KR102152310B1 - Multi-residue and multi-class analytical method for agricultural chemical, veterinary drug and mycotoxin in feed - Google Patents

Abstract

The present invention relates to a simultaneous analysis method of pesticide residues in feed, veterinary medicine, and mycotoxin and provides a method of simultaneous analysis of each feed sample by methods such as GC-MS/MS and LC-MS/MS with only two pretreatments using the polarity difference of each material remaining in the feed. Through the present invention, it is possible to construct an effective analysis method that can save time, cost, etc. for monitoring harmful substances in feed by providing an easier analysis method compared to the existing technology using about 40 methods.

Description

{Multi-residue and multi-class analytical method for agricultural chemical, veterinary drug and mycotoxin in feed}

The present invention relates to a method for simultaneous analysis of pesticide residues, veterinary medicines and mycotoxins in feed. In more detail, the present invention provides a method of simultaneously analyzing each feed sample by a method such as GC-MS/MS and LC-MS/MS with only two pretreatments using the polarity difference of each substance remaining in the feed. .

Mass cultivation is also taking place in agricultural technology through the current rapid development of industrial technology. This becomes possible through large-scale injection of pesticides, and generally, pesticides administered to agricultural products are distributed without being completely treated. In recent years, interest in the environment has been increasing, and in particular, as the effects of pesticides on the human body have been reported one after another, the residual amount regulation has been enforced. Veterinary medicines are used for the purpose of treating, preventing or diagnosing diseases of livestock, but due to abuse, veterinary medicines are introduced into the body of the animal and remain in livestock/aquatic products, processed products, and foods such as honey. Since veterinary medicines, unlike medicines for humans, are mainly aimed at preventing and treating diseases of livestock and fish, there is a possibility of threatening human health through livestock/fishery food. Mycotoxins are secondary metabolites produced by fungi, and over 400 species are known so far. These residual pesticides, veterinary medicines, and mycotoxins are pollutants that can easily be introduced into feeds manufactured using animal and plant raw materials, and there is a need to prevent the distribution of feeds containing toxic ingredients by preventing their inflow and detecting residual ingredients. It is emerging.

Therefore, the present inventors were developing a method to make the analysis of harmful substances such as pesticide residues in feed easier, and immediately applied GC-MS/MS and LC-MS/MS through a simple two-sample preparation method. By building a method that can be done, the present invention could be completed.

Republic of Korea Patent Registration No. 10-1825950 (Name of invention: Simultaneous analysis of melittin, apamine and phospholipase A2 in bee venom using ultra-high performance liquid chromatography, Applicant: Korea, registration date: January 31, 2018) Republic of Korea Patent Publication No. 10-2010-0009940 (Name of the invention: pyrethroid pesticide detection method and residual concentration analysis method contained in animal biological samples, Applicant: Chonnam National University Industry-Academic Cooperation Foundation, Publication Date: January 29, 2010 ) Republic of Korea Patent Publication No. 10-2013-0039215 (Name of the invention: Analysis method for residual pesticides of bean sprouts using acetonitrile, Applicant: Korea (National Agricultural Products Quality Management Institute), Publication date: April 19, 2013)

An object of the present invention is to provide a method for simultaneous analysis of pesticide residues, veterinary medicines and mycotoxins in feed. In more detail, an object of the present invention is to provide a method of simultaneously analyzing each feed sample by a method such as GC-MS/MS and LC-MS/MS with only two pretreatments using the polarity difference of each material remaining in the feed. In having to.

The present invention relates to a method for simultaneous analysis of pesticide residues, veterinary medicines and mycotoxins in feed. The simultaneous analysis method may include the following process.

Preferably, the pretreatment step of the feed sample for the first analysis and the second analysis;

Perform the first analysis by performing GC (gas chromatography) and LC (liquid chromatography) using the feed sample pretreated for the first analysis, and then perform the LC (liquid chromatography) using the feed sample pretreated for the second analysis. Chromatography) to perform a second analysis;

Summing the results of the first analysis and the second analysis to analyze the residual amounts of pesticides, veterinary medicines and mycotoxins in the feed; And,

It may include.

The compound analyzed through the first analysis is a substance having an octanol-water partition coefficient (log P) of -1 or more, and the substance analyzed through the second analysis has a value of the octanol-water partition coefficient (log P) It is characterized in that the material is less than -1.

The GC of the first analysis is GC-MS/MS (gas chromatography-mass spectrometry/mass spectrometry), and the LC of the first and second analyzes is LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry). Analysis).

Among the substances to be analyzed in the first analysis, the detection limit concentration of residual pesticide is 0.12 ~ 1.54 µg/kg, the detection limit concentration of veterinary medicine is 5.01 ~ 20.13 µg/kg, and the detection limit concentration of mycotoxin is 0.51 ~ 6.11. It may be μg/kg.

Among the substances to be analyzed in the first analysis, the quantitative limit concentration of residual pesticide is 0.40 ~ 5.13 ㎍/kg, the quantitative limit concentration of veterinary medicine is 16.69 ~ 67.03 ㎍/kg, and the quantitative limit concentration of mycotoxin is 1.70 ~ 20.35 It may be μg/kg.

