KR100948589B1 - Method for assaying mycotoxin - Google Patents

Abstract

The present invention relates to the measurement of low concentration mycotoxin, and more particularly, to isolate and purify mycotoxins present in low concentrations in various foods, and then adsorb onto a metal substrate to analyze mycotoxins with a secondary ion mass spectrometer. It is about.

According to the present invention, it is possible to screen the harmful toxic substances in the human body in a fast and accurate manner in advance, which will greatly contribute to the improvement of health care and the study of toxic substances.

Mycotoxin, Aflatoxin, Isolation, Purification, Secondary Ion Mass Spectrometry, Food, Toxicity

Description

Method for measuring mycotoxin at low concentration {Method for assaying mycotoxin}

The present invention relates to the measurement of low concentration mycotoxin, and more particularly, to isolate and purify the low concentration of mycotoxin present in various foods and then adsorbed on a metal substrate to analyze the mycotoxin with a secondary ion mass spectrometer. It is about.

Mycotoxin is a secondary metabolite that is caused by contamination of various grains and fruits with fungi. According to data released by the Food and Agriculture Organization (FAO), more than 25% of the world's crops are contaminated with mycotoxins, which have been reported to be fatal to cancer or disease-associated agents (World Wide). Regulations for Mycotoxins in Food and Feed in 2003.FAO Food and Nutrit. 2003, 81, 3.1.-3.5.). One of the most notable mycotoxins is that of Aspergillus , a type of yeast fungus. Aflatoxin is known as a major causative agent of liver cancer (IARC. In Mycotoxins; International Agency for Research on Cancer: Lyon, France, 1993, Vol. 56, pp 249-395; JM Toxicology 2001, 167, 101-134 ). As another example, ocartoxin, which occurs in species such as Aspergillus and Penicillium , which are parasitic in grains, has been reported to cause chronic kidney disease in humans ( Mol . Nutr . Food . Res . 2006, 50, 519). -529).

Therefore, efforts have been made to develop various measurement methods for analyzing the amount of mycotoxin present in grains, fruits, and the like.

The method by chromatography is widely used as a measuring method of mycotoxin. Specifically, methods such as liquid chromatography (HPLC), fluorescence analysis liquid chromatography (HPLC with fluorescence detection (HPLC-FD), thin-layer chromatrography (TLC), etc. (USA patent No. 4285698; J. Nat. Toxins 1999, 7, 347-352), but these methods have the disadvantages of long measurement times and lack of reproducibility by users.

Enzyme-linked immunosorbent assay (ELISA), electrochemical immunoassay, etc. ( Anal. Bioanal. Chem . 2006, 384, 286-294; Biosens . Bioelectron . 2005, 21, 588-596) has been introduced, but due to the non-specific signal value, the accuracy in the quantitative analysis is somewhat reduced and it is very difficult to simultaneously analyze a variety of mycotoxins.

Recently, mass spectrometry methods have been tried to improve the accuracy of mycotoxin detection in samples and to simultaneously analyze various mycotoxins in the mixture. Specifically, matrix-assisted-laser desorption ionization mass spectrometry ( Anal. Chem. , 2005, 77, 24, 8155-8157), mass spectrometry with electrospray and atmospheric pressure chemical ionization, ESI / APCI MS) ( J. Mass Spectrom . 2000, 35, 23-32), Liquid chromatography with electrospray ionization tandem mass spectrometr, LC / ESI / MS / MS (Mass Spectrom. 2006, 20, 771-776). However, these methods require complex pretreatment and highly skilled techniques, and in particular MALDI, liquid phase matrices can be used to remove noise caused by matrices because they can interfere with low molecular mass signals. A cumbersome procedure involving the use of ion matrices and the addition of additives should be involved.

In contrast, recently, secondary ion mass spectrometry (SIMS) has been known as a method for analyzing the lower mass range more effectively. This is a method that can perform qualitative analysis of the target material very accurately by directly irradiating the primary ion beam on the surface and analyzing the secondary ion from the sample. In addition, the relatively small size of the ion beam without the use of a matrix allows reproducible measurements at high sensitivity (Biomaterials, 24, 3635-3653). However, the measurement is limited because the analyte is fragmented by the primary ion beam and the secondary ion mass signal of the analyte changes according to the type of the substrate. It is difficult to analyze.

