MHLW Japan Notification No. 499

7/31/2019 MHLW Japan Notification No. 499

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Provisional Translationfrom Japanese Original

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Ministry of Health, Labour and Welfare Notif ication No. 499

The Minister of Health, Labour and Welfare has par t ia l ly revised the

Specif icat ions and Standards for Food, Food Addit ives, Etc . (Ministry of Health

and Welfare Notif icat ion No. 370, 1959) , as given below, based on the provision

of Paragraph 1, Art ic le 11 of the Food Sanita t ion Law; this revision wil l take

effect on May 29, 2006.

Notwithstanding this notif icat ion, food products that are manufactured or

processed on or before May 28, 2006 may observe the exist ing regulat ions,

instead of the regulat ions t o be applied f rom the given date .

November 29, 2005

Jiro Kawasaki

Minister of Health, Labour and Welfare

I tem 1 in Section A General Composi t ional Standards for Food, Par t I Food

shal l be revised as fol lows:

1. Foods shall not contain any antibiot ics or chemically synthesized

antibacter ia l substances (substances obta ined by inst igat ing chemical

reactions to e lements and/or compounds through chemical methods,

except for decomposit ion; this applies hereinaf ter in this paragraph) ,

except for the fol lowing cases:

(1) When the substance concerned is identical to the food addit ive

determined by the Minister of Health, Labour and Welfare as having no

potentia l to cause damage to human health under Arti c le 10 of the Food

Sanita t ion Law (Law No. 233, 1947, hereinaf ter the Law) .

(2) When composit ional s tandards are se t for th in 5, 6 , 7 , 8 or 9 below for

the substance concerned.

(3) When the food product concerned has been manufactured or processed

using a food ingredient that meets the composit ional s tandards given in

5, 6, 7 , 8 or 9 below (except for foods containing antibiot ics or

chemically synthesized antibacter ia l substances for which

composit ional s tandards are not se t for th in 5, 6 , 7 , 8 or 9 below.)

In Section A General Composi t ional Standards for Food, Par t I Food, I tem 2

shall be dele ted and replaced by I tem 3, and I tems 4 and 5 sh all be renumberedto 3 and 4, respectively, to be fol lowed by the new I tem 5 shown below.


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5. Foods shall not contain substances ( including substances produced by

chemical t ransformation: this applies hereinaf ter in this paragraph) used

as ingredients of agr icultura l chemicals and other chemical substances

l is ted in the table in (1) below. The agr icultura l chemicals and other

chemical substances s ta ted above, here and a lso la ter in this paragraph,

refer to substances used for purposes designated by the Agriculture ,

Forestry and Fisher ies Minister ia l Ordinance according to the provision

of Paragraph 3 of Art ic le 2 of the Law Concerning Safety Assurance and

Quali ty Improvement of Agricultura l Chemicals and Feeds (Law No. 35,

1953) , which is s t ipula ted under Paragraph 1 of Art ic le 1-2 of the

Agricultura l Chemicals Regulat ion Law (Law No. 82, 1948) , with such

aims as adding to, mixing with, or inf i l t ra t ing into feeds ( the feeds

st ipula ted under Paragraph 2 of Art ic le 2 of Law No. 35) , or medical

products to be used for animals , which are s t ipula ted in Paragraph 1 of

Art ic le 2 of the Pharmaceutical Affairs Law (Law No. 145, 1960) . In

associa t ion with this regulat ion, a sample of the foods l is ted in the

foods column in the table in (2) below shall be tested using the par t

l is ted in the samples column in the table by the test ing methods

descr ibed in (3) to (15) below. No ingredients of agr icultura l chemicals

or other chemical substances shall be detected in these tests .

(1) Substances used as ingredients of agr icultura l chemicals and other

chemical substances that are s t ipula ted to be Not detected in foods

1. 2, 4 , 5-T

2. Azocyclotin and cyhexatin

3. Amitrol

4. Captafol

5. Carbadox

6. Coumaphos

7. Chloramphenicol

8. Chlorpromazine

9. Diethylst i lbestrol

10. Dimetr idazole

11. Daminozide

12. Nitrofurans

13. Propham

14. Metronidazole

15. Ronidazole


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(2) Samples

Food Sample mater ia l

Barley and buckwheat Threshed seeds

Wheat and rye Husked seeds

Rice (brown r ice) Husked seeds

Corn (maize) Seeds with the husks, the s i lk and

the cores removed

Other cereal gra ins Threshed seeds

Peas, beans (dry)( including butter

beans, cowbeans ( red beans) , lenti l ,l ima beans, pegia , sul tani , sul tapya

and white beans)*, broad beans and

soybeans (dry))

Seeds without the pods

Peanuts With the shells removed

Other legumes/pulses Seeds without the pods

Apricot , mume plum, cherry,

Japanese plum ( including prune)

and nectar ine

With the peduncle and the seeds

removed

Peach With the skins and the seeds

removed

Orange ( including navel orange) ,

grapefruit , c i t rus natsudaidai

(whole) , l ime and lemon

Whole f rui t

Citrus natsudaidai (pulp) and unshu

orange (pulp)

With the peels removed

Citrus natsudaidai , peels With the calyxes removed

Other c i trus f rui ts Whole f rui t

Pear , Japanese pear , quince and

apple

With the blossom scars , the cores

and the peduncles removed

Loquat With the peduncles, the skins and

the seeds removed

Avocado and mango With the seeds removed

Kiwifruit With the skins removed

Guava With the calyxes removed


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Date With the calyxes and the seeds

removed

Pineapple With the tops removed

Passion f rui t and papaya Whole f rui t

Banana With the pedicels removed

Strawberry, cranberry, huckleberry,

blackberry and blueberry

With the calyxes removed

Raspberry Whole f rui t

Other berr ies With the calyxes removed

Japanese persimmon With the calyxes and the seeds

removed

Watermelon, makuwauri melon and

melons

With the r inds removed

Grape With the peduncles removed

Other f rui ts Edible por t ions

Turnip ( roots) and Japanese radish

(roots , including radish)

With the dir t l ightly r insed off with

water

Turnip ( leaves) , watercress , kale ,

Japanese radish ( leaves, including

radish) , and brussels sprouts

With the decayed leaves removed

Caulif lower and broccoli With the leaves removed

Cabbage and Chinese cabbage A sample consist ing of one por t ion

from each of four heads, with each

head equally cut into four por t ions,

without the decayed outer leaves

and the cores

Kyona and komatsuna (Japanese

mustard spinach)

With the roots and the decayed

leaves removed

Horseradish Roots with the dir t l ightly r insed

off with water

Qing-geng-cai and other

cruciferous vegetables

Edible por t ions

Sweet pota to, konjac , taro, pota to,

yam and other pota toes

With the dir t l ightly r insed off with

water

Pumpkin ( including squash) ,

cucumber ( including gherkin) and

orienta l pickling melon (vegetable)