Among the substances to be analyzed in the second analysis, the detection limit concentration of pesticide residues is 8.21 ~ 13.76 ㎍/kg, the detection limit concentration of veterinary drugs is 1.27 ~ 21.00 ㎍/kg, and the detection limit concentration of mycotoxin is 3.42 ~ 4.66 ㎍/kg/ May be kg.

Among the substances to be analyzed in the second analysis, the quantitative limit concentration of residual pesticide is 27.34 ~ 43.29 ㎍/kg, the quantitative limit concentration of animal medicine is 4.23 ~ 69.93 ㎍/kg, and the quantitative limit concentration of mycotoxin is 11.39 ~ 37.93 ㎍/kg/kg. May be kg.

On the other hand, in the first pre-processing step of the feed for analysis, then a solution of the sample feed to the Na ₂ EDTA- maekil vane (Na ₂ EDTA-McIlvaine) buffer solution, by addition of Acetonitrile by solvent extraction to analyze the various assay components It is preferable to use. At this time, after adding the extraction solvent to Acetonitrile, it is recommended to add NaCl and MgSO ₄ to increase the recovery rate and selectivity. The EDTA (Na ₂ EDTA-McIlvaine) is characterized in that EDTA is added to secure the safety of the chelating compound among the analyzed components. After that, it is preferable to perform LC with the first supernatant collected by centrifugation. In addition, it is preferable to perform GC with the second supernatant obtained by centrifugation by adding PSA (Primary-secondary Amine) and MgSO ₄ for removing impurities such as fatty acids, pigments, and sugars to the first supernatant.

It is preferable to filter the first supernatant and the second supernatant with a syringe filter of 0.2 μm or less.

In the pretreatment step of the feed for the second analysis method, it is preferable to perform LC with the supernatant obtained by mixing the homogenized feed sample in a methanol solution in which formic acid is dissolved, adding NaCl and MgSO ₄ , stirring, and centrifuging. Do.

In this case, the second analysis supernatant is better to be obtained by mixing with C18 silica powder, stirring, and centrifuging, and filtering through a syringe filter of 0.2 μm or less is preferable.

The mobile phase solution of the LC in the first analysis method is A solution, which is an aqueous solution of ammonium formate containing formic acid; And, A solution B, which is a methanol solution of ammonium formate containing formic acid; It is characterized by using a mixture, and it is preferable to use a C8 silica column for extensive compound analysis. .

In addition, the mobile phase solution of the LC according to the second analysis method includes: Solution A, an aqueous solution containing ammonium formate and ammonium hydroxide; And, it is characterized by using a mixture of B solution, which is an acetonitrile solution containing formic acid, and using a HILIC column to analyze components having an octanol-water partition coefficient of -1 or less. It is desirable.

More preferably, the mobile phase solution of the LC of the first analysis method is a solution A, which is an aqueous solution of 3 to 10 mM ammonium formate containing 0.05 to 2 (w/v)% formic acid; And, 3 ~ 10 mM ammonium formate containing 0.05 ~ 2 (w / v)% formic acid (formic acid) 3 ~ 10 mM ammonium formate (ammonium formate) methanol solution B solution; characterized in that a mixture of.

In addition, the mobile phase solution of the LC of the second analysis method is a solution A, which is an aqueous solution containing 5 to 15 mM ammonium formate and 10 to 30 mM ammonium hydroxide; And, it is characterized in that using a mixture of the solution B, which is an 85-95% (v/v) acetonitrile solution containing 0.1-0.3% (w/v) formic acid (formic acid).

The present invention relates to a method for simultaneous analysis of pesticide residues, veterinary medicines, and mycotoxins in feed, wherein each feed sample is converted to GC-MS/MS and LC-with only two pretreatments using the polarity difference of each substance remaining in the feed. It provides a method of simultaneous analysis by methods such as MS/MS. Through the present invention, it is possible to construct an effective analysis method that can save time, cost, etc. for monitoring harmful substances in feed by providing an easier analysis method compared to the existing technology using about 40 methods.

1 shows the structure of a metal-EDTA compound.
2 shows the results of a total ion chromatogram (TIC) of LC-MS/MS according to the first analysis method.
3 shows TIC (Total Ion Chromatogram) results of GC-MS/MS according to the first analysis method.
4 shows TIC (Total Ion Chromatogram) results according to the second analysis method.

The present invention relates to a method for simultaneous analysis of pesticide residues, veterinary medicines, and mycotoxins in feed, and preferably, a pretreatment step of a feed sample for a first analysis and a second analysis; Perform the first analysis by performing GC-MS/MS and LC-MS/MS using the feed sample pretreated for the first analysis, and LC-MS/MS using the feed sample pretreated for the second analysis. Performing a second analysis by performing a second analysis; And, summing the results of the first analysis and the second analysis to analyze the residual amount of pesticide residues, veterinary medicines, and mycotoxins in the feed; It is about simultaneous analysis.

At this time, the compound analyzed through the first analysis is a substance having an octanol-water partition coefficient (log P) of -1 or more, and the substance analyzed through the second analysis is an octanol-water partition coefficient (log P) It may be a material having a value of less than -1.

In the GC-MS/MS of the first analysis, the GC column is an HP-5MS column, in the LC-MS/MS, the LC column is a C8 silica column, and in the LC-MS/MS of the second analysis, the LC column is a HILIC column. It is preferable to use.

