through dog milk.
The bitch requires the same amount of additional energy to produce milk as is contained in the excreted milk. Therefore, the energy value of the diet will primarily depend on the milk supply of the bitch. The calorie content of the feed also depends on the lactation period. It is known that in the first and second weeks of lactation, the bitch produces less milk than in the third and fourth. Therefore, the diet should be designed in such a way that in the first half of the lactation period the energy intensity of the feed increased by 2 times, in the second - by 3 times compared to the bitch's need in the normal state. In practice, it looks like this: for the formation of milk in the first half of lactation, a lactating bitch with a body weight of 10 kg needs an additional diet with a calorie content of 750 kcal in addition to the main one, and in the next two weeks - 1500 kcal daily.
Immediately after giving birth, the female mammary glands secrete colostrum. It is necessary to ensure that each newborn puppy must receive colostrum containing specific antibodies.
Of great importance for the formation of milk are minerals, the lack of which causes various kinds of osteodystrophic diseases not only in lactating bitches, but also in offspring. At the same time, the backbone of lactating females is depleted in minerals and becomes porous, fragile, osteoporosis appears, and rickets in newborn puppies. It is important to prevent the accumulation of mineral reserves during pregnancy, and in young bitches - during the period of growth and preparation for the first lactation. Lactating bitches need more salt than non-lactating bitches.
Qualitative composition and daily energy intensity of diets of lactating bitches weighing 5 kg in the first-second and third-fourth weeks of lactation
Qualitative composition and daily energy intensity of the diets of lactating bitches weighing 15 kg in the first-second and third-fourth weeks of lactation
Qualitative composition and daily energy intensity of the diet of lactating bitches weighing 25 kg in the first-second and third-fourth weeks of lactation
Methods for the determination of mycotoxins
Modern methods for detecting and determining the content of mycotoxins in food and feed include screening methods, quantitative, analytical and biological methods. Mycotoxin methodology is developing very rapidly. The number of developed methods and various modifications has already reached several hundred and continues to grow.
Screening methods, characterized by simplicity and speed of analysis, allow you to quickly and reliably "screen out" uncontaminated samples. These include commonly used methods such as the mini-column assay for aflatoxins, ochratoxin A and zearalenone; TLC methods for the simultaneous determination of up to 30 different mycotoxins; fluorescent method for detecting contamination with aflatoxins, etc.
Quantitative analytical methods for the determination of mycotoxins can be subdivided into chemical, radioimmunochemical and enzyme immunoassay. Chemical methods are currently the most common and include successive stages of isolation and proper quantification of mycotoxins. The isolation stage consists of two stages: extraction - separation of the mycotoxin from the substrate, and purification - separation of the mycotoxin from compounds with similar physical and chemical characteristics. The final separation of mycotoxins is carried out using one- or two-dimensional thin layer chromatography (TLC) on silica gel plates in various solvent systems, gas and gas-liquid chromatography, high performance liquid chromatography and mass spectrometry. Quantitative determination of mycotoxins is usually carried out by direct comparison of the intensity of fluorescence on TLC in ultraviolet light with standards of known concentration, both visually and densitometrically. To improve the reliability of the methods, various confirmatory tests are used, based on the preparation of mycotoxin derivatives with other chromatographic, colorimetric or fluorimetric characteristics.
Recent years have been characterized by increased attention to the development of highly sensitive and highly specific radioimmunochemical and enzyme immunoassay methods for the detection, identification and quantification of mycotoxins. These methods are based on obtaining antisera to mycotoxin conjugants with bovine serum albumin. Their advantage is their exceptional sensitivity, which makes it possible to detect picograms of mycotoxins and to carry out developments in the direction of automating the determination process. Biological methods, which are usually not very specific and sensitive, are used mainly for the detection of mycotoxins for which chemical methods of analysis are not available, or as confirmatory tests. Various microorganisms, chicken embryos, many laboratory animals, cell and tissue cultures serve as test objects.
To determine the content of mycotoxins in the composition of food products, sample preparation is carried out by the method of solid-phase extraction on concentrating cartridges "Diapak" (p. 3.2.3). Separation, identification and quantitative determination of mycotoxins in prepared samples is carried out by high performance liquid chromatography on a microcolumn chromatograph of the Milichrome-5 series in a modular design (Fig. 12).
Rice. 12. Microcolumn chromatograph
The paper considers a reversed-phase version of HPLC for the determination of patulin. Detection is carried out at a wavelength of 276 nm. For accurate identification of patulin, multiwavelength detection is also used, which makes it possible to use spectral ratios as an additional identification parameter. The separation is carried out in the gradient elution mode, which improves the resolution and sensitivity of the analysis.
Rice. 13. Liquid chromatograph:
1 - pump; 2 – sample injection unit; 3 – chromatographic column; 4 - detector;
5 - drain for eluate or collector for fractions; 6 - registrar (recorder,
integrator or personal computer)
6
4
Sample analysis using a liquid chromatograph (Fig. 13) is carried out as follows. A certain volume of the analyzed sample solution is introduced into the upper part of the chromatographic column (3) using the sample injection unit (2). Using a pump (1), the analyzed mixture is pumped with eluent through a chromatographic column (3), in which the analyzed mixture is separated into separate fractions (components). The eluate flowing from the column, containing the separated components, is analyzed by the detector (4), the readings of which are recorded by the recorder (6).
Methodological foundations of HPLC
Eluotropic series. The eluting power of the eluent is the ability of the eluent (solvent or mixture of solvents) to displace the adsorbate from the surface of the adsorbent. In this case, the stronger the eluent molecules are adsorbed on the active sites of the sorbent, the higher its eluting power. Solvents arranged in a row in increasing eluting strength form an eluotropic series.
As eluents for reverse-phase chromatography, mixtures of solvents containing water and organic compounds that modify the eluting strength are used - n-alcohols, acetonitrile, tetrahydrofuran, and others that form true solutions with water. In normal-phase chromatography, polar modifiers are used as eluents - linear and cyclic hydrocarbons (hexane, cyclohexane, heptane, etc.).
Detectors for liquid chromatographs. Currently, more than 20 HPLC detectors have been developed. Five are the most widely used, three of which are optical and two are electrochemical. These five detectors allow you to analyze all classes of organic and inorganic substances.
Optical detectors include a spectrophotometric detector (ultraviolet (UV), operating in the wavelength range
200–360 nm and visible with a wavelength range of 360–780 nm), fluorimetric and refractive index detectors; to electro-chemical - voltammetric and conductometric detectors. Of particular importance is the mass spectrometric detector, which has a unique information content, as it allows the identification of chromatographically separated components using databases of mass spectra of substances.
Spectrophotometric detector is the most common HPLC detector. The principle of its operation is based on the well-known Bouguer-Lambert-Beer light absorption law. A spectrophotometric detector records the spectra of selective absorption absorption of radiation by a substance. The absorption spectra depend on the structure of the substance under study. This allows you to identify a substance by its spectrum, having a library of spectra or standards. According to the Bouguer-Lambert-Beer law, the intensity of the spectral bands depends on the concentration of a substance, which is the basis for quantitative analysis, i.e., determining the concentration of a substance.
