A blank feed shall be analysed to check that neither diclazuril nor interfering substances are present. The blank feed shall be similar in type to that of the sample and on analysis diclazuril or interfering substances shall not be detected.
A recovery test shall be carried out by analysing the blank feed which has been fortified by addition of a quantity of diclazuril similar to that present in the sample. To fortify at a level of 1 mg/kg add 0,1 ml of the stock standard solution (3.8.1) to 50 g of a blank feed, mix thoroughly and leave for 10 min. mixing again several times before proceeding (5.2).
Alternatively, if a blank feed similar in type to that of the sample is not available (see 5.1.1), a recovery test can be performed by means of the standard addition method. In this case, the sample to be analysed is fortified with a quantity of diclazuril, similar to that already present in the sample. This sample is analysed, together with the unfortified sample and the recovery can be calculated by subtraction.
Weigh to the nearest 0,01 g approximately 50 g of the sample. Transfer to a 500 ml conical flask, add 1,0 ml internal standard solution (3.9.2), 200 ml extraction solvent (3.12) and stopper the flask. Shake the mixture on the shaker (4.1) overnight. Allow to settle for 10 minutes. Transfer a 20 ml aliquot of the supernatant to a suitable glass container and dilute with 20 ml water. Transfer this solution on an extraction cartridge (3.11), and pass through by applying vacuum (4.5). Wash the cartridge with 25 ml of a mixture of extraction solvent (3.12) and water, 65 + 35 (V + V). Discard the collected fractions and elute the compounds with 25 ml of a mixture of extraction solvent (3.12) and water, 80 + 20 (V + V). Evaporate this fraction until it had just reached dryness by means of the rotary evaporator (4.3) at 60 oC. Dissolve the residue in 1,0 ml DMF (3.6), add 1,5 ml of water (3.1) and mix. Filter through a membrane filter (4.4). Proceed to the HPLC determination (5.3).
Weigh to the nearest 0,001 g approximately 1 g of the sample. Transfer to a 500 ml conical flask, add 1,0 ml internal standard solution (3.9.3), 200 ml extraction solvent (3.12) and stopper the flask. Shake the mixture overnight on the shaker (4.1). Allow to settle for 10 minutes. Transfer an aliquot of 10 000/p ml (p = nominal content of diclazuril in the premix in mg/kg) of the supernatant to a round bottomed flask of suitable size. Evaporate until it had just reached dryness, under reduced pressure at 60 oC by means of the rotary evaporator (4.3). Redissolve the residue in 10,0ml DMF (3.6), add 15,0 ml water (3.1) and mix. Proceed to the HPLC determination (5.3).
The following conditions are offered for guidance, other conditions may be used provided that they give equivalent results.
| Liquid chromatographic column (4.2.1) | 100 mm × 4,6 mm, Hypersil ODS, 3 μm packing, or equivalent | |
| Mobile phase: | Eluent A (3.13.1): | Aqueous solution of ammonium acetate and tetrabutyl-ammonium hydrogen sulphate |
| Eluent B (3.13.2): | acetonitrile | |
| Eluent C (3.13.3): | methanol | |
| Elution mode: |
Flush with B during 10 min. | |
| Flow rate: | 1,5-2 ml/min. | |
| Injection volume: | 20 μl | |
| Detector wavelength: | 280 nm. | |
Check the stability of the chromatographic system, injecting several times the calibration solution (3.10), containing 2 μg/ml, until constant peak heights and retention times are achieved.
Inject 20 μl of the calibration solution (3.10) several times and determine the mean peak height (area) of the diclazuril and internal standard peaks.
Inject 20 μl of the sample solution (5.2.1 or 5.2.2) several times and determine the mean peak height (area) of the diclazuril and internal standard peaks.