As a substance to be analyzed in the first analysis, the detection limit concentration of pesticide residues is 0.12 ~ 1.54 ㎍/kg, the quantitative limit concentration is 0.40 ~ 5.13 ㎍/kg, and the detection limit concentration of veterinary drugs is 5.01 ~ 20.13 ㎍/kg, quantitative quantification. The limit concentration is 16.69 ~ 67.03 μg/kg, the detection limit concentration of mycotoxin is 0.51 ~ 6.11 μg/kg, and the quantitative limit concentration is 1.70 ~ 20.35 μg/kg.

As a substance to be analyzed in the second analysis, the detection limit concentration of pesticide residues is 8.21 ~ 13.76 ㎍/kg, the quantitative limit concentration is 27.34 ~ 43.29 ㎍/kg, the detection limit concentration of veterinary drugs is 1.27 ~ 21.00 ㎍/kg, and the quantitative limit The concentration is 4.23 ~ 69.93 ㎍ / kg, the detection limit concentration of the mycotoxin is 3.42 ~ 4.66 ㎍ / kg, the quantitative limit is characterized in that the concentration is 11.39 ~ 37.93 ㎍ / kg.

In the pretreatment step of the feed for the first analysis method, the feed sample is mixed with Na ₂ EDTA-McIlvaine buffer, left for 30 minutes to 1 hour, added Acetonitrile, shaken and stirred, and then NaCl and MgSO ₄ were added. To obtain a supernatant obtained by shaking agitation and centrifugation, of which, for LC-MS/MS, a process of filtering the supernatant may be performed, and for GC-MS/MS analysis, the supernatant is PSA (Primary- secondary Amine) and MgSO ₄ in dSPE (Dispersive Solid Phase Extraction) tube, shaking, centrifugation, and then filtering may be performed.

Even more preferably, in the pretreatment step of feed for the first analysis method, based on 5 g of feed sample, 8 to 20 ml of 50 to 200 mM Na ₂ EDTA-McIlvaine buffer is mixed and left for 30 minutes to 1 hour. After adding 5~20 ml of Acetonitrile and stirring, 1g NaCl, 3~5g MgSO ₄ were added, agitated again, and the supernatant obtained by centrifugation was obtained. Among them, the supernatant was used for LC-MS/MS. Filtration can be performed, and for GC-MS/MS analysis, the supernatant is placed in a dSPE (Dispersive Solid Phase Extraction) tube containing 100-200 mg MgSO ₄ based on 25 mg PSA (Primary-secondary Amine) and shaken. And the process of filtering after centrifugation can be performed. 0.5 to 2 ml of the supernatant may be appropriately added to dSPE.

At this time, each centrifugation is preferably performed at 3,000 to 13,000 rpm for 1 to 5 minutes, and the shaking agitation or shaking process is preferably 30 seconds to 2 minutes.

In the preparation of the sample for the first analysis method, a diluted solution adjusted to pH 4.5-5.5 using a formic acid solution by dissolving 50-200mM ammonium formate in water may be used.

In addition, the following protective agent solution may be used for the analysis of GC-MS/MS. To this end, 400-600 mg of sorbitol was dissolved in 10 ml of Acetonitrile aqueous solution, and 400-600 mg of D-(+)-gluconic acid-δ-lactone was dissolved in 10 ml of D-(+). -gluconic acid-δ-lactone stock solution, shikimic acid stock solution in which 400-600 mg of shikimic acid is dissolved in 10 ml of Acetonitrile aqueous solution, and 2 g of 3-ethoxy-1,2-propanediol in powder form were prepared. Each stock solution or powder compound can be prepared by mixing in the following process. Preferably, based on 2 g of 3-ethoxy-1,2-propanediol, D-(+)-gluconic acid-δ-lactone stock solution 1~3 ml, sorbitol stock solution 0.5~2 ml, shikimic acid stock solution 0.2~ It may be prepared by mixing 2 ml and adding an aqueous Acetonitrile solution to the flask to adjust the volume of the solution to 10 ml. At this time, the Acetonitrile aqueous solution can be suitably used in a concentration of 30 to 70 (v/v)% of Acetonitrile, respectively.

In the pretreatment step of the feed for the second analysis method, the homogenized feed sample is mixed by adding a methanol solution in which formic acid is dissolved, followed by shaking and stirring, adding NaCl and MgSO ₄ to shaking, stirring, and centrifuging. The supernatant obtained by separation may be mixed with C18 silica powder, shaken and centrifuged again, and then filtered.

Even more preferably, in the pretreatment step of the feed for the second analysis method, 8 to 20 ml of a methanol solution in which 0.05 to 2% (w/v) formic acid is dissolved based on 5 g of a homogenized feed sample is added and mixed. After shaking and stirring, adding 1g NaCl and 3~5g MgSO ₄ to shaking and centrifuging, mixing 25~100 mg of C18 silica powder based on 4 ml of the supernatant, shaking and centrifuging again and filtering You can do the process.

At this time, each centrifugation is preferably performed at 3,000 to 13,000 rpm for 1 to 5 minutes, and the shaking agitation or shaking process is preferably 30 seconds to 2 minutes.

