AOAC SPIFAN ERP & Working Group Method Reviews (July 10, 2019)

water. The solution was stored at ambient temperature for up to 1 week. ( g ) Phosphate buffer (0.2 M, pH 7.6, with 10 mM MgCl 2 ).— Prepared by dissolving 2 g magnesium chloride hexahydrate in a 1 L volumetric flask with purified water. Then 31 mL 1 M potassium phosphate monobasic and 168 mL 1 M potassium phosphate dibasic were added, and the solution was diluted to volume with purified water. The solution was prepared fresh daily just before analysis. ( h ) Glycine buffer (0.2 M, pH 10.2, with 1 mM MgCl 2 ).— Prepared by dissolving 1.5 g glycine and 20 mg magnesium chloride hexahydrate in a 100 mL volumetric flask and bringing to volume with purified water. The solution was stored refrigerated for up to 1 week. ( i ) ATP (100 mM).— Prepared by dissolving approximately 0.275 g in a 5 mL volumetric flask with purified water and then bringing to volume. The solution was prepared fresh daily just before analysis. E. Mobile Phase Preparation All mobile phases were prepared with vacuum-degassed purified water and stored on the system in polypropylene bottles under pressurized nitrogen gas. Mobile phases A (50 mM NaOH) and B (1 M NaOH) were prepared by adding the appropriate amount of 50% (w/w) NaOH to just under 2 L purified degassed water, bringing to volume, then gently mixing. Mobile phase A was prepared with 8 g 50% (w/w) NaOH. Mobile phase B was prepared with 160 g 50% (w/w) NaOH. The mobile phases were kept for 1 month at ambient conditions. F. Preparation of Standard Solutions The purity on the certificate of analysis was used for the myo-inositol reference material. A 0.1 g portion of myo-inositol was weighed into a 100 mL volumetric flask and dissolved with purified water to create a 1 mg/mL stock solution. Working level standards that ranged from 0.1 to 20.0 mg/L were prepared from the stock solution. To prepare each working level standard, an appropriate aliquot of stock standard or working standard solution was diluted to volume with purified water. Standards were stored for up to 3 months in a refrigerator set to maintain 5 ± 3°C. Reference standard material was kept in a desiccator at ambient temperature. G. Sample Preparation ( a ) Free analysis .—Each sample was prepared by weighing 2 g milk-based infant formula (powder basis) into a beaker and adding approximately 50 mL purified water and a stir bar. Samples were mixed on a stir plate for 20 min; then the pH was adjusted to 4.5 with HCl (6, 1, 0.1 M) and/or NaOH (1, 0.1 M) to precipitate any protein. Samples were quantitatively transferred to a 100 mL volumetric flask with purified water, brought to volume, and filtered through a GFA filter. An aliquot of the filtrate was diluted appropriately with purified water and filtered through a 0.45 μm PTFE syringe filter into an injection vial. ( b ) Total analysis (autoclave) .—Each sample was prepared by weighing 1 g soy-based infant formula (powder basis) into a 25 × 150 mm screw-top test tube and adding approximately 10 mL purified water. The test tubes were mixed by vortexing to homogenize, and 10 mL concentrated HCl was added to each tube. The samples were then incubated in an autoclave for 6 h at 120°C. Samples were allowed to cool and then neutralized with KOH in a fume hood. The samples were then quantitatively transferred with purified water to a 100 mL volumetric flask, brought to volume, and

Table 2012.12A. Microwave system conditions

Power, W, % a Ramp, min PSI (max) b Control, ° C Hold, min

Stage

1 2

800, 100 0 800, 100 10

400 400

100 120

10 60

a The recommended W for eight vessels.

Maximum psi value.

b

filtered through a GFAfilter. Appropriate dilutions were done on the filtrates with purified water, and solutions were then syringe-filtered with 0.45 μm PTFE filters into injection vials (VWR). ( c ) Total analysis (microwave/enzyme) .—Each sample was prepared by weighing 1 g soy-based infant formula (powder basis) into a microwave liner and adding approximately 30 mL purified water along with approximately 10 mL concentrated HCl. Samples were mixed with a stir bar on a stir plate to homogenize, and the stir bar was removed before microwaving. The microwave conditions are outlined in Table 2012.12A . The samples were allowed to cool and then neutralized with KOH in a fume hood. The samples were quantitatively transferred with purified water into a 100 mL volumetric flask, brought to volume, and filtered through a GFA filter. A 2.5 mL aliquot of filtrate, 2.5 mL 0.1 M acetate buffer pH 5.0, and a stir bar were added to a 16 × 100 mm screw-top test tube. The test tubes were swirled to mix before adding 0.2 mL phytase (1000 U/mL). The tubes were capped and incubated at 37°C with stirring for 1 h in a water bath made out of a crystallizing dish filled with water and containing a test tube rack that was placed on a heated stir plate. The test tubes were removed, and 2.5 mL 0.2 M glycine buffer, pH 10.2, was added. The test tubes were swirled to mix before adding 0.2 mL alkaline phosphatase (1000 U/mL). The tubes were capped and incubated again with stirring in the same 37°C water bath for 1 h. The tubes were removed and enzymes quenched in a boiling water bath for 5 min. Appropriate dilutions were performed with purified water and then syringe-filtered with 0.45 μm PTFE filters into injection vials. H. Sample Preparation (Glycerol Kinase Treatment) ( a ) Free analysis for samples with high glycerol background .— Each sample was prepared by weighing an appropriate amount of sample into a 100 mL volumetric flask and adding approximately 50 mL purified water and a stir bar. Samples were mixed for 30 min with low heat (approximately 40°C). Samples were cooled, the stir bar was removed, and the solution was brought to volume with 0.2 M phosphate buffer, pH 7.6. Samples were filtered through a GFA filter. A 2 mL aliquot of filtrate was transferred into a 7 mL scintillation vial, along with 0.2 mL ATP (0.1 M), 0.2 mL glycerol kinase (20 U/mL), and a micro stir bar, and the vial was capped. Samples were incubated for 2 h with stirring at ambient temperature on a stir plate and then placed in a boiling water bath for 5 min to quench the enzyme. Samples were filtered through a 0.45 μm PTFE syringe filter into an injection vial. ( b ) Total analysis for sampleswithhighglycerol background .—A 2.5 mL aliquot from the final treated extracts after either total analysis method was added into a 16 × 100 mm screw-cap test tube, along with 5 mL 0.2 M phosphate buffer, pH 7.6. The tubes were swirled to mix before adding 0.2 mL 0.1 M ATP, 0.2 mL glycerol kinase (20 U/mL), and a micro stir bar. The test tubes were capped

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