Methods to be Reviewed by OMB in 2016

(d)

Vitamin K

(phytonadione) intermediate I standard

solution.—

Dilute 1.0 mL vitamin K

stock standard to 100 mL

with iso-octane. Prepare fresh each time new working standards

are made.

(e)

Vitamin K

(phytonadione) intermediate II standard

solution.—

Dilute 10.0 mL vitamin K

intermediate I standard

to 50 mL with iso-octane. Prepare fresh each time new working

standards are made.

(f)

Vitamin K

(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

is light-sensitive, all samples must

be prepared, handled, and stored in the dark or under yellow-

shielded lighting,

B(q)

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

.—(

) 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.

(

) 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.

(

) 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.

Figure 2015.09C. Chromatogram of SRM 1849a.

Figure 2015.09B. Chromatogram of vitamin K

standard.

Candidates for 2016 Method of the Year

106