Commission Regulation (EC) No 152/2009Show full title

E. DETERMINATION OF ROBENIDINE U.K.

1,3-bis [(4-chlorobenzylidene)amino]guanidine — hydrochloride

1. Purpose and scope U.K.

This method makes it possible to determine the levels of robenidine in feed. The limit of quantification is 5 mg/kg.

2. Principle U.K.

The sample is extracted with acidified methanol. The extract is dried and an aliquot portion subjected to a clean-up on an aluminium oxide column. Robenidine is eluted from the column with methanol, concentrated, and made up to a suitable volume with mobile phase. The content of robenidine is determined by reversed-phase high-performance liquid chromatography (HPLC) using an UV detector.

3. Reagents U.K.

3.1. Methanol. U.K.

3.2. Acidified methanol. U.K.

Transfer 4,0 ml hydrochloric acid (ρ20 = 1,18 g/ml) into a 500 ml graduated flask, make up to the mark with methanol (3.1) and mix. This solution shall be freshly prepared before use.

3.3. Acetonitrile, equivalent to HPLC grade. U.K.

3.4. Molecular sieve. U.K.

Type 3A, 8 to 12 mesh beads (1,6-2,5 mm beads, crystalline alumino-silicate, diameter of pores 0,3 mm).

3.5. Aluminium oxide acidic activity grade I for column chromatography. U.K.

Transfer 100 g aluminium oxide into a suitable container and add 2,0 ml of water. Stopper and shake for approximately 20 minutes. Store in a well stoppered container.

3.6. Potassium dihydrogen phosphate solution, c = 0,025 mol/l. U.K.

Dissolve 3,4 g of potassium dihydrogen phosphate in water (HPLC grade) in a 1 000 ml graduated flask, make up to the mark and mix.

3.7. Di-sodium hydrogen phosphate solution, c = 0,025 mol/l. U.K.

Dissolve 3,55 g of anhydrous (or 4,45 g of dihydrate or 8,95 g of dodecahydrate) di-sodium hydrogen phosphate in water (equivalent to HPLC grade) in a 1 litre graduated flask, make up to the mark and mix.

3.8. HPLC mobile phase. U.K.

Mix together the following reagents:

• 650 ml acetonitrile (3.3),

• 250 ml water (equivalent to HPLC-grade),

• 50 ml potassium di-hydrogen phosphate solution (3.6),

• 50 ml di-sodium hydrogen phosphate solution (3.7).

Filter through a 0,22 μm filter (4.6) and degas the solution, (e.g. by ultrasonification for 10 minutes).

3.9. Standard substance. U.K.

Pure robenidine: 1,3-bis [(4-chlorobenzylidene)amino]guanidine — hydrochloride.

3.9.1. Robenidine stock standard solution: 300 μg/ml U.K.

Weigh to the nearest 0,1 mg, 30 mg of robenidine standard substance (3.9). Dissolve in acidified methanol (3.2) in a 100 ml graduated flask, make up to the mark with the same solvent and mix. Wrap the flask with aluminium foil and store in a dark place.

3.9.2. Robenidine intermediate standard solution: 12 μg/ml U.K.

Transfer 10,0 ml of the stock standard solution (3.9.1) into a 250 ml graduated flask, make up to the mark with the mobile phase (3.8) and mix. Wrap the flask with aluminium foil and store in a dark place.

3.9.3. Calibration solutions U.K.

Into a series of 50 ml calibrated flasks, transfer 5,0, 10,0, 15,0, 20,0 and 25,0 ml of the intermediate standard solution (3.9.2). Make up to the mark with mobile phase (3.8) and mix. These solutions correspond to 1,2, 2,4, 3,6, 4,8 and 6,0 μg/ml of robenidine respectively. These solutions must be freshly prepared before use.

3.10. Water equivalent to HPLC grade. U.K.

4. Apparatus U.K.

4.1. Glass column. U.K.

Constructed of amber glass fitted with a stopcock and a reservoir of approximately 150 ml capacity, internal diameter 10 to 15 mm, length 250 mm.

4.2. Mechanical shaker or magnetic stirrer. U.K.

4.3. Rotary film evaporator. U.K.

4.4. HPLC equipment with variable wavelength ultraviolet detector or diode array detector operating in the range of 250 to 400 nm. U.K.

4.4.1. Liquid chromatographic column: 300 mm x 4 mm, C₁₈ 10 μm packing or equivalent. U.K.

4.5. Glass fibre filter paper (Whatman GF/A or equivalent). U.K.

4.6. Membrane filters, 0,22 μm. U.K.

4.7. Membrane filters, 0,45 μm. U.K.

5. Procedure U.K.

Note:

Robenidine is light-sensitive. Amber glassware shall be used in all operations.

5.1. General U.K.

5.1.1. A blank feed shall be analysed to check that neither robenidine nor interfering substances are present. U.K.

5.1.2. A recovery test shall be carried out by analysing the blank feed (5.1.1) which has been fortified by addition of a quantity of robenidine, similar to that present in the sample. To fortify at a level of 60 mg/kg, transfer 3,0 ml of the stock standard solution (3.9.1) to a 250 ml conical flask. Evaporate the solution to ca. 0,5 ml in a stream of nitrogen. Add 15 g of the blank feed, mix and wait for 10 minutes before proceeding with the extraction step (5.2). U.K.

Note:

For the purpose of this method, the blank feed shall be similar in type to that of the sample and on analysis robenidine shall not be detected.

5.2. Extraction U.K.

Weigh to the nearest 0,01 g, approximately 15 g of the prepared sample. Transfer to a 250 ml conical flask and add 100,0 ml of acidified methanol (3.2), stopper and shake for one hour on the shaker (4.2). Filter the solution through a glass fibre filter paper (4.5) and collect the whole filtrate in a 150 ml conical flask. Add 7,5 g molecular sieve (3.4), stopper and shake for five minutes. Filter immediately through a glass-fibre filter paper. Retain this solution for the purification step (5.3).

