Commission Regulation (EC) No 152/2009 of 27 January 2009 laying down the methods of sampling and analysis for the official control of feed (Text with EEA relevance) (c. 152)

ANNEX VIII U.K. METHODS OF ANALYSIS TO CONTROL ILLEGAL PRESENCE OF NO LONGER AUTHORISED ADDITIVES IN FEED

B. DETERMINATION OF OLAQUINDOX U.K.

2-[N-2'-(hydroxyethyl)carbamoyl]-3-methylquinoxaline-N¹,N⁴-dioxide

1. Purpose and scope U.K.

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

2. Principle U.K.

The sample is extracted by a water-methanol mixture. The content of olaquindox 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. Methanol, equivalent to HPLC grade. U.K.

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

3.4. Mobile phase for HPLC. U.K.

Water (3.3)-methanol (3.2) mixture, 900 +100 (V + V).

3.5. Standard substance: pure olaquindox 2-[N-2'-(hydroxyethyl)carbamoyl]-3-methylquinoxaline-N¹,N⁴-dioxide, E 851. U.K.

3.5.1. Olaquindox stock standard solution, 250 μg/ml U.K.

Weigh to the nearest 0,1 mg 50 mg of olaquindox (3.5) in a 200 ml graduated flask and add ca. 190 ml water. Then place the flask for 20 min. into an ultrasonic bath (4.1). After ultrasonic treatment bring the solution to room temperature, make up to the mark with water and mix. Wrap the flask with aluminium foil and store in a refrigerator. This solution must be prepared fresh each month.

3.5.2. Olaquindox intermediate standard solution, 25 μg/ml U.K.

Transfer 10,0 ml of the stock standard solution (3.5.1) into a 100 ml graduated flask, make up to the mark with the mobile phase (3.4) and mix. Wrap the flask with aluminium foil and store in a refrigerator. This solution must be prepared fresh each day.

3.5.3. Calibration solutions U.K.

Into a series of 50 ml graduated flasks transfer 1,0, 2,0, 5,0, 10,0, 15,0 and 20,0 ml of the intermediate standard solution (3.5.2). Make up to the mark with the mobile phase (3.4) and mix. Wrap the flasks with aluminium foil. These solutions correspond to 0,5, 1,0, 2,5, 5,0, 7,5 and 10,0 μg of olaquindox per ml respectively.

These solutions must be prepared fresh each day.

4. Apparatus U.K.

4.1. Ultrasonic bath U.K.

4.2. Mechanical shaker U.K.

4.3. HPLC equipment with variable wavelength ultraviolet detector or diode array detector U.K.

4.3.1. Liquid chromatographic column, 250 mm × 4 mm, C₁₈, 10 μm packing, or equivalent U.K.

4.4. Membrane filters, 0,45 μm U.K.

5. Procedure U.K.

Note:

Olaquindox is light sensitive. Carry out all procedures under subdued light or use amber glassware.

5.1. General U.K.

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

5.1.2. A recovery test shall be carried out by analysing the blank feed which has been fortified by addition of a quantity of olaquindox, similar to that present in the sample. To fortify at a level of 50 mg/kg, transfer 10,0 ml of the stock standard solution (3.5.1) to a 250 ml conical flask and evaporate the solution to ca. 0,5 ml. Add 50 g of the blank feed, mix thoroughly and leave for 10 min. mixing again several times 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 olaquindox must not be detected.

5.2. Extraction U.K.

Weigh to the nearest 0,01 g, approximately 50 g of the sample. Transfer to a 1 000 ml conical flask, add 100 ml of methanol (3.1) and place the flask for 5 min. in the ultrasonic bath (4.1). Add 410 ml water and leave in the ultrasonic bath for further 15 min. Remove the flask from the ultrasonic bath, shake it for 30 min. on the shaker (4.2) and filter through a folded filter. Transfer 10,0 ml of the filtrate into a 20 ml graduated flask, make up to the mark with water and mix. An aliquot is filtered through a membrane filter (4.4). (see 9. Observation) Proceed to the HPLC determination (5.3).

5.3. HPLC determination U.K.

5.3.1. Parameters: U.K.

The following conditions are offered for guidance, other conditions may be used provided that they give equivalent results.

Analytical column (4.3.1)
Mobile Phase (3.4):	water (3.3)-methanol (3.2) mixture, 900 + 100 (V + V)
Flow rate:	1,5-2 ml/min.
Detection wavelength:	380 nm
Injection volume:	20 μl –100 μl

Check the stability of the chromatographic system, injecting several times the calibration solution (3.5.3) containing 2,5 μg/ml, until constant peak heights and retention times are achieved.

5.3.2. Calibration graph U.K.

Inject each calibration solution (3.5.3) several times and determine the mean peak heights (areas) for each concentration. Plot a calibration graph using the mean peak heights (areas) of the calibration solutions as the ordinates and the corresponding concentrations in μg/ml as the abscissae.

5.3.3. Sample solution U.K.

Inject the sample extract (5.2) several times using the same volume as taken for the calibration solutions and determine the mean peak height (area) of the olaquindox peaks.

6. Calculation of the results U.K.

From the mean height (area) of the olaquindox peaks of the sample solution determine the concentration of the sample solution in μg/ml by reference to the calibration graph (5.3.2).

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

in which:

c

=

olaquindox concentration of the sample extract (5.2) in μg/ml

m

=

weight of the test portion in g (5.2).

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 (5.2) and the calibration solution (3.5.3) containing 5,0 μg/ml are compared.

7.1.1. Co-chromatography U.K.

A sample extract (5.2) is fortified by addition of an appropriate amount of calibration solution (3.5.3). The amount of added olaquindox must be similar to the amount of olaquindox found in the sample extract.

Only the height of the olaquindox 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 height, must be within ± 10 % of the original width of the olaquindox peak of the unfortified sample extract.

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 ± 2 nm.

(b)

Between 220 and 400 nm, the sample and standard spectra recorded at the peak apex of the chromatogram, must not be different for those parts of the spectrum within the range 10 %-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 220 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 %-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 peak 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 15 % relative to the higher result for olaquindox contents between 10 and 200 mg/kg.

7.3. Recovery U.K.

For a fortified blank sample the recovery shall be at least 90 %.

8. Results of a collaborative study U.K.

An EC collaborative study was arranged in which four piglet feed samples including one blank feed were analysed by up to 13 laboratories. The results are given below:

	Sample 1	Sample 2	Sample 3	Sample 4
L	13	10	11	11
n	40	40	44	44
mean [mg/kg]	—	14,6	48,0	95,4
Sr [mg/kg]	—	0,82	2,05	6,36
SR [mg/kg]	—	1,62	4,28	8,42
CVr [%]	—	5,6	4,3	6,7
CVR [%]	—	11,1	8,9	8,8
Nominal content
[mg/kg]	—	15	50	100
recovery %	—	97,3	96,0	95,4

L

=

number of laboratories

n

=

number of single values

S_r

=

standard deviation of repeatability

S_R

=

standard deviation of reproducibility

CV_r

=

coefficient of variation of repeatability

CV_R

=

coefficient of variation of reproducibility.

9. Observation U.K.

Although the method has not been validated for feeds containing more than 100 mg/kg of olaquindox, it may be possible to obtain satisfactory results by taking a smaller sample weight and/or diluting the extract (5.2) to reach a concentration within the range of the calibration graph (5.3.2).