OMB Meeting Book_9-11-14

8 8 with most products; however, if products on an RTF basis contain high levels of protein (>5–6%) or high levels of hydrolyzed proteins (>4%), 900 mg Alltech C or C cartridges should be used. Note : Alltech C and C cartridges can be used interchangeably. 8 18 Condition each cartridge with at least 20 mL acetonitrile b y a l lowi n g a c e ton it r il e t o g rav i t y fi l t e r th rou gh th e c a r t r id ge and rinse each cartridge with at least 10 mL laboratory water. Using volumetric pipets, transfer sample filtrates to cartridges using the guidelines in Table 2011.10B . If necessary apply enough vacuum so that the samples drip steadily through the cartridges. Discard eluant. After all of the sample filtrate has passed through the cartridge, rinse each cartridge with 5 mL laboratory water and discard eluant. Air-dry each cartridge by pulling a vacuum until no more effluent is observed. Close each stopcock. Place a 5 or 10 mL volumetric flask under each cartridge. Add 4.4 mL 25% acetonitrile to all Alltech 600 mg cartridges and 4.4 mL 30% acetonitrile to all Alltech 900 mg cartridges. Open KCN to each volumetric flask. Place prepared samples in a 95°C oven for at least 1.5 h, but for no more than 4 h. After at least 1.5 h, remove samples from the oven and cool to room temperature. Dilute to volume with laboratory water. Filter an aliquot of each standard and prepared sample through a 0.45  m syringe filter into an autosampler vial. ( b ) HPLC analysis.— ( 1 ) System setup and configuration.—See Figures 2011.10A and B for configurations. ( 2 ) Instrument operation conditions.— ( a ) Run time .—30 min. ( b ) Injection volume .— 900 uL to 2.0 mL. ( c ) System configuration.—See Table 2011.10C . ( d ) Isocratic pump.— Mobile phase D: 2.5% acetonitrile. 18 up and concentrated, insert a 600– 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. As a general guideline, 600 mg Alltech C or C cartridges have enough capacity for use each stopcock and elute vitamin B Final dilution .—For samples 12 into the volumetric flasks. collected in 10 mL volumetric flasks, dilute to volume with water. For samples collected in 5 mL volumetric flasks, in a hood add 0.1 mL freshly prepared 0.4% 18

Table 2011.10C .

System configuration

Time, min

Valve configuration

0.00–10.5

Configuration 1

10.5–14.5

Configuration 2

14.5–30.0

Configuration 1

give a stock standard concentration of 10 000  g/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 reference standard that should be weighed:

12

S

= 10000 × 0.1 × 1/P

w

where S

= amount of vitamin B

standard to be weighed in

w

12

mg; 10 000 = 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

intermediate standard (1000 μg/L).— Dilute

12

10 mL vitamin B stock standard solution to 100 mLwith laboratory

12

water. Expiration 1 week.

( 3 ) Vitamin B

working standards (2.5–25 μg/L).— Dilute 0.5,

12

1, 2, 3, 4, and 5 mL vitamin B

intermediate standard solution to

12

200 mL with 10% acetonitrile. Expiration 1 month. E. Procedure

**A section describing a procedure for evaluating SPE cartridge performance will be added here.** Prepare all samples under UV shielded fluorescent lights. Store prepared product samples up to 14 days after preparation (store at 2–8°C in tightly stoppered volumetric flasks). Mix or stir products before sampling to ensure all product samples are uniform and representative. ( a ) Sample preparation for infant and adult nutritional products.— ( 1 ) Sampling.— Mix all products thoroughly before sampling. Reconstitute nonhomogeneous powders per label instructions. Weigh the appropriate amount of sample (±10%) into a 100 mL volumetric flask and record the weight to at least 4 significant figures. Typical weights are 20 g for adult and pediatric and – 3025 g for infant ready-to-feed (RTF) liquids and reconstituted powders and 3 g for unreconstituted powders. Add 25 mL laboratory water to all unreconstituted powder samples and mix until all of the powder dissolves. Add 1 mL of 6% taka-diastase if samples contain significant levels of starch. Allow taka-diastase to react with samples for at least 30 min before continuing with the extraction. ( 2 ) Extraction.— Add 30 mL 0. 125 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.) After at least 60 min, remove samples from oven and immediately cool in ice bath. Dilute samples to volume with laboratory water. Mix well.

Flow rate: Adjust so that vitamin B exclusion column between 10.5 and

elutes from the size- 12 14.5 min. Typical flow rates,

2 phase A, 0.4% TEA in laboratory water, pH 5–7; mobile phase B, 0.4% TEA and 25% acetonitrile in H O, pH 5–7; mobile phase C, 0.4% TEA and 75% acetonitrile in H O, pH 5–7. Gradient to 12 Gradient pump .—Mobile phase compositions: mobile 1.1–1.2 mL/min. Note : To determine an appropriate flow rate, connect the size-exclusion column directly to the UV-Vis detector and inject the high standard. Adjust flow rate as necessary so that vitamin B elutes between 10.5 and 14.5 min. ( e )

2

Table 2011.10D. C18 column gradient

Mobile phase, %

Time, min

A

B

C

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

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