BS EN 16006:2011 pdf download – Animal feeding stuffs — Determination of the Sum of Fumonisin B1 & B2 in compound animal feed with immunoaffinity clean-up and RP-HPLC with fluorescence detection after pre- or post- column derivatisation
These calibration levels are recommendations and may be adjusted to the individual needs. The exactconcentrations of the calibration levels should be calculated based on the final dilution and the exactconcentration of the stock solution (4.20).
NOTE The above solutions may be stored for up to 5 days at 6 °C±2°C in the dark.
4.23 IAC (lmmunoaffinity column)
The immunoaffinity columns must contain a stationary phase with immobilized monoclonal antibodies specificto, at least,Fumonisin B, and B To be suitable for this method they must meet the requirements statedbelow:
An aliquot of more than 5 ml of an extract of a fumonisin-free representative compound animal feed material isspiked with FB, & FBz in equal parts at either 920(high) ng/ml or 110 (low) ng/ml for the sum of both. Thendilute 5,0 ml of that spiked extract to a total volume of 50,0 ml(see 6.2).
Following the procedures described in 6.3 and 6.4 this will result in expected concentrations in the injectionsolutions of either 460 ng/ml or 55 ng/ml for the sum of FB,& FB2.
After measuring (Clause 7) these solutions the observed concentrations of FB,& FB2 can be calculated withEquation (1) and Equation (2) of Clause 8. Dividing the sum of the observed concentrations of FB, &FBz bythe expected concentrations will result in the yield of the immunoaffinity columns.
These yields must be 99 %±18 %(U, k=2) at the high level and 118 %±18 %(U,k=2) at the low level.
The above column test should be performed for each level on at least three randomly selected columns ofevery new batch of immunoaffinity columns which will be used.Should the tested batch not meet the aboverequirements either a new batch which does should be obtained or the conditions described in 6.3 need to beadjusted such that the requirements are met (the user instructions supplied with the columns are a goodstarting point).
Any changes to the clean-up procedures will necessitate a revalidation of the clean-up and allsubsequent 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 rotating drum with internal fins and paddels or moving a closed container 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= 0,01g)
5.9 Glass micro fibre filter, binder-free with ca. 2 μm 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 Gastight glass syringes and/or direct displacement pipettors
Capable of precisely dispensing the following volumes: 5 μl, 50 μl, 125 μl, 160 μl, 500 μl, and 1 000 μl.
5.15 Support stand for immunoaffinity 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 derivatization, capable of mixing reagent and sample solution before injection.
5.16.3 Chromatographic column
Any column which provides symmetric peak (peak asymmetry factor 0,9< A s <1,4 at 10% of full height),sufficient retention (k > 2), and resolution (R s >1) for FB 1 & FB 2..
5.16.4 Fluorescence detector
Capable of providing the required excitation and emission wavelengths and equipped with a flow cell of appropriate size.
5.16.5 Post-column derivatisation system (not necessary if pre-column derivatisation is used)
Either a commercial unit or self-assembled. If self-assembled the following is needed:
• Reagent pump: capable of delivering a constant pulsation-free flow of the derivatisation reagent against the required pressures.
• Peek tubing: of the outer diameter required by the HPLC system in use and varying inner diameters, e.g.1/16″OD and 0,04″ID, 0,02″ID, 0,01″ID, and/ or 0,005″ ID.
• Mixing Tee: small internal volume PEEK.