BS/EN 17299-2019 pdf download.Animal feeding stuffs: Methods of sampling and analysis — Screening and determination of authorized coccidiostats at additive and 1 % and 3 % cross-contamination levels, and of non- registered coccidiostats and of one antibiotic at sub-additive levels, in compound feed with High Performance Liquid Chromatography — Tandem Mass Spectrometry detection (LC-MS/MS).
9.2.2 Positive control sample PCS
9.2.2.1 Target feed
Accurately weigh to the nearest 0,1 g 5.0 g of a target compound feed blank into a 50 ml polypropylene stoppered tube (6.14).
Accurately pipette (6.8), 400 jd of the target feed coccidiostats spiking solution (5.15.1). Mix thoroughly to ensure the contact with the feed. Open at least I h before submitting to (9.3).
9.2.2.2 Non-target feed
Accurately weigh to the nearest 0,1 g 5,0 g of a non-target compound feed blank into a 50 ml polypropylene stoppered tube (6.14).
Accurately pipette (6.8), 400 id of the non-target feed coccidiostats spiking solution (5.15.3). Mix thoroughly to ensure the contact with the feed. Open at least 1 h before submitting to (9.3).
9.2.3 Negative control sample NCS
Accurately weigh to the nearest 0,1 g 5,0 g of a compound feed blank into a 50 ml polypropylene stoppered tube (6.14). Submit to (9.3).
9.3 Sample preparation
9.3.1 Extraction
Prepare 2 replicate test portIons (8.4) from each unknown sample. Add 40 ml of solvent mixture for the feed extraction (5.11). Close tightly the polypropylene centrifuge tube (6.14) and check that there is no leak through the cap. Shake vigorously to ensure all the feed is suspended in the extraction solvent and then put it in an ultrasonic bath (6.5) set at 22 °C for 0.5 h. Then perform a solid-liquid extraction by shaking the mixture head to head for 1 h on the shaker (6.6) followed by centrifugation at 1 850g and 20°C during 10 mm (6.20).
The PCS and NCS are submitted to the same procedure but as single analysis (no replicates).
NOTE The unknown samples for which a simple screening is desired, are sublected to the same procedure (9.3)
but with a single analysis (no replicates) as for the PCS and NCS.
9.3.2 Filtration
Filter the supernatant obtained in 9.3.1 (for the compound feeds and for the PCS and the NCS) through a 0,2 urn polyamide 6.6 syringe filter (6.17) into a clean polypropylene tube (6.14).
For compound feeds containing coccidiostats at cross-contamination levels and potentially nonregistered coccidiostats (ethopabate. clopidol. ronidazole, dimetridazole and/or amprolium) and the banned antibiotic furazolidone at sub-additive levels and the PCS, proceed directly to 9.4. For the NCS, proceed to 9.5.2.
9.3.3 Dilution
For compound feeds containing the coccidiostats at additive levels, dilution is necessary.
Accurately pipette (6.8) 200 id of the compound feed sample filtrate obtained in 9.3.2 in a 20 ml round bottom volumetric flask (6.9) and make up to 20 ml with the solvent mixture for feed extraction (5.11). Proceed to 9.4.
NOTE This standard Is suitable for the screening and for the determination of the coccidiostats and of the furazolidone antibiotic. If screening is the sole purpose, it is advised to proceed to 9.5.
9.4 Quantthcation through standard addition
Accurately pipette with an appropriate automatic pipette (6.8). seven 2 mI-aliquots of the final filtered (9.3.2) or diluted extract (9.3.3) into glass tubes (6.12) and label as SOa, Sob, Si, S2, S3. S4 and S5, respectively.
Accurately pipette with an appropriate automatic pipette (6.8). 50 iI of target feed spiking solution of 9 internal standards (5.15.2) or non-target feed spiking solution of 9 internal standards (5.15.4), depending on which compound feeds are subjected to analysis, and add to each of the glass tubes labelled as SOa, SOb, Si, S2, S3, S4 and S5, respectively.
Accurately pipette with an appropriate automatic pipette (6.8). 0,0, 10, 20,30,40 and 50 p1 of target feed spiking solution of 11 coccidiostats (5.15.1) or non-target feed spiking solution of Ii coccidiostats (5.15.3), depending on which compound feeds are subjected to analysis, and add to the glass tubes labelled as SOa, Sob. Si. S2, S3, S4 and S5. respectively.
Accurately pipette with an appropriate automatic pipette (6.8) 50, 50. 40, 30, 20, 10 and 0 iii of acetonitrile (5.2) and add to the glass tubes labelled as SOa. Sob. Si, S2. 53, 54 and S5. respectively.
In the case of non-target feed extracts accurately additionally pipette with an appropriate automatic pipette (6.8) 4 ml of methanol (5.3) and add to the glass tubes with the extracts and mix thoroughly.
NOTE If peak shapes issues are noticed when proceeding to the chromatography for non-target feed extracts, the dilution can be performed with 4 ml of the solvent mixture for feed extraction (5.11) instead of the 4 ml of methanol (5.3).
In the case of target feed extracts the later step is not required.
Transfer separately 1 ml of each final sample contained in the glass tubes into HPLC 1.5 ml vials respectively labelled SOa. SOb. Si. S2. S3, S4 and 55 (6.4), cap them and shake vigorously the contents. For the target feed extracts, mix carefully before transferring the 1 ml of each final sample.
Submit all HPLC vials to analysis (9.5.2).
For the sole purpose of screening. only inject the SO extracts and identify the potential analytes of interest. Estimate the mass fraction content of these analytes against a calibration range of coccidiostats and / or antibiotic of interest, individually built or as a mixture. The calibration range should be prepared from the solutions 5.15.1. 5.15.2 or 5.15.3, 5.15.4 in acetonitrile.
9.5 HPLC analysis
9.5.1 Analytical conditions
The following conditions are provided for guidance. Other conditions may be used provided they yield to equivalent results.
9.5.1.1 HPLC column: as in 6.3
9.5.1.2 Guard column: as in 6.2
9.5.1.3 Mobile phase: as in 5.12, flow rate: 0,35 mI/mm
Elution programme: step gradient mode. Mobile phases A and B are used to apply the following gradient elutlon: hold 90 % mobile phase A for 2 mm, decrease to 10 % at 2 mm and hold for 9 mm. At 11 mm, increase mobile phase A to 90 % and hold for 4 mm. The total run time is 15 mm.BS/EN 17299-2019 pdf download.