Let monochromatic light from a source with intensity I 0 falls on a ditch with a length l (optical path). The cuvette is filled with a solution of a substance with concentration With. A substance is capable of absorbing monochromatic radiation. The measure of the ability to absorb a given monochromatic radiation is the value ε is the molar absorption coefficient, or extinction coefficient. A weakened light beam emerges from the cuvette with an intensity I. According to the law of light absorption at a wavelength λ= const
I= I 0 ∙10 -ε Cl , (7)
where I is the intensity of the light flux after passing through the cuvette;
I 0 is the intensity of the incident light flux; ε
is the extinction coefficient; With is the molar concentration of the substance in the cuvette; l is the length of the cuvette.
Attitude I to I 0 , expressed as a percentage, is called transmission T, and the value A=lg(I 0 / I) – optical density.
A=lg(I 0 /I)= ε С∙l. (8)
The optical density of a substance is directly proportional to the concentration of the analyte. In spectrophotometric detectors, the analytical quantity is the optical density BUT. Formula (8) serves as the basis for quantitative analysis when using a spectrophotometric detector, since the optical density BUT substance is directly proportional to the height or area of the chromatographic peak.
Optical density dependence BUT from the wavelength of the light incident on the cuvette with the substance in the wavelength range from 190 to 360 nm is called the ultraviolet absorption spectrum (Fig. 14).
200 230 260 290 320 350 λ , nm
BUT, f.r.p.
Rice. 14. Ultraviolet absorption spectrum of an aqueous solution
substances X
3.2.3. Sample preparation
Any substance has a wavelength of maximum absorption(λ , nm). When using this wavelength, the detector has the lowest threshold for detecting a substance.
Using a spectrophotometric detector (SPD), a large number of substances of various classes that absorb ultraviolet light are detected.
The use of a spectrophotometric detector in chromatography greatly facilitates identification. The multi-wavelength detector in combination with the software allows you to get a chromatogram at several wavelengths at the same time and calculate an additional parameter for identifying components - spectralattitude (Q) is the ratio of the heights of the chromatographic peak at different wavelengths
where BUT 1 and BUT 2 - coefficients of optical density at wavelengths 1 and 2. The value Q for each substance is a constant characteristic and does not depend on its concentration. The accuracy of the spectral ratios depends on the values of the optical density of the substance and the design of the detector.
Sample preparation using the solid phase extraction method on concentrating cartridges saves time and labor costs. Solid-phase extraction allows you to concentrate the sample and purify it from accompanying impurities. The complex scheme of solid-phase extraction provides for the sequential use of two cartridges:
– universal concentrating cartridge “DIAPAK P-Z” – reusable cartridge (up to 50 samples) with a set of upper and lower filters (10 pieces);
– universal cartridge for fine cleaning “DIAPAK S” – disposable cartridge.
For preparation of concentrating cartridges you need to perform the following operations.
For preparation of the concentrating cartridge "DIAPAK P-Z":
- pump 10 ml of a mixture of water-acetonitrile (58:42) through the cartridge;
– immediately before sample preparation, pump 10 ml of distilled water through the cartridge.
For preparation of the concentrating cartridge "DIAPAK S":
- pump 5 ml of benzene through the cartridge and plug the cartridge at both ends.
Preparing the food product for concentration
Place a sample weighing 10.0 g in a glass beaker, mix with a small amount of distilled water and quantitatively transfer to a 50 ml volumetric flask. Add 6.0 ml of Carrez I solution and Carrez II solution to the flask. Bring the contents of the flask to the mark with distilled water, mix thoroughly and filter into a graduated cylinder through a paper pleated filter. Measure the volume of the clear filtrate P.
When preparing clarified juices and drinks, filter the sample through a dense paper filter until 20 ml of a clear filtrate P is obtained.
Concentration of the sample on the cartridge "DIAPAK P-Z"
Apply the entire volume of the filtrate P to the previously prepared cartridge at a rate of 1–2 drops per second.
Rinse the cartridge with 5 ml of bidistilled water, discarding all washes.
Elute the patulin from the 10 ml ethyl acetate cartridge into a flask or stoppered volumetric tube containing 5 ml
1.5% aqueous sodium carbonate solution, stopper, mix vigorously and allow to exfoliate.
Assemble the drying column by filling the filter housing with approximately 2 g of anhydrous sodium sulfate, compact the drying agent by tapping on the wall of the column and fix with a cotton swab.
Decant the upper ethyl acetate layer with a pipette, filter through a drying column, collecting the filtrate in a 50 ml heart-shaped flask; additionally extract an aqueous solution of sodium carbonate first with 10 ml and then with 5 ml of ethyl acetate and, after separation, successively filter the decanted volumes of ethyl acetate through a drying column into the same flask.
Evaporate ethyl acetate in vacuum at a temperature not exceeding 40°C to a volume of about 0.5 ml (do not evaporate to dryness!), Add 2.5 ml of benzene (keep volume ratio 1:5).
Cleaning the sample on the cartridge "DIAPAK S"
Remove the caps from the prepared cartridge and pass the benzene-ethyl acetate sample solution at a rate of 1-2 drops per second. Wash the flask with another 0.5-1.0 ml of benzene-ethyl acetate (85:15) and apply to the cartridge, discarding the washings.
Elute the patulin from the 6 ml cartridge with benzene:ethyl acetate (7:3), collecting the eluate into a core stripping flask.
Evaporate the eluate to dryness in a dry-air bath at a temperature not exceeding 40°C.
Immediately! After evaporation, re-dissolve the sample in 0.2–0.25 ml of eluent A (section 3.2.4.2), cooled to 5–8°C.
3.2.4. Work order
Objective– determination of the content of mycotoxin patulin in the composition of food products by high-performance liquid chromatography on a microcolumn chromatograph of the Milichrome-5 series.3.2.4.1. Measurement technique
Measurements include the following main steps:- calibration of the chromatograph according to solutions with a known mass concentration of patulin;
- preparation of a food sample by the method of solid-phase extraction according to paragraphs. 3.2.3;
– analysis of the extract by HPLC with signal registration by a UV detector;
– identification of patulin by retention parameters and spectral ratios;
– calculation of the mass concentration of patulin in the sample based on the registered analytical signal (peak height) and the calibration graph;
- calculation of the mass fraction of patulin in the composition of the food product.
