JP4998151B2 - Purification method of pesticide residues in crude drug samples - Google Patents

Description

  The present invention relates to a method for purifying residual agricultural chemicals in herbal medicine samples.

  Conventionally, various agricultural chemicals have been used to increase the productivity of agricultural products. In recent years, interest in residual substances in agricultural products has increased and importance has been placed on measurement of residual substances. Corresponding to this, the Ministry of Health, Labor and Welfare is also trying to set a standard for residual substances remaining in agricultural products (Non-Patent Document 1).

  Non-Patent Document 1 describes a “simultaneous test method for agricultural chemicals by GC / MS (agricultural products)” as a simultaneous test method, and extraction of test solution preparation in the case of fruits, vegetables, herbs, tea and hops The process is described as follows.

“For fruits, vegetables and herbs, weigh 20.0 g of sample. For tea and hops, add 20 mL of water to 5.00 g of sample and leave for 15 minutes.
50 mL of acetonitrile is added to this and homogenized, followed by suction filtration. Add 20 mL of acetonitrile to the residue on the filter paper, homogenize, and suction filter. The obtained filtrates are combined, and acetonitrile is added to make exactly 100 mL.
Take 20 mL of the extract, add 10 g of sodium chloride and 20 mL of 0.5 mol / L phosphate buffer (pH 7.0), and shake. After standing, discard the separated aqueous layer. Anhydrous sodium sulfate is added to the acetonitrile layer for dehydration, and anhydrous sodium sulfate is filtered off, and then the filtrate is concentrated at 40 ° C. or lower to remove the solvent. Add 2 mL of a mixture of acetonitrile and toluene (3: 1) to the residue and dissolve. "

When the present inventors examined the ratio of acetonitrile and water in the acetonitrile aqueous solution to be subjected to column chromatography, the volume ratio of 2: 3 was optimal considering only the addition recovery rate of agricultural chemicals. In 2: 3, there were many impurities and it became difficult to analyze.
Notification of Food Safety No.0124001, Director of Food Safety Department, Food and Drug Administration, Ministry of Health, Labor and Welfare

  An object of the present invention is to provide a method for purifying a residual agricultural chemical in a herbal medicine sample that can increase the removal rate of impurities while maintaining the addition and recovery rate of the agricultural chemical in the herbal medicine sample.

The gist of the present invention is as follows.
(1) Next step:
(I) extracting a crude drug sample with an aqueous acetonitrile solution having a volume ratio of acetonitrile to water of 1: 1 to 19: 1;
(Ii) a step of adjusting the volume ratio of acetonitrile and water in the aqueous acetonitrile solution after extraction to 2: 3 to 3: 2, and (iii) a solution in which the volume ratio of acetonitrile and water is adjusted by the step (ii). A method for purifying a pesticide residue in a herbal medicine sample, comprising a step of subjecting to column chromatography.
(2) The method according to (1) above, wherein the herbal medicine sample is a herbal medicine or a Chinese medicine preparation.
(3) The method according to (1) above, wherein the herbal medicine sample is selected from Soyo, Senna, Biwayo, Kankyo, Saishin, Salamander, Kyokats, Zenko, Clove, Moko, Ryokyo and Wakakatsu.
(4) The method according to any one of (1) to (3), wherein the residual agricultural chemical is a pyrethroid agricultural chemical.

  ADVANTAGE OF THE INVENTION According to this invention, the removal rate of a foreign material can be raised, maintaining the addition collection | recovery rate of an agricultural chemical, and mixing of the foreign material seen with a normal refinement | purification method can be prevented.

