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Bidlack et al.:
J
ournal of
AOAC I
nternational
V
ol.
98, N
o.
5, 2015
1385
naturally fluoresce or form fluorescent derivatives when
exposed to zinc.
Acceptance Criteria
Previously established method performance requirements for
vitamin K (SMPR 2014.001) are summarized in Table 1.
Results
The SRM repeatability and recovery requirements were met
for all 12 matrixes. Repeatability data are summarized in Table
2. Repeatability precision (RSD
r
) and intermediate precision
(RSD
IP
) ranged from 0.6 to 3.5% and 1.1 to 6.0%, respectively.
Pooled across all samples, the RSD
r
was 2.0% and the RSD
IP
was 2.8%. Mean spike recovery data are summarized in Table 3
and ranged from 91.9 to 106%, averaging 97.8% across all
samples. The method demonstrated good linearity over a
standard range of approximately 2–90 µg/L
trans
vitamin K
1
with r
2
averaging 0.99995. These data are summarized in
Table 4. Average calibration errors were <1% and are illustrated
graphically in Figure 1.
The LOQ requirement wasmet. LODand LOQwere estimated
to be 0.1 and 0.4 µg/L, respectively, with standards and 0.03
and 0.09 µg/100 g ready-to-feed (RTF) liquids, respectively, for
products assuming a 4 g sample diluted to 10 mL.
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 2 and 3 for infant, pediatric, and nutritional
matrixes for which the method has been validated.
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; 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) 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.
(d)
Isopropanol (isopropyl alcohol)
.—HPLC grade.
(e)
Methanol
.—HPLC grade.
Figure 1. Vitamin K
1
standard curve calibration errors; L = level.
94