Instruments, reagents and materials, needed to do the job:
– chromatograph of the Milichrome-5 series or any other HPLC chromatograph with WinXrom or Multichrome software;
– HPLC chromatographic column: Diasfer–110–С10СN (5 µm, 2×80 mm, TU 4215–001–05451931–94);
– variable volume dispenser for 1-100 µl;
– variable volume dispenser for 100–1000 µl;
- a conical flask with a tightly ground thin section with a capacity of up to 10 ml;
– membrane filters;
- a set of standard solutions of patulin with a concentration of 1, 2, 5 and 10 mg/l;
- phosphoric acid 85%;
– acetonitrile for liquid chromatography (special purity specification TU 6–09–3513–86, UV absorption up to 200 nm);
– hexane, chemically pure (rectified);
– trifluoroacetic acid;
– Carrez solution I – dissolve 15.0 g of potassium hexacyanoferate II in 100 ml of water.
- Carrez II solution - dissolve 30.0 g of zinc acetate in 100 ml of water;
- eluents A, B and C.
Preparation of eluents
Eluent A. 20 ml of acetonitrile and 0.5 ml of a 10% solution of trifluoroacetic acid are added to a volumetric flask with a tightly ground glass section with a capacity of 100 ml. The solution is thoroughly mixed and brought to the mark with distilled water, previously filtered through a nylon membrane filter (0.2 µm). Then the eluent is degassed by evacuation for 1 minute at a rarefaction of 1 kg∙s/cm 2 .
Eluents B and C prepared in the same way as eluent A, only 10 and 0 ml of acetonitrile are taken per 100 ml of solution, respectively.
3.2.4.2. Setting the chromatographic separation modes
and registration of results
To carry out measurements, it is necessary to set the operating modes of the chromatograph. Dispenser modes:
– regeneration – 300 µl;
– sample volume – 5 µl;
– eluent consumption – 150 µl/min;
– gradient elution modes: 800 µl – eluent A,
500 µl - eluent B, 300 µl - eluent C;
- thermostat temperature - 35 0 С.
Detection modes:
– number of wavelengths – 3;
– wavelengths – 250, 276, 290 nm;
– measurement time – 0.04 s.
Conditioning of the chromatographic system is carried out 30 minutes before measurements by performing a “blank” analysis, in which the standard mixture containing patulin is replaced with 5–10 µl of eluent, and then the standard mixture of mycotoxins is separated.
Exercise 1. To study the method of sample preparation of food products to determine the content of patulin. Prepare samples in accordance with paragraph 3.2.3.
Task 2. Calibrate the chromatograph. The chromatograph is calibrated by sequentially introducing standard solutions of patulin with concentrations of 1, 2, 5, and 10 mg/l.
Under the guidance of a teacher, it is proposed to enter the parameters of the chromatographic separation (clause 3.2.4.3) from the PC keyboard into the program (WinXrom) and start 2-4 chromatographic analyzes in automatic mode. The results are presented in the form of a table. 6.
T a b l e 6
On the basis of the obtained chromatographic profiles, build a calibration graph (the concentration is plotted along the ordinate axis - mg / l, along the abscissa axis is the optical density of mycotoxin BUT– s.o.p.).
Task 3. Identify patulin in the test sample of the food product and determine its concentration.
Under the guidance of a teacher, start the measurement of the prepared food product sample in automatic mode
(with chromatographic separation parameters selected in task 2). Upon completion of the measurement, identify the patulin mycotoxin according to the standard file (“StandardDD–MM–YY.001”) obtained in task 2. Using the calibration graph built in task 2, determine the concentration of patulin in the food sample.
Record the results in Table. 7.
Table 7
The mass concentration of patulin in a food sample is calculated by the formula
where With– mass concentration of patulin in the sample, mg/l (calculated according to the calibration dependence, based on the height of the analytical peak); V p is the sample volume, ml; R - the degree of extraction of mycotoxin at the stage of sample preparation (equal to 60%); M pr is the mass of the food product sample used for purification and subsequent chromatographic determination, g.
The result of measuring the mass fraction of mycotoxin in the object being determined is presented in the following form: X± mg/kg; at R\u003d 0.95 and recorded in the protocol (Appendix 1), where X i , is the mass concentration of patulin in the sample, mg/kg; R- probability; is the absolute error limit, calculated by the formula
After receiving the result, it is necessary to evaluate the values of the standards for the operational control of convergence, which are given in the relevant GOSTs for control (analysis) methods.
Make a conclusion about the compliance (or non-compliance) of the content of patulin in the studied food product with the permissible levels established by SanPiN 2.3.2.1078–01.
Questions for self-control
1. What principles underlie the classification of chromatographic methods of analysis?
2. What is the essence of chromatographic separation? How is qualitative identification and quantitative analysis carried out?
3. What food products contain mycotoxins? What mycotoxins are determined in the composition of food products in accordance with the requirements of SanPiN?
4. What chromatographic method is used to determine the content of mycotoxins in food products?
5. What is spectrophotometric detection based on? What are spectral ratios and what are they used for?
6. How is food sample preparation carried out to determine the content of patulin by HPLC?
7. What operations are included in the HPLC method for determining patulin?
8. What is eluent and gradient elution? What eluents are used in the determination of patulin by HPLC?
9. How is patulin identified and quantified?
10. How is the accuracy of HPLC determination of patulin ensured?
Methods for the determination of mycotoxins
Modern methods for detecting and determining the content of mycotoxins in food and feed include screening methods, quantitative analytical and biological methods.
Screening methods are fast and convenient for serial analysis, allow you to quickly and reliably separate contaminated and uncontaminated samples. Screening methods include thin layer chromatography (TLC methods), a fluorescent method for determining grain contaminated with aflatoxins.
Quantitative analytical methods for the determination of mycotoxins are represented by chemical, radioimmunological and enzyme immunoassay methods. Today, the most common are chemical methods, which include two stages: the isolation stage and the stage of quantitative determination of mycotoxins. The isolation stage includes extraction (separation of mycotoxin from the substrate) and purification (separation of mycotoxin from compounds with similar physical and chemical characteristics). The final separation and quantification of mycotoxins is carried out using various chromatographic methods. A universal method for determining all types of mycotoxins is thin layer chromatography (TLC).
When sampling from a batch of product, the main objective is to obtain an average sample or an average sample that is representative of the entire batch in terms of mycotoxin concentration (the samples taken should characterize the quality of the entire batch). The fulfillment of this task depends on the nature and distribution of mycotoxins, the characteristics of the product (raw, processed, free-flowing, liquid, pasty, etc.), the method of sample preparation. For example, contamination of peanuts with aflatoxins has a pronounced heterogeneous character: in individual peanut grains, their content can vary from thousandths of a milligram to tens or more milligrams per 1 kg, i.e., differ by 5-6 orders of magnitude. For this reason, the contribution of sampling error to the total analysis error in the determination of aflatoxins in peanuts is the main one and in some cases can be more than 90%.
From the point of view of the uniformity of mycotoxin contamination, all products can be divided into two groups: 1) products with a high degree of heterogeneity (shelled and unshelled peanuts, oilseeds, whole or coarse grains, nuts); 2) products with a uniform nature of contamination (liquids: milk, vegetable oils, juices, purees; flour, ground meals).