  The herbal medicines that are the subject of the present invention include, for example, Soyo, Senna, Biwayo, Kankyo, Saishin, Salamander, Kyokats, Zenko, Clove, Moko, Ryokyo, Tokyo Katsu, Akyo, Ireisen, Inchinkou, Fennel, Engosaku, Ogi, Ogon , Grasshopper, auren, onji, gaiyou, cashew, kakkon, kasseki, carocon, caroten, licorice, kyoukyo, kikuka, pheasant, kisokon, kyounin, kuzin, kei gai, keihi, koka, kojin, kobushi, kobayoku, gosh , Burdock, sesame, trash, psycho, hawthorn, sanshishi, sanshuyu, sansounin, sanjak, kangzhou, jikoppi, sicon, tsutsuri, peonies, shazenshi, jukujiou, sukusha, shi Scarlet, shrimp, ginger, shinny, gypsum, senkyu, senkotsu, centai, sardine, scorpion, soboku, daiou, taiyou, takusha, chikujo, chikutsujinjin, chimo, chayou, butterfly, chorei, chimpi, tennansho, tenma , Tenmondou, Capsicum, Toki, Tounin, Spruce, Tokon, Tochu, Dokatsu, Carrot, Nindo, Baimo, Bakuga, Bakumondo, Hakka, Hamaboufu, Hange, Beekou, Bakushi, Bakujutsu, Binrouji, Bokufu, Ayumu , Boxok, Buttonpi, Borei, Maou, Machinin, Mokutsu, Yokuinin, Ryuganiku, Ryukotsu, Ryutan, Forsythia, Rennik, preferably Soyo, Senna, Biwayo , The environment, up-to-date, pepper, extortion, all houses, clove, woodworking, Ryo today include the Wakyoukatsu.

  The pesticide to be purified in the present invention is not particularly limited, and examples thereof include tephritrin, cinerine I, cinerine II, cyhalothrin, cypermethrin, jasmolin I, jasmolin II, pyrethrin I, pyrethrin II, flucitrinate, fulvalinate, Pyrethroid pesticides such as deltamethrin, acrinathrin, permethrin I, permethrin II, cyfluthrin, silafluophene, fenvalerate, esfenvalerate, fipronil, bifenthrin, fenpropatoline, tralomethrin, etc .; Son, bifenox, cyfluthrin, fulvalinate, tefluthrin, propizzamide, dicohol, binapacril, chlorbenzilate, quin Organochlorine pesticides such as Zen, Endosphan, Prosimidone, Chlorpropyrate, Bromopropyrate, Tetradiphone, Halfenprox, Fluorimide, Chlorophenetol, Holpet, Endrin; Metolachlor, Triadimenol, Quinomethionate, Paclobutra Zole, pretilachlor, flusilazole, propiconazole, lenacyl, tenyl chlor, acetamiprid, flutolanil, mefenacet, phenarimol, vitertanol, pyridaben, pyrimidifene, EPTC, esprocarb, pendimethalin, microbutanyl, tricyclazole, cyproconazole, mepronate Pyriproxyfen, difenoconazole, imibenconazole, triflura Nitrogen-containing pesticides such as phosphorus, metribuzin, trichlamide, hexaconazole, etoxazole, cihalohop butyl, and cavenstrol; methylenedioxy pesticides such as piperonyl butoxide; acetanilide pesticides such as alachlor; butyrate, isoprocarb, dietofencarb, Examples include carbamate pesticides such as methiocarb, chloroprofam, pyrimicarb, thiobencarb, piributichalbu, bendiocarb, etiophencarb, fenobucarb, and carbaryl; and organic sulfur pesticides such as dimethipine and benfrecetate.

In the extraction process in the present invention, an acetonitrile aqueous solution having a volume ratio of acetonitrile to water of 1: 1 to 19: 1 is used as an extraction solvent from the viewpoint of herbal characteristics (water content is 10% or less). The ratio of acetonitrile to water in the acetonitrile aqueous solution is preferably a volume ratio of 7: 3 to 9: 1, more preferably 4: 1. The usage-amount of acetonitrile aqueous solution is 8-12 mL normally per 1g of samples, Preferably it is 9-11 mL. During the extraction, there is no restriction on the mixing order of the sample and the solvent. An aqueous acetonitrile solution may be prepared in advance and then mixed with the sample. After mixing the sample with one of acetonitrile and water, the other solvent is added. May be.
The amount of the sample used is not particularly limited, but is usually 1 to 4 g.

  The sample and solvent mixture is then shaken thoroughly. Thereafter, the mixture is centrifuged and the supernatant is taken. Preferably, the above acetonitrile aqueous solution is added to the residue (usually 10 to 30 mL, preferably 15 to 25 mL per 1 g of residue), and after shaking, the operation of centrifuging and collecting the supernatant is repeated one or more times. Combine the supernatants.

  In the present invention, water is added to the extracted acetonitrile aqueous solution (the supernatant) to adjust the volume ratio of acetonitrile to water in the solution to 2: 3 to 3: 2. By performing this process, contamination can be prevented. The amount of water added here is usually 6 to 20 mL, preferably 9 to 15 mL, per 1 g of the sample.

