EN 16158-2012 pdf download.Animal feeding stuffs – Determination of semduramicin content -Liquid chromatographic method using a “tree” analytical approach.
4.5.4 Nigerlcin sodium intermediate standard solution (for the LC-MS method), Ca. 100 pg/mi
Accurately pipette 1,0 ml of the nigericin sodium stock standard solution (4.5.2) into a 10 ml volumetric flask (5.7). Make up to 10 ml of with acetonitrile (4.1.2). Store at -20 tmC and protected from light. Prepare fresh intermediate solutions monthly.
4.5.5 Semduramicln spiking solution (for the LC-MS method), Ca. 2 pg/mI.
Accurately pipette 200 p1 of the semduramicin intermediate solution (4.5.3) with an appropriate automatic pipette (5.6) into a 10 ml volumetric flask (5.7). Make up to 10 ml with acetonitrile (4.1.2). Store at -20 C and protected from light. Prepare fresh spiking solutions weekly.
4.5.6 Nigencin sodium spiking solution (for the LC-MS method), Ca. 2 pg/mI.
Accurately pipette 200 p1 of the nigericin sodium intermediate solution (4.5.4) with an appropriate automatic pipette (5.6) into a 10 ml volumetric flask. Make up to 10 ml with acetonitnle (4.1.2). Store at -20°C and protected from light. Prepare fresh spiking solutions weekly.
5 Apparatus
Usual laboratory apparatus and, in particular, the following:
5.1 LC-MS method HPLC system consisting of the following:
5.1.1 Pump, pulse free, flow capacity 0.1 mI/mm 102,0 mI/mm.
5.1.2 Injection system, manual or autosampler, with a loop suitable for 10 p1 Injections.
5.1.3 Single quadrupole mass spectrometer or triple quadrupole mass spectrometer used in the MS configuration able to operate in a mass range at least between 200 m!z to 1 000 mlz.
NOTE For the MS detector the vacuum should be as stable as possible to ensure satisfactory repeatabiIity The system should therefore be pumped at least 48 h before starting the measurements.
5.1.4 Computer data system.
5.1.5 Analytical column, Alitima HP C18, 5 pm 150 mm x 2.1 mm, 190 A. 12 % C load and 200 m2/g or equivalent.
5.1.6 Guard column, Ailtima HP C18. 5 pm, 7.5 mm x 2,1 mm. 190 A, 12% C load and 200 m2/g or equivalent.
5.2 LC-PCD-IJV method HPLC system consisting of the following:
5.2.1 Pump, pulse free, flow capacity 0,1 mI/mm to 2.0 mI/mm.
5.2.2 Injection system, manual or autosampler, with a loop suitable for 100 p1 injections.
5.2.3 UVN1S detector, variable wavelength, suitable for reliable measurements at 598 nm, or UVNIS photodiode array detector (DAD).
5.2.4 Computer data system.
5.2.5 Post-column reactor, with a 1,5 ml to 2,0 ml reaction coil, for operation at 92 °C.
The coil may be a commercially available coil or it may be made using 1/16 316 SS tubing, 0,020w <>0,5 mm ID (between 8 m and 10 m length) folded to fit the reactor heating chamber. To ensure effective mixing of reagent and column effluent, use a vortex or static mbchg tee (not a regular tee) before the reaction coil.
5.2.6 Post column reagent pump, pulse free, flow capacity from 0,5 mI/mm to 2,0 mI/mm.
5.2.7 Analytical column, lnertsil ODS-3 C18. 5 pm, 150 x 4,6 mm 100 A 15% carbon load and 450m2/g or equivalent.
5.2.8 Guard column, Prevail C18, 5 pm, 7,5 x 4,6 mm or equivalent.
5.3 Glass vials, 1,5 ml HPLC glass vials.
5.4 Shaker, head-over-head or equivalent.
5.5 Balances, one analytical, of 10 g capacity or greater with 0,1 mg readability.
5.6 VarIable-volume positive displacement piston pipettes, suitable for pipetting volumes ranged from 20 p1 to 1 000 p1.
5.7 VolumetrIc flasks, 10 ml, 20 ml. 500 ml and 1 000 ml.
5.8 Glass graduated cylinders, 100 ml and 1 000 ml.
5.9 Polypropylene centrifuge tubes, 50 ml capacity. stoppered (Falcon or equivalent).
5.10 Disposable syringes, 20 ml.
5.11 Solvent filtration system, all glass filter apparatus suitable for 47 mm filters.
5.12 Membrane filters, Durapore® membrane filters, PVDF, hydrophobic, of 47 mm diameter and pore size
0,45 pm or equivalent.
5,13 Nylon filters, 47 mm diameter and pore size 0,22 pm or equivalent.
5.14 Syringe filters Nylon 0,2 pm or equivalent i.e. full chemical compatibility with acetonitrile.
5.15 pH meter.
5.16 Grinding instrument.
5.17 Sieve, with 1 mm apertures.
6 Sampling
It is important that the laboratory receives a sample that is truly representative and has not been damaged or changed during transport or storage.
Sampling is not part of the method specified in this European standard. A recommended sampling method is given in EN ISO 6497 [1].
7 Preparation of test sample
7.1 General
Prepare the test sample in accordance with prEN ISO 6498.EN 16158-2012 pdf download.