To obtain a representative average sample of products of the l-th group, the size of the initial sample should be as large as possible (at least 2 kg), while the average laboratory sample should be isolated from the ground (homogenized) average sample.
For homogeneous products of the 2nd group (jam, marmalade, fruit juices in small tin containers, condensed milk, dry dairy products, etc.), samples should be taken in the number of packaging units corresponding to the size of the average sample (100-200 g), provided that the product comes from the same batch.
Chemical methods for the detection and identification of individual aflatoxins are based on their specific fluorescence in UV light (about 365 nm), on differences in mobility in thin layer chromatography, on the specificity of their absorption and fluorescence spectra.
Unlike aflatoxins, trichothecenes do not have absorption or fluorescence in the visible part of the spectrum, which makes it difficult to detect them with thin layer chromatography. At the same time, trichothecenes can be detected by TLC using methods based on the treatment of TLC plates with special reagents that form colored or fluorescent derivatives with trichothecenes. For example, T -2 toxin when processing plates; concentrated sulfuric acid forms spots with blue fluorescence;) in UV light.
Arbitration methods for the quantitative determination of mycotoxins are as follows:
‣‣‣ gas-liquid chromatography (for T-2 toxin);
‣‣‣ high performance liquid chromatography (HPLC) using a UV photometric detector (for deoxynivalenol and patulin);
‣‣‣ HPLC using a fluorescent detector (for aflatoxins; and zearalenone).
On fig. 2 shows the device of a modern liquid chromatograph in the simplest design.
The mobile phase from the tank 1 through the inlet filter 9 is supplied by a high-pressure pump 2 to the sample input system 3 - a manual injector or an autosampler, where the sample is also introduced. Then, through the filter 8, the sample with the current of the mobile phase enters the separation column 4 through the pre-column. Then the flow of the mobile phase leaving the column and containing the components of the mixture to be separated (eluate) enters the detector 5 and is removed into the overflow tank 7. When the eluate flows through the measuring the detector loop, the chromatogram is registered and the data is transferred to the recorder 6 or to a computer.
Liquid chromatograph device (isocratic system):
1 - capacity; 2 - high pressure system; 3 - manual injector or autosampler; 4 - separating column; 5 - detector; b - registrar or computer; 7 - drain tank; 8 - filter; 9 - input filter
The system shown in fig. 2 is isocratic: the composition of the mobile phase does not change during chromatography. If during the chromatographic analysis it is extremely important to change the concentration of one or more components of the mobile phase, then so-called gradient systems are used, usually consisting of two or more pumps. In the case of gradient elution, each solvent is fed from a separate vessel into a special mixing chamber with a magnetic stirrer, where, according to a certain program, they are mixed with a given volume ratio.
For the analysis of mycotoxins, gradient systems are more often used, where solutions of acetonitrile in water with a concentration that changes linearly with time are used as the mobile phase.
The chromatographic column is a metal tube 150 to 250 mm in diameter with an inner diameter of 4.6 mm, filled with a special sorbent based on silica gel with grafted hydrocarbon radicals. The guard column serves to protect the chromatographic column from contamination.
The UV photometric detector is the most common type of HPLC detector. The principle of operation of the detector is similar to that of a conventional spectrophotometer: it registers the optical density of a solution. The difference is that the UV detector is a flow detector, instead of a cuvette with a solution, it uses a photometric cell. The eluent stream flows through the working cell and the pure mobile phase flows through the reference cell. The light source is a mercury lamp, which produces intense UV radiation. Light with the desired wavelength is selected using suitable optical filters, passes through the cells, is partially absorbed by the molecules of the mobile phase and the components to be separated, and is captured by a photodetector. The light absorption (optical density) of the eluate is continuously recorded by a chart recorder or computer, recording the chromatogram. Separated components of the mixture (for example, mycotoxins) are presented in the chromatogram as peaks. The peak position on the chromatogram is used to identify the substance, and the peak area is used for quantitative determination.
A more complex device is a fluorescent (fluorimetric) detector.
Hosted on ref.rf
Such a detector uses the ability of organic compounds, in particular aflatoxins and zearalenone, to fluoresce when exposed to UV or visible radiation. The fluorescent detector has a flow cell with two mutually perpendicular optical channels. One of them serves to supply exciting radiation, the other allows measuring the fluorescence intensity. In the case of the analysis of aflatoxins B 1 and M 1 the excitation wavelength is 360 nm and the emitted wavelength is 420 nm.
It should be noted that a UV detector can also be used for the analysis of aflatoxins, but its sensitivity is an order of magnitude lower than that of a fluorimetric detector; therefore, fluorescence detection is preferable when analyzing low concentrations of aflatoxins (at the MPC level and below).
Methods for determining mycotoxins - concept and types. Classification and features of the category "Methods for the determination of mycotoxins" 2017, 2018.
GOST 32835-2014
INTERSTATE STANDARD
JUICE PRODUCTS
Determination of mycotoxins by tandem high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS)
Juice products. Determination of mycotoxins by tandem high performance liquid mass spectrometry (HPLC-MS/MS)
MKS 67.080.01
Introduction date 2016-01-01
Foreword
The goals, basic principles and basic procedure for carrying out work on interstate standardization are established by GOST 1.0-92 "Interstate standardization system. Basic provisions" and GOST 1.2-2009 "Interstate standardization system. Interstate standards, rules and recommendations for interstate standardization. Rules for the development, adoption, application, renewal and cancellation
About the standard
1 DEVELOPED by the Federal State Educational Institution of Higher Professional Education "Moscow State University of Food Production" (FGBOU VPO "MGUPP")
2 INTRODUCED by the Federal Agency for Technical Regulation and Metrology
3 ADOPTED by the Interstate Council for Standardization, Metrology and Certification (Minutes of June 25, 2014 N 45-2014)
Voted to accept:
Short name of the country according to MK (ISO 3166) 004-97 | Abbreviated name of the national standards body |
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Armenia | Ministry of Economy of the Republic of Armenia |
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Belarus | State Standard of the Republic of Belarus |
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Kyrgyzstan | Kyrgyzstandart |
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Russia | Rosstandart |
4 By order of the Federal Agency for Technical Regulation and Metrology dated August 19, 2014 N 896-st GOST 32835-2014 was put into effect as a national standard of the Russian Federation from January 1, 2016.
5 This standard has been developed taking into account the provisions of the following international documents:
- CODEX STAN 247-2005* Codex General Standard For Fruit Juices And Nectars of the Codex Alimentarius Commission;
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* Access to international and foreign documents mentioned hereinafter in the text can be obtained by clicking on the link to the site http://shop.cntd.ru. - Database manufacturer's note.
- Regulation of the Commission of the European Union of 23.02.2006 r. no. 406/2006/EC Laying down the sampling methods and methods of analysis for the official control of the levels of mycotoxins in foodstuffs for the official control of mycotoxin levels in foods");
- AIJN Code of Practice for Evaluation of Quality and Authenticity of Fruit and Vegetable Juices of the European Fruit Juice Association.