In LC-MS/MS of the first analysis method, the mobile phase solution of LC is A solution of 3-10 mM ammonium formate containing 0.05-2 (w/v)% formic acid, and 0.05-2 (w/v) A mixture of 3-10 mM ammonium formate methanol solution B solution containing% formic acid may be used. At this time, Solution A and Solution B are mixed in a volume ratio of 85:15 at a volume ratio of 10:90 until 12 minutes, and a volume ratio of 85:15 at a volume ratio of 2:98 until 20 minutes from exceeding 12 minutes. desirable. More preferably, in units of 0 minutes, 1 minute, 1.5 minutes, 10 minutes, 12 minutes, 12.1 minutes, 16 minutes, and 20 minutes, 85:15, 85:15, 40:60, 10:90, 10:90, It is better to mix in a volume ratio of 2:98, 2:98, 85:15, 85;15.

In LC-MS/MS of the second analysis method, the mobile phase solution of LC is 85 containing solution A, an aqueous solution containing 5 to 15 mM ammonium formate and 10 to 30 mM ammonium hydroxide, and 0.1 to 0.3% (w/v) formic acid. A mixture of ~95% (v/v) Acetonitrile solution B solution may be used. At this time, Solution A and Solution B are mixed in a volume ratio of 70:30 at a volume of 2:98 until 10 minutes, and a volume ratio of 2:98 at a volume ratio of 70:30 from exceeding 10 minutes to 16 minutes. It is desirable. More preferably, in units of 0 minutes, 3 minutes, 10 minutes, 13 minutes, 13.1 minutes, and 16 minutes, in a volume ratio of 2:98, 2:98, 70:30, 70:30, 2:98, 2:98 Better to be mixed.

Hereinafter, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, it is provided to sufficiently convey the spirit of the present invention to those skilled in the art so that the contents introduced herein are thorough and complete.

<Example 1. Preparation of sample and standard product to be analyzed (residual pesticide, veterinary medicine, mycotoxin)>

Example 1-1. Preparation of the sample

Animal feed used as a sample was analyzed using AAFCO (Association of American Feed Control Official) certified feed.

Example 1-2. Preparation of standard products (residual pesticide, veterinary medicine, mycotoxin)

Standard solutions of 340 pesticides, 137 veterinary medicines, and 21 mycotoxins, which may remain in feed, were purchased and prepared as standard solutions.

For the first analysis method, each standard was diluted with Acetonitrile to prepare a working solution, and for the second analysis method, each standard was prepared with a methanol solution containing 0.1% (w/v) formic acid and stored in a refrigerator. And used for analysis.

<Example 2. Sample preparation>

The sample of Example 1-1 was pretreated for the following first analysis method and second analysis method.

Example 2-1. Pretreatment of the first sample for analysis

5 g of a homogenized sample was weighed into a 50 ml tube, and 10 ml of Na ₂ EDTA-McIlvaine buffer (pH 4.0) (100 mM) was added, mixed, and left for 1 hour.

At this time, the reason for adding Na ₂ EDTA-McIlvaine buffer (pH 4.0) is that some of the veterinary drugs such as Tetracycline, Ionophore, Penicillin, and Quinolone have the property of binding with polyvalent metal ions, and this property is the recovery rate during the extraction process. This is because it results in a reduction in In order to solve this problem and improve extraction efficiency, when EDTA (FIG. 1), which is a chelating agent, is added, it serves to remove metal ions present in the sample in the form of a complex.

The pretreatment of the sample in the first analysis method serves to remove unnecessary polar substances through hydration treatment, pH control, etc., and to stably remain dissolved in water in the form of dissociation. Most veterinary drugs are acidic ( Since extraction was performed at a high recovery rate under pH 2-4) conditions, and most of the pesticides were stable under acidic conditions, EDTA (pH 4, 100 mM) was allowed to react to the sample using EDTA-Mcilvaine buffer to proceed with hydration. . In the preliminary experiment, it was confirmed in advance that the overall recovery rate of the standard product was increased by more than 10% as a result of the addition of 100mM Na ₂ EDTA-McIlvaine buffer (pH 4.0) than the actual EDTA-Mcilvaine buffer was added.

Next, after adding 10 ml of Acetonitrile to the tube containing the sample, it was shaken vigorously for 1 minute using a stirrer. In order to increase the recovery rate of residual substances that may be included in the sample again, 4g MgSO ₄ and 1g NaCl were added to the tube containing the sample, and the mixture was shaken strongly for 1 minute using a stirrer.

After shaking, centrifugation at 3,000 rpm/min for 5 minutes using a centrifuge to obtain a supernatant.

In order to prepare a sample for LC-MS/MS analysis of the supernatant, 1 ml of the supernatant was filtered through a 0.2 μm syringe filter and used as a test solution.

To prepare a sample for GC-MS/MS analysis, take 1 ml of the supernatant and add 25 mg PSA (Primary-secondary Amine [which serves to purify pigments such as fatty acids, organic acids, sugars, anthocyanins, etc.]) and 150 mg MgSO ₄ It was put into the contained Dispersive SPE (Solid Phase Extraction) tube, shaken for 1 minute, centrifuged at 13,000 rpm/min for 2 minutes, filtered through a 0.2 μm syringe filter, and used as a test solution.