With the vines removed


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Other cucurbitaceous vegetables Edible por t ions

Artichoke, endive and chicory With the decayed leaves removed

Burdock and sals ify A sample thinly s l iced then groundwith a meat gr inder , the l eaves

having been removed and the dir t

having been l ightly r insed off with

water

Shungiku With the roots and the decayed

leaves removed

Lettuce ( including cos le t tuce and

leaf le t tuce)

With the decayed outer leaves and

the cores removed

Other composite vegetables Edible por t ions

Shii take mushroom, button

mushroom and other mushrooms

Edible por t ions

Celery, parsley and mitsuba With the roots and the decayed

leaves removed

Carrot and parsnip With the dir t l ightly r insed off with

water

Other umbell iferous vegetables Edible por t ions

Tomato, egg plant and pimiento

(sweet pepper)

With the calyxes removed

Other solanceous vegetables Edible por t ions

Asparagus Stems

Onion, gar l ic , welsh ( including leek)

and mult iplying onion

With the outer skins and the root

hair removed

Nira a nd o the r l i l ia c eous ve ge tab les Ed ib le po rt ions

Green soybeans, kidney beans( immature , with pods) and peas

( immature , with pods)

With the pedicels removed

Okra With the calyxes removed

Sugarcane With the husks removed

Ginger With the leaves removed, and with

the dir t l ightly r insed off with

water

Sugar beet With the dir t l ightly r insed off withwater


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Spinach With red roots lef t on, and with the

root hair and the decayed leaves

removed

Bamboo shoots and o the r vege tables Edib le por t ions

Sesame seeds, rapeseeds, sunf lower

seeds, saff lower seeds, cotton seeds

and other oi l seeds

Seeds only

Almond, ginkgo nut , chestnut ,

walnut , pecan and other nuts

With the husks removed

Cacao beans and coffee beans Beans without the pods

Tea Tea leaves

Hop Dried f lowers

Other spices and other herbs Edible por t ions

(3) 2, 4 , 5-T analytical method

1. Equipment

Gas chromatograph with an e lectron capture detector (GC-ECD) and a

gas chromatograph-mass spectrometer are used.

2. Reagents/Test solut ions

In addit ion to the reagents and test solut ions l is ted below, those l is ted

in Section C Reagents /Tes t So lut ions, Etc. , Par t I I Food addi t ives are

to be used.

Reagents designated as specia l grade in this sect ion must meet the

requirements for specia l grade specif ied in the Japan Industr ia l

Standards for the reagents .

Acetonitr i le : Three hundred ml of acetonitr i le is concentra ted using a

rotary vacuum evaporator . After removing the acetonitr i le , the

residue is dissolved in 5 ml of n-hexane. When 5 l of the dissolved

residue is injected into the GC-ECD for analysis , the heights of peaks

for compounds other than n-hexane on the gas chro matogram must be

the same as or lower than the peak for gamma-BHC at 2x10- 11

g.

Acetone: Three hundred ml of acetone is concentra ted using a rotary

vacuum evaporator . After removing the acetone, the residue is

dissolved in 5 ml of n-hexane. When 5 l of the dissolved residue is

injected into the GC-ECD for analysis , the heights of peaks for

compounds other than n-hexane on the gas chromatogram must be the

same as or lower than the peak for gamma-BHC at 2x10- 11

g.


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Ether : Three hundred ml of die thyl e ther is concentra ted using a rotary

vacuum evaporator . After removing the die thyl e ther , the residue is





g.

Sodium chlor ide: Sodium chlor ide (specia l grade) . In cases where a

substance that interferes with the analysis of an ingredient of the

agr icultura l chemical product concerned is contained, i t should be

r insed out with a solvent such as n-hexane.

Synthetic magnesium si l ica te (Flor is i l) for column chromatography:

Flor is i l (par t ic le s ize: 150-250 m) produced for column

chromatography is heated a t 130C for more than 12 hours before

being a l lowed to cool in a desiccator .

Diatomaceous ear th: Diatomaceous ear th for chemical analysis is us ed.

Ethyl aceta te : Three hundred ml of e thyl aceta te is concentra ted using

a rotary vacuum evaporator . After removing the e thyl aceta te , the



for compounds other than n-hexane on the gas chromatogram must bethe same as or lower than the peak for g amma-BHC at 2x10

- 11g.

Reagent for butyl ester if icat ion: Ten grams of boron tr if luor ide e ther

complex is dissolved in 25 ml of n-butanol .

n-Hexane: Three hundred ml of n-hexane is concentra ted to 5 ml using

a rotary vacuum evaporator. When 5 l of the concentra ted sample is




g.

Water : Dist i l led water is used. In cases where a substance that

interferes with analysis of an ingredient of the agr icultura l chemical

product concerned is contained, i t should be r insed out with a solvent

such as n-hexane.

Sodium sulfa te (anhydrous) : Sodium sulfa te (anhydrous) (specia l

grade) . In cases where a substance that interferes with analysis of an

ingredient of the agr icultura l chemical product concerned is

contained, i t should be r insed out with a solvent such as n-hexane.

Methanol: Three hundred ml of methanol is concentra ted using a rotary


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vacuum evaporator . After removing the methanol, the residue is





g.

3. Reference mater ia l

2,4,5-T: This product should consist of 98% or more 2,4,5-T.

Melt ing point: 156C

4. Preparat ion of test solut ions

a . Extract ion methods

i . Cereal gra ins, legumes/pulses and seeds

Cereal gra ins, legumes/pulses and seeds are crushed so as to pass

through a s tandard mesh sieve (420 m) before being weighed to

prepare a 10.0-gram sample . Twenty ml of water is added to the

obtained sample and lef t to s tand for two hours.

Then, acetone (100 ml) and 4 mol/ l hydrochlor ic acid (5 ml) are

added. After homogenizing for three minutes, the mixture is f i l tered

by suction into a rotary vacuum evaporator using f i l ter paper

covered with a one-centimeter- thick layer of dia tomaceous ear th.

The residue on the surface of the f i l ter paper is collected andacetone (50 ml) is added before homogenizing for three minutes.

The above procedure is repeated and the f i l t ra te is added to the

rotary vacuum evaporator and concentra ted to approximately 30 ml

at 40C or l ower.

The concentra ted solution is t ransferred to a 300-ml separat ing

funnel a lready containing 100 ml of 10% sodium chlor ide solution.