6 INTRODUCED FOR THE FIRST TIME
Information about changes to this standard is published in the annual information index "National Standards", and the text of changes and amendments - in the monthly information index "National Standards". In case of revision (replacement) or cancellation of this standard, a corresponding notice will be published in the monthly information index "National Standards". Relevant information, notification and texts are also posted in the public information system - on the official website of the Federal Agency for Technical Regulation and Metrology on the Internet
1 area of use
1 area of use
This standard applies to juices and other juice products from fruits and vegetables, with the exception of citrus fruit cells, and establishes a method for the determination of mycotoxins - patulin and ochratoxin A - using tandem high-performance liquid chromatomass spectrometry in the measurement range of mass concentration of patulin from 0.1 to 100.0 µg/dm and ochratoxin A from 0.1 to 20.0 µg/dm.
NOTE This International Standard is recommended to be applied for the purpose of approbation and accumulation of additional information in terms of its application.
2 Normative references
This standard uses normative references to the following interstate standards:
GOST 12.1.004-91 Occupational safety standards system. Fire safety. General requirements
GOST 12.1.007-76 Occupational safety standards system. Classification and general safety requirements
GOST 12.1.010-76 Occupational safety standards system. Explosion safety. General requirements
GOST 12.1.019-79 Occupational safety standards system. Electrical safety. General requirements and nomenclature of types of protection
GOST 61-75 Reagents. Acetic acid. Specifications
GOST OIML R 76-1-2011 State system for ensuring the uniformity of measurements. Non-automatic scales. Part 1. Metrological and technical requirements. Tests
GOST 1770-74 (ISO 1042-83, ISO 4788-80) Measuring laboratory glassware. Cylinders, beakers, flasks, test tubes. General specifications
GOST ISO 3696-2013 Water for laboratory analysis. Technical requirements and control methods
GOST ISO 5725-1-2003 Accuracy (correctness and precision) of measurement methods and results. Part 1. Basic provisions and definitions
GOST ISO 5725-2-2003 Accuracy (correctness and precision) of measurement methods and results. Part 2: Basic method for determining the repeatability and reproducibility of a standard measurement method
GOST 5789-78 Reagents. Toluene. Specifications
GOST 16317-87 Household electric refrigeration appliances. General specifications
GOST 20015-88 Chloroform. Specifications
GOST 25336-82 Glassware and laboratory equipment. Types. Main parameters and dimensions
GOST 26313-84 Processed products of fruits and vegetables. Acceptance rules, sampling methods
GOST 26671-85 Processed products of fruits and vegetables, canned meat and meat and vegetables. Sample preparation for laboratory analysis
GOST 29030-91 Fruit and vegetable processing products. Pycnometric method for determining the relative density and content of soluble solids
GOST 29227-91 (ISO 835/1-81) Laboratory glassware. Pipettes graduated. Part 1. General requirements
GOST ISO/IEC 17025-2009 General requirements for the competence of testing and calibration laboratories
GOST 32689.1-2014 Food products of plant origin. Multimethods for gas chromatographic determination of pesticide residues. Part 1. General Provisions
GOST 32689.2-2014 Food products of plant origin. Multimethods for gas chromatographic determination of pesticide residues. Part 2: Extraction and Purification Methods
GOST 32689.3-2014 Food products of plant origin. Multimethods for gas chromatographic determination of pesticide residues. Part 3. Determination and validation of results
Note - When using this standard, it is advisable to check the validity of reference standards in the public information system - on the official website of the Federal Agency for Technical Regulation and Metrology on the Internet or according to the annual information index "National Standards", which was published as of January 1 of the current year, and on issues of the monthly information index "National Standards" for the current year. If the reference standard is replaced (modified), then when using this standard, you should be guided by the replacing (modified) standard. If the referenced standard is canceled without replacement, the provision in which the reference to it is given applies to the extent that this reference is not affected.
3 Abbreviations
HPLC-MS/MS - tandem high-performance liquid chromatography-mass spectrometry;
OTA, ochratoxin A;
PAT - patulin;
ESI- sputter ionization in an electric field ( Electrospray Ionization);
IARC- International Agency for Research on Cancer;
LOD- detection limit;
LOQ- limit of quantitative determination;
SRM- identification of components in the mode of control of selective reactions ( Selected Reaction Monitoring).
4 Essence of the method
The essence of the method lies in the preliminary extraction of PAT and OTA mycotoxins with acetonitrile in the presence of anhydrous magnesium sulfate, concentration, re-dissolution in acetonitrile, and quantitative determination of the mass concentration of mycotoxins using HPLC-MS/MS with spray ionization in an electric field and identification of components in the control mode of selective reactions.
5 Measuring instruments, auxiliary equipment, reference materials, reagents and glassware
Analytical HPLC-MS/MS* system with a three-quadrupole mass detector for measurements in the mass range from 10 to 3000 atomic mass units (a.m.u.), with a mass measurement accuracy of at least 0.1 a.m.u., ionization sputtering in an electric field, the ability to work in the mode of control of selected reactions and scanning of child and parent ions, a minimum signal-to-noise ratio of 1000:1. The analytical system should include an HPLC module consisting of a binary pump with a mixer, a chromatographic column thermostat providing a heating temperature of up to 50°C, and a chromatographic column with a reversed-phase sorbent with a grain size of 5 μm C, 150 mm long and 3 mm in inner diameter. The system used must ensure the detection of mycotoxins in the range from 0.1 to 100.0 µg/dm.
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* See Appendix A for more information on recommended LC/MS/MS systems.
A spectrophotometer with a measurement range that allows testing at a wavelength from 250 to 400 nm, allowed by an absolute error in optical density measurements of not more than 0.1%.
Scales in accordance with GOST OIML R 76-1, providing weighing accuracy with the limit of permissible absolute error of a single weighing of not more than ±0.01 mg.
Bath ultrasonic.
Centrifuge with a rotor speed of 4000-5000 rpm for test tubes with a capacity of 50 ml.
Centrifuge with a rotor speed of 10000-12000 rpm for test tubes of the Eppendorf type with a capacity of 1.5-2.0 ml.
Drying cabinet providing temperature maintenance up to 200°С.
Household refrigerator according to GOST 16317.
Shaker for mixing.
Devices for dosing liquid samples with a constant or variable capacity of 20-1000 mm with a relative error in dosing the actual volume of not more than 2.5%.
Microfilter - nozzle on a syringe (regenerated cellulose, diameter 13 mm, pore size 0.2-0.4 microns).
Quartz cuvettes with a working length of 1 cm.
PAT and OTA mycotoxins for use as reference samples with the content of the main substance of at least 98%.
Glacial acetic acid according to GOST 61, analytical grade.
Acetonitrile for Gradient HPLC.
Methanol for Gradient HPLC.
Magnesium sulfate anhydrous, chemically pure
Calcium chloride anhydrous, granular, chemically pure
Chloroform according to GOST 20015, chemically pure
Toluene according to GOST 5789, chemically pure
Ethyl alcohol, absolute.