  In this invention, it is preferable to adjust pH of the extract obtained as mentioned above to 3.5-4.5 from the point which improves the collection rate of an agrochemical. The pH is more preferably 3.8 to 4.2, and most preferably 4.0. Although there is no restriction | limiting in particular as a solution used in order to adjust pH, Usually, acidic aqueous solution, such as phosphoric acid aqueous solution, acetic acid aqueous solution, formic acid aqueous solution, paratoluenesulfonic acid aqueous solution, sodium hydrogencarbonate aqueous solution, sodium carbonate aqueous solution, sodium hydroxide aqueous solution, etc. These alkaline aqueous solutions are used alone or in combination. The volume ratio of acetonitrile to water in the acetonitrile aqueous solution after pH adjustment is preferably 2: 3 to 3: 2, more preferably 9:11 to 11: 9.

  In the present invention, the solution adjusted in pH as described above is subjected to column chromatography.

  The column chromatography used in this step is not particularly limited. For example, C18 column chromatography, PSA column chromatography, graphite carbon black (GCB) column chromatography, SAX / PSA column chromatography, Florisil column chromatography, etc. are used alone. Or in combination.

  According to the present invention, it is possible to increase the removal rate of contaminants while maintaining the addition and recovery rate of agricultural chemicals even in a crude drug sample that is difficult to analyze with a large amount of contamination by a normal purification method. By analyzing the sample pretreated by the method using, for example, GC / MSD (Gas Chromatograph Device with Mass Spectrometer), it is possible to analyze the residual pesticide in the crude drug with high accuracy.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, the scope of the present invention is not limited to a following example.

(Example 1 and Comparative Example 1)
1. Sample Weighing 2.0 g (1.95 to 2.04 g) of Soyo powder was accurately weighed into a 50 mL centrifuge tube with a drain plug.

2. Extraction 2-1. 20 mL of acetonitrile / water mixture (volume ratio 4: 1) was added to the centrifuge tube.
2-2. The centrifuge tube was capped and shaken under the following conditions.
Shaking time: 10 minutes, shaking speed: 200 times / minute 2-3. After shaking, the mixture was centrifuged under the following conditions.
Rotational speed: 3000 rpm, centrifugation time: 5 minutes 2-4. A funnel with a cotton plug was placed on a 100 mL Erlenmeyer flask and washed with about 2 mL of acetonitrile / water mixture (volume ratio 4: 1).
2-5. The funnel with a cotton plug was placed on a 200 mL Erlenmeyer flask, the centrifuge tube was tilted, and the supernatant was transferred to the funnel.
2-6. The operation of 2-1 to 3 was performed once again on the sample in the centrifuge tube. Transfer to a 2-5 funnel and combine with previous extract.

3. Adjust the pH of the extract. Wash the funnel of 3-1.2-6 with 10 mL of water. After removing the funnel, 15 mL of water (Example 1) or 30 mL (Comparative Example 1) was added to the extract and mixed well.
3-2. When the pH of the 3-1 extract was measured using a pH test paper, the pH was about 5.7.
A 10% phosphoric acid aqueous solution or a saturated aqueous sodium hydrogen carbonate solution was added to the extract to adjust the pH to 4.

4). C18 (5g) -PSA-GCB column treatment (preparation under C18 cartridge)
4-1. An injection needle was attached to a C18 (5 g) cartridge, placed on an assembly funnel, and a beaker was placed at the bottom. 20 mL of acetonitrile was added to the top of the cartridge. When the solvent at the top of the cartridge was exhausted, 20 mL of water and 20 mL of a water / acetonitrile mixture (volume ratio 3: 2) were sequentially added.

(Adsorption of pesticides)
The adjustment solution of 4-2.3-2 was added to a C18 (5 g) cartridge. A reservoir cartridge equipped with an adapter was attached to a C18 (5 g) cartridge, and about 65 mL (Example 1) or about 80 mL (Comparative Example 1) of 3-2 adjustment liquid was added to the reservoir cartridge. When the adjustment liquid at the top of the C18 (5 g) cartridge was exhausted, the reservoir cartridge with the adapter attached was removed, and the solvent in the C18 (5 g) cartridge was pushed out using a syringe. The introduced liquid was discarded.
4-3. A syringe needle was attached to the PSA cartridge, placed on an assembly funnel, and a beaker was placed at the bottom. 10 mL of acetonitrile was added to the top of the cartridge. The introduced liquid was discarded.