Example 2-2. Pretreatment of the second sample for analysis

5 g of a homogenized sample was weighed into a 50 ml tube, 10 ml of a methanol solution in which 0.1% (w/v) formic acid was dissolved was added, mixed, and then shaken vigorously for 1 minute using a stirrer. In order to increase the recovery rate of residual substances that may be included in the sample, 4g MgSO ₄ and 1g NaCl were added to the tube containing the sample, and then the mixture was shaken vigorously for 1 minute using a stirrer. After shaking, centrifugation was performed for 5 minutes at 3,000 rpm/min using a centrifuge. Next, 4 ml of the centrifuged supernatant was added to a 15 ml tube containing 50 mg of C18 silica powder in advance, shaken for 1 minute, and centrifuged again for 5 minutes at 3,000 rpm/min. At this time, C18 silica plays a role of pre-purifying substances other than residual substances to be analyzed. The centrifuged supernatant was filtered through a 0.2 μm syringe filter and used as a test solution.

On the other hand, in the process of preparing samples or standards in all the processes up to this point, some components such as penicillin among the residual substances (standard products) to be analyzed in the first analysis and the second analysis are adsorbed to glass and filters. The experiment was conducted using a polypropylene (pp) material, and no filters other than polypropylene were used.

<Example 3. Media correction calibration>

Feed contains a variety of media such as fat, protein, and moisture. Accordingly, in order to correct various media effects, a matrix matched calibration method is generally used, which was applied before performing the analysis after pretreatment of the sample in the first and second analysis methods of the present invention.

In order to use the calibration calibration method, it is common to prepare a calibration curve by mixing the target material to be calibrated and the matrix. However, when the media is mixed with a standard material, the destruction of standard materials such as Cephalosphorin and Ionophore is quick. It could be confirmed that it occurred. Therefore, in both the first analysis method and the second analysis method, the destruction of standard materials was minimized by using the auto addition function of the analysis device.

The auto addition function refers to the function of mixing and injecting according to the amount set just before injection of the device, even if the medium and the standard are not mixed in advance. For example, it refers to mixing 1.8 µl of Matrix and 0.2 µl of a standard material and injecting it into the device just before injection of the analysis device.

[Schematic diagram of auto addition function]

Example 3-1. Dilution of first analysis sample and standard

Since substances that can be detected in feed, such as pesticides, are stable at pH 5.0, if the pH is changed in the preparation stage or analysis stage of the sample, the analysis result can be confirmed differently. Thus, a diluted solution for the sample was prepared as a buffer solution of pH 5.0. To this end, 100 mM Ammonium formate was dissolved in water and adjusted to pH 5.0 using formic acid solution, and the diluted solution of the first analysis method was used. Used. Table 1 shows the conditions for preparing a diluted solution for the analysis of pesticides (LC-MS/MS) in the first analysis method, and Table 2 shows the conditions for preparing a diluted solution for analysis of the pesticides (GC-MS/MS) in the first analysis method.

division Standard material for calibration curve preparation Sample solution One 2 3 4 5 Target concentration 0.5㎍/ℓ 1㎍/ℓ 2.5㎍/ℓ 5㎍/ℓ 10㎍/ℓ Preparation method 600 µl of buffer solution + 200 µl of Acetonitrile 600 µl of buffer solution + 300 µl of Acetonitrile STD
5㎍/ℓ
100 μl STD
10㎍/ℓ
100 μl STD
25㎍/ℓ
100 μl STD
50㎍/ℓ
100 μl STD
100㎍/ℓ
100 μl Blank matrix 100µl 100 µl of sample extract

Meanwhile, prior to the analysis, the following protective agent solution was prepared to improve the analysis efficiency of GC-MS/MS. For the preparation of the protective agent, a stock solution was first prepared and then mixed. This process is as follows.

500 mg of sorbitol was dissolved in 10 ml of Acetonitrile:DW (1:1, v:v), and 500 mg of D-(+)-gluconic acid-δ-lactone was dissolved in 10 ml of Acetonitrile:DW (6: 4, D-(+)-gluconic acid-δ-lactone stock solution dissolved in v:v), shikimic acid 500 mg is stored in 10 ml of Acetonitrile:DW (6:4, v:v). A solution and 2 g of 3-ethoxy-1,2-propanediol in powder form were prepared. At this time, each compound was first dissolved in water and a solution was prepared by adding an organic solvent.

Each stock solution or powder compound thus prepared was mixed in the following procedure. To a 10 ml flask, add 2 g of 3-ethoxy-1,2-propanediol, 2 ml of D-(+)-gluconic acid-δ-lactone stock solution, 1 ml of sorbitol stock solution, and 1 ml of shikimic acid stock solution to the flask. Acetonitrile:DW (7:3, v:v) was added to adjust the solution volume to 10 ml, and then stored at 4°C, and mixed as shown in Table 2, and used. *DW: Distilled water.

division Standard material for calibration curve preparation Sample solution One 2 3 4 5 Target concentration 5㎍/ℓ 10㎍/ℓ 25㎍/ℓ 50㎍/ℓ 100㎍/ℓ Preparation method Protective agent 15µl + formic acid 5µl + IS 1000µg/ℓ 50µl 15 µl of protective agent
+ 5µl formic acid
+ Acetonitrile 75µl STD
100㎍/ℓ
25µl STD
200㎍/ℓ
25µl STD
500㎍/ℓ
25µl STD
1000㎍/ℓ
25µl STD
2000㎍/ℓ
25µl Blank matrix 405µl 405 μl of sample extract

For veterinary medicines and mycotoxins of the first analysis method, samples and standards were mixed with Matrix in a volume ratio of 1:9 using the Auto Add function.