Water for laboratory analysis, purity 1 according to GOST ISO 3696.
Graduated pipettes of the 2nd accuracy class with a capacity of 1, 2, 5, 10 cm 2 of the 2nd accuracy class according to GOST 29227.
Volumetric flasks of the 2nd class of accuracy with a capacity of 5, 10, 25, 50, 100 and 1000 cm 2 or 2a according to GOST 1770.
Sharp bottom flasks with a capacity of 10.25 cm3.
Eppendorf type centrifuge tube with a capacity of 1.5-2.0 ml.
Microtest tube with a capacity of 100-400 mm.
Measuring cylinders of the 2nd class of accuracy with a capacity of 25, 50, 250 cm3 of any design in accordance with GOST 1770.
Centrifuge tube with screw cap, 50 cm
Porcelain cup with a diameter of 125-150 mm.
Laboratory desiccator with a capacity of 3 dm.
Funnels laboratory in accordance with GOST 25336.
Flat-bottom flasks with a capacity of 50, 100, 250 cm3 according to GOST 25336.
Chemical glasses with a capacity of 10, 20, 50, 100 and 200 cm3 in accordance with GOST 25336.
It is allowed to use other measuring instruments, auxiliary equipment, utensils that are not inferior to the above in terms of metrological and technical characteristics and provide the necessary measurement accuracy, as well as standard samples, reagents and materials in quality no worse than the above.
6 Sampling
Sampling - according to GOST 26313. Preparation and storage of samples - according to GOST 26671, GOST 32689.1, GOST 32689.2 and GOST 32689.3.
7 Preparation for testing
7.1 General requirements
Before testing, preliminary preparation of laboratory glassware is carried out, as well as quality control of reagents and auxiliary materials in accordance with the requirements of GOST 32689.1, GOST 32689.2 and GOST 32689.3.
7.2 Preparation of auxiliary solutions
7.2.1 Preparation of mobile phase A
In a volumetric flask with a capacity of 1000 ml with a tightly closed ground glass or fluoroplastic stopper, pipette 1 ml of glacial acetic acid, 100 ml of methanol and bring to the mark with bidistilled water. The mixture is thoroughly mixed.
Shelf life of the mobile phase A at room temperature - no more than one month.
7.2.2 Preparation of mobile phase B
In a volumetric flask with a capacity of 1000 ml with a tightly closed ground glass or fluoroplastic stopper, 1 ml of glacial acetic acid is placed with a pipette and brought to the mark with methanol. The mixture is thoroughly mixed.
Shelf life of the mobile phase B at room temperature - no more than one month.
NOTE The mobile phase must not come into contact with rubber and polymeric materials [with the exception of polytetrafluoroethylene (PTFE)].
7.2.3 Preparation of solvent 1
In a suitable container, mix 99 parts by volume of toluene and one part by volume of glacial acetic acid. The mixture is thoroughly mixed.
The shelf life of solvent 1 at room temperature is no more than 6 months.
7.3 Preparation of magnesium sulfate
The anhydrous magnesium sulphate used in the extraction as a sorbent must be dried even within the expiration date to remove the absorbed moisture from the air. The sorbent is calcined at a temperature of 180°C - 200°C for 6-10 hours and stored in a desiccator over anhydrous calcium chloride. The criterion for the suitability of the reagent is the absence of an additional aqueous layer when the solution is heated, the extraction stage is carried out to a temperature of 30°C - 40°C and after 2-3 minutes the reaction mass is stirred.
7.4 Preparation of mycotoxin stock solutions
7.4.1 Preparation of PAT solutions
7.4.1.1 Preparation of PAT stock solution, mass concentration 200 µg/cm
Take 2.0 mg of pure crystalline PAT, weighed with an accuracy of 0.01 mg, dissolve in a 10 ml volumetric flask in a small amount of chloroform, and then bring the volume of the solution to the mark with chloroform.
The shelf life of the initial PAT solution at a temperature of 0°C in a glass volumetric flask with a ground stopper tightly wrapped in aluminum foil is no more than 1 month.
7.4.1.2 Preparation of PAT solution, mass concentration 20 µg/cm
Transfer 1 ml of the resulting PAT stock solution (see 7.4.1.1) to a 10 ml volumetric flask and dilute to the mark with chloroform. To determine the exact mass concentration of PAT in the solution, 5.0 ml of the resulting PAT standard solution is taken and transferred to a container with a capacity of about 15 cm, then chloroform is removed by nitrogen purge until a dry substance is obtained. Immediately after obtaining the dry matter, 5.0 ml of absolute ethanol is added to the container. Completely dissolve PAT. The resulting PAT solution is introduced into a quartz cuvette with an optical path length of 1 cm, then the spectrum of the solution is recorded on a spectrophotometer in the wavelength range from 250 to 350 nm, using absolute ethanol in the reference cuvette as a control.
The mass concentration of PAT in solution, µg/cm, is calculated by the formula
where is the maximum value of the optical density of the spectrum (wavelength about 275 nm), units. OP;
- molecular weight of PAT, equal to 153.1 g/mol;
- conversion factor;
- molar coefficient of optical absorption (extinction), equal to 14600, m/mol.
7.4.1.3 Preparation of 100 µg/cm PAT solution
5 ml of the initial solution of PAT in chloroform with a mass concentration of 200 µg/ml (see 7.4.1.1) is transferred into a volumetric flask with a capacity of 10 ml, concentrated to a dry residue at room temperature under a stream of nitrogen and immediately redissolved in acetonitrile, bringing it to the volume in the flask to labels.
7.4.1.4 The shelf life of PAT solutions according to 7.4.1.2 and 7.4.1.3 at 0°C in a glass volumetric flask with a ground stopper tightly wrapped in aluminum foil is no more than 24 hours.
Before use, the temperature of the solutions is brought to room temperature (it is not allowed to remove the aluminum foil from the volumetric flask until the contents reach room temperature). Due to the destruction of PAT, it is not allowed to store reference samples in the form of a thin film of dry matter obtained after removal of the solvent -.
7.4.2 Preparation of OTA stock solution
7.4.2.1 Preparation of 20 µg/ml OTA stock solution
Dissolve 2.0 mg of pure crystalline OTA, weighed to the nearest 0.01 mg, in a 25 ml beaker with solvent 1 (see 7.2.3) and quantitatively transfer to a 100 ml volumetric flask and dilute with solvent 1 to the mark.
To determine the exact mass concentration of OTA in a solution, the obtained initial OTA solution is introduced into a quartz cuvette with an optical path length of 1 cm, then the spectrum of the solution is recorded on a spectrophotometer in the wavelength range from 300 to 370 nm, using solvent 1 as a reference cuvette.
The mass concentration of OTA in the initial solution, μg/cm, is calculated by the formula
where is the maximum value of the optical density of the spectrum (wavelength is about 333 nm), units. OP;
- molecular weight of OTA, equal to 402.7 g/mol;
- conversion factor;
- correction factor determined in accordance with Appendix A;
- molar coefficient of optical absorption (extinction), equal to 544, m/mol.