The present invention is to solve the problems of the prior art as described above, and an object of the present invention is to provide a quantitative analysis method of mycotoxin of low concentration easily and accurately using secondary ion mass spectrometry.

It is also an object of the present invention to specifically measure only specific mycotoxins to be analyzed in a mixture.

It is also an object of the present invention to provide a method capable of simultaneously detecting a plurality of mycotoxins in a mixture.

That is, according to the present invention, the presence or absence (quality) and content (quantity) of mycotoxins can be accurately determined by selectively separating / purifying low molecular weight mycotoxins which are difficult to measure due to low concentrations in food and obtaining high quality mass spectrometry signals. It aims to provide a new way to analyze.

The present invention for achieving the above object, (A) extracting the mycotoxin (mycotoxin) from the analyte is separated / purified (B) adsorbing the separated / purified mycotoxin to the metal substrate (C) the It relates to a low concentration mycotoxin measurement method comprising the; analyzing the mycotoxin adsorbed on the metal substrate with a secondary ion mass spectrometer (SIMS).

Mycotoxins measurable in the present invention include aflatoxin, arachatoxin, steigmatosystin, patulin, rubratoxin, fusariumtoxin, and ziralenone. (zearalenone), deoxynivalenol, and the like, but are not limited thereto.

In the present invention, the mycotoxin separation / purification method in step (A) may be used by selecting any one of a solid phase extraction method, a reverse phase separation method, an ionized resin exchange separation method, or an antibody affinity separation method. However, it is preferable to use antibody affinity separation method for the accuracy of the measurement. In the following examples, antibody affinity separation was applied.

In the present invention, a plurality of mycotoxins may be analyzed at the same time by using an antibody affinity column for different mycotoxins in step (A).

In the present invention, the metal substrate in the step (B) is preferably a gold or silver material, if necessary, by dividing the metal substrate into 10 ~ 500 μm ² area to minimize the amount of the reaction solution at the same time to analyze multiple samples It is good to be able to.

That is, in order to solve the problem of quantitative analysis of mycotoxin using SIMS, the present invention uses a metal surface such as gold and silver that has a signal amplification effect after appropriately separating and purifying the mycotoxin to be analyzed in the mixture. To effectively and accurately analyze mycotoxins.

According to the present invention, mycotoxins contained in trace amounts in foods such as cereals and fruits can be measured effectively, accurately and quickly. That is, by separating and purifying mycotoxin present in low concentrations in food (mixture), it is possible to simultaneously identify and quantify a plurality of types.

Therefore, by being able to quickly and accurately screen harmful toxic substances in the human body present in food in advance, it will be able to greatly contribute to the improvement of health care and the study of toxic substances.

In the following description, the antibody-affinity separation method was applied for the separation / purification of mycotoxin, but it will be obvious that the same result can be obtained even if other conventionally known separation methods are applied. In addition, in the examples, only aflatoxin was tested, but other mycotoxins such as okratoxin (ochratoxin), steigmatosystin, patulin, rubratoxin, fusariumtoxin, Toxic substances such as zearalenone and deoxynivalenol may also be separated / purified in the same manner by an antibody-affinity column composed of corresponding monoclonal antibodies.

In addition, although not mentioned in the examples, when the concentration is very low, the concentration of an additional antibody-affinity column can be used to detect a very low amount of aflatoxin (a few ppb or less) from a large amount of samples, thereby increasing the measurement sensitivity. Can be. For example, the amount of sample used in the above-mentioned antibody-affinity column is 10 mL, and 1 mL of the final solution is extracted so that 10 times is concentrated and extracted, but 1 mL of the extracted solution and 10 mL of fresh sample are obtained. A 2 mL sample containing 1 mL can be concentrated to an additional 10-fold if diluted to 10 mL and flowed back into a new antibody-affinity column. As a result, the concentration of aflatoxin in trace amounts can be measured by showing the final concentration of 100 times.

The present invention is largely divided into two stages.

(A ) Mycotoxin pretreatment step using antibody-affinity column: A pretreatment step for effectively separating and purifying samples with an antibody-affinity column to obtain high-quality mass signals before obtaining secondary ion mass spectrometry spectra. Extraction process, and purification process using an antibody-affinity column. More specifically, the process of pre-treating aflatoxin B ₁ using corn as a reference sample was illustrated. Corn samples performed in this step is only an example for mycotoxin detection is not limited thereto.

