6. AOACSPIFANMethods-2018Awards

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1386 Bidlack et al.: J ournal of AOAC I nternational V ol. 98, N o. 5, 2015

Isocratic Mobile Phase Pump w/Helium Sparge

Autosampler

Silica HPLC Column

Mixing Tee

Zinc Post Column Reactor Column

Fluorescence Detector

Post Column Electrolyte Pump w/Helium Sparge

Figure 2015.09A. Vitamin K system configuration.

(f)  Phytonadione/phylloquinone ) .—U.S. Pharmacopeia Reference, Official Lot. Store per label instructions. (g)  Laboratory water . (h)  Reagent alcohol .—ACS. (i)  Sodium acetate anhydrous .—ACS. (j)  Zinc .—Sigma-Aldrich (St. Louis, MO) Part No. 324930, <150 µm, 99.995% or equivalent. (k)  Zinc chloride .—ACS. D. Standard and Solution Preparation Note : Since vitamin K 1 is light-sensitive, all standards must be prepared, handled, and stored in the dark or under yellow shielded lighting ( see B ) unless otherwise stated. If the standards must be transported through or into an area without yellow shielded lighting, they must be wrapped tightly in foil. All standard solutions must be prepared using Class A volumetric glassware. (a)  Mobile phase.— Add about 900 mL iso-octane to a 1000 mL volumetric flask. Add 0.3–0.4 mL isopropanol to the volumetric flask and dilute to volume with iso-octane. ( Note : The isopropanol concentration in the mobile phase can be adjusted slightly until baseline resolution of cis and trans vitamin K 1 from other peaks present in some samples is achieved. See Figures 2015.09B–D ). (b)  Postcolumn electrolyte solution.— Transfer 0.25 (±0.02) g zinc chloride and 0.10 (±0.05) g sodium acetate anhydrous to a 1000 mL volumetric flask with reagent alcohol. Add 75 (±5) µL concentrated acetic acid and dilute to volume with reagent alcohol. Add a stir bar and stir solution for about 30 min or until solution is clear and all salts are dissolved. (c)  Vitamin K 1 (phytonadione) stock standard solution.— Accurately weigh to 0.00001 g about 0.05500 g vitamin K 1 (phytonadione) into a 250 mL volumetric flask. Dissolve oil and dilute to volume with iso-octane. Store refrigerated in a tightly stoppered container protected from light. Expiration 6 months. (d)  Vitamin K 1 (phytonadione) intermediate I standard solution.— Dilute 1.0 mL vitamin K 1 stock standard to 100 mL with iso-octane. Prepare fresh each time new working standards are made. (e)  Vitamin K 1 (phytonadione) intermediate II standard solution.— Dilute 10.0 mL vitamin K 1 intermediate I standard (vitamin K 1

to 50 mL with iso-octane. Prepare fresh each time new working standards are made. (f)  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.) is light-sensitive, all samples must be prepared, handled, and stored in the dark or under yellow-shielded lighting ( B ) unless otherwise stated. If the samples must be transported through or into an area without yellow-shielded lighting, they must be wrapped tightly in foil.)] (a)  Sample preparation .—( 1 ) Accurately weigh to 0.0001 g, up to 0.5 g homogeneous powder or up to 4 g of ready-to-feed (RTF) liquids or nonhomogeneous powders diluted to RTF concentrations into 50 mL centrifuge tubes. To powders weighed directly into the 50 mL centrifuge tubes, add 4 mL water and mix well. To liquids with sample weights that are less than 4 g, add enough water to the tubes so that the sample weight plus the amount of added water 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 allow samples to sit undisturbed for at least 10 min, but no more than 40 min, after vortexing with methanol, or add a magnetic stir bar to each sample, place each capped sample onto a magnetic stir plate, and stir each sample for at least 10 min, but not more than 40 min, at a spin rate that causes a vortex. ( 3 ) 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. ( 4 ) Add 5 (±1) mL laboratory water to each sample and cap tubes. Laboratory water can be added to all the samples prior to vortexing or stirring. Vortex or shake each sample for at least E. Procedure [( Note : Because vitamin K 1