The shelf life of the original OTA solution at a temperature of minus 18°C in a glass volumetric flask with a ground stopper tightly wrapped in aluminum foil is no more than four years.
7.4.2.2 Preparation of 5 µg/ml OTA solution
Withdraw 2.5 ml of the OTA stock solution (7.4.2.1), transfer to a 10 ml volumetric flask and dilute with solvent 1 (7.2.3) to the mark.
The shelf life of an OTA solution at a temperature of 4°C in a glass volumetric flask with a ground stopper tightly wrapped in aluminum foil is no more than 24 hours.
Before use, the temperature of the solution is brought to room temperature (it is not allowed to remove the aluminum foil from the volumetric flask until the contents reach room temperature) -.
7.5 Preparation of PAT and OTA calibration solutions
PAT and OTA calibration solutions are prepared by mixing certain volumes of their stock solutions (see 7.4.1.1 and 7.4.2.1) with clarified apple juice that does not contain the analytes to be determined.
7.5.1 Preparation of PAT calibration solutions
7.5.1.1 Preparation of an intermediate solution of PAT mass concentration of 1000 ng/cm (solution n-1)
1 ml of the PAT solution (see 7.4.1.3) or 1 ml of the standard sample of the PAT composition with a mass concentration of PAT of 100 µg/cm is transferred into a volumetric flask with a capacity of 100 ml and the volume of the solution is adjusted to the mark with acetonitrile.
7.5.1.2 Preparation of an intermediate solution of PAT mass concentration of 10 ng/cm (solution n-2)
Transfer 1 ml of solution -1 (see 7.5.1.1) into a 100 ml volumetric flask and dilute to the mark with acetonitrile.
7.5.1.3 Preparation of PAT calibration solutions
Selected in accordance with table 1 certain volumes of intermediate solutions n-1 and n-2 (see 7.5.1.1 and 7.5.1.2) and dispense into 10 ml volumetric flasks.
Table 1 - Volumes of solutions n-1 and n-2 for preparation of PAT calibration solutions
Name of indicator | Calibration solutions |
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Solution volume n-2 cm | |||||||
Solution volume n-1 cm | |||||||
Amount of injected PAT, ng | |||||||
Mass concentration of PAT in solution, ng/cm | |||||||
7.5.2 Preparation of OTA calibration solutions
7.5.2.1 Preparation of the 200 ng/ml OTA intermediate solution (solution A-1)
Concentrate 1 ml of the 5 µg/ml OTA solution (see 7.4.2.2) to dryness under nitrogen flow at room temperature and immediately transfer with acetonitrile to a 25 ml volumetric flask.
7.5.2.2 Preparation of the 10 ng/ml OTA intermediate solution (solution BUT-2)
0.5 ml OTA intermediate solution BUT-1 (see 7.5.2.1) is transferred to a 10 ml volumetric flask and diluted to the mark with acetonitrile.
7.5.2.3 Preparation of OTA calibration solutions
Selected in accordance with table 2 certain volumes of intermediate solutions BUT-1 and BUT-2 (see 7.5.2.1 and 7.5.2.2) and dispense into 10 ml volumetric flasks.
Table 2 - Volumes of solutions BUT-1 and BUT-2 for preparation of OTA calibration solutions
Name of indicator | Calibration solutions |
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Solution volume BUT-2 cm | |||||||
Solution volume BUT-1 cm | |||||||
Amount of administered OTA, ng | |||||||
Mass concentration of OTA in solution, ng/cm | |||||||
Bring the volume of the solution in the flasks to the mark with clarified apple (or other filtered) juice.
For testing in HPLC-MS/MS, the system is injected with 10 mm of PAT and OTA calibration solutions prepared according to 7.5.1.3 and 7.5.2.3 and calibrated according to 7.7, taking into account the conditions of 8.3.1.
The shelf life of the calibration solution at a temperature of 0°C - 4°C in a glass volumetric flask with a ground stopper is no more than 24 hours.
7.6 Preparing the LC/MS/MS system
The preparation of the HPLC-MS/MS system for measurements is carried out in accordance with the manual (instruction) for operation and the information given in Appendix B.
When setting up the operating modes of the mass spectrometer, it is recommended to use the MS/MS parameters for the determination of mycotoxins given in Appendix B.
In this case, the following conditions must be met:
- ambient air temperature from 20°С to 25°С;
- atmospheric pressure from 84 to 106 kPa;
- voltage in the mains (220±10) V;
- frequency of current in the mains from 49 to 51 Hz;
- relative air humidity from 40% to 80%.
7.7 Calibration of the HPLC-MS/MS system
The calibration of the system with solutions of mycotoxins in juices according to 7.5 is carried out in accordance with the operating manual (instruction) for the HPLC-MS/MS system and taking into account the conditions according to 8.3.1 once a month. Peak areas of PAT and OTA are determined on chromatograms and a calibration dependence is established from the peak area in the concentration range according to 7.5. Calculate the correlation coefficient and the deviation of the calculated values of the mass concentration of mycotoxins at each calibration point from the actual value in accordance with the procedure for preparing calibration solutions (see 7.5). The calibration is considered acceptable if the correlation coefficient is at least 0.999 (for PAT) and 0.965 (for OTA), and the relative deviation of the calculated value of the mass concentration from the actual value is not more than ±10%.
Instead of a relative deviation, the acceptability of a calibration characteristic can be assessed by a relative standard deviation, which should not exceed 5%.
8 Testing
8.1 Extraction
10 ml () of preliminarily thoroughly mixed juice products are placed in a centrifuge tube with a screw cap with a capacity of 50 ml. 20 ml of acetonitrile and 15 g of anhydrous magnesium sulfate are added to the test tube. The mixture is intensively stirred for three to five minutes manually or with a shaker. After stirring, the obtained extract is centrifuged for 10 minutes at 4000-5000 rpm at room temperature or 5 minutes in the presence of a centrifuge with cooling at a temperature of 5°C. Measure the total volume of the extract after centrifugation (). 18-19 cm () of the extract, taken with a pipette or dosing device, is transferred into a sharp-bottomed flask with a capacity of 25 cm. 1 cm () acetonitrile.
If there is an insoluble caramel film on the walls of the vessel, it is destroyed in an ultrasonic bath for three to five minutes. The solution is transferred into an Eppendorf-type tube with a capacity of 1.5-2.0 cm3 and centrifuged at 10,000-12,000 rpm for 3-5 minutes. The upper layer is taken and filtered through a microfilter with a pore size of 0.2-0.4 µm directly into a microtube with a capacity of 100-400 mm. For the HPLC-MS/MS test, 10 mm of the prepared sample is injected into the system.