The eggplant-shaped f lask of the above rotary vacuum evaporator is

washed with 100 ml of e thyl aceta te to obta in the washings, which

are added to the separat ing funnel above. The mixture is shaken

vigorously for f ive minutes using a shaker before being lef t to s tand,

and then the e thyl aceta te layer is t ransferred to a 300-ml conical

f lask. Fif ty ml of e thyl aceta te is added to the aqueous layer , and,

af ter repeating the above procedure , the e thyl aceta te layer is added

to the conical f lask. An adequate amount of sodium sulfa te

(anhydrous) is a lso added to the f lask, which is lef t to s tand for 15

minutes and shaken from t ime to t ime. The content of the f lask is

then f i l tered into a rotary vacuum evaporator . The conical f lask is


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then washed with 20 ml of e thyl aceta te to obta in the washings, with

which the residue on the surface of the f i l ter paper is washed twice .

The washings obta ined f rom the repeated washing are then added to

the rotary vacuum evaporator and concentra ted to approximately 1

ml a t 40C or lower , and then the solvent is evaporated to near

dryness in a ni trogen st ream at room temperature .

Thir ty ml of n-hexane is added to the dr ied residue and the mixture

is t ransferred to a 100-ml separat ing funnel , to which 30 ml of

n-hexane-saturated acetonitr i le is added. After shaking the mixture

vigorously for f ive minutes usin g a shaker , the funnel is lef t to s tand

and the acetonitr i le layer is t ransferred to a 200-ml separat ing

funnel . After adding n-hexane-saturated acetonitr i le (30 ml) to the

n-hexane layer , the above procedure is repeated twice and the

acetonitr i le layer is added to the separat ing funnel above. Fif ty ml

of n-hexane sa turated with acetonitr i le is a lso added into the

separat ing funnel , which is l ightly shaken and lef t to s tand. The

acetonitr i le layer is then transferred into a rotary vacuum

evaporator and concentra ted to approximately 1 ml a t 40C or lower ,

and then the solvent is evaporated to near dryness in a ni trogen

stream at room temperature .i i . Fruit , vegetables, matcha and hops

Fruit and vegetables are weighed accurate ly to prepare a sample of

about one ki logram. An appropria te amount of water is measured

and added to the sample , i f required. After homogenizing, a sample

equivalent to 20.0 g is measured out .

Matcha is weighed to prepare a 5.00-gram sample , to which 20 ml of

water is added and lef t to s tand for two hours.

Hops are f irs t crushed into pieces and weighed to prepare a

5.00-gram sample , to which 20 ml of water is added and lef t to s tand

for two hours.

Acetone (100 ml) and 4 mol/ l hydrochlor ic acid (5 ml) are added to

the obta ined sample . The mixture , af ter homogenizing for three

minutes, is f i l tered by suction into a rotary vacuum evaporator using

f i l ter paper covered with a one-centimeter- thick layer of

dia tomaceous ear th. The residue on the surface of the f i l ter paper is

collected and acetone (50 ml) is added before homogenizing for

three minutes. The above procedure is repeated, and the f i l t ra te is


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added to the rotary vacuum evaporator and concentra ted to

approximately 30 ml a t 40C or lower .




washed with 100 ml of e thyl aceta te to obta in the washings, which

are combined in the separat ing funnel above. The mixture is shaken

vigorously for f ive minutes using a shaker before being lef t to s tand

and the e thyl aceta te layer is t ransferred to a 300-ml conical f lask.

Fif ty ml of e thyl aceta te is added to the aqueous layer and, af ter

repeating the above procedure , the e thyl aceta te layer is combined

in the conical f lask. An adequate amount of sodium sulfa te




then washed with 20 ml of e thyl aceta te to obta in the washings, with



the rotary vacuum evaporator and concentra ted to approximately 1

ml a t 40C or lower , and then the solvent is evaporated to neardryness in a ni trogen stream at room temperature .

i i i . Teas other than matcha

A 9.00-gram sample soaked in 540 ml of water a t 100C is lef t to

stand a t room temperature for f ive minutes before being f i l tered.

From the cooled f i l t ra te , 360 ml is t ransferred into a 500-ml conical

f lask, to which 18 g of sodium chlor ide and 4 mol/ l hydrochlor ic

acid are added to adjust the pH to 1 or lower . This solut ion is

transferred to a 1,000-ml separat ing funnel a lready containing 100

ml of e thyl aceta te before shaking vigorously for f ive minutes using

a shaker . The shaken mixture is lef t to s tand and the e thyl aceta te

layer is t ransferred to a 300-ml conical f lask. One hundred ml of

e thyl aceta te is added to the aqueous layer and, af ter repeating the

above procedure , the e thyl aceta te layer is combined in the conical

f lask. An adequate amount of sodium sulfa te (anhydrous) is a lso

added to the f lask, which is lef t to s tand for 15 minutes and shaken

from time to t ime. The content of the f lask is then f i l tered into a

rotary vacuum evaporator. The conical f lask is then washed with 20


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ml of e thyl aceta te to obta in the washings, with which the residue on

the surface of the f i l ter paper is washed twice . The washings

obtained f rom the repeated washing are then added to the rotary

vacuum evaporator and concentra ted to approximately 1 ml a t 40C

or lower , and then the solvent is evaporated to near dryness in a

nitrogen stream at room temperature .

iv. Foods other than those l is ted in i to i i i above

Extracts are obta ined by the methods descr ibed in i or i i .

b . Hydrolysis

The residue obta ined by the extract ion method descr ibed in a .

Extract ion methods is dissolved in 20 ml of methanol and

transferred to a 100-ml eggplant-shaped f lask, to which 10 ml of a 1.5

mol/ l sodium hydroxide solution is added. A ref lux condenser is

a t tached to the f lask, which is then heated for 30 minutes in a water

bath a t 80C and a l lowed to cool . The solution is t ransferred to a

rotary vacuum evaporator and most of the methanol is removed a t

40C or lower . The residue is then f i l tered by suction through a glass

f i l ter (pore s ize G3) . The f i l t ra te is t ransferred to a 300-ml separat ing

funnel ( I ) . The residue on the glass f i l ter is washed with a small

amount of acetone and water and the washings are added to theseparat ing funnel above, to which 50 ml of e ther and 100 ml of 10%

sodium chlor ide solution are a lso added. The mixture is shaken


and then the aqueous layer is t ransferred to a 300-ml separat ing

funnel ( I I ) , to which 4 mol/ l hydrochlor ic acid is added to adjust the

pH to 1 or lower . Fif ty ml of e thyl aceta te is added to the adjusted

solution before being vigorously shaken for f ive minutes using a

shaker and lef t to s tand. The e thyl aceta te layer is then transferred to

a 300-ml conical f lask. Fif ty ml of e thyl aceta te is added to the

aqueous layer and, af ter repeating the above procedure , the e thyl

aceta te layer is combined in the conical f lask descr ibed above. An

adequate amount of sodium sulfa te (anhydrous) is a lso added to the

f lask, which is lef t to s tand for 15 minutes and shaken from t ime to

t ime. The content of the f lask is then f i l tered into a rotary vacuum

evaporator . The conical f lask is then washed with 20 ml of e thyl

aceta te to obta in the washings, with which the residue on the surface

of the f i l ter paper is washed. The washings are added to the rotary


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vacuum evaporator and concentra ted to approximately 1 ml a t 4 0C or

lower.