Example 3-2. Dilution of second analysis sample and standard

The second analysis method was used by mixing a sample and a standard material with a matrix in a volume ratio of 1:9 using the auto addition function.

<Example 4. Performance of analysis>

During sample analysis, an adduct is formed when multiple reaction monitoring (MRM) is set according to mobile phase conditions, matrix effect, ESI mode of MS/MS, column conditions, etc. In particular, in the case of veterinary drugs such as Ionophore, it is easily combined with cations such as Li ⁺ , NH ₄ ⁺ , Ca ₂ ⁺ , and Mg ₂ ⁺ . Among them, potassium and sodium are commonly present in the analysis environment, so adduct formation is more difficult. easy. Considering these adducts, multiple reaction monitoring (MRM) was set for each laboratory-specific analysis component. Representative examples of adducts are shown in Table 3 below.

Positive adducts Negative adducts +H +1 -H ₂ OH -19 +H ₂ O +18 -H -One +Na +23 +Na-2H +20 +CH ₃ OH+H +33 +Cl +35 +K +39 +K-2H +37 +ACN+H +42 +FA-H +45

Example 4-1. Performing the first analysis

For the first analysis, GC-MS/MS and LC-MS/MS were performed under the following conditions.

○ GC-MS/MS conditions

As the carrier gas of GC, helium was used at a flow rate of 1.5 ml per minute. The injection part used the splitless mode and the temperature was set at 250℃. Transfer line temperature and ion source temperature were set to 300℃ and 230℃, respectively. The oven temperature was analyzed by injecting the sample at 90°C, maintaining it for 3 minutes, increasing it to 120°C at a rate of 20°C per minute, and then raising the temperature to 300°C at a rate of 8°C per minute and maintaining it for 3 minutes.

GC Agilent, 7890A GC System Column HP-5MS UI (30 m × 0.25 mm(i.d), 0.25 ㎛) Flow rate 1.5 ml/min, Helium (99.9999%) Injector -Temperature: 250 ℃
-Pulsed splitless mode 15psi, 3 min Injection volume: 1µl Oven 90 ℃ (3 min hold) → 20 ℃/min → 120 ℃ → 8 ℃/min → 300 ℃ (3 min hold)
※ Total run time: 30 min MS/MS Agilent, GCMS-TQ7000 Ionization Electron ionization (EI), -70 eV MS conditions · Multiple reaction monitoring (MRM) mode
Electron multiplier voltage: tuning value
Loop time: 1 sec
· Transfer line: 300 ℃, Ion source 230 ℃

○ LC-MS/MS conditions

As the mobile phase of LC, an aqueous solution in the form of 5mM Ammonium formate and 0.1% (w/v) Formic acid and a methanol solution in the form of 5mM Ammonium formate and 0.1% (w/v) Formic acid were mixed and used as a mobile phase. In this case, the sensitivity to classify/analyze compounds was remarkably better than when an individual solution of Ammonium formate or Formic acid was used as a mobile phase. In this case, it showed higher sensitivity and selectivity compared to the experiment using water or methanol containing 5-10 mM Ammonium formate and water or methanol containing 0.1-0.5% Formic acid as mobile phases, respectively. In addition, it was confirmed that tailing appeared in Tetracycline series when each was used rather than when Ammonium formate or Formic acid was used together.

Example 4-2. Performing the second analysis

While performing the first analysis, the second analysis was performed under the following conditions.

Polar substances, which are the components to be analyzed in the second analysis method, can have different analysis results depending on the pH. Therefore, the pH of the mobile phase is adjusted using Ammonium formate, Ammonium hydroxide, and Formic acid to obtain 10 mM Ammonium formate and 20 mM Ammonium hydroxide. By mixing an aqueous solution (mobile phase A) and 90% (v/v) Acetonitrile solution (mobile phase B) of 0.2% (w/v) Formic acid, the mobile phase composition was set to pH 2.8-7.9 to improve selectivity and sensitivity. .

<Example 5. Classification of analyte compounds>

When the compounds were analyzed by the above method, compounds suitable for GC-MS/MS and LC-MS/MS of the first analysis, and LC-MS/MS of the second analysis were classified as follows.

<Example 6. Characteristics of the analyte compound>

As a result of analyzing the properties of the compounds classified as described above, a component having an octanol-water partition coefficient (log P) of -1 or more is a compound that can be analyzed by the first analysis method, and the octanol-water partition coefficient (log P) Components with a value of less than -1 were found to be classified by the second analysis method.

Log P represents the ratio of the concentration of the compound dissolved in the water and octanol layer when the compound is dissolved in two unmixed solvents, water and octanol. Through this value, the compound is hydrophilicity. It can be predicted whether or not it has hydrophobicity. The higher the value, the greater the distribution of the compound in the highly hydrophobic octanol layer, so it can be expected that the hydrophobicity is large.

