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riboflavin solution with the highest concentration vitamin B 12 working standard. Expiration: 1 week. ( 12 )  0.25 M Sodium acetate buffer .—Dissolve 41 g sodium acetate anhydrous or 68 g sodium acetate trihydrate in approximately 1800 mL laboratory water. Adjust pH to 4.50 with concentrated acetic acid. Dilute to 2000 mL with laboratory water. Expiration 3 months. ( 13 )  6% Taka-diastase .—Dissolve 0.6 g taka-diastase in 10 mL water. Prepare fresh daily before use. (b)  Standards .—Prepare all standards in volumetric glassware under UV shielded fluorescent lights and store at 2–8°C in tightly stoppered volumetric flasks. ( 1 )  Vitamin B 12 stock standard (10 000 μ g/L) .—Accurately weigh, to 0.00001 g, the appropriate amount of vitamin B 12 USP reference standard to give a stock standard concentration of 10 000 μg/L. Dissolve in and dilute to 100 mL with 25% ethanol. Expiration 6 months. Use the following equation to calculate the amount of vitamin B 12 reference standard that should be weighed: S w = 10 000 × 0.1 × 1/P where S w = amount of vitamin B 12 standard to be weighed in mg; 10000 = desired stock standard concentration in μg/L; 0.1 = dilution volume in L; P = purity of the USP reference standard in μg cyanocobalamin/mg of the standard. See standard label. ( 2 )  Vitamin B 12 intermediate standard (1000 μg/L) .—Dilute 10 mL vitamin B 12 stock standard solution to 100 mL with laboratory water. Expiration 1 week. ( 3 )  Vitamin B 12 working standards (2.5–25 μg/L) .—Dilute 0.5, 1, 2, 3, 4, and 5 mL vitamin B 12 intermediate standard solution to 200 mL with 10% acetonitrile. Expiration 1 month. E. Procedure Prepare all samples under UV shielded fluorescent lights. Mix or stir products before sampling to ensure all product samples are uniform and representative. Store prepared product samples up to 14 days after preparation in tightly stoppered volumetric flasks at 2–8°C. (a)  SPE cartridge qualification .—To establish SPE cartridge equivalency or to verify the suitability of new lots of cartridges: ( 1 ) Prepare a solution containing 160 µg/L vitamin B 12 in water. ( 2 ) Prepare three samples from one representative product that contains the highest amount of protein of any product that will be analyzed with this method following steps E(b) ( 1 ) and ( 2 ) of the sample preparation procedure described below. ( 3 ) Combine all extracted sample filtrates. Accurately transfer 1 mL solution Table 2011.10D. Guidelines for loading sample filtrates onto SPE cartridges a Vitamin B 12 concentration in RTF product, µg/kg Volume of filtrate loaded onto SPE cartridge, mL Final dilution volume, mL

prepared in step ( 1 ) to 80 or 100 mL of sample filtrate (spiked sample), and accurately transfer 1 mL water to 80 or 100 mL of sample filtrate (unspiked sample). ( 4 ) Continue preparing the spiked and unspiked sample using the sample cleanup and concentration, E(b) ( 3 ), and final dilution, E(b) ( 4 ), procedures described in the sample preparation procedure below. ( 5 ) Analyze the two samples chromatographically. ( 6 ) Calculate the vitaminB 12 concentration of the spiked and unspiked samples and calculate the spike recovery. ( 7 ) In order for the cartridges to be considered acceptable, spike recoveries should be ≥90%. (b)  Sample preparation for infant, pediatric, and adult nutritional products .—( 1 ) Sampling .—Mix all products thoroughly before sampling. Reconstitute nonhomogeneous powders per label instructions. Weigh the appropriate amount of product (±10%) into a 100 mL volumetric flask and record the weight to at least four significant figures. Typical weights are 20 g for adult and pediatric RTF liquids and reconstituted powders, 25 g for infant RTF liquids and reconstituted powders, and 3 g for unreconstituted powders. Add 25 mL laboratory water to flasks containing unreconstituted powders and mix until all of the powder dissolves. Add 1 mL of 6% taka-diastase to products containing starch. Allow taka-diastase to react with samples for at least 30 min before continuing with the extraction. Note : Add 0.5 g milk protein such as calcium caseinate to nutritional products that do not contain any intact protein (i.e., infant elemental powders) and reconstitute or add water to the powder immediately before the extraction step. ( 2 )  Extraction .—Add 30 mL 0.25 M sodium acetate buffer (pH 4.5) to each sample and swirl to mix. In a hood, add 1 mL freshly prepared 1% KCN to each sample and swirl to mix. Heat samples in a 105°C oven for at least 60 min, but for no more than 120 min. (Oven temperature will drop when the door is opened. Start timing when oven temperature returns to 105°C.) Remove samples from the oven and immediately cool in an ice bath. Dilute samples to volume with laboratory water. Mix well. Filter samples through Whatman 2V filter paper (www. whatman.com) into 125 mL Erlenmeyer flasks or equivalent glassware. Note : If prepared samples are milky and contain very small insoluble particles, centrifuge samples and then transfer liquid layer to funnels lined with Whatman 2V filter paper. Note : Do not heat samples to which 0.5 g milk protein has been added, but continue with the dilution and filtration steps. ( 3 )  Sample cleanup and concentration .—For each sample, insert a 900 mg SPE cartridge onto the stopcock of the vacuum manifold and attach a 30 mL disposable syringe barrel to the top of each cartridge. Note : Alltech C 8 and C 18 cartridges can be used interchangeably. Condition each cartridge with at least 20 mL acetonitrile by allowing acetonitrile to pass by gravity through the cartridge and rinse each cartridge with at least 10 mL laboratory water. Using volumetric pipets, transfer sample filtrates to cartridges using the guidelines in Table 2011.10D . If the vitamin B 12 concentration is unknown, use guidelines for RTF products containing 1 – 10 µg/L. If necessary apply enough vacuum so that the samples drip steadily through the cartridges. Sample filtrates should pass through the cartridges at a rate of no more than 120 drops/min. Discard eluent. After all of the sample filtrate has passed through the cartridge, rinse each cartridge with 5 mL

<1

80

5

1–10

70–80 50–60 20–40

10 10

11–20 21–50

10 a  Do not load more than 60 mL adult and pediatric nutritionals onto an Alltech C 8 or C 18 cartridge.