5.3. Purification U.K.

5.3.1. Preparation of the aluminium-oxide column U.K.

Insert a small glass-wool plug into the lower end of a glass column (4.1) and tamp it down using a glass rod. Weigh out 11,0 g of the prepared aluminium oxide (3.5) and transfer to the column. Care shall be taken to minimise the exposure to the atmosphere during this stage. Gently tap the loaded column at its lower end to settle the aluminium oxide.

5.3.2. Sample purification U.K.

Transfer onto the column by pipette 5,0 ml of the sample extract prepared in (5.2) Rest the pipette tip close to the column wall and allow the solution to be absorbed onto the aluminium oxide. Elute the robenidine from the column using 100 ml methanol (3.1), at a flow rate of 2 to 3 ml/minute and collect the eluate in a 250 ml round bottomed flask. Evaporate the methanol solution to dryness under reduced pressure at 40 ^oC by means of a rotary film evaporator (4.3). Re-dissolve the residue in 3 to 4 ml of mobile phase (3.8) and transfer quantitatively to a 10 ml graduated flask. Rinse the flask with several 1 to 2 ml portions of mobile phase and transfer these rinsings to the graduated flask. Make up to the mark with the same solvent and mix. An aliquot is filtered through a 0,45 μm membrane filter (4.7). Reserve this solution for HPLC determination (5.4).

5.4. HPLC determination U.K.

5.4.1. Parameters U.K.

The following conditions are offered for guidance, other conditions may be used provided they yield equivalent results:

Liquid chromatographic column (4.4.1),

HPLC mobile phase (3.8),

Flow rate: 1,5 to 2 ml/minute,

Detector wavelength: 317 nm,

Injection volume: 20 to 50 μl.

Check the stability of the chromatographic system, injecting the calibration solution (3.9.3) containing 3,6 μg/ml several times, until constant peak heights and retention times are achieved.

5.4.2. Calibration graph U.K.

Inject each calibration solution (3.9.3) several times and measure the peak heights (areas) for each concentration. Plot a calibration curve using the mean peak heights or areas of the calibration solutions as the ordinates and corresponding concentrations in μg per ml as abscissae.

5.4.3. Sample solution U.K.

Inject the sample extract (5.3.2) several times, using the same volume as taken for the calibration solutions and determine the mean peak height (area) of the robenidine peaks.

6. Calculation of results U.K.

From the mean height (area) of the robenidine peaks of the sample solution determine the concentration of the sample solution in μg/ml by reference to the calibration graph (5.4.2).

The content of robenidine w (mg/kg) in the sample is given by the following formula:

in which:

c

=

robenidine concentration of the sample solution in μg/ml,

m

=

weight of the test portion in grams.

7. Validation of the results U.K.

7.1. Identity U.K.

The identity of the analyte can be confirmed by co-chromatography, or by using a diode-array detector by which the spectra of the sample extract and the calibration solution (3.9.3) containing 6 μg/ml are compared.

7.1.1. Co-chromatography U.K.

A sample extract is fortified by addition of an appropriate amount of calibration solution (3.9.3). The amount of added robenidine must be similar to the estimated amount of robenidine found in the sample extract.

Only the height of the robenidine peak shall be enhanced after taking into account both the amount added and the dilution of the extract. The peak width, at half of its maximum height, must be within approximately 10 % of the original width.

7.1.2. Diode-array detection U.K.

The results are evaluated according to the following criteria:

(a)

the wavelength of maximum absorption of the sample and of the standard spectra, recorded at the peak apex on the chromatogram, must be the same within a margin determined by the resolving power of the detection system. For diode-array detection, this is typically within approximately 2 nm;

(b)

between 250 and 400 nm, the sample and standard spectra recorded at the peak apex on the chromatogram, must not be different for those parts of the spectrum within the range 10 % to 100 % of relative absorbance. This criterion is met when the same maxima are present and at no observed point the deviation between the two spectra exceeds 15 % of the absorbance of the standard analyte;

(c)

between 250 and 400 nm, the spectra of the upslope, apex and downslope of the peak produced by the sample extract must not be different from each other for those parts of the spectrum within the range 10 % to 100 % of relative absorbance. This criterion is met when the same maxima are present and when at all observed points the deviation between the spectra does not exceed 15 % of the absorbance of the spectrum of the apex.

If one of these criteria is not met the presence of the analyte has not been confirmed.

7.2. Repeatability U.K.

The difference between the results of two parallel determinations carried out on the same sample must not exceed 10 % of the higher result for robenidine content higher than 15 mg/kg.

7.3. Recovery U.K.

For a fortified blank sample the recovery shall be at least 85 %.

8. Results of a collaborative study U.K.

An EC collaborative study was arranged in which four samples of poultry and rabbit feed, in meal or pelleted form were analysed by 12 laboratories. Duplicate analyses were performed on each sample. The results are given in the table below:

	Poultry		Rabbit
	Meal	Pellet	Meal	Pellet
Mean [mg/kg]	27,0	27,99	43,6	40,1
sr [mg/kg]	1,46	1,26	1,44	1,66
CVr [%]	5,4	4,5	3,3	4,1
SR [mg/kg]	4,36	3,36	4,61	3,91
CVR [%]	16,1	12,0	10,6	9,7
Recovery [%]	90,0	93,3	87,2	80,2

s_r

=

standard deviation of repeatability,

CV_r

=

coefficient of variation of repeatability, %

S_R

=

standard deviation of reproducibility,

CV_R

=

coefficient of variation of reproducibility. %