(Preparation for GCB cartridge)
4-4. A syringe needle was attached to the GCB cartridge, placed on an assembly funnel, and a beaker was placed at the bottom. Hexane 10 mL, acetone 10 mL, and acetonitrile 10 mL were sequentially added to the top of the cartridge. The introduced liquid was discarded.
4-5. Add 3 tablespoons of anhydrous sodium sulfate to a 100 mL Erlenmeyer flask.

(Elution of adsorbed pesticides)
A reservoir cartridge with an adapter attached to a 4-6.4-2 C18 (5 g) cartridge was attached. A 4-3 PSA cartridge was placed under the C18 (5 g) cartridge, and a 4-4 GCB cartridge was placed under the PSA cartridge. A 4-5 100 mL Erlenmeyer flask was placed under the GCB cartridge.
4-7. 50 mL of acetonitrile was weighed and added to the reservoir cartridge at the top of the C18 (5 g) cartridge.
4-8. When the solvent at the top of the C18 (5 g) cartridge was exhausted, the reservoir cartridge with the adapter attached was removed, and the solvent in the C18 (5 g) cartridge was pushed out using a syringe.
4-9. When the solvent at the top of the PSA cartridge was exhausted, the solvent in the cartridge was pushed out using a syringe, and the cartridge was removed.

5. Separation of dehydrating agent 5-1. The funnel with the fold filter paper was placed on a 100 mL Erlenmeyer flask and washed with about 5 mL of acetonitrile.
5-2. A funnel with pleated filter paper was placed on top of a 200 mL eggplant-shaped flask.
The eluate of 5-3.4-9 was transferred to a funnel on which a fold filter paper was placed. About 5 mL of acetonitrile was added to a 4-9 100 mL Erlenmeyer flask and washed. The same operation was performed twice more.

6). The eluate of eluate concentration 6-1.5-3 was concentrated under reduced pressure to dryness using a rotary evaporator.
Hexane 5mL was added to the concentrated liquid of 6-2.6-1, and it concentrated under reduced pressure to 2mL.

7). SAX / PSA-florisil (1 g) column treatment (preparation of Florisil cartridge)
7-1. An injection needle was attached to the SAX / PSA cartridge, placed on an assembly funnel, and a beaker was placed at the bottom. 10 mL of hexane was added to the top of the cartridge. When the solvent at the top of the cartridge was exhausted, 10 mL of ethyl acetate and 10 mL of hexane were sequentially added. The introduced liquid was discarded.
7-2. An injection needle was attached to a Florisil (1 g) cartridge, placed on a prefabricated funnel, and a beaker placed on the bottom. 10 mL of hexane was added to the top of the cartridge. When the solvent at the top of the cartridge was exhausted, 10 mL of ethyl acetate and 10 mL of hexane were sequentially added. The introduced liquid was discarded.

(Preparation for SAX / PSA cartridge)
A 7-2 Florisil (1 g) cartridge was placed under the 7-3.7-1 SAX / PSA cartridge and a beaker was placed under the Florisil (1 g) cartridge.
2 mL of hexane was added to the concentrated solution of 7-4.6-2 and dispersed uniformly using an ultrasonic cleaner. The paste was added to the top of the SAX / PSA cartridge using a Pasteur pipette.
4 mL of hexane was added to the 200 mL eggplant type flask of 7-5.7-4. It was dispersed uniformly using an ultrasonic cleaner and added to the top of the SAX / PSA cartridge. The same operation was performed again. The introduced liquid was discarded.
7-6. When the solvent at the top of the Florisil (1 g) cartridge was exhausted, a 100 mL eggplant-shaped flask was placed at the bottom of the Florisil (1 g) cartridge. 30 mL of a hexane / ethyl acetate mixture (3: 1) was added to the top of the SAX / PSA cartridge.
7-7. When the solvent at the top of the SAX / PSA cartridge was exhausted, the solvent in the cartridge was pushed out using a syringe, and the cartridge was removed.
7-8. When the solvent at the top of the Florisil (1 g) cartridge was exhausted, the solvent in the cartridge was extruded using a syringe.

8). The eluate of eluate concentration 8-1.7-8 was concentrated under reduced pressure to about 2 mL at 40 ° C. or lower using a rotary evaporator.
Acetone 5 mL was added to 8-2.8-1 concentrated solution, and concentrated under reduced pressure to about 1 mL. The same operation was performed again.

