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© 2015 AOAC INTERNATIONAL
AOAC Official Method 2015.09
Trans
Vitamin K
1
in Infant, Pediatric, and Adult Nutritionals
HPLC with Fluorescence Detection
First Action 2015
(Applicable to the determination of
trans
vitamin K
1
in infant,
pediatric, and adult nutritional formulas.)
Caution
: Refer to Material Safely Data Sheets (MSDS) of
chemicals prior to use and use the suggested personal
protective equipment. Zinc powder should be handled in
a fume hood; it is self-heating and may catch fire. Zinc
powder should not be allowed to come into contact with
water, which may release flammable gases that may
spontaneously ignite.
See
Tables
2015.09A
and
B
for infant, pediatric, and nutritional
matrixes for which the method has been validated; and for study
results supporting acceptance of the method.
A. Principle
This normal-phase (NP) HPLC method with postcolumn
reduction and fluorescence detection allows for the quantitative
determination of
trans
vitamin K
1
in infant, pediatric, and adult
nutritionals. Vitamin K
1
is extracted from products with iso-octane
after precipitation of proteins and release of lipids with methanol.
Prepared samples are injected onto a silica HPLC column where
cis
and
trans
vitamin K
1
are separated with an iso-octane–isopropanol
mobile phase. The column eluent is mixed with a dilute ethanolic
solution of zinc chloride, sodium acetate, and acetic acid, and
vitamin K
1
is reduced to a fluorescent derivative in a zinc reactor
column. The resulting fluorescent compound is then detected by
fluorescence at an excitation wavelength of 245 nm and an emission
wavelength of 440 nm.
B. Apparatus and Materials
(a)
HPLC system
.—Two isocratic pumps; autosampler capable
of injecting 20 µL; fluorescence detector; high-pressure mixing tee;
and postcolumn reactor column 20 × 4 mm stainless steel (Waters,
Milford, MA, USA; Part No. WAT084550 or equivalent). The
system should be configured as shown in Figure
2015.09A
.
(b)
Analytical column
.—Silica 150 × 3.0 mm, 3 µm, 60 Å, or
equivalent.
(c)
Analytical balance
.—Capable of weighing to the nearest
0.00001 g.
(d)
Beakers
.—Glass, assorted sizes.
(e)
Centrifuge
.
(f)
Centrifuge tubes and caps
.—50 mL glass tubes with Teflon-
lined caps.
(g)
Cylinders
.—Graduated, glass, assorted sizes.
(h)
Gas regulator
.—Helium.
(i)
Helium sparge
.—Tubing and filtering assembly.
(j)
Magnetic stirrer.—
Variomag Telesystem (Daytona Beach,
FL, USA) HP 60 S (Part No. HP 46040U) with tube rack (Part
No. HP 86029) and egg-shaped Teflon stirring bars (Part No.
PI20110) or equivalent.
(k)
Pipet.—
Disposable glass, Pasteur.
(l)
Pipet.—
Mechanical, variable volume, 0.5–5 mL and
10–100 µL.
(m)
Pipet
.—Repeating 5 and 25 mL or equivalent.
(n)
Volumetric flasks.—
Glass, Class A, assorted sizes.
(o)
Volumetric pipets.—
Glass, Class A, assorted sizes.
(p)
Vortex mixers
.
(q)
Yellow lights or yellow shields with cutoff of at least 440 nm
.
C. Reagents
(a)
Acetic acid
.—Glacial, ACS.
(b)
Helium
.—Zero grade or equivalent
.
(c)
Iso-octane (2,2,4-trimethylpentane)
.—HPLC grade.
Table 2015.09A.
Trans
vitamin K
1
SLV data—precision
Sample type
No. of replicates
(duplicates on multiple days)
Mean, µg/100 g
RTF
SD
r
RSD
r
, % SD
IP
RSD
IP
, %
Child formula powder, placebo
6
2.03
0.040
2.0
0.045
2.2
Infant elemental powder, placebo
6
2.03
0.020
1.0
0.025
1.2
Adult nutritional RTF high protein, placebo
6
3.47
0.021
0.6
0.040
1.2
Adult nutritional RTF high fat, placebo
6
3.07
0.034
1.1
0.034
1.1
Infant formula RTF milk based, placebo
6
2.16
0.038
1.8
0.038
1.8
SRM 1849a
12
1.11
a
0.022
2.0
0.025
2.3
Adult nutritional powder milk protein based
12
3.26
0.097
3.0
0.142
4.4
Infant formula powder partially hydrolyzed milk based
12
7.69
0.123
1.6
0.170
2.2
Infant formula powder partially hydrolyzed soy based
12
8.99
0.141
1.6
0.226
2.5
Adult nutritional powder low fat
12
2.92
0.102
3.5
0.102
3.5
Child formula powder
12
2.66
0.049
1.8
0.050
1.9
Infant elemental powder
12
7.57
0.129
1.7
0.451
6.0
Infant formula powder milk based
12
6.09
0.105
1.7
0.131
2.2
Infant formula powder soy based
12
6.26
0.105
1.7
0.211
3.4
Infant formula RTF milk based
12
9.01
0.168
1.9
0.189
2.1
Adult nutritional RTF high protein
16
9.10
0.283
3.1
0.299
3.3
Adult nutritional RTF high fat
12
10.7
0.120
1.1
0.179
1.7
a
Results reported as mg/kg powder.
Candidates for 2016 Method of the Year
104