c . Butyl ester if icat ion

The solution obta ined by the hydrolysis descr ibed in b. Hydrolysis

is t ransferred to a 20-ml eggplant-shaped f lask, and then the solvent

is evaporated to near dryness in a ni trogen stream at room

temperature . After the desiccation, one ml of a butyl ester if icated

agent is added. A ref lux condenser is a t tached to the eggplant-shaped

flask descr ibed above, which is then heated for 30 minutes in a water

bath a t 90C and a l lowed to cool . The cooled mixture is t ransferred to

a 200-ml separat ing funnel a lready containing 50 ml of 10% sodium

chlor ide solution and 50 ml of n-hexane before shaking vigorously

for f ive minutes using a shaker . The shaken mixture is lef t to s tand

and the n-hexane layer is t ransferred to a 200-ml conical f lask. Fif ty

ml of n-hexane is added to the aqueous layer and, af ter repeating the

above procedure , the n-hexane layer is combined in the conical f lask

descr ibed above. An adequate amount of sodium sulfa te (anhydrous)

is a lso added to the f lask, which is lef t to s tand for 15 minutes and

shaken from t ime to t ime. The content of the f lask is then f i l t ered into

a rotary vacuum evaporator . The conical f lask is then washed with 10ml of n-hexane to obta in the washings, with which the resi due on the

surface of the f i l ter paper is washed. The washings are added to the

rotary vacuum evaporator and concentra ted to approximately 2 ml a t

40C or lower.

d. Clean-up

Five grams of f lor is i l for column chromatography suspended in

n-hexane is added to a chromatograph tube ( inner diameter : 15 mm

and length: 300 mm), over which approximately 5 g of sodium sulfa te

(anhydrous) is fur ther added. The n-hexane is then spil t out of the

column unti l only a small amount remains on the packing of the

column, into which the solut ion obta ined by the butyl ester if icat ion

descr ibed in c . Butyl ester if icat ion is poured. Then, 50 ml of a

mixture of e ther and n-hexane (1:19) is a lso poured into the column

and the eff luent is discarded. In addit ion, 150 ml of a mixture of e ther

and n-hexane (3:17) is a lso poured into the column and the e luate is

collected in a rotary vacuum evaporator and concentra ted to

approximately 1 ml a t 40C or lower , and then the solvent is


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evaporated to near dryness in a ni trogen stream at room temperature .

The residue obta ined is dissolved in n-hexane to make exactly 10 ml

of solut ion, which is used as the sample solution.

5. Determination

a. Quali ta t ive tests

Quali ta t ive tests are performed under the fol lowing condit ions. Test

results obta ined must be the same as the results obta ined in the

reference mater ia l under the procedure descr ibed in c . Butyl

ester if icat ion in 4. Preparat ion of test solut ions.

Testing condit ions

Column: A si l ica te glass capil lary column ( inner diameter : 0 .25 mm

and length: 30 m) coated with 5% phenyl methyl s i l icone for gas

chromatography to a thickness of 0.25 m is used.

Column temperature: The column temperature is held a t 50C for one

minute , fol lowed by an increase of 25C every minute unti l reaching

125C, af ter which the temperature is increased by 10C every

minute unti l reaching 300C, where i t is held for f ive minutes.

Inle t temperature: 260C

Detector : Should be operated a t 300C

Gas f low ra te : Nitrogen or hel ium is used as the carr ier gas. The f lowrate should be adjusted so that n-butyl (2,4,5- tr ichlorophenoxy)

aceta te f lows out in approximately 15 minutes.

b. Quanti ta t ive tests

The quanti ty is determined from the test results obta ined under the

condit ions descr ibed in a . Quali ta t ive tests , using e i ther the peak

height or peak area method.

c . Confirmation tests

Gas chromatography/mass spectrometry is performed under the same

condit ions as those descr ibed in a . Quali ta t ive tests . Test results

obta ined in the reference mater ia l must be the same as the results

obta ined under the procedure descr ibed in c . Butyl ester if icat ion in

4. Preparat ion of test solut ions. The quanti ty may be determined by

either the peak height or peak area method, i f requi red.

(4) Analytical method for azocyclotin and cyhexatin

1. Equipment

A gas chromatograph with a f lame photometr ic detector ( interference

f i l ter for t in determination, wavelength: 610nm) and a gas


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chromatograph-mass spectrometer are used.




used.










g.






same as or lower than the peak for gamma-BHC at 2x10

- 11

g.3 mol/ l e thylmagnesium bromide-ethereal solut ion: 3 mol/ l

e thylmagnesium bromide-ethereal solut ion.

Ether : Three hundred ml of die thyl e ther is concentra ted using a rotary

vacuum evaporator . After removing the die thyl e ther , the residue is





g.


substance that interferes with analysis of an ingredient of the



Synthetic magnesium si l ica te (Flor is i l) for column chromatography:

Flor is i l (par t ic le s ize: 150-250 m) produced for column



Diatomaceous ear th: Diatomaceous ear th for chemical analysis is used.


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Cyhexatin s tandard solution: A mixture of acetic acid and e thyl aceta te

(1:99) is added to 10.0 mg of cyhexatin to make a 100-ml solution. Of

the 100-ml solution, 10 ml is taken out and n-hexane is added to make

100 ml of mix.

Sodium dodecyl sulfa te : A reagent with a pur i ty of 85% or higher is

used.






g.




such as n-hexane.





3. Reference mater ia lCyhexatin: This product should consist of 99% or more cyhexatin.

Melt ing point: 195-198C



i . Legumes/pulses and seeds

Legumes/pulses and seeds are crushed so as to pass through a

standard mesh sieve (420 m) before being weighed to prepare a

10.0-gram sample . Twenty ml of water is added to the obta ined

sample and lef t to s tand for two hours.

Then, 100 ml of a mixture of acetone and acetic acid (99: 1) is added.