<Example 7. Confirmation of differences in analysis results according to column selection>

The inventors of the present invention attempted to select a column for LC that satisfies most of the criteria for validating components while being easy to analyze for non-polar substances, which are components to be analyzed, in LC-MS/MS of the first analysis.

Accordingly, through a prior experiment, a C18 silica column and a C8 silica column were used for comparison. C18 Silica Column It is well known that it is commonly used for simultaneous multi-component analysis. It consists of 18 carbons, and is mainly used for separation of non-polar substances based on log P values. However, since the components of the compound to be analyzed of the present invention have log P values ranging from non-polar to polar, a C8 silica column and a C18 silica column were compared to analyze all of them.

As a result, it was confirmed that the use of a Waters ACQUITY UPLC BEH C8 silica column for the analysis of various types such as ionophore, quinolone, and sulfonamide is effective in classifying and analyzing compounds in the present invention. That is, when using the C8 column than the C18 column, a wider range is possible, and since the column of small particles (1.7 μm) was used, it was excellent in terms of analysis speed, resolution and sensitivity, and the advantage of reducing the medium effect. There was this.

Accordingly, as a result of the first analysis using a C8 silica (octasilane) column, in the case of residual pesticides, log P could be included as an analysis component until log P is -1 or more, and in the case of veterinary drugs, Ionophore-based, Quinolone-based, It was confirmed that the analysis of 4 groups of mycotoxins, such as sulfonamide, was possible. On the other hand, when a C18 silica (octadecylsilane) column or a dodecylsilane (C-12) column was used, the selectivity and sensitivity of the Ionophore system and the Tetracycline system were lowered, making analysis difficult.

Since the LC-MS/MS of the second analysis method is a method for analyzing polar substances, it was analyzed using a HILIC column for LC. In general, HILIC columns have a structure in which a hydroxy group is attached to a silica base, and the hydroxy group is hydrogen bonded to -OH of a water molecule, and the hydroxy group of a water molecule retains a polar substance. The HILIC column has disadvantages that it cannot be used for many materials because it cannot select methanol as the mobile phase according to its characteristics, the water ratio cannot be more than 50%, and the pH is limited. Since some components such as Tetracycline system are detected according to the pH of the mobile phase in the target materials of the second analysis method, it is necessary to select a column that has a wide pH range and a polarity retention. Therefore, in the second analysis method, a SeQuant PEEK coated ZIC-cHILIC (pH 3~8) column was selected, which is a positive/anionic column at the end of the chain because ZIC treatment is performed, which has a wide pH control range and most components to be analyzed are effective. It was confirmed to be suitable for Analysis 2 in accordance with the verification guidelines. As a result of performing the second analysis method using the column, polar pesticides, Tetracycline, Macrolide, Fumonisin, etc. were included as components to be analyzed.

<Example 8. Final analysis result-Method validation>

The verification of each method performed as described above is based on the criteria set by the European Committee of Standardization (CEN) 2008 Standard method EN 156222 CEN and SANCO guideline/12571/2013, based on linearity, sensitivity, and selectivity. ), accuracy, and precision to confirm that the analysis was successful.

In addition, it is confirmed that the concentration is appropriate for the limit of detection (LOD) and limit of quantification (LOQ) for each compound, and it can be seen that individual separation and analysis of each compound is possible as shown in FIGS. 2 to 4.

Group Compound r ² LOD (㎍/kg) LOQ (㎍/kg) Analysis 1 Residual pesticide 0.9993 ~ 0.9999 0.12 ~ 1.54 0.40 ~ 5.13 Veterinary medicine 0.9991 ~ 0.9999 5.01 ~ 20.13 16.69 ~ 67.03 Mycotoxin 0.9996 ~ 0.9999 0.51 ~ 6.11 1.70 ~ 20.35 Analysis 2 Residual pesticide 0.9992 ~ 0.9999 8.21 ~ 13.76 27.34 ~ 43.29 Veterinary medicine 0.9993 ~ 0.9999 1.27 ~ 21.00 4.23 ~ 69.93 Mycotoxin 0.9997 ~ 0.9999 3.42 ~ 4.66 11.39 ~ 37.93

Group Compound Precisions(%RSD) Recovery intra-day inter-day level 1 level 2 level 3 Analysis 1 Residual pesticide 1.3~8.3 2.2~7.0 66.1~100.5 79.5-108.2 77.8~110.0 Veterinary medicine 1.3~8.2 1.2~9.4 63.0~101.2 68.9-108.6 71.7~105.5 Mycotoxin 2.4~7.3 6.3~8.5 67.5-100.3 70.3~101.1 70.9~103.8 Analysis 2 Residual pesticide 2.3~3.1 2.6~3.8 77.2~98.9 78.3~97.2 76.5~100.2 Veterinary medicine 1.3~10.2 2.0~9.8 61.2~95.0 66.5~100.1 70.6~99.5 Mycotoxin 3.1~5.9 2.3~4.8 70.3~79.3 73.3~78.1 73.2~80.8

Claims (15)

