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1658 Butler-Thompson et al.: J ournal of AOAC I nternational V ol. 98, N o. 6, 2015

(c)  Drierite .—Desiccant, anhydrous calcium sulfate, 8 mesh. (d)  Ethanol .—Reagent alcohol, 95%, denatured. (e)  Formic acid .—88%, ACS. (f)  Laboratory water .—≥15 MΩ·cm. (g)  Potassium cyanide .—≥97%, ACS. (h)  Riboflavin .—≥96%, ACS. (i)  Sodium acetate anhydrous or sodium acetate trihydrate .—ACS. (j)  Taka-diastase .—Accurate Chemical Co. (www. accuratechemical.com) or equivalent. (k)  Triethylamine (TEA) .—HPLC grade. (l)  Vitamin B 12 (cyanocobalamin) standard .—USP reference, official lot number (refer to USP catalog for current lot). Store in desiccator protected from white light. ( Note : See standard label for purity). D. Solution and Standard Preparation All solutions can be scaled up or down for convenience provided good laboratory practices are observed. Solutions can be stored at 2–30°C in tight, inert containers unless otherwise noted. (a)  Solutions .—( 1 )  Mobile phase A .—4.0 mL TEA diluted with 1000 mL water and pH adjusted to 5–7 with about 1.25 mL concentrated formic acid. Expiration 1 week. ( 2 )  Mobile phase B .—4.0 mL TEA and 250 mL acetonitrile diluted with 750 mL laboratory water and pH adjusted to 5–7 with about 1.25 mL concentrated formic acid. Expiration 1 week in tightly stoppered container. ( 3 )  Mobile phase C .—4.0 mL TEA and 750 mL acetonitrile diluted with 250 mL laboratory water and pH adjusted to 5–7 with about 1.25 mL concentrated formic acid. Expiration 1 week in tightly stoppered container. ( 4 )  Mobile phase D (2.5% acetonitrile in water) .—50 mL acetonitrile diluted to 2000 mL with laboratory water. Expiration 1 week in tightly stoppered container. ( 5 )  10% Acetonitrile in water .—150 mL acetonitrile diluted to 1500 mL with laboratory water. Expiration 1 month in tightly stoppered container. ( 6 )  30% Acetonitrile in H 2 O SPE elution solvent .— 30.0 mL acetonitrile diluted to 100 mL with laboratory water. Expiration 1 month in tightly stoppered container. ( 7 )  50% Acetonitrile in water, column cleaning and storage solution .—500 mL acetonitrile diluted to 1000 mL in a volumetric flask. Expiration 6 months. ( 8 )  25% Ethanol .—50 mL ethanol diluted to 200 mL with laboratory water. Expiration 1 year in tightly stoppered container. ( 9 )  0.40% Potassium cyanide for samples with 5 mL final dilution volume .—Dissolve 0.02 g potassium cyanide in and dilute to 5 mL with 0.25 M sodium acetate buffer. Make fresh daily before use. ( 10 )  1% Potassium cyanide .—Dissolve 0.25 g potassium cyanide in and dilute to 25 mL with laboratory water. Prepare fresh daily before use. ( 11 )  Resolution test solution for determining appropriate gradient conditions when a new analytical column is installed .— Weigh about 0.005 g riboflavin onto a weighing paper. Transfer to a 100 mL volumetric flask and dilute to volume with 10% acetonitrile solution. Stir to dissolve. Mix equal amounts of

local, state, and federal regulations. See Tables 2011.10A–C for results of the single-laboratory validation and interlaboratory 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 SPE cartridges and analyzed with size-exclusion and RP chromatography. Determination of B 12 is made by LC with UV at 550 nm. B. Apparatus and Materials (a)  HPLC system .—Gradient systemwith 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. (b)  Column .—Analytical size-exclusion column 4 μm, 250 × 9.4 mm (Zorbax GF-250, P/N 884973-901; www.chem. agilent.com), 5 μm, 300 × 8 mm (Showa Denko America, Inc., New York, NY) Protein KW-802.5, P/N F6989000), or equivalent. (c)  Column .—Analytical C 18 column 3 μm, 100 × 4.6 mm (Thermo Scientific Aquasil P/N 77503-104630; www. thermoscientific.com) with C 18 drop-in guard cartridges 3 μm, 10 × 4.6 mm (Thermo Scientific Aquasil P/N 77503-014001), Epic phenyl hexyl, 3 µm, 120 Å, 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 to 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 (www.whatman.com) 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, Bannockburn, IL; P/N 20966), C 18 900 mg (Alltech/Grace Davison; P/N 20942), or equivalent. See E for SPE cartridge qualification. (r)  Syringes .—Disposable, assorted sizes. (s)  Syringe filters .—0.45 μm nylon. (t)  Vacuummanifold .—24 ports with stopcocks or equivalent.

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

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