JP5045282B2 - 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 “Simultaneous testing method for agricultural chemicals and the like by GC / MS (agricultural products)” as a simultaneous testing method. In this method, after extracting a sample with an acetonitrile aqueous solution, the pH of the extract is adjusted to 7.0, and then subjected to column chromatography.

  However, when the present inventors applied this method to a crude drug, the recovery rate of the agrochemical decreased, and sufficient results could not be obtained.

Notification of Food Safety No.0124001, Director of Food Safety Department, Food and Drug Administration, Ministry of Health, Labor and Welfare, “A test method for substances that are ingredients of agricultural chemicals, feed additives or veterinary drugs remaining in food”

  An object of the present invention is to provide a method capable of purifying a residual agricultural chemical with a high recovery rate in a herbal medicine sample.

The gist of the present invention is as follows.
(1) Next step:
(I) a step of extracting a crude drug sample with an aqueous acetonitrile solution;
(Ii) adjusting the pH of the extract to 3.5 to 4.5, and (iii) subjecting the solution adjusted in the above step (ii) to column chromatography, the residual pesticide in the crude drug sample The purification method of
A method for purifying a residual agricultural chemical in a herbal medicine sample, wherein the residual agricultural chemical is a compound having an ester structure .
(2) The method according to (1) above, wherein the herbal medicine sample is a herbal medicine or a Chinese medicine preparation.
(3) The above-mentioned herbal medicine sample is selected from citrus, licorice, keihi, sanshi, chimpi, biwayo, carrot, tonin, chorei, soyo, diou, taiso, senna, peony, salamander, sanshuyu, kojin, buttonpi, onji and ogi The method according to (1).
( 4 ) The method according to any one of (1) to (3), wherein the compound having an ester structure is a pyrethroid pesticide.

  According to the present invention, it is possible to purify residual agricultural chemicals in herbal medicine samples with a high recovery rate. Therefore, the method of the present invention is useful as a pretreatment method in a method for analyzing residual agricultural chemicals in herbal medicine samples.

  The herbal medicines that are the subject of the present invention include, for example, citrus, apricot, irresen, inchinkou, fennel, engosac, ogi, ogon, oak, ouren, onji, gaiyou, kashi, kakon, caseki, calocon, caronin, licorice, kikuka, kikuka , Pheasant, kissokon, kyoukatsu, kyounin, kujin, kugai, keihi, kouka, kojin, kobushi, koubay, koboku, goshitsu, goshuyu, goboushi, sesame, trash, saiko, saisin, hawthorn, saoshishi, sasyuyu, salamander Sanyaku, Kanjio, Jikopi, Shikon, Shitsuri, Peonies, Shazenshi, Zukujiou, Shukusha, Shokyo, Shobakaku, Shouma, Shinyi, Gypsum, Senkyu, Zenko, Senkotsu, Ningtai, Senna, Sojutsu, Sakuhaku, Soboku, Soyo, Dai-o, Taiso, Takusha, Chikujo, Chikusetinjinjin, Chimo, Chayou, Clove, Butterflyfish, Chorei, Chimpi, Tennansho, Tenma, Tenmondo, Togashi, Toki, Tonin , Spruce, tocon, eucommia, dokatsu, carrot, nindo, baimo, bakuga, bakakumondo, mint, kingfisher, hangage, sandalwood, peony, sandalwood, biwayo, betel, beak, bushi, jumbo , Maow, machinin, mokutsu, mokko, yokoinin, ryugannik, ryukko, ryutan, ryokyou, forsythia, rennik, kyoko katsu, preferably kankyo, licorice, Torquecontrol, Sanshishi, citrus unshiu peel, Biwayou, carrots, the person, Chorei, sophistication, rhubarb, gymnastics, senna, peony, pepper, cornel, red ginseng, moutan bark, Onji, include the Radix Astragali.

