E tg/166/4(proj. 3) Original




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Дата канвертавання25.04.2016
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8.Explanations on the Table of Characteristics


8.1 Explanations covering several characteristics



Characteristics containing the following key in the second column of the Table of Characteristics should be examined as indicated below:
(a) 10-12 true leaves stage (prior to internode elongation);

(b) The pedicel is in hook stage;

(c) Time of flowering (when the first flower opens on the main stem at 10 % of the plants);

(d) Full blossom;

(e) 10-14 days after the petals drop down on main stem;

(f) Mature, dry capsule of main stem.




8.2 Explanations for individual characteristics



Ad. 2: Rosette leaf: white spots

Ad. 3: Rosette leaf: color

Ad. 4: Rosette leaf: waxiness
The observations of white spots, color and waxiness should be made on the upper side of the rosette leaf.


Ad. 5: Rosette leaf: depth of lobes








1

2

3

absent or shallow

medium

deep



Ad. 6: Main stem:  length




Ad. 7: Stem: anthocyanin coloration

Ad. 8: Stem: hairiness
The observation of anthocyanin coloration and hairiness should be made between the capsule and the upper stem leaf.


Ad. 9: Flower bud: anthocyanin coloration








1

2

3

absent

ring around stem

bottom part towards stem end


Ad. 11: Petal: blotch




1

none




2

entire blotch



3

band




4

radial stripes


Ad. 13: Petal: extension of blotch
The measurement should be made at the widest point of petal.








1

2

3

below the widest point

at the widest point

above the widest point


Ad. 14: Petal: incisions






1

9

absent

present



Ad. 18: Capsule: shape of longitudinal section









1

2

3

oblate

truncate

circular







4

elliptic







5

conical

Ad. 19: Capsule: shape of base

Ad. 20: Capsule: length

length







1

2

3

pointed

truncate

depressed


Ad. 22: Capsule: ribbing
For the observation of ribbing, the capsule should be touched.


Ad. 23: Capsule: dehiscence
For the observation of dehiscence the capsule should be held upside-down and shaken. If seeds do not fall out, the capsule is indehiscent (1). If seeds fall out, the capsule is dehiscent (2).
Ad. 24: Stigmatic disc: shape



1

2

erect

semi-erect




3

horizontal





4

5

declined

decumbent








1

2

erect

semi-erect







3

5

horizontal

decumbent

Ad. 26: Stigmatic disc: apex of lobe




1

2

pointed

rounded




3

truncate

Ads. 29-32: Capsule: determination of alkaloid contents: morphine, codeine, thebaine and narcotine
Determination of Morphine, Codeine, Thebaine, Papaverine

and Narcotine content in poppy capsule

HPLC method, MS detection

1. Scope
Determination of Morphine, Codeine, Thebaine, Papaverine and Noscapine content in poppy

capsule for qualification purposes.

Limit Of Detection (LOD): 10 mg/kg/component

Limit Of Quantitation (LOQ): 50 mg/kg/component


2. Principle
The sample is extracted with methanol containing 1 ml of cc. hydrochloric acid/litre. The alkaloid content of the extract is determined by HPLC-MS system using RP C18 column. External standards are used for qualitative and quantitative determination.
3. Procedure
3.1. Sample preparation

The receipt sample is weighted and dried to air-dry condition. The stalk, the crop and the seeds are separated. The stalk is ground using 0,5 mm sieve.

3.2. Extraction and clean-up

Weigh 0,2 g of grinded sample and add 100 ml methanol-HCl solution (1 ml cc. HCl/litre methanol. Keep in ultrasonic bath 30 minutes. Filter and this solution inject to the HPLC column.

3.3. HPLC measurement

The determination of the alkaloid content is performed by MS detection (SIM mode) after separation using reversed phase C18 column.


HPLC conditions

The HPLC conditions are advised listed below, but any other conditions can be used if those give suitable results.


Chromatographic column: NUCLEODUR C-18 Gravity 150*4.6mm*5μm or equivalents.
Mobile phase

A eluent: HPLC grade methanol

B eluent: 2 g Ammonium-acetate/litre HPLC grade water

Gradient: 0-4 min. 70% B

4-14 min. 10% B-ig linear gradient

14-20 min. 10% B

Post time: 5 min.
Flow rate

0.9 cm3/min.


Detector

MS SIM APCI: 2-20 perc: 286.0 AMU Positive

300.0 AMU Positive

312.0 AMU Positive

340.0 AMU Positive

414.0 AMU Positive



Injected volume: 2 l
For qualitative and quantitative determination used analytical grade standard solutions in HCL methanol (1 ml cc. HCl/litre methanol) solvent. Calibrate according to ESTD method.
4. Expression of the results
The results are expressed in mg/kg referred to air-dry material.
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