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© 2015 AOAC INTERNATIONAL
(d)
Isopropanol (isopropyl alcohol)
.—HPLC grade.
(e)
Methanol
.—HPLC grade.
(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, USA) 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(q)
] 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.
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
Table 2015.09B.
Trans
vitamin K
1
SLV data–accuracy
Sample type
Spike level
100%
50%
No. of replicates
(duplicates on multiple days)
Native level,
µg/100 g RTF Recovery, % RSD, %
Recovery, % RSD, %
Child formula powder
6
2.66
98.2
5.9
96.2
7.1
Infant elemental powder
6
7.57
93.2
7.6
94.0
2.6
SRM 1849a
6
1.11
a
104
2.9
95.5
1.8
Adult nutritional powder milk protein based
6
3.26
96.4
2.0
95.1
1.8
Infant formula powder partially hydrolyzed milk based
6
7.69
96.6
3.6
91.9
3.0
Infant formula powder partially hydrolyzed soy based
6
8.99
97.9
1.0
96.1
2.6
Adult nutritional powder low fat
6
2.92
98.0
2.5
95.2
2.6
Infant formula powder milk based
6
6.09
97.6
1.0
102
2.3
Infant formula powder soy based
6
6.26
97.9
1.7
102
0.3
Infant formula RTF milk based
6
9.01
100
1.3
104
0.4
Adult nutritional RTF high protein
6
9.10
96.7
2.7
106
0.7
Adult nutritional RTF high fat
6
10.7
98.2
1.2
93.8
4.0
a
Results reported as mg/kg powder.
Candidates for 2016 Method of the Year
105