BS ISO 23305:2020 pdf download – Fortified milk powders, infant formula and adult nutritionals — Determination of total biotin by liquid chromatography coupled with immunoaffinity column clean-up extraction
6 Apparatus
Usual laboratory glassware and equipment and, in particular, the following.
6.1 HPLC system, consisting of a PDA detector, low pressure gradient pump system, a sample injector unit, a degasser unit, and a column oven.
6.2 Column, Kinetex Phenyl-Hexyl (Cat. No. 00F-4495-E0, Phenomenex 2) ),
150 mm × 4,6 mm × 2,6 μm × 10 nm.
6.3 Glass microfibre filters (Cat. No. 1820-125, Whatman ®2) ).
6.4 Immunoaffinity column pack (R-Biopharm Rhone EASI-EXTRACT ® BIOTIN P82/P82B 2) or equivalent).
6.5 SPE manifold, with accessories.
6.6 Autoclave, set at 121 °C.
6.7 Centrifuge, variable speed.
6.8 Analytical balance, four decimal places.
6.9 Amber glass screw-cap bottle, 100 ml.
6.10 Horizontal shaker.
6.11 Volumetric flasks, 1 l, 250 ml, 100 ml and 10 ml.
6.12 Pipettors, calibrated, 10,0 ml, 5,0 ml, 1,0 ml, 200 µl, 100 µl and 50 µl.
6.13 Measuring cylinder, 1000 ml, 100 ml and 50 ml.
6.14 Reaction vial, Reacti-Vials (Cat. No. 13223, Thermo Scientific 2) ).
6.15 Heating block, Reacti-therm, with nitrogen blow down (Thermo Scientific 2) ).
6.16 Ultrasonic bath, set at 50 °C and room temperature.
6.17 Centrifuge tubes, 50 ml.
6.18 Vortex mixer.
6.19 Syringe filter, polytetrafluoroethylene (PTFE) 0,45 µm.
6.20 Disposable syringes, 10 ml and 1 ml.
6.21 HPLC vials, 2 ml with 200 µl glass inserts.
7 Procedure
7.1 Sample preparation
7.1.1 For mass and loading volumes for the different ranges of product, see Table 1. A slurry may be used wherever product homogeneity is suspected or unknown.
For the slurry, reconstitute 25 g of powder (m 1 ) with warm water (~50 °C) to a total mass of 200 g (m 2 ).
Mix thoroughly on a horizontal shaker for 20 min and then sonicate at 50 °C for 10 min. Cool to room temperature. For liquid samples, mix well to ensure homogeneity of the sample portion and weigh the specified quantity.
7.1.2 Weigh the sample/slurry (m 3 ) into a 100 ml amber glass screw-cap bottle. See Table 1.
7.1.3 Add 0,15 mol/l sodium phosphate buffer to an approximate volume of 50 ml.
7.1.4 Swirl gently to mix.
7.1.5 Autoclave the sample preparation at 121 °C for 25 min.
7.1.6 Cool the sample to room temperature. Quantitatively transfer the extract into a 100 ml volumetric flask and make up to the mark with 0,15 mol/l sodium phosphate buffer, mix well.
7.1.7 Transfer the extract into a centrifuge tube and centrifuge the sample at 4 000 rpm for 15 min.
7.1.8 Filter the sample using the glass microfibre filter paper (6.3) and collect the filtrate.
7.1.9 Set up the SPE manifold (6.5). Attach the immunoaffinity column (IAC) connected to a 10 ml reservoir. Drain off buffer just above the gel.
7.1.10 Load the sample filtrate onto the column in accordance with Table 1 and initialize the flow with the help of a vacuum pump.
7.1.11 Turn off the vacuum and let the solution pass through the column by gravity at a rate of one drop per second.
7.1.12 Wash the column by passing 10 ml of PBS (5.1.8) through the column, followed by 10 ml of water.
Initialize the flow with the help of vacuum at every step and then leave it to flow by gravity.
7.1.13 Remove any residual liquid from the column by introducing a gentle vacuum.
7.1.14 Introduce a reaction vial (6.14) and elute the analyte under gravity with 2 ml methanol. Elute further with an additional 1 ml of methanol. Backflush at least three times when eluting; this can be achieved by a gentle up and down motion of the syringe plunger to maximize the elution.
7.1.15 Evaporate the eluate to dryness using a heating block (6.15) set at 85 °C ± 5 °C under a gentle nitrogen blow down.
7.1.16 Remove from the heating block and cool down to room temperature (about 15 min).
7.1.17 Re-dissolve with 1 ml water and then cap the reaction vial (6.14) and vortex for 30 s. Filter by using a syringe filter into a clean glass insert in a HPLC vial for the HPLC analysis.
7.2 Chromatography
7.2.1 Set-up the HPLC system with the following configuration. Examples of chromatograms of a calibration standard and an infant formula sample used in the interlaboratory study are given in Annex A.
7.2.2 Mobile phase A, 0,1 % phosphoric acid.
7.2.3 Mobile phase B, 100 % acetonitrile.
7.2.4 Mobile phase C, 80 % acetonitrile.