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Bidlack et al.:
J
ournal of
AOAC I
nternational
V
ol.
98, N
o.
5, 2015
(f)
Phytonadione/phylloquinone
(vitamin
K
1
)
.—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
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.)
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
) 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
Figure 2015.09A. Vitamin K system configuration.
Isocratic Mobile
Phase Pump
w/Helium Sparge
Post Column
Electrolyte Pump
w/Helium Sparge
Autosampler
Silica HPLC Column
Mixing
Tee
Zinc Post Column
Reactor Column
Fluorescence
Detector
Candidates for 2016 Method of the Year
113