BS EN 16006:2011 pdf download

BS EN 16006:2011 pdf download.Animal feeding stuffs —Determination of the Sum of Fumonisin B1& B2 incompound animal feed within munoaffinity clean-up and RP-HPLC with fluorescence detection after pre- or post-column derivatisation.
These levels are recommendations and may be adpusted to the individual needs. The exact concentrations of the calibration levels should be calculated based on the final dilution and the exact concentration of the stock solution (4.20).
NOTE The above solubons may be stored for upto 5 days at 6 C a 2’C In the darx
4.23 IAC (lrnmunoaffinity column)
The immunoaffinily columns must contain a stationary phase with immobilized rnonoclonal antibodies specific to, at least. Fumonisin B1 and B2 To be suitable for this method they must meet the requirements stated below:
An aliquot of more than 5 ml of an extract of a tumonrsln-free representative compound animal teed material Is spded with FB1 8. FB2 In equal parts at elthe 920 (h.gh) ng!ml or 110 (low) nglml for the sum of both. Then dIlute 5.0 ml of that spiked extract to a total volume of 50.0 ml (see 6.2).
Following the procedures descflbed in 6.3 and 6,4 this will result in expected concentrations in the injection solutions of either 460 nglml or 55 ng/ml for the sum of FB1 & FB2.
After measuring (Clause 7) these solutions the observed concentrations of FB1 & FB can be calculated with Equation (1) and Equation (2) of Clause 8. Dividing the sum of the observed concentrations of FB, & FB2 by the expected concentrations will result In the aeld of the Immunoaffinity columns.
These yields must be 99% ± 18% (U, k—2) at the high level and 118% ± 18% (U. k2) at the low level.
The above cokimn test should be performed for each level on at least three randomly selected columns of every new batch of immunoattinhty columns which will be used. Should the tested batch not meet the above requirements either a new batch whicti does should be obtained or the conditions described in 6.3 need to be adjusted such that the requwernents are met (the user instructions supplied with the columns are a good starting powit).
Any changes to th. cl.an.up proc.dur.s will necessItat, a r.valldatlon of th. clean-up and all subsequent steps (chromatography).
5 Apparatus
Usual laboratory equipment and, in particular, the following:
5.1 Mill
5.2 Tumble mixer
Creates a folding motion of the material through, for instance, a rOtatJn9 drum with internal fins and paddels or moving a dosed contamer head-over.heels.
5.3 Vortex mixer
5.4 Laboratory shaker
5.5 250 ml flasks with screw caps
5.6 Graduated cylinders. 5 ml, 50 ml, 1 000 ml and 2 000 ml
5.7 Graduated pipettes (Class A, EN ISO 835) 2 ml, 10 ml and 50 ml
5.8 AnalytIcal balance (d 0,Olg)
5.9 Glass micro fibre filter, binder-free with Ca. 2 pm pore size
5.10 Filter funnel, of appropriate size
5.11 Auto sampler vials, of appropriate size with caps
5.12 Reservoirs for IACs
Of appropriate size with adapter for connection to top of IACs.
5.13 Volumetric flasks (Class A, EN ISO 1042) 2 ml, 5 ml, 10 ml and 20 ml
5.14 Gastlght glass syringes andlor direct displacement pipettors
Capable of precisely dispen5ing the fOllowing volumes: 5 il, 50 il. 125 i, 160 p1, 500 p1. and 1 000 i.
5.15 Support stand for Immunoafflnlty columns, of appropriate size
5.16 HPLC instrumentation, comprising the following:
5,16.1 Solvent delivery system
Capable of generating a binary gradient with sufficient precision at the required pressures,
5.16.2 Auto sampler
Capable of injecting sufficient volumes of injection solution with sufficient repeatability and, for pre-column decivatization, capable of mixing reagent and sample solution before injection,
6 Sample preparation
6.1 Sample preparation
It is important that the laboratory receives a sample which is truly representative and has not been damaged or changed during transport or storage. Samples should be taken and prepared in accordance with European legislation where applicable (1J. Samples should be finely ground and thoroughly mixed using a mill (5.1) and a tumble mixer (5.2) or another process that has been shown to give complete homogenisation before a test portion is removed for analysis.
In all instances: If the sample has been frozen allow it tO thaw completely before sampling. Mix the sample thoroughly before removing an analytical test portlon
NOTE The materials for the inteilaboratory study (3J were milled to a pertido size of Ca. 0,5 mm.
6.2 Extraction of FB1 & FB2
• Weigh 20,0 g of the test sample into a large enough container with lid, e.g. 250 ml flask (5.5);
• add 200 ml of extraction sotvent (4.18). cap. and shake vigorously by hand, so that the material disperses evenly;
• put on a shaker (5.4) for 120 mm: choose speed such that the material Is mixed well without collecting In the top of the flask;
• allow the extracted sample to settle after shaking;
• of the supematant take 5.0 ml and dilute with PBS (4.16) to a total volume of 50,0 nil and mix.