9. The concentrated solution of analytical sample preparation 9-1.8-2 was transferred to a 2 mL volumetric flask and made up to 2 mL with 0.01% polyethylene glycol acetone.
9-2. Using a Pasteur pipette, the inside of the volumetric flask was well stirred.
9-3. About 1 mL of the sample solution was placed in a vial and a cap was set.
9-4. The cap was closed using a crimper.

10. GC / MSD Measurement According to the following conditions, analysis by GC / MSD was performed.
Detector: MSD
Column: DB-1MS 15m long, 0.25mm inner diameter, 0.25μm film thickness
Column temperature: 100 ℃ (2min hold)-(20 ℃ / min)-194 ℃-(5 ℃ / min) -220 ℃-(15 ℃ / min)-300 ℃ (5.77min hold)
Inlet temperature: 250 ° C
Interface: 300 ° C
Injection volume: 2.0 μL
Injection method: Pulsed splitless pulse pressure: 20.0 psi
Pulse time: 1.00 min
Carrier gas: helium average linear velocity: 63 cm / sec

11. Setting of selected ion monitoring (SIM) and ion setting of target pesticide The ions for quantification and fragment ions (qualifier ions 1 and 2) were selected from the elution order of the target pesticide.
Table 1 shows the SIM settings and the target pesticide ion settings.

  The analysis results are shown in Table 2. Cypermethrin is a mixture of isomers, and the number of peaks to be confirmed differs depending on the detection method or the measurement conditions of the equipment used, and four types of peaks are detected under the current measurement conditions. Therefore, they are designated as cypermethrin I, cypermethrin II, cypermethrin III, and cypermethrin IV, respectively. Similarly, fenvalerate is a mixture of isomers, and two peaks are detected under the current measurement conditions, so that fenvalerate I and fenvalerate II are used.

(Criteria)
(A) A recovery rate of 70-120% was determined to be applicable.
The recovery rate outside the range is marked with a strikethrough.
(B) Qualifier ion determination:
Target ions (calibration curves are created when performing quantitative analysis; ions to be quantified; quantification ions), qualifier ions (Qualifier ions; target ions to confirm whether the substance is really the substance when performing quantitative analysis) "I" for calculating the ratio of ions) 1 (confirmation ions 1) and qualifier ions 2 (confirmation ions 2) can be confirmed "○", target ions and qualifier ions 1 or qualifier ions 2 can be confirmed Is shown in Table 2 as “Δ”. In addition, the case where the target ion could not be confirmed and the case where the target ion could be confirmed but neither the qualifier ion 1 nor the qualifier ion 2 could be confirmed was shown as “x”.

  From Table 2, both Example 1 and Comparative Example 1 showed good results in the addition recovery rate, but in the blank qualifier ion determination, Example 1 had less “△” It turns out that there is little mixing. For cypermethrin II, the peak of the crude drug-derived contaminant is detected at the same position, so the blank qualifier of the example is ○.

Claims (5)

Next step:
(I) extracting a crude drug sample with an aqueous acetonitrile solution having a volume ratio of acetonitrile to water of 7: 3 to 19: 1;
(Ii) a step of adjusting the volume ratio of acetonitrile and water in the aqueous acetonitrile solution after extraction to 9:11 to 3: 2, and (iii) a solution in which the volume ratio of acetonitrile and water is adjusted by the step (ii). A method for purifying a pesticide residue in a herbal medicine sample, comprising a step of subjecting to column chromatography. The volume ratio of acetonitrile and water in the acetonitrile aqueous solution used in step (i) is 7: 3 to 9: 1, and the volume ratio of acetonitrile and water in the acetonitrile aqueous solution after extraction in step (ii) is 9:11 to 11: The method of claim 1, wherein the method is adjusted to 9. The method according to claim 1 or 2 , wherein the herbal medicine sample is a herbal medicine or a Chinese medicine preparation. 3. The method according to claim 1 or 2 , wherein the herbal medicine sample is selected from Soyo, Senna, Biwayo, Kankyo, Saishin, Salamander, Kyokats, Zenko, Clove, Mokko, Ryokyo and Oyakukatsu. The method according to any one of claims 1 to 4 , wherein the residual agricultural chemical is a pyrethroid agricultural chemical.