  The pesticide to be purified in the present invention is not particularly limited, and examples thereof include teflutrin, cinerine I, cinerin II, cyhalothrin, cypermethrin I, cypermethrin II, cypermethrin III, cypermethrin IV, jasmolin I, jasmolin II, Pyrethrin I, pyrethrin II, flucitrinate, fulvalinate, deltamethrin, acrinatrin, permethrin I, permethrin II, cyfluthrin, silafluophene, fenvalerate I, fenvalerate II, esfenvalerate, fipronil, bifenthrin, fenpropatorin, tralomethrin, etc. Pyrethroid pesticides; dichlorane, bromobutide, chloromethoxynil, swep, diclofluuride, chlorfenson, bifenox, cyfluthrin, fulvalinate, te Organochlorine pesticides such as lutoline, propyzamide, dicohol, binapacryl, chlorbenzilate, quintozen, endosphan, procymidone, chlorpropyrate, bromopropyrate, tetradiphone, halfenprox, fluoroimide, chlorophenetol, holpet, endrin; Metolachlor, triadimenol, quinomethionate, paclobutrazole, pretilachlor, flusilazole, propiconazole, lenacyl, tenyl chlor, acetamiprid, flutolanil, mefenacet, phenarimol, vitertanol, pyridaben, pyrimidifene, EPTC, esprocarbura, pendimethaclazole, pendimethatriazole Cyproconazole, mepronil, tebuconazole, iprodione, tebufenpi Nitrogenous pesticides such as rad, pyriproxyfen, difenoconazole, imibenconazole, trifluralin, metribudine, trichlamide, hexaconazole, etoxazole, cihalohop butyl, cavenstrol; benzodioxole pesticides such as piperonyl butoxide Acetanilide pesticides such as chlore; Carbamate pesticides such as butyrate, isoprocarb, dietofencarb, methiocarb, chloroprofam, pirimicarb, thiobencarb, piributicarb, bendiocarb, etiofencarb, fenobucarb, carbaryl, etc .; organic sulfur pesticides such as dimethipine, benfrecetate, etc. Is mentioned.

  In the extraction step of the present invention, an acetonitrile aqueous solution is used as an extraction solvent from the viewpoint of the characteristics of herbal medicine (water content is 10% or less). The ratio of acetonitrile to water in the acetonitrile aqueous solution is preferably 1: 1 to 95: 5, more preferably 7: 3 to 9: 1, and most preferably 4: 1 in volume ratio. 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.

  Although the usage-amount of a sample does not have a restriction | limiting in particular, Usually, 1-20g, Preferably it is 1-4g.

  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.

  Preferably, water is added to the obtained supernatant in order to precipitate a water-insoluble solid content of the herbal medicine and reduce the viscosity of the extracted supernatant. The amount of water added here is usually 10 to 50 mL, preferably 15 to 25 mL, per 1 g of the sample.

  In the pH adjustment step in the present invention, the pH of the extract obtained as described above is adjusted to 3.5 to 4.5. When the pH is less than the lower limit, the compound having an ester structure such as pyrethroid type agrochemicals has an ester bond breakage, so that the recovery rate is remarkably reduced. The recovery rate is remarkably reduced due to the incorporation of. The pH is preferably 3.8 to 4.2, 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.

  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, the pesticide residue is purified with a high recovery rate even in a crude drug sample. By analyzing this by, for example, GC / MSD (Gas Chromatograph Device with Mass Spectrometer), the residual pesticide in the crude drug is removed. It is possible to analyze 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 Examples 1-3)
1. Sample Weighing 2.0 g (1.95 to 2.04 g) licorice 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, 24 mL of water 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 sodium hydrogen carbonate aqueous solution was added to the extract to adjust the pH to 1, 4, 6 or 7.
At pH 1, the extract was colored yellow and slightly turbid. At pH 4, no change was seen. At pH 6, turbidity was generated from the upper layer, and when it was left for a while, sediment was formed, so the experiment was stopped. At pH 7, there was no significant change with a slight turbidity.

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. About 20 mL of acetonitrile was added to the top of the cartridge. When the solvent at the top of the cartridge was exhausted, about 20 mL of water and 10 mL of a water / acetonitrile mixture (volume ratio 1: 1) were sequentially added.

(Adsorption of pesticides)
The adjustment solution of 4-2.3-2 was added to a C18 (5 g) cartridge. The reservoir cartridge with the adapter attached was attached to a C18 (5 g) cartridge, and about 64 mL of 3-2 adjustment solution 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. Elution was performed with 20 mL of a water / acetonitrile mixture (volume ratio 1: 1).

(Preparation under PSA cartridge)
4-3. A syringe needle was attached to the PSA cartridge, placed on an assembly funnel, and a beaker was placed at the bottom. About 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. 10 mL of acetonitrile was added to the top of the cartridge. The introduced liquid was discarded.

(Elution of adsorbed pesticides)
A reservoir cartridge with an adapter attached to a 4-5.4-2 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 100 mL Erlenmeyer flask was placed under the GCB cartridge.
4-6. 50 mL of acetonitrile was weighed and added to the reservoir cartridge at the top of the C18 (5 g) cartridge.
4-7. 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.
4-8. 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.
4-9. When the solvent at the top of the GCB cartridge was exhausted, 20 mL of hexane saturated acetonitrile was added to the top of the GCB cartridge. When there was no solvent at the top of the cartridge, the solvent in the cartridge was pushed out using a syringe and left as it was for 5 minutes.