8.2 Sample preparation from concentrated products
Concentrated juices (puree) are reconstituted with water to the minimum level of soluble solids stipulated by regulatory documents for a particular type of juice product. Concentrated juice products, for which the minimum levels of soluble solids are not provided, are restored with bidistilled water to a content of soluble solids of 11.2%. The content of soluble solids is controlled according to GOST 29030.
Extraction of reconstituted samples is carried out according to 8.1.
8.3 Taking measurements
8.3.1 General conditions
Samples and calibration solutions prepared according to 8.1 are injected in the chosen sequence. The most common method is when the injection of calibration solutions starts and ends a series of sample injections.
The HPLC-MS/MS system must be set to SRM with transitions providing selective detection of analyzed mycotoxins. The retention times and peak areas are determined using the analysis software for recording and calculating the results of the analysis, attached to the HPLC-MS/MS system. Examples of HPLC/MS/MS systems, separation conditions and mass spectrometric detection are given in Appendix B.
Samples are tested under repeatability conditions for two parallel determinations in accordance with GOST ISO 5725-1 (subsection 3.14) and GOST ISO 5725-2.
8.3.2 Identification of mycotoxins
To identify mycotoxins, the retention times obtained from the sample solutions are compared with the retention times of the corresponding mycotoxins from the calibration solutions. To confirm the presence of mycotoxins, a comparison is made between the ratio of signal intensities from the first and second m/z-transition with the ratio of intensities of mycotoxin signals from calibration solutions.
The ratio of peaks for one mycotoxin should not differ by more than 20% from the expected ratio of signal intensities.
9 Processing and presentation of test results
9.1 Quantification
Quantification of mycotoxins in the injected volume of the prepared extract (see 8.1) is carried out by comparing the area (or height) of the mycotoxin peak with the corresponding calibration characteristic for this mycotoxin.
The mass concentration of mycotoxins in the tested products, µg/dm, is calculated by the formula
where is the conversion factor from cubic centimeters to cubic decimeters;
- concentration of mycotoxin in the extract volume of 10 mm, injected into the HPLC-MS/MS system, determined by the calibration dependence, ng;
is the volume of acetonitrile in which the extract was redissolved after concentration, cm;
- the total volume of the extract from which the volume is taken for concentration, cm;
- sample volume introduced into the chromatograph (=10 mm), mm;
- volume of a sample of juice products taken for testing, cm;
is the volume of extract selected for concentration, see
When calculating the amount of mycotoxins in concentrated juice products, the degree of dilution with water is taken into account in accordance with 8.2.
The measurement result is taken as the arithmetic mean of the results of three parallel determinations, if the acceptance condition is met
where , are the maximum and minimum values from the obtained three results of parallel determinations, µg/dm;
, , - results of three parallel determinations, µg/dm;
- value of the critical range, %.
The discrepancy between three parallel determinations (as a percentage of the mean value) performed in the same laboratory should not exceed the repeatability (convergence) limit equal to 3.6· with a probability = 0.95. If this condition is met, the final measurement result is taken as the arithmetic mean of three parallel determinations, rounded up to the third decimal place.
The measurement results are recorded in the protocol in accordance with GOST ISO / IEC 17025.
9.2 If the result obtained shows that the mycotoxin content exceeds the upper limit of the range of the calibration dependence, prepare a new sample by increasing its dilution with water, and re-measure.
10 Metrological characteristics
The metrological characteristics of the method for PAT and OTA correspond to the conditions given in Table 3.
Table 3 - HPLC-MS/MS Method Precisions
Measurement range, µg/dm | Relative standard deviation of repeatability, % | Relative standard deviation of reproducibility, % |
when determining PAT (no more) |
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when determining OTA (no more) |
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The limits of detection of PAT and OTA in samples of juice products are: LOD- 0.03 mcg/dm, LOQ- 0.1 mcg / dm.
11 Quality control of measurement results
The quality control of the measurement results in the laboratory involves monitoring the stability of the measurement results using the stability check of the standard deviation of the intermediate precision. Stability testing is carried out using Shewhart's control charts. The frequency of monitoring the stability of the results of the measurements performed is regulated in the internal documents of the quality system. In case of unsatisfactory control results, for example, when the action limit is exceeded or the warning limit is regularly exceeded, the reasons for these deviations are found out, including changing reagents, checking the work of the operator.
GOST 12.1.007.
ATTENTION! When working with mycotoxins, it should be taken into account that PAT and OTA have strong toxic properties with pronounced nephrotoxic, immunotoxic, teratogenic and genotoxic effects. According to the classification IARC OTA refers to carcinogens potentially hazardous to humans (group 2B). Greater safety precautions must be observed when working with mycotoxins. Laboratory personnel should wear protective clothing, including a face shield, gloves, and goggles. All operations with mycotoxins are carried out in a fume hood. After completion of the work, the used laboratory glassware and waste are subjected to decontamination.
Annex A (mandatory). Verification of the spectrophotometer and determination of the correction factor CF for calculating mass concentrations of mycotoxins in standard solutions
Annex A
(mandatory)
Verification of the spectrophotometer and determination of the correction factor for calculating the mass concentrations of mycotoxins in standard solutions
A.1 To determine the mass concentrations of mycotoxins in stock solutions (see 7.4.1.1 and 7.4.2.1), use a spectrophotometer suitable for measuring the optical density of solutions in a quartz cuvette with a 1 cm optical path length in the wavelength range from 200 nm to 400 nm.
Calibration of the spectrophotometer is carried out as follows.
Measure the optical density of three solutions of potassium dichromate (KCrO) in sulfuric acid (HSO) - 0.25; 0.125 and 0.0625 mmol / dm at the maximum absorption point (wavelength about 350 nm), using as a control a solution of sulfuric acid (HSO) with a concentration of 0.009 mmol / dm.
Then the value of the molar coefficient of optical density is calculated, m/mol, for each concentration of potassium dichromate according to the formula
where is the measured value of the optical density of a solution of potassium dichromate in sulfuric acid for the corresponding concentration, units. OP;
- concentration of potassium dichromate solution in sulfuric acid, mmol/dm.
If the difference between the three measured values is outside the guaranteed range of optical density measurement accuracy, then the calibration procedure or equipment should be checked. Calculate the arithmetic mean.
Determine the correction factor (dimensionless value) for specific equipment (spectrophotometer and cuvette) according to the formula
where is the characteristic value of the molar optical density coefficient for solutions of potassium dichromate (KCrO), m/mol;
- molar coefficient of optical density, calculated according to the formula (A.1), m/mol.
If the resulting correction factor value is less than 0.95 or greater than 1.05, then the calibration procedure or equipment must be checked to eliminate deviations (the same set of cuvettes is used for calibration and purity) -.
Appendix B (informative). Examples of HPLC-MS/MS systems for the determination of mycotoxins in juices and other juice products*
Annex B
(reference)
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* These examples are recommended and are provided for the convenience of users of this International Standard.
B.1 HPLC-MS/MS System No. 1
Hardware platform: Varian 320-MS LC/MS/MS.
Ionic