The mixture , af ter homogenizing for three minutes, is f i l tered by

suction into a rotary vacuum evaporator using f i l ter paper covered

with a one-centimeter- thick layer of dia tomaceous ear th. The

residue on the surface of the f i l ter paper is collected and 50 ml of a

mixture of acetone and acetic acid (99:1) is added before

homogenizing for three minutes. The above procedure is repeated,

and the f i l t ra te is added to the rotary vacuum evaporator and


16/126


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concentra ted to approximately 30 ml a t 40C or lower .




washed with 100 ml of n-hexane to obta in the washings, which are

added to the separat ing funnel above. The mixture is shaken


and then the n-hexane layer is t ransferred to a 30 0-ml conical f lask.

Fif ty ml of n-hexane is added to the aqueous layer and, af ter

repeating the above procedure , the n-hexane layer is added to the

conical f lask. An adequate amount of sodium sul fa te (anhydrous) is

a lso added to the f lask, which is lef t to s tand for 15 minutes and

shaken from t ime to t ime. The content of the f lask is then f i l tered

into a rotary vacuum evaporator . The conical f lask is then washed

with 20 ml of n-hexane to obta in the washings, with which the

residue on the surface of the f i l ter paper is washed twice . The

washings obta ined f rom the repeated washing are then added to the

rotary vacuum evaporator , and n-hexane is removed a t 40C or

lower.

Twenty ml of n-hexane is added to the residue and the mixture istransferred to a 100-ml separat ing funnel , to which 40 ml of


vigorously for f ive minutes usin g a shaker , the funnel is lef t to s tand

and the acetonitr i le layer is t ransferred to the rotary vacuum

evaporator. After adding n-hexane-saturated acetonitr i le (40 ml) to

the n-hexane layer , the above procedure is repeated twice . The

acetonitr i le layer is then added to the rotary vacuum evaporator , and

the acetonitr i le is removed a t 40C or lower . The residue is

dissolved in n-hexane to make exactly 5 ml of solut ion.

i i . Cereal gra ins, f rui t and vegetables

Cereal gra ins are crushed so as to pass through a s tandard mesh

sieve (420 m) before being weighed to prepare a 10.0-gram sample .

Twenty ml of water is added to the obta ined sample and then lef t to

stand for two hours.


about one ki logram. Appropria te amount of water is measured and

added to the sample , i f required. After homogenizing, a sample


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One hundred ml of a mixture of acetone and acetic acid (99:1) is

added to the 20-gram sample before being f inely crushed for three

minutes, and f i l tered by suction into a rotary vacuum evaporator

using f i l ter paper covered with a one-centimeter- thick layer of


collected and 50 ml of a mixture of acetone and acetic acid (99:1) is

added before homogenizing for three minutes. The above procedure

is repeated and the f i l t ra te is added to the rotary vacuum evaporator

and concentra ted to approximately 30 ml a t 40C or lower.










conical f lask. An adequate amount of sodium sul fa te (anhydrous) isa lso added to the f lask, which is lef t to s tand for 15 minutes and






rotary vacuum evaporator , and the n-hexane is removed a t 40C or

lower . The residue is dissolved in n-hexane to make exactly 10 ml of

solut ion.

i i i . Matcha and hops


water is added and lef t to s tand for two hours.


5.00-gram sample , to which 20 ml of water is added and lef t to s tand

for two hours.

One hundred ml of a mixture of acetone and acetic acid (99:1) is

added to this sample . The mixture , af ter homogenizing for three


18/126


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minutes, is f i l tered by suction into a rotary vacuum evaporator using

f i l ter paper covered with a one-centimeter- thick layer of


collected and 50 ml of a mixture of acetone and acetic acid (99:1) is

added before homogenizing for three minutes. The above procedure

is repeated, and the f i l t ra te is added to the rotary vacuum evaporator

and concentra ted to approximately 30 ml a t 40C or lower.






vigorously for f ive minutes using a shaker before being lef t to s tand

and the n-hexane layer is t ransferred to a 300-ml conical f lask. Fif ty

ml of n-hexane is added to the aqueous layer and, af ter repeating the

above procedure , the n-hexane layer is added to the conical f lask.

An adequate amount of sodium sulfa te (anhydrous) is a lso added to

the f lask, which is lef t to s tand for 15 minutes and shaken from t ime

to t ime. The content of the f lask is then f i l tered into a rotary

vacuum evaporator . The conical f lask is then washed with 20 ml of n-hexane to obta in the washings, with which the residue on the

surface of the f i l ter paper is washed twice . The washings obta ined

from the repeated washing are added to the rotary vacuum

evaporator and concentra ted to approximately 5 ml a t 40C or lower ,

to which n-hexane is added to make exactly 10 ml of s olut ion.

iv. Teas other than matcha



From the cooled f i l t ra te , 360 ml is t ransferred into a 500-ml

separat ing funnel .

To this conical f lask, 30 g of sodium chlor ide , 2 ml of 2% sodium

dodecyl sulfa te solut ion and 100 ml of n-hexane are a lso added. The

mixture is shaken vigorously for f ive minutes using a shaker and

lef t to s tand. The n-hexane layer is then transferred to a 300-ml

conical f lask. One hundred ml of n-hexane is added to the aqueous

layer and, af ter repeating the above procedure , the n-hexane layer is

added to the conical f lask. An adequate amount of sodium sulfa te


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then washed with 20 ml of n-hexane to obta in the washings, with



the rotary vacuum evaporator , and n-hexane is removed a t 40C or

lower . The residue is dissolved in n-hexane to make exactly 6 ml of

solut ion.

v. Foods other than those l is ted in i to iv above

Extracts are obta ined by the methods descr ibed in i , i i , or i i i .

b . Ethyla t ion

One ml (2 ml for cereal gra ins, teas and hops) of the solut ion

obtained by the method descr ibed in a . Extract ion methods is

transferred to a 50-ml test tube with a glass s topper , to which 1 ml (2

ml for cereal gra ins, teas and hops) of a 3 mol/ l e thylmagnesium

bromide-ethereal solut ion is added. The mixture is lef t to s tand for 20

minutes a t room temperature .

Then, 10 ml of 0.5 ml/ l su lfur ic acid is gradually added, fol lowed by

the addit ion of 10 ml of water to be mixed in. Ten ml of n-hexane isadded to the mixture before being vigorously shaken for one minute .

