AOAC Final Action Methods in 2018

Figure 2015.09D. Chromatogram of an adult nutritional.

( f ) Vitamin K 1 (phytonadione) intermediate II standard solution.— Dilute 10.0 mL vitamin K 1 intermediate I standard to 50 mL with iso-octane. Prepare fresh each time new working standards are made. ( g ) Vitamin K 1 (phytonadione) working standard solutions.— Dilute 1.0, 3.0, 6.0, 10.0, and 20.0 mL intermediate II standard to 100 mL with iso-octane. Store refrigerated in tightly stoppered containers protected from light. Expiration 3 months. ( Note : Transfer working standards to autosampler vials with Pasteur pipets or equivalent glass. Do not pour the standards from the volumetric flasks into vials.) E. Procedure [( Note : Because vitamin K 1 is light-sensitive, all samples must be prepared, handled, and stored in the dark or under yellow- shielded lighting, B(r) , unless otherwise stated. If samples must be transported through or into an area without yellow-shielded lighting, they must be wrapped tightly in foil.)] (a)  Sample preparation .—( 1 ) Sampling. —If a powder sample homogeneity is unknown, assume that it is nonhomogenous and proceed as for dry-blended/nonhomogenous powder samples. ( a ) Liquid samples. —Thoroughly mix or homogenize liquid. Accurately weigh to 0.0001 g, up to 4 g of ready-to-feed (RTF) liquid into 50 mL centrifuge tubes. To liquids with sample weights of less than 4 g, add enough water so that the sample weight plus the amount of water added (g or mL) equals about 4 and mix well. ( b ) Wet-blended powders .—Accurately weigh to 0.0001 g, up to 0.5 g wet-blended powder into 50 mL centrifuge tubes. Add 4 mL water and mix well. ( c )  Dry-blended/nonhomogeneous powders. —Accurately weigh 25 g powder and add approximately 200 g water. Record all weights. Mix until a homogeneous suspension is obtained. Homogenize if needed. Accurately weigh to 0.0001 g, up to 4 g of homogeneous suspension into 50 mL centrifuge tubes. To homogeneous suspensions with sample weights of less than 4 g, add enough water so that the sample weight plus the amount of water added (g or mL) equals about 4 and mix well. ( 2 ) Add 25 (±2.0) mL methanol to each sample just prior to vortexing or stirring. Methanol should not be added to more than

two samples consecutively without vortexing or stirring. Cap each centrifuge tube. Vortex each sample at high speed for at least 30 s, and then allow samples to set undisturbed for at least 10 min, but no more than 40 min, or add a magnetic stir bar to each tube, cap tubes and place on a magnetic stir plate, and stir samples for at least 10 min, but not more than 40 min at a spin rate that causes a vortex. Begin timing after vortex forms in the tubes. ( 3 ) Accurately add 10 (±0.05) mL iso-octane to each sample with a volumetric pipet and cap tubes. Iso-octane can be added to all samples before vortexing or stirring any of the samples. Vortex each sample for at least 45 s or stir each sample for at least 45 s at a spin rate that causes a vortex to form within the sample. Begin timing after vortex forms in the tubes. ( 4 ) Add 5 (±1) mL laboratory water to each sample and cap tubes. Water can be added to all the samples prior to vortexing or stirring. Vortex or shake each sample for at least 20 s or stir each sample for at least 20 s at a spin rate that causes a vortex to form within the sample. Begin timing after vortex forms in the tubes. ( 5 ) Centrifuge the samples until a clean separation of the iso- octane and water–methanol layers results. The iso-octane layer should be a clear layer at the top of the centrifuge tube, and the water–methanol layer should be a cloudy layer below the iso-octane layer. In some samples there may be a small emulsion layer between the iso-octane and water–methanol layers. (A good separation of solvent layers can usually be achieved by centrifuging samples for approximately 10 min at 800 relative centrifugal force.) ( 6 ) Remove samples from the centrifuge and inspect each sample to verify that the iso-octane and water–methanol layers are separated. With a glass pipet, carefully rinse down the upper walls of the centrifuge tube with a portion of the iso-octane layer. If the layers become mixed together, centrifuge the sample again. Pipet a portion of the clear iso-octane layer into a labeled autosampler vial and cap the vial. (b)  HPLC analysis .—( 1 )  Instrumental operating conditions .— ( a )  HPLC analytical column mobile phase flow rate .— 0.4 mL/min. ( b )  Post-column flow rate .—0.4 mL/min. ( c )  Injection volume .—20 µL. ( d )  Run time .—20 min.

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