Pretreatment of a feed sample for a first analysis and a second analysis;
Perform the first analysis by performing GC (gas chromatography) and LC (liquid chromatography) using the feed sample pretreated for the first analysis, and then perform the LC (liquid chromatography) using the feed sample pretreated for the second analysis. Chromatography) to perform a second analysis; And,
Summing the results of the first analysis and the second analysis to analyze the residual amounts of pesticides, veterinary medicines and mycotoxins in the feed;
As an analysis method comprising,
In the first pre-processing step of the feed for the analysis method, the feed sample Na ₂ EDTA- maekil vane (Na ₂ EDTA-McIlvaine) were mixed in buffer solution, stirred by the addition of NaCl and MgSO ₄ in acetonitrile as an extraction solvent, Simultaneous analysis of pesticide residues, veterinary medicines and mycotoxins in feed, characterized in that LC is performed with the first supernatant obtained by centrifugation. The method of claim 1,
The compound analyzed through the first analysis is a substance with an octanol-water partition coefficient (log P) of -1 or more, and the substance analyzed through the second analysis has a value of the octanol-water partition coefficient (log P). Simultaneous analysis of pesticide residues, veterinary medicines and mycotoxins in feed, characterized in that the material is less than -1. The method of claim 1,
The GC of the first analysis is GC-MS/MS (gas chromatography-mass spectrometry/mass spectrometry), and the LC of the first and second analyzes is LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry). Analysis) simultaneous analysis of pesticide residues, veterinary medicines, and mycotoxins in feed. The method of claim 1,
Among the substances to be analyzed in the first analysis, the detection limit concentration of pesticide residues is 0.12 ~ 1.54 ㎍/kg, the detection limit concentration of veterinary drugs is 5.01 ~ 20.13 ㎍/kg, and the detection limit concentration of mycotoxin is 0.51 ~ 6.11 ㎍ Simultaneous analysis of pesticide residues, veterinary medicines and mycotoxins in feed, characterized by /kg. The method of claim 1,
Among the substances to be analyzed in the first analysis, the quantitative limit concentration of residual pesticide is 0.40 ~ 5.13 ㎍/kg, the quantitative limit concentration of veterinary medicine is 16.69 ~ 67.03 ㎍/kg, and the quantitative limit concentration of mycotoxin is 1.70 ~ 20.35 Simultaneous analysis of pesticide residues, veterinary medicines and mycotoxins in feed, characterized in that ㎍ / kg. The method of claim 1,
Among the substances to be analyzed in the second analysis, the detection limit concentration of pesticide residues is 8.21 ~ 13.76 ㎍/kg, the detection limit concentration of veterinary drugs is 1.27 ~ 21.00 ㎍/kg, and the detection limit concentration of mycotoxin is 3.42 ~ 4.66 ㎍/kg/ Simultaneous analysis of pesticide residues, veterinary medicines and mycotoxins in feed, characterized in that in kg. The method of claim 1,
Among the substances to be analyzed in the second analysis, the quantitative limit concentration of residual pesticide is 27.34 ~ 43.29 ㎍/kg, the quantitative limit concentration of animal medicine is 4.23 ~ 69.93 ㎍/kg, and the quantitative limit concentration of mycotoxin is 11.39 ~ 37.93 ㎍/kg/kg. Simultaneous analysis of pesticide residues, veterinary medicines and mycotoxins in feed, characterized in that in kg. delete The method of claim 1,
Simultaneous analysis of pesticide residues in feed, veterinary medicines and mycotoxins, characterized in that GC is performed with the second supernatant obtained by centrifugation by adding PSA (Primary-secondary Amine) and MgSO ₄ to the first supernatant . The method of claim 1 or 9,
The first supernatant and the second supernatant are simultaneously analyzed for residual pesticides, veterinary medicines and mycotoxins in feed, characterized in that filtering through a syringe filter of 0.2 μm or less. The method of claim 1,
In the pretreatment step of the feed for the second analysis method, the homogenized feed sample is mixed with a methanol solution in which formic acid is dissolved, followed by adding NaCl and MgSO ₄ , stirring, and performing LC with the supernatant obtained by centrifugation. Simultaneous analysis of pesticide residues, veterinary medicines, and mycotoxins in feed. The method of claim 11,
The supernatant is mixed with C18 silica powder, stirred, and obtained by centrifugation. A method for simultaneous analysis of pesticide residues in feed, veterinary medicines and mycotoxins. The method of claim 12,
The supernatant is filtered through a syringe filter of 0.2 µm or less. Simultaneous analysis of pesticide residues in feed, veterinary medicines and mycotoxins. The method of claim 1,
The mobile phase solution of the LC in the first analysis method is A solution, which is an aqueous solution of ammonium formate containing formic acid; And, B solution, a methanol solution of ammonium formate containing formic acid; Simultaneous analysis of pesticide residues in feed, veterinary medicines and mycotoxins, characterized in that using a mixture of ammonium formate containing (formic acid). The method of claim 1,
The mobile phase solution of the LC in the second analysis method is a solution A, which is an aqueous solution containing ammonium formate and ammonium hydroxide; And, A method of simultaneous analysis of pesticide residues in feed, veterinary medicines and mycotoxins, characterized in that using a mixture of B solution, which is an acetonitrile solution containing formic acid.

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