After being lef t to s tand, the n-hexane layer is t ransferred to a 50 ml

conical f lask. Five ml of n-hexane is added to the aqueous layer and,

af ter repeating the above procedure twice , the n-hexane layer is

added to the conical f lask. An adequate amount of sodium sulfa te


minutes and shaken from t ime to t ime. The content of the f lask is then

f i l tered into a rotary vacuum evaporator . The conical f lask is then

washed with 5 ml of n-hexane to obta in the washings, with which the


washings f rom the repeated washing are then added to the rotary

vacuum evaporator and concentra ted to 2 ml a t 40C or l ower.

c . Clean-up

Five grams of f lor is i l for column chromatography suspended in

n-hexane is added to a chromatograph tube ( inner diameter : 15 mm

and length: 300 mm), over which approximately 5 g of sodium sulfa te

(anhydrous) is fur ther added. The n-hexane is then spil t out unti l only


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a small amount remains on the packing of the column, into which the

solution obta ined by the e thyla t ion descr ibed in b. Ethyla t ion is

poured. Then, the eggplant-shaped f lask of the rotary vacuum

evaporator is washed with 15 ml of n-hexane to obta in the washings,

which are transferred to a column. The e luate is collected in the

rotary vacuum evaporator . Then, 50 ml of a mixture of e ther and

n-hexane (1:99) is poured in and the e luate is collected in the rotary

vacuum evaporator , and the e ther and n-hexane are removed a t 40C

or lower . The residue obta ined is dissolved in n-hexane to make

exactly 2 ml of solut ion, which is used as the sample solution.

5. Determination




cyhexatin reference mater ia l under the procedure descr ibed in b.

Ethylat ion in 4. Preparat ion of test solut ions. Azocyclotin is

modif ied by e thyla t ion into th e same substance as cyhexatin.

Test ing condi t ions

Column: A si l ica te glass capil lary column ( inner diameter : 0 .32

mm-0.53 mm and length: 30 m) coated with 5% phenyl methylsi l icone for gas chromatography to a thickness of 1.5 m is used.

Column temperature: The column temperature is held a t 120C for

two minutes, fol lowed by an increase of 10C every minute unti l

reaching 200C, af ter which the temperature is increased by 20C

every minute unti l reaching 300C, where i t is held for f ive minutes.



Gas f low ra te : Helium is used as the carr ier gas. The f low ra te should

be adjusted so that cyhexatin f lows out in approximately 13-15

minutes. The f low ra tes of a ir and hydrogen are adjusted to optimal

condit ions.



same condit ions descr ibed in a . Quali ta t ive tests , using e i ther the

peak height or peak area method.




21/126


21/126

condit ions descr ibed in a . Quali ta t ive tests . Test results obta ined

must be the same as the results obta ined in the reference mater ia l

under the procedure descr ibed in b. Ethyla t ion in 4. Preparat ion of

test solut ions. The quanti ty may be determined by e i ther the peak

height or peak area method, i f required.

(5) Amitrole analytical method

1. Equipment

A high-performance l iquid chromatograph with a f luorescence detector

and l iquid chromatograph mass spectrometer are used.




used.







g.

Ethanol: Three hundred ml of e thanol is concentra ted using a rotary

vacuum evaporator . After removing the e thanol , the residue is





g.


Acetic acid buffer solut ion: 0.05 mol/ l sodium aceta te solut ion is

added to 800 ml of 0.05 mol/ l acet ic acid solu t ion to make a 1,000-ml

solution.

Weak acid cat ion exchange resin: Weak acid cat ion exchange resin

produced for column choromatography is f irs t washed with 1 mol/ l

hydrochlor ic acid, secondly with a 2.8% aqueous ammonia , and

thirdly with 1 mol/ l hydrochlor ic acid again. Thereaf ter the washings

are fur ther washed with water unti l they become neutra l .

Fluorescamin: A reagent with a pur i ty of 98% or higher is used.

Water : Dist i l led water is used. In cases where a substance thatinterferes with analysis of an ingredient of the agr icultura l chemical


22/126


22/126


such as n-hexane.

Phosphate buffer solut ion: 10% phosphoric acid solut ion is added to a

0.05 ml/ l monosodium phosphate solut ion to adjust the pH to 3.0.


Amitrole : This product should consist of 98% or more amitrole .




i . Cereal gra ins, legumes/pulses, seeds, f rui t , vegetables, matcha and

hops



prepare a 30.0-gram sample .


about one ki logram. An appropria te amount of water is measured

and added to the sample , i f required. After homogenizing, a sample


Matcha is weighed to prepare a 30.0-gram sample .

Hops are crushed into pieces and weighed to prepare a 30.0-gramsample .

Eighty ml of e thanol is added to the obta ined sample before being

crushed f inely for three minutes. The crushed sample is f i l tered by


with a one-centimeter- thick layer of dia tomaceous ear th and the

f i l t ra te is t ransferred to a 20 0-ml graduated cylinder. The residue on

the surface of the f i l ter paper is collected and 40 ml of 60% ethanol

is added before homogenizing for three minutes. After repeating the

above procedure , the f i l t ra te is added to the graduated cylinder and

the amount of f i l t ra te is measured.

Ten ml of the f i l t ra te is t ransferred to a 200-ml round bottom f lask,

to which 1 ml of hydrogen peroxide solution is added. A ref lux

condenser is a t tached to the f lask, which is then heated for 30

minutes in a water bath a t 75C and a l lowed to cool .

i i . Teas except matcha




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From the cooled f i l t ra te , 12 ml is t ransferred into a 200-ml round

bottom f lask, to which 1 ml of hydrogen peroxide solution is added.

A ref lux condenser is a t tached to the f lask, which is then heated for

30 minutes in a water bath a t 75C and a l lowed to cool .

i i i . Foods except those l is ted in i and i i above

Extracts are obta ined by the methods descr ibed in i .

b . Clean-up

One ml of s trong acid cat ion exchange resin (par t ic le s ize:

0.063-0.156 m) in a water suspension is poured into a

chromatograph tube ( inner diameter : 10 mm and length: 300 mm).

The water is then spil t out unti l only a small amount remains on the

packing of the column, into which 5 ml of water is poured and the

eff luent is discarded. The solution obta ined by the method descr ibed

in a . Extract ion methods is then poured into the column. The round

bottom f lask descr ibed above is then washed with 10 ml of water and

the washings are a lso poured into the column and the eff luent is

discarded. Subsequently, 12 ml of 2.8% aqueous ammonia is added to

the column. The e luate is collected in a rotary vacuum evaporator and

30 ml of n-propanol is added before removing the water and

n-propanol a t 45C or lower . Five ml of water is added to the residueto dissolve i t .

In a chromatograph tube ( inner diameter : 10 mm and length: 300 mm),

5 ml of weak acid cat ion exchange resin (par t ic le s ize: 0.33-0.50 m)

in a water suspension is poured. The water is then spil t out unti l only

a small amount remains on the packing of the column, into which 10

ml of water is poured and the eff luent is discarded. The above

solution is poured in and the eff luent is discarded. Fif ty ml of water

is a lso poured in and the eff luent is discarded. Subsequently, 35 ml of

2.8% aqueous ammonia is added to the column. The e luate is

collected in a rotary vacuum evaporator and 100 ml n-propanol is

added before removing the aqueous ammonia and n-propanol a t 45C

or lower.

c . Derivatizat ion

Two ml of acetic acid buffer solut ion is added to the residue to

dissolve i t well . To 1 ml of this solut ion, 100 l of 0.25%

fluorescamin-acetone solution is added, which is then shaken well

before being a l lowed to s tand for one hour . Then, 0.5 ml of 0.05 mol/ l


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sodium borate solut ion is a lso added to prepare the test solut ion.

