BS EN 15835:2010 pdf download
BS EN 15835:2010 pdf download.Food stuffs -Determination of ochratoxin A in cereal based foods for in fants and young children HPLC method with immunoaffinity column cleanup and fluorescence detection.
4.19 Methanol, gradient grade
4.20 Toluene
4.21 Mixture of methanol and acetic acid solution
Mix 72 parts per volume of methanol (4.19) with 28 parts per volume of acetic acid solution (4.17).
422 Tert-butyl methyl ether
WARNING — Tert-butyl methyl ether Is hazardous and samples shall be blended using an explosion proof blender which is housed within a fume cupboard. Centrifugation of extracts shall be performed at cool temperature (4 C to 8 C).
4.23 Mixture of toluene and glacial acetic acid
Mix 99 parts per volume of toluene (4.20) with one part per volume of glacial acetic acid (4,16),
424 HPLC mobile phase A
Acetic acid solution (4.17).
425 HPLC mobile phase B
Methanol (4.19).
Degas the mobile phases A and B with for example helium (4.2). Helium can be pumped into the reservoirs of both mobile phases A and B. The pumping rate should be 50 rnllmin to 100 mt!min. The use of a degasser is also an acceptable option.
426 Immunoaffinity columns
The mimunoaffirnty column contain antibodies raised against ochratoxln A. The column shall have a capacity of not less than 100 ng of ochratoxiri A and shall give a recovery of not less than 85 % when applied as a standard solution of ochratoxin A in a mxture of 15 parts per volume of methanol (4.19) and 85 parts per volume of PBS solution (4.14) containing 3 ng of odiratoxin A.
4.27 Ochratoxln A, In crystal form or as a film In ampoules
WARNING — Ochratoxin A Is a potent nephrotoxin with immunotoxic, teratogenic and potential genotoxic properties. The International Agency for Research on Cancer (IARC) has classified ochratoxin A as a possible human carcinogen (group 2B). Protective clothing, gloves and safety glasses should be worn at all times, and all standard and sample preparation stages should be can-led out in a fume cupboard.
428 Ochratoxln A stock solution
Prepare a stock solution of ochratoxln A m the mixture of toluene and acial acetic acid (4.23) with a nominal concentration of 10 iJgIml.
To deterrnme the exact concentration, record the absorption curve between a wavelength of 300 nm and 370 nm in 5 nm steps wi 1 cm quartz cells (5.22) in a spectrometer with the solvent mixture (4.23) as reference. Identify the wavelength for maxwnum absorption and calculate the mass concentration of odiratoxin A. p.. in micrograms per millilitre. using Equation (1):
5.9 CentrIfuge bottles, of 250 ml capacity with screw cap, chemically resistant to teil-butyl methyl ether and
able to work at 15 300 g without deformation
5.10 Rotary evaporator, with water bath
5.11 Disposable syringe barrels, to be used as reservoirs, of 5 ml. 20 ml and 50 ml capacity, luer locks and
attachments to fit to mm unoatfinity columns
5.12 Mlcrosyrlng.s, of 25 p1, 50 p1. 100 p1, 500 p1 and 1 000 p1 capacity
5.13 CalIbrated volumetric flasks, e.g of 10 ml, 50 ml and 1 000 ml capacity
5.14 Vacuum system
5.15 Round bottomed flasks, of 100 ml capacity
5.16 Calibrated volumetric pipettes
5.17 Displacement pipettes, of 100 p1 and 1 000 p1 capacity, with appropnate tips
5.18 Glass vials, of GC autosampler type. approximately 1,8 ml capacity and crimp caps
5.19 Separating funnel, of 250 ml capacity
5.20 Vortex mixer
5.21 HPLC apparatus, comprising the following
5.21.1 Injection system, capable of injecting e.g. 50 p1
5.21.2 Eluent and post column reagent reservoirs
5.21.3 MobIl. phas. pump, gradient, capable of rnaintaerung a volume flow rate of I mllmin pulse free
5.21.4 Fluorescence detector, able to provide A = 390 nm excitation and A = 440 nm emission wavelengths
521.5 Recorder, integrator or computer based data processing system
5.21.6 AnalytIcal revers..phas. HPLC separating column, for example C, base deactivated octadecyl silane (ODS). length of 25 cm, Inner diameter of 4.7 mm and a particle size of 5 pm, which ensures resolution of octiratoxan A from al other peaks. The maximum overlap of peaks shall be less then 10 %. A suitable corresponding reverse-phase guard column should be used
5.21.7 Degasser, optional, for degassing HPLC mobile phases (4,24) and (4.25) and the ammonium hydroxide solution 14.9)
5.21.8 Column oven, capable to operate at (50 i 1) C
5.21.9 Post-column derivatitatlon system. comprslng a second LC pulseless pump, zero dead volume T-plece. reacbon stainless steel tubing mm. 10 cm 0.25 mm internal diameter
5.22 UV spectrometer, with suitable quartz cels.
