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

( b ) Column.— Analytical size-exclusion column 4 μ, 250 × 9.4 mm (Zorbax GF-250, P/N 884973-901; www.chem.agilent. com), 5 μ, 300 × 8 mm (Shodex Protein KW-802.5, P/N F6989000) or equivalent. ( c ) Column.— Analytical C 18 column 3 μ, 100 × 4.6 mm (Thermo Scientific Aquasi1 P/N 77503-104630; www.thermoscientific. com) with C 18 drop-in guard cartridges 3 µ, 10 × 4.6 mm (Thermo Scientific Aquasil P/N 77503-014001), Epic Phenyl Hexyl, 3 µ, 120A, 100 × 4.6 mm (ES Industries P/N 125191-EPHX; www. esind.com) with appropriate guard cartridge; or equivalent. ( d ) Oven.— Capable of maintaining temperatures of 95 ± 5°C and 105 ± 5°C. ( e ) pH meter.— With calibration buffer . ( f ) Analytical balance.— Capable of weighing 0.00001 g. ( g ) Beakers.— Glass, assorted sizes. ( h ) Bottle top dispenser.— Capable of dispensing 30 mL. ( i ) Cylinders.— Graduated glass, assorted sizes. ( j ) Desiccator. ( k ) Erlenmeyer flasks.— 125 mL or equivalent glassware. ( l ) Filter paper.— Whatman 2V or equivalent. ( m ) Funnels.— Plastic, suitable to use with filter paper. ( n ) Gloves. ( o ) Pipettor.— Variable volume, 100–1000 μL. ( p ) Shields.— Yellow or clear shields with a cutoff of at least 385 nm. ( q ) SPE cartridges.— C 8 900 mg (Alltech/Grace Davison P/N 20966), C 18 900 mg (Alltech/Grace Davison P/N 20942), or equivalent. See E , procedure for SPE cartridge qualification. ( r ) Syringes.— Disposable, assorted sizes. ( s ) Syringe filters.— 0.45 μm nylon. ( t ) Vacuum manifold — 24 ports with stopcocks or equivalent.

AOAC Official Method 2011.10 12 in Infant and Pediatric Formulas and Adult Nutritionals High-Performance Liquid Chromatography First Action 2011 Final Action 2014

Vitamin B

ISO–AOAC Method* (Applicable to the determination of vitamin B 12 pediatric formulas and adult nutritionals.) Caution: Refer to Material Safety Data Sheets (MSDS) of chemicals prior to use and follow the suggested personal protective equipment. Potassium cyanide is highly toxic. When handling this chemical, wear gloves and appropriate personal protective equipment. Weigh chemical and dispense solutions in a fume hood. Perform test in a well-ventilated area. Treat sample waste with sodium hypochlorite and dispose of waste according to local, state, and federal regulations. See Tables 2011.10A–C for results of the single-laboratory validation and multilaboratory study supporting acceptance of the method. A. Principle Vitamin B 12 is extracted from samples using sodium acetate buffer (pH 4.5) and potassium cyanide at 105°C. Extracts are purified and concentrated with C 8 or C 18 solid-phase extraction (SPE) cartridges and analyzed with size-exclusion and reversed- phase chromatography. Determination of B 12 is made by liquid chromatography with visible detection at 550 nm. B. Apparatus and Materials ( a ) HPLC system.— Gradient system with switching valve and additional isocratic pump and a UV-Vis detector equipped with a tungsten lamp (capable of monitoring at 550 nm wavelength). Autosampler capable of injecting 900 μL to 2 mL sample. in infant and

( u ) Volumetric pipets.— Assorted sizes. ( v ) Volumetric flasks.— Assorted sizes. C. Reagents

( a ) Acetic acid.— Glacial, ACS. ( b ) Acetonitrile.— HPLC grade.

Table 2011.10A. Single-laboratory validation: Repeatability precision data for vitamin B 12

No. of replicates (duplicates on multiple days)

Sample type

Mean (µg/100 g RTF)

SD

RSD r

, %

r

Infant formula (NIST SRM 1849a) Infant formula powder soy-based Infant formula powder milk-based Infant formula RTF milk-based Infant formula powder partial  hydrolyzed milk-based Infant formula powder partial  hydrolyzed soy-based Adult nutritional powder milk-based Adult nutritional RTF high protein Adult nutritional RTF high fat Infant elemental powder Adult nutritional powder low fat Child formula powder

18 12 12 12 12

42.4 a 0.415 0.236 0.355 0.377

1.37

3.27 3.62 3.52 2.34 3.50

0.0150 0.0083 0.0083 0.0132

12

0.257

0.0090

3.51

12 12 12 12 12 12

0.299

0.0141 0.0250 0.0225 0.0463 0.0223 0.0187

4.72 2.21 2.36 2.81 4.13 2.81

1.13

0.955

1.65

0.540 0.666

a  Results in µg/kg powder.

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