5. The eluate of 5-1.4-9 eluate concentration was concentrated under reduced pressure to 2 mL using a rotary evaporator.
2 mL of hexane was added to the concentrated solution of 5-2.5-1, and concentrated under reduced pressure to 1 to 2 mL.

6). Florisil-SAX / PSA column treatment (preparation of Florisil cartridge)
6-1. A syringe was attached to a Florisil (1 g) cartridge (Supelco), placed on a prefabricated funnel, and a beaker placed on the bottom. 5 mL of hexane was added to the top of the cartridge. When the solvent at the top of the cartridge was exhausted, 5 mL of ethyl acetate and 5 mL of hexane were sequentially added. The introduced liquid was discarded.
(Preparation for SAX / PSA cartridge)
6-2. A syringe was attached to a SAX / PSA cartridge (0.5 g, 0.5 g, 6 mL), placed on a prefabricated funnel, and a beaker placed on the bottom. 10 mL of a hexane / ethyl acetate mixture (volume ratio 9: 1) was added to the top of the cartridge. When the solvent at the top of the cartridge was exhausted, 1 mL of a hexane / ethyl acetate mixture (volume ratio 9: 1) was added twice. The introduced liquid was discarded.
A 6-2 SAX / PSA cartridge was placed under the 6-3.6-1 florisil cartridge and a beaker was placed under the SAX / PSA cartridge.
6-4. Using a Pasteur pipette, 5-2 concentrate was added to the top of the Florisil cartridge.
6-5. When the solvent at the top of the SAX / PSA cartridge was exhausted, a 100 mL eggplant-shaped flask was placed at the bottom of the SAX / PSA cartridge. 20 mL of a hexane / ethyl acetate mixture (volume ratio 9: 1) was added to the top of the Florisil cartridge.
6-6. When the solvent at the top of the Florisil cartridge was exhausted, the solvent in the cartridge was pushed out using a syringe, and the cartridge was removed.
6-7. When the solvent at the top of the SAX / PSA cartridge was exhausted, the solvent in the cartridge was pushed out using a syringe.

7). Eluate concentration 7-1. The entire eluate was collected and concentrated under reduced pressure to about 2 mL using a rotary evaporator.
Acetone 5 mL was added to the concentrated solution of 7-2.7-1, and concentrated under reduced pressure to about 1 mL. The same operation was performed again.

8). The concentrated solution of analytical sample preparation 8-1.7-2 was transferred to a 2 mL volumetric flask and made up to 2 mL with 0.01% polyethylene glycol acetone.
8-2. Using a Pasteur pipette, the inside of the volumetric flask was well stirred.
8-3. About 1 mL of the sample solution was placed in a vial and a cap was set.
8-4. The cap was closed using a crimper.

9. 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
The analysis results are shown in Table 1.

(Criteria)
(A) It was judged that an additive recovery rate of 70 to 120% was applicable. The recovery rate outside the range is marked with a strikethrough.
(B) Qualifier ion determination:
Target ion (calibration curve is created when quantitative analysis is performed), and qualifier ion (qualifier ion) is used to calculate the ratio of the target ion to confirm that it is really the substance. Table 1 shows the case where both of 1 and the qualifier ion 2 can be confirmed as “◯”, and the case where the target ion and the qualifier ion 1 or the qualifier ion 2 can be confirmed as “Δ”. .

Claims (4)

Next step:
(I) a step of extracting a crude drug sample with an aqueous acetonitrile solution;
(Ii) adjusting the pH of the extract to 3.5 to 4.5, and (iii) subjecting the solution adjusted in the above step (ii) to column chromatography, the residual pesticide in the crude drug sample The purification method of
A method for purifying a residual agricultural chemical in a herbal medicine sample, wherein the residual agricultural chemical is a compound having an ester structure .   The method according to claim 1, wherein the herbal medicine sample is a herbal medicine or a Chinese medicine preparation.   The herbal medicine sample is selected from licorice, licorice, cinnamon, chimpanzee, chimpi, biwayo, carrot, tonin, chorei, soyo, daiou, taiso, senna, peony, salamander, sanshuyu, kojin, buttonpi, onji and ogi. the method of. The method according to any one of claims 1 to 3, wherein the compound having an ester structure is a pyrethroid pesticide.