(B) Analysis of secondary ion mass signal of mycotoxin: This is the process of analyzing the measurement sample pre-treated with antibody affinity column by the actual mass spectrometry spectrum.

Specifically, after the measurement solution from the unknown concentration of the pre-treatment is adsorbed on the gold substrate for 1 hour to obtain a mass spectrum to convert the quantitative concentration from the calibration curve (calibration curve). The standard curve of mass spectrometry according to the amount of mycotoxin can be obtained from the mass peak value obtained from known concentrations of mycotoxin. Since the standard curve for the sample may be different from the average residual range in the food presented by the World Health Organization (WHO), it is desirable to obtain a standard curve for each concentration in the appropriate range.

Hereinafter, the measurement method will be described in more detail through the method of secondary ion mass spectrometry, examples, and preliminary comparative experiments. This example illustrates one aspect of various embodiments of the invention and is not intended to limit the scope or scope of the invention.

Secondary ion mass spectrometry

The secondary ion mass spectrometry was performed under static conditions (<10 ¹³ ion / cm ² ) of the surface with Bi ₁ ⁺ primary ion (25 KeV) using TOF-SIMS V (ION-TOF GmbH). Mass calibration was adjusted to peaks of H ⁺ , H ₂ ⁺ , CH ₃ ⁺ , C ₂ H ₃ ⁺ , and C ₃ H ₅ ⁺ . _{^{CH 3 +, C 2 H 3}} +, the resolution of the peak of the Au ₁ ⁺ were all in the more than 5,000, a specific surface area measurement was performed in a 500 × 500 μm ^2. The measurement time was about 100 seconds per sample. Therefore, when 10 samples are measured, the time required on average is about 20-30 minutes when the samples required for the standard curve are included. Including the separation / purification time performed in step (a), the total time required per 10 samples is only within 1 hour. This is about 1/4 to 1/5 times shorter than HPLC.

As a substrate for measuring samples, gold and silver materials are good. Especially for quantitative analysis, the same gold substrate should be analyzed on the same substrate, so that the surface of the substrate before the adsorption of the sample is irradiated with pyranic acid (sulfuric acid: hydrogen peroxide solution = 4: 1, v / v) and the like to keep the surface clean for 5-10 minutes at room temperature is very important. In particular, the mass peak from the measurement sample may change little by little depending on the intensity of the primary ion, the measurement time, and the current value when the ion is released every time the measurement is performed. The reference substrate is a gold substrate without a sample when a sample is measured using a gold substrate, and the mass peak value of [Au ₁ ] ⁺ from the reference substrate is determined from the mass peak of [M + H] ⁺ value of each sample. By dividing and converting, it is possible to accurately correct different errors in every measurement. If a silver substrate is used, the mass peak value of [Ag ₁ ] ⁺ should be equally corrected.

Since the analytical area for secondary ion mass spectrometry is basically about 500 × 500 μm ² , a size of 1 × 1 cm ² is sufficient. However, if the reaction solution is small enough to deposit all of the substrate or if the size of the substrate needs to be adjusted, the amount of solution can be reduced by reducing the size of the substrate to the minimum size (about 5 × 5 mm ² ) that can be mounted in the holder. And substrates can be saved. In this case, the shape of the substrate does not necessarily have to be a rectangular shape, but is sufficient if it is deposited in the reaction solution.

In addition, the size of the analysis area for performing secondary ion mass spectrometry can be adjusted to 10 × 10 μm ² . In this case, the mass signal value can be amplified within the same measurement time as compared to when analyzing a large area. When the same mass signal value is obtained, the measurement time can be further shortened. However, if the analysis area is small, the reproducibility may be deteriorated depending on the surface position. Therefore, it is preferable to perform mass spectrometry by repeating the measurement while moving the position on the same surface.

