BS ISO 20639 pdf free download

BS ISO 20639-2015 pdf free download.Infant formula and adult nutritionals — Determination of pantothenic acid by ultra high performance liquid chromatography and tandem mass spectrometry method (UHPLC-MS/MS).
4.1 Standards
4.1.1 Calcium d-pantothenate, Sigma 1) or equivalent CAS 137-08-6.
4.1.2 Calcium pantothenate-[ 13 C 6 , 15 N 2 ], IsoSciences 1) or equivalent CAS 356786-94-2.
4.2? α-Amylase, Sigma A3176 1) , from porcine pancreas, about 25 U/mg or equivalent.
4.3 Solvents
4.3.1 Acetonitrile, LC grade or equivalent.
4.4 Ammonium acetate, ACS grade, > 98 % (Fluka 9690) 1) .
4.5 Acetic acid, ACS grade.
4.6 Formic acid, ACS grade.
4.7 1 % Formic acid in water, ACS grade.
4.8 Preparation of standard solutions
4.8.1 Pantothenic acid (PA) stock solution, ρ = 250 μg/ml. Weigh 54,5 mg of calcium pantothenate (4.1.1) into a 200 ml volumetric flask (take into account the moisture content given in the supplier’s certificate or dry to constant mass at 105 °C) and dilute to volume with water. Store aliquots at −20 °C.
4.8.2 Pantothenic acid intermediate solution, ρ = 10 μg/ml. Transfer 1 ml of PA stock solution (4.8.1) into a 25 ml volumetric flask and dilute to volume with water. Store aliquots at –20 °C.
4.8.3 Calcium pantothenate-[ 13 C 6 , 15 N 2 ] solution [IS (Internal Standard)] stock solution, ρ = 20 μg/ml. Weigh 5,0 mg of calcium pantothenate-[ 13 C 6 , 15 N 2 ] (4.1.2) into a 250 ml volumetric flask and dilute to volume with water. Store aliquots at –20 °C.
4.8.4 Solutions for the five-level standard curve. Transfer appropriate volumes of the PA intermediate solution (10 μg/ml) (4.8.2) into 10 ml volumetric flasks to obtain five different concentrations of PA (0,08 μg/ml, 0,16 μg/ml, 0,32 μg/ml, 0,64 μg/ml and 1,2 μg/ml). Add 500 μl of the IS stock solution (20 μg/ml) (4.8.3) and dilute to volume with water. The concentration of IS in each standard solution is 1 μg/ml. Store aliquots of these solutions at –20 °C for no longer than one month before use.
4.8.5 Ammonium acetate solution, c = 400 mmol/l, pH = 3,8 (used for sample extraction). Into a 500 ml beaker, add (30,8 ± 0,10) g ammonium acetate. Add about 300 ml water and stir to dissolve with a magnetic stirrer. Adjust to pH = 3,8 ± 0,1, carefully adding glacial acetic acid (about 150 ml is needed). Transfer into a 1 000 ml volumetric flask and make up to volume with water. This solution is stable for one month at 4 °C.
5 Apparatus Usual laboratory glassware and equipment and, in particular, the following.
5.1 Balances, with readability of 0,1 mg, capacity 210 g; with readability of 0,1 g, capacity 4 100 g.
5.2 pH-meter, with readability of 0,01 pH unit.
5.3 Homogenizer 2) .
5.4 Stir plate with magnetic stirrers.
5.5 Filters. Syringe filters, 0,22 μm pore size, 33 mm internal diameter, Millex-GV PVDF (Millipore) 3) . Membrane disc filters, 0,45 μm pore size (Millipore) 3) or equivalent. 5.6 UHPLC-MS/MS system, UPLC column, e.g. ACQUITY UPLC ®3) coupled with triple quadrupole detector equipped with electrospray ionization (ESI) source and T3 column (1,8 μm, 100 mm × 2,1 mm internal diameter; Waters Corp.) 3) or equivalent. 6 Procedure 6.1 Sample preparation 6.1.1 General If the product contains starch, add 50 mg α-amylase to the suspensions and incubate for 15 min at 40 °C to decrease viscosity and facilitate handling. Mix liquid samples well to ensure homogeneity and continue directly to extraction. If the powder sample homogeneity is unknown, assume that it is non- homogenous and proceed with 6.1.2.
6.1.3 Wet blended powder samples For wet blended homogenous powder samples, accurately weigh approximately 2,0 g of sample (m s ) into a 50 ml volumetric flask. Add 14 g of water at 40 °C. Mix until a homogeneous suspension is obtained. 6.1.4 Liquid samples For liquid sample samples, accurately weigh approximately 20,0 g (m s ) into a 50 ml volumetric flask. 6.2 Extraction Using the prepared sample (6.1), add a 25 ml volume of a 0,4 mol/l ammonium acetate solution, pH = 3,8. Dilute the sample extract to volume with water. Add a stir bar and stir for 10 min. Filter a 20 ml portion through folded paper (Grade 597½). Run chromatographic analysis. 6.3 Analysis 6.3.1 Chromatographic analysis Transfer a 1,0 ml aliquot of the filtrate obtained in 6.2 into a 15 ml polypropylene tube containing 500 μl of the IS stock solution (4.8.3). It is critical to use the same IS solution as used in the preparation of the standard curve (4.8.4). Dilute the solution to 10 ml with water, cap and mix. Filter through a 0,22 μm syringe filter (5.5). Inject into the UHPLC-MS/MS system.BS ISO 20639 pdf download.