OMB Meeting Book_9-11-14

18 Scientific Aquasi1 P/N 77503-104630; www.thermoscientific. com) with C drop-in guard cartridges 3 μ, 10 × 4.6 mm (Thermo Scientific Aquasil P/N 77503-014001) , Epic Phenyl Hexyl, 3u, 120A, 100 x 4.6 mm (ES Industries 125191-EPHX) 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. ( l ) Filter paper.— Whatman 2V or equivalent. ( m ) Funnels.— Plastic, suitable to use with filter paper.

12 AOAC Official Method 2011.10 Vitamin B in Infant Formula and Adult Nutritionals High-Performance Liquid Chromatography First Action 2011

(Applicable to the determination of vitamin B and adult nutritionals.)

in infant formula

12

Caution: Refer to Material Safety Data Sheets (MSDS) of chemicals prior to use and follow the suggested personal protective equipment. NOTE: Potassium cyanide is highly toxic. When handling this chemical, wear gloves and appropriate personal protective equipment. Weigh chemical and dispense solutions in a 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 Table 2011.10A for infant formula and adult nutritional matrixes for which the method was validated. A. Principle

( n ) Gloves.

Vitamin B

is extracted from the sample using sodium acetate

12

8 ( o ) Pipettor.— Variable volume, 100–1000 μL. ( p ) Shields.— Yellow or clear shields with a cutoff of 385 nm. ( q ) SPE cartridges.— C 600 mg (Alltech/Grace Davidson P/N 20958; www.discoverysciences.com), C 900 mg (Alltech/Grace 18 Davidson P/N 20966), C 600 mg (Alltech/Grace Davidson P/N 20934), C 900 mg (Alltech/Grace Davidson P/N 20942), or equivalent. ( r ) Syringes.— Disposable, assorted sizes. ( s ) Syringe filters.— 0.45 μm nylon. ( t ) Vacuum manifold — 24 ports with stopcocks or equivalent. 18 8

buffer (pH 4.5) and potassium cyanide at 105°C. Extracts are purified and concentrated with C8 or C18 solid-phase extraction (SPE) cartridges and analyzed with size-exclusion and reversed-

12 phase chromatography. Determination of B chromatography with visible detection at 550 nm.

is made by liquid

B. Apparatus and Materials

( a ) HPLC system.— Gradient system with switching valve and isocratic pump on side and a UV-Vis detector equipped with a tungsten lamp (capable of monitoring at 550 nm wavelength). Autosampler capable of injecting 900 uL or 2 mL sample. ( 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 column 3 μ, 100 × 4.6 mm (Thermo

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

C. Reagents

( a ) Acetic acid.— ACS.

( b ) Acetonitrile.— HPLC grade.

18

Table 2011.10A. Infant formula and adult nutritional matrixes for which method was validated

Product type

HPLC method, μg/kg (RSD, %) Microbiological method, μg/kg

HPLC/micro, %

n = 9

Hypoallergenic infant formula powder

51.4 (4.00) n = 12 47.8 (5.30) n = 15 59.5 (5.19) n = 12 4.30 (4.42) n = 9 42.9 (4.70) n = 12 7.04 (5.27) n = 9 51.7 (5.66) n = 12 11.2 (4.85) n = 21

Adult diabetic nutritional beverage

2

n = 9

Soy-based infant formula powder

Metabolic child/adult nutritional powder

8

n = 14

High-calorie milk-based infant formula powder

Disease-specific adult nutritional beverage

1

n = 9

Milk-based infant formula liquid

Lactose-free infant formula powder

Child nutritional powder

Adult nutritional powder

Adult nutritional beverage

Metabolic child/adult nutritional powder

48.0 (8.88)

© 2012 AOAC INTERNATIONAL