<Preliminary Comparative Experiment with Conventional Technology>

First, take 50g sample from corn, put it in a vertical cutter mixer (OMNI), add 5g NaCl to increase the extraction effect, add 100 mL of methanol / water (80:20) mixed solvent, Grind for 5 minutes at speed. The pulverized product was filtered with filter paper (Whatman, pore size 0.4 μm), and only 10 mL of the solution was used for the antibody affinity column. 10 mL of the extracted solvent was flowed into the antibody affinity column (Immunoaffinity Column, Aflatest ^TM , Vicam, USA) to which aflatoxin B ₁ monoclonal antibody was fixed at 1-2 drops per second at room temperature, adsorbed sufficiently, and then 10 mL of water was flowed. After washing with water once, 1 mL of methanol was flowed at the same rate to elute aflatoxin, and the extract was recovered in a glass bottle. The total recovery time was about 30-40 minutes from the initial milling time to the end of recovery.

To determine how much aflatoxin was extracted from the extracted solution, use an HPLC (Alliance 2690, WATERS, USA) with a fluorescence detector (474 fluorescence detector, WATERS, USA) (initial excitation wavelength 360 nm / emission wavelength 440 nm) of the detector. Comparing the concentration of aflatoxin B ₁ with the final sample of corn, the recovery was about 98% and the concentration of the initial reaction solution (10 mL) and the recovery solution (10 mL) was about 10 times concentrated. .

On the other hand, the wavelength of the fluorescence detector may vary depending on the measurement sample and is not limited to the scope or area of the present invention. For example, the conditions for setting a fluorescence detector for aflatoxin M1 are excitation wavelength 365 nm and measurement wavelength 400 nm, and the measurement of patulin is measured by absorption wavelength at 276 nm using a UV detector instead of fluorescence. The method of measuring by HPLC has a long time-consuming disadvantage (average analysis time for 10 samples is about 4-5 hours) and was used as a reference tool for verifying the measured value by mass spectrometry. It is not a necessary process in Esau.

<Example>

Example 1 Analysis of Secondary Mass Mass Signal of Aflatoxin

Aflatoxin (Sigma), one of the representative mycotoxins, was dissolved directly in a solvent (acetone: methanol = 4: 1, v / v) without pretreatment, and the mass signal was confirmed on the secondary ion mass spectrometry spectrum. In other words, when there was only one type of aflatoxin under unconcentrated conditions (FIG. 1A) and when mixed (FIG. 1B), the mass signals were compared.

Samples with the aflatoxin was diluted to a concentration of _{(B 1, B 2, G} 1, G 2, Sigma) for use were acetone and methanol, respectively, a mixed solution 100 ng / mL (= 100 ppb ) , and then, 1 × 1 cm ² After reacting by depositing at room temperature for 1 hour on a gold substrate, secondary ion mass spectrometry was performed. Qualitative analysis of each aflatoxin was performed through the intensity of the mass signal ([M + H] ⁺ ) for each mass (M) on the mass spectrometry spectrum. As shown (FIGS. 1A and 1B), the sample was accurately detected at the position corresponding to [M + H] ⁺ of each mass (B ₁ : 313.286, B ₂ : 315.302 G ₁ : 329.286 G ₂ : 331.301) No mass signal was detected on the gold substrate without. In addition, even when the four types of aflatoxins were mixed in a ratio of 1: 1: 1: 1 (molar ratio), mass signals were simultaneously detected on one spectrum. These results indicate that mass signals of single or mixed species of aflatoxin can be detected simultaneously on the chip.

Example 2 Preparation of Aflatoxin Standard Concentration Curve

To quantify aflatoxin B ₁ , a standard curve was prepared by performing mass spectrometry on a reference solution for each concentration. Since aflatoxin B ₁ has a food residue allowable range of about 10 to several hundred ppb, a standard curve for each concentration was prepared.

As a sample, aflatoxin (B ₁ , Sigma) was diluted in acetone and methanol mixed solution at concentrations of 10 ppb, 100 ppb, 1 ppm, and 10 ppm, respectively, and then deposited on a 1 × 1 cm ² gold substrate at room temperature for 1 hour. After reacting, secondary ion mass spectrometry was performed. The amount of aflatoxin was converted through the intensity of the mass signal ([M + H] ⁺ ) for each molecular weight (M: 312.3) on the mass spectrometry spectrum. For accurate quantitation, the mass size was corrected by dividing the [M + H] ⁺ value from each sample by the mass peak value of [Au ₁ ] ⁺ on a gold substrate without any sample. As illustrated in FIG. 2, the concentration and the intensity of the mass signal were linearly proportional to each other in the concentration range of 100 ppb to 10 ppm, and the minimum measurable concentration range was 10 ppb. In the present embodiment, only a wide concentration range was examined, but in the case of measuring only a range of 10 to 100 ppb, a separate standard curve can be prepared by increasing the number of samples for each concentration within the range. You will get a graph of.