5. Determination




reference mater ia l under the procedure descr ibed in c .

Derivatizat ion in 4. Preparat ion of test solut ions.

Testing condit ions

Column packing: Octadecylsi lane-bonded si l ica gel (par t ic le s ize: 5

m) i s used .

Column: A sta inless tube ( inner diameter : 4 .6 mm and length: 150

mm) is used.

Column temperature: 40C

Detector : Should be operated with an excita t ion wavelength of 380

nm and a f luorescent wavelength of 484 nm.

Mobile phase: A mixture of acetonitr i le and phosphate buffer solut i on

(3:7) is used. The f low ra te should be adjusted so that the amitrole

f lows out in approximately 15 minutes.


The quanti ty is determined from the test results obta ined under thesame condit ions descr ibed in a . Quali ta t ive tests , using e i ther the



Liquid chromatography/mass spectrometry is performed under the

same condit ions descr ibed in a . Quali ta t ive tests . Test results

obta ined in the reference mater ia l must be the same as the results

obta ined under the procedure descr ibed in c . Derivatizat ion in 4.

Preparat ion of test solut ions. The quanti ty may be determined by

either the peak height or peak area method, i f requi red.

(6) Captafol analytical method

1. Equipment

A gas chromatograph with an e lectron capture detector (GC-ECD) and

a gas chromatograph-mass spectrometer are used.




used.


25/126


25/126










g.







g.





Synthetic magnesium si l ica te (Flor is i l) for column chromatography:Flor is i l (par t ic le s ize: 150-250 m) produced for column





a rotary vacuum evaporator . After removing the e thyl aceta te , the





g.






g.




26/126


26/126


such as n-hexane.






Captafol: This product should consist of 98% or more captafol .




i . Cereal gra ins, legumes/pulses and seeds



prepare a 10.0-gram sample . Twenty ml of 3% phosphoric acid

solution is added to the obta ined sample , which is then lef t to s tand

for two hours.

Acetone (100 ml) is then added. The mixture , af ter homogenizing

for three minutes, is f i l tered by suction into a rotary vacuum

evaporator using f i l ter paper covered with a one-centimeter- thicklayer of dia tomaceous ear th. The residue on the surface of t he f i l ter

paper is collected and acetone (50 ml) is added before homogenizing

for three minutes. The above procedure is then repeated and the

f i l t ra te is added to the rotary vacuum evaporator and concentra ted to

approximately 30 ml a t 40C or lower .







and then the n-hexane layer is t ransferred to a 300-ml conical f lask.



conical f lask. An adequate amount of sodium sulfa te (anhydrous) is




27/126


27/126






lower.

Thir ty ml of n-hexane is added to the residue and the mixture is then

transferred to a 100-ml separat ing funnel , to which 30 ml of


vigorously for f ive minutes using a shaker , the funnel is lef t to s tand

and the acetonitr i le layer is t ransferred to the rotary vacuum

evaporator. After adding n-hexane-saturated acetonitr i le (30 ml) to

the n-hexane layer , the above procedure is repeated twice . The

acetonitr i le layer is then added to the rotary vacuum evaporator , and

the acetonitr i le is removed a t 40C or lower . The residue is

dissolved in 5 ml of n-hexane.

i i . Fruit , vegetables, matcha and hops


about one ki logram, to which 500 ml of 10% phosphoric acid

solution is added before homogenizing. Then, a sample equivalentto 20.0 g is measured out .


3% phosphoric acid solut ion is added and lef t to s tand for two

hours.


5.00-gram sample , to which 20 ml of 3% phosphoric acid solut ion is

added and lef t to s tand for two hours.

One hundred ml of acetone is then added to the mixture and f inely

crushed for three minutes. The crushed mixture is f i l tered by


with a one-centimeter- thick layer of dia tomaceous ear th. The

residue on the surface of the f i l ter paper is collected and acetone

(50 ml) is added before homogenizing for three minutes. The above

procedure is repeated and the f i l t ra te is added to the rotary vacuum

evaporator and concentra ted to approximately 30 ml a t 40C or

lower.



28/126


28/126

















lower . The residue is dissolved in 5 ml of n-hexane.

iii . Teas except matcha


stand a t room temperature for f ive minutes before being f i l tered.From the cooled f i l t ra te , 360 ml is t ransferred into a 500-ml conical

f lask.

Thir ty ml of phosphoric acid, 100 ml of acetone and 2 ml of

sa turated lead aceta te solut ion are a lso added to this conical f lask.

The mixture is lef t to s tand for one hour a t room temperature before

being f i l tered by suction using f i l ter paper covered with a

one-centimeter- thick layer of dia tomaceous ear th. The f i l t ra te is

transferred to a 1,000-ml separat ing funnel . The conical f lask is

then washed with 50 ml of acetone to obta in the washings, with

which the residue on the surface of the f i l ter paper is washed. The

washings are added to the separat ing funnel , to which 30 g of

sodium chlor ide and 100 ml of n-hexane are a lso added. The mixture

is shaken vigorously for f ive minutes and lef t to s tand. The

n-hexane layer is then transferred to a 300-ml conical f lask. One

hundred ml of n-hexane is added to the aqueous layer , and af ter




29/126


29/126

also added to the f lask, which is lef t to s tand for 15 minutes and






rotary vacuum evaporator , and n-hexane is removed a t 40C or lower .

The residue is dissolved in 5 ml of n-hexane.

iv. Foods except those l is ted in i to i i i above

Extracts are obta ined by the methods descr ibed in i or i i .

b . Clean-up

In a chromatograph tube ( inner diameter : 15 mm and length: 300 mm),

5 g of f lor is i l for column chromatography suspended in n-hexane is

added, over which approximately 5 g of sodium sulfa te (anhydrous) is

fur ther added. The n-hexane is then spil t out unti l only a small

amount remains on the packing of the column, into which the solut ion

obtained by the extract ion method descr ibed in a . Extract ion

methods is poured. Then, 100 ml of n-hexane is a lso poured in and

the eff luent is discarded. Subsequently, 150 ml of a mixture of e thyl

aceta te and n-hexane (1:9) is added and the e luate is collected in arotary vacuum evaporator , and the e thyl aceta te and n-hexane are

removed a t 40C or lower . The residue is dissolved in n-hexane to

make exactly 5 ml of solut ion, which is used as the sample solution.

