BS ISO 20637:2015 pdf odwnload – Infant formula and adult nutritionals — Determination of myo-inositol by liquid chromatography and pulsed amperometry

BS ISO 20637:2015 pdf odwnload – Infant formula and adult nutritionals — Determination of myo-inositol by liquid chromatography and pulsed amperometry
6.1.1.3 Dry blended powder samples For dry blended/non-homogenous powder samples, reconstitute per the product label instructions. Accurately weigh 0,5 g to 5 g reconstituted product into a 100 ml volumetric flask. Record the mass to the nearest 0,000 1 g.
6.1.1.4 Wet blended powder samples For wet blended/homogenous powder samples, accurately weigh 0,25 g to 1,5 g powder into a 100 ml volumetric flask and record the mass to the nearest 0,000 1 g. Add approximately 10 ml to 15 ml water to the volumetric flask and swirl or stir to completely dissolve the powder.
6.1.2 Extraction Add enough 0,5 % hydrochloric acid (4.2.5) to each sample to adjust the sample pH to 4,5 ± 0,2 and swirl to mix. Allow the samples to react with 0,5 % hydrochloric acid for a minimum of 2 min and then dilute to volume with water. Mix well. Filter samples through filter paper (5.15) into 125 ml conical flasks or appropriate glassware. NOTE Although some samples will filter cloudy, the filtrates can still be used. Filter an aliquot of sample filtrate through a 0,45 μm syringe filter (5.14) into an autosampler vial.
6.2 Myo-inositol bound as phosphatidylinosito
6.2.1 Sample preparation
6.2.1.1 General Prepared samples that are constantly stored at 1 °C to 8 °C in closed containers are stable for up to 5 days. After 5 days, samples shall be prepared again. Thoroughly mix or stir products prior to sampling. Mix liquid samples well to ensure homogeneity. If the powder sample homogeneity is unknown, assume that it is non-homogenous and proceed with the preparation of dry blended/non-homogenous powder samples given in 6.2.1.3.
6.2.1.2 Liquid samples For ready-to-feed liquid samples, accurately weigh (4 ± 0,4) g of product into a 50 ml centrifuge tube and record the mass to the nearest 0,000 1 g.
6.2.1.3 Dry blended powder samples For dry blended/non-homogenous powder samples, reconstitute per the product label instructions. Accurately weigh (4 ± 0,4) g reconstituted sample into a 50 ml centrifuge tube. Record the mass to the nearest 0,000 1 g.
6.2.1.4 Wet blended powder samples For wet blended homogenous powder samples, accurately weigh (1 ± 0,1) g powder into a 50 ml centrifuge tube and record the mass to the nearest 0,000 1 g. Add 4 ml water centrifuge tube and mix well.
6.2.2 Extraction In a fume hood, add 10 ml methanol to each sample and stir for at least 20 min or vortex for at least 1 min and allow samples to set for at least 20 min. Add 20 ml chloroform and stir for at least 5 min or vortex for at least 1 min and allow samples to set for at least 5 min. If large clumps form when chloroform is added, cap tube and shake well for at least 1 min to mix sample. Add 5 ml 6 % metaphosphoric acid (4.2.9) and 1 ml 1 mol/l NaCl (4.2.6) and mix well. Centrifuge until layers separate. Using a 25 ml glass gas-tight syringe with a stainless steel needle (5.21), transfer the bottom chloroform layer to a clean 50 ml centrifuge tube and evaporate the chloroform with nitrogen in a 60 °C water bath.
6.2.3 Cleanup In a fume hood, condition a 1 g silica SPE cartridge (5.20) with 6 ml hexane. Dissolve residue in the bottom of the centrifuge tube in 1 ml chloroform:methanol (2:1). Quantitatively transfer dissolved residue to the conditioned silica SPE cartridge. Rinse the 50 ml centrifuge tube with 3 ml hexane:diethyl ether (80:20) and then transfer to the SPE cartridge. Discard the eluent. Rinse the 50 ml centrifuge tube with 3 ml hexane:diethyl ether (50:50) and then transfer to the SPE cartridge. Collect eluent in a clean 50 ml centrifuge tube. Rinse 50 ml centrifuge tube with 4 ml methanol and then transfer to the SPE cartridge. Collect eluent in the same 50 ml centrifuge tube. Rinse 50 ml centrifuge tube with 4 ml methanol:chloroform:water (75:15:10) and transfer to the SPE cartridge. Collect eluent in the same 50 ml centrifuge tube. Evaporate eluents collected from SPE cartridge with nitrogen in a 60 °C water bath.
6.2.4 Hydrolysis In a fume hood, add 40 μl glacial acetic acid (4.1.1) and 2 ml concentrated hydrochloric acid (4.1.7) to the residue in the centrifuge tube from the sample cleanup step. Tightly cap tube. Heat in a 120 °C oven for 2 h. Cool. Add approximately 10 ml of water and swirl to mix. Add 1,25 ml 50 % (m/m) sodium hydroxide (4.1.12). Transfer sample to a 50 ml volumetric flask and dilute to volume with water. Filter an aliquot of sample filtrate through a 0,45 μm syringe filter into an autosampler vial.