Example 3 Quantitative Analysis of Aflatoxin in Corn Mixtures

In order to analyze aflatoxins in cereals, mass spectrometry was performed after the separation and purification of aflatoxins in corn mixtures.

First, 50 g of corn samples without aflatoxin detected were added to a vertical cutter mixer (OMNI), and 5 g NaCl was added to increase the extraction effect. Then, 100 mL of a methanol / water (80:20) mixed solvent was added thereto and then aflatoxin was added. B ₁ was mixed in the sample to a final concentration of 10, 50, 100 ppb. Thereafter, the mixture was ground for 5 minutes at a speed of 3500 rpm. The ground mixture was filtered with filter paper (Whatman, pore size 0.4 μm), and only 10 mL of the solution was used for the antibody-affinity column. 10 mL of the extracted solvent was flowed to the antibody affinity column (Immunoaffinity Column, Aflatest ^TM , Vicam, USA) to which aflatoxin B ₁ monoclonal antibody was fixed at 1-2 drops per second at room temperature for sufficient adsorption. The mixture was flushed once with water, and 1 mL of methanol was poured at the same rate to elute aflatoxin to recover the extract in a glass bottle. The extract was reacted by depositing the extract at room temperature for 1 hour on a gold substrate of 1 × 1 cm ² , followed by secondary ion mass spectrometry. The comparison through the intensity of the mass signal ([M + H] ⁺ ) for each molecular weight (M: 312.3) on the mass spectrometry is as in Example 2 above.

To ensure correct quantitation by mass spectrometry, the same amount of aflatoxin containing corn sample was measured using HPLC (Alliance 2690, WATERS, USA) equipped with a fluorescence detector (474 fluorescence detector, WATERS, USA) 360nm / emission wavelength 440nm) was compared. In the case of HPLC, a standard curve was prepared using the fluorescence value detected in the reference solution for each concentration of aflatoxin, and the fluorescence value of the corn extract was measured and quantitatively analyzed. As shown in FIG. 3, the comparison between the HPLC and the mass spectrometry showed a high correlation ( R ² = 0.9812). This proves that mass spectrometry can be effectively used for quantitative analysis of aflatoxins.

1 is a mass spectrometry graph of a single species or mixed species of aflatoxin.

Figure 2 is a graph showing the mass spectrometry and standard curve according to the concentration of aflatoxin.

Figure 3 is a graph showing the correlation between the HPLC and mass spectrometry of aflatoxin contained in corn.

Claims (6)

(A) extracting mycotoxin from the analyte to isolate / purify; (B) adsorbing the separated / purified mycotoxin to the metal substrate; (C) analyzing the mycotoxin adsorbed on the metal substrate with a secondary ion mass spectrometer; low concentration mycotoxin measurement method comprising a. The method of claim 1, The mycotoxin is aflaxtoxin (aflaxtoxin), okratoxin (ochratoxin), steigmatosystin (sterigmatosystin), patulin (patulin), rubratoxin (fubraumtoxin), ziralenone (zearalenone) or dera Oxynivalenol (deoxynivalenol) low concentration mycotoxin measurement method characterized in that any one or a mixture selected from the group consisting of. The method of claim 1, Mycotoxin separation / purification method in step (A) is a low concentration mycotoxin measurement method, characterized in that one of the solid phase extraction method, reverse phase separation method, ionized resin exchange separation method or antibody-affinity separation method. The method of claim 3, wherein The mycotoxin separation / purification method in step (A) is an antibody affinity separation method, wherein a low concentration of mycotoxins can be analyzed simultaneously by using antibody-affinity columns for different mycotoxins. Toxin measurement method. The method of claim 1, The metal substrate in step (B) is a low concentration mycotoxin measurement method, characterized in that the gold or silver material. The method of claim 1, Analysis method of low concentration mycotoxin, characterized in that the analysis area on the metal substrate in step (B) is 10 ~ 500 μm ² .

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