5. Determination



results obta ined under any of the condit ions must be the same as the

results obta ined in the reference mater ia l .

Test ing condit ions 1


and length: 10-30 m) coated with methyl s i l icone for gas





minute unti l reaching 300C, where i t is held for f ive minutes.



30/126


30/126



be adjusted to the optimal condit ion.

Test ing condit ions 2


and length: 10-30 m) coated with 5% phenyl methyl s i l icone for gas





minute unti l reaching 300C, where i t is held for three minutes.




be adjusted to the optimal condit ion.





c . Confirmation testsGas chromatography/mass spectrometry is performed under the same

condit ions descr ibed in a . Quali ta t ive tests . Test results obta ined

must be the same as the results obta ined in the reference mater ia l .

The quanti ty may be determined by e i ther the peak height or peak

area method, i f required.

(7) Carbadox analytical method

Quinoxaline-2-carboxylic acid is analyzed.

1. Equipment

A high-performance l iquid chromatograph with an ul traviole t

spectrophotometr ic detector and a l iquid chromatograph mass

spectrometer are used.

2. Reagents/ test solut ions



used.




31/126


31/126


Octadecylsi lane-bonded si l ica gel minicolumn (500 mg): A

polyethylene column with an inner diameter of 8-9 mm packed with

500 mg of octadecylsi lane-bonded si l ica gel or one with the same

separat ion character is t ics is used.

Strong basic anion exchanger minicolumn (360 mg): A polyethylene

column with an inner diameter of 8-9 mm packed with 360 mg of

tr imethylamino acrylamide copolymer s i lane-bonded si l ica gel or one

with the same separat ion character is t ics is used.

Water : Water produced for l iquid chromatography is used.





Phosphate buffer solut ion (pH 2.5) : Monopotassium dihydrogen

monophosphate (1.36 g) is dissolved in water to make a 800-ml

solution, to which phosphoric acid is added to adjust the pH to 2.5.

Water is added to the adjusted solution to make a 1,000-ml solutio n.


Quinoxaline-2-carboxylic acid: This product should consist of 99% ormore quinoxaline-2-carboxylic acid.

Melt ing point: 208C



After homogenizing, a sample of 5.00 g is measured out . For muscle ,

the fa t layer should be removed as much as possible before chopping.

One hundred ml of a mixture of methanol and 0.3% metaphosphoric

acid solut ion (3:7) is added to the measured-out sample . The mixture ,

af ter homogenizing, is f i l tered by suction into a rotary vacuum

evaporator using f i l ter paper covered with a two-mill imeter- thick

layer of dia tomaceous ear th. The residue on the surface of the f i l ter

paper is then washed with 10 ml of a mixture of methanol and 0.3%

metaphosphoric acid solut ion (3:7) to collect the washings, which are

then f i l tered by suction and added into the rotary vacuum evaporator.

The mixture is concentra ted to 30 ml a t 45C or lower , and 0.1 ml of

phosphoric acid is added.

b. Clean-up


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32/126

i . Octadecylsi lane-bonded si l ica gel column chromatography

Five ml of methanol is added to an octadecylsi lane-bonded si l ica gel

minicolumn (500 mg), fol lowed by 10 ml of water . The eff luent is

discarded. The solution obta ined by the extract ion method descr ibed

in a . Extract ion methods is poured into this column and

subsequently 20 ml of phosphate buffer solut ion (pH 2.5) is a lso

added. The eff luent is discarded. Ten ml of methanol is poured into

the column. The e luate is collected in a rotary vacuum evaporator ,

and the methanol is removed a t 40C or lower . The residue is

dissolved in 5 ml of water .

i i . S trong basic anion exchanger column chromatography

Five ml of water is poured into a s trong basic anion exchanger

minicolumn (360 mg) and the eff luent is discarded. The solution

obtained in i . Octadecylsi lane-bonded si l ica gel column

chromatography is poured into this column. Two ml of water , 10 ml

of e thanol and then 5 ml of water are a lso added in that order . The

eff luent is discarded. Five ml of 0.1 mol/ l hydrochlor ic acid is

poured into this column and the e luate is collected in a 50-ml test

tube. Three ml of 3 mol/ l hydrochlor ic acid and 15 ml of e thyl

aceta te are a lso added before shaking vigorously using a shaker forf ive minutes. After leaving to s tand, the e thyl aceta te layer is

transferred into a 100-ml conical f lask. Fif teen ml of e thyl aceta te is

added to the aqueous layer , and af ter repeating the above procedure ,

the e thyl aceta te layer is added to the conical f lask. An adequate

amount of sodium sulfa te (anhydrous) is a lso added to the f lask,

which is lef t to s tand for 15 minutes and shaken from t ime to t ime.

The content of the f lask is then f i l tered into a rotary vacuum

evaporator , and the e thyl aceta te is removed a t 40C or lower . The

residue is dissolved in 1.0 ml of a mixture of acetonitr i le and

phosphate buffer solut ion (pH 2.5) (1:4) , which is used as the test

solut ion.

5. Determination




reference mater ia l .

Test ing condi t ions


33/126


33/126

Column packing: Octadecylsi lane-bonded si l ica gel (par t ic le s ize: 5

m) i s used .

Column: A sta inless tube ( inner diameter : 4 .0-6.0 mm and length: 150

mm) is used.

Column temperature: 40C

Detector : Should be operated a t an absorption wavelength of 245 nm.

Mobile phase: A mixture of acetonitr i le and phosphate buffer solut i on

(pH 2.5) (1:4) is used. The f low ra te should be adjusted so that

quinoxaline-2-carboxylic acid f lows out in approximately 10

minutes.







condit ions descr ibed in a . Quali ta t ive tests , but the mobile phase

should be a mixture of acetonitr i l and water (1:4) . Test results

obta ined must be the same as the results obta ined in the reference

mater ia l . The quanti ty may be determined by e i ther the peak height orpeak area method, i f required.

(8) Coumaphos analytical method

1. Equipment

A gas chromatograph with an a lkali f lame ionizat ion detector , a f lame

photometr ic detector ( interference f i l ter for phosphorus determination,

wavelength: 526 nm), or a highly-sensi t ive ni trogen phosphorus

detector , and a gas chromatograph-mass spectrometer are used.




used.








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g.







g.





Sil ica gel for column chromatography (par t ic le s ize: 63-200 m):

Sil ica gel (par t ic le s ize: 63-200 m) produced for column





a rotary vacuum evaporator . After removing the e thyl aceta te , theresidue is dissolved in 5 ml of n-hexane. When 5 l of the dissolved




g.






g.




such as n-hexane.






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3. Reference m

Documents

MHLW Japan Notification No. 499