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Bidlack et al.
: J
ournal of
AOAC I
nternational
V
ol.
98, N
o.
5, 2015
INFANT FORMULA AND ADULT NUTRITIONALS
Received May 20, 2015. Accepted by SG July 8, 2015.
This method was approved by the Expert Review Panel for Infant
Formula and Adult Nutritionals as First Action.
The Expert Review Panel for Infant Formula and Adult Nutritionals
invites method users to provide feedback on the First Action methods.
Feedback from method users will help verify that the methods are
fit-for-purpose and are critical for gaining global recognition and
acceptance of the methods. Comments can be sent directly to the
corresponding author or
methodfeedback@aoac.org.1
Corresponding author’s e-mail:
karen.schimpf@abbott.comDOI: 10.5740/jaoacint.15-130
This normal-phase 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 hydroquinone is then detected
by fluorescence at an excitation wavelength of
245 nm and an emission wavelength of 440 nm.
During a single-laboratory validation of this
method, repeatability and intermediate precision
ranged from 0.6 to 3.5% RSD and 1.1 to 6.0% RSD,
respectively. Mean overspike recoveries ranged
from 91.9 to 106%. 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
and average calibration errors of <1%. LOQ and
LOD in ready-to-feed nutritionals were estimated to
be 0.03 and 0.09 µg/100 g, respectively. The method
met AOAC Stakeholder Panel on Infant Formula and
Adult Nutritionals
Standard Method Performance
Requirements
®
and was approved as a first action
method at the 2015 AOAC Mid-Year Meeting.
V
itamin K
1
is an antihemorrhagic vitamin first isolated
in 1939 after it was discovered that chicks fed diets
previously extracted with nonpolar solvents developed
subdural or muscular hemorrhages. Vitamin K
1
, which is
also known as phylloquinone and phytonadione, consists of a
methyl-substituted naphthoquinone nucleus attached to a side
chain of three saturated and one unsaturated isoprene units and
is a yellow viscous oil. Although vitamin K
1
occurs naturally
in the
trans
form, during synthesis of vitamin K
1
both the
cis
and
trans
isomers are formed with the
trans
isomer being the
major product. Vitamin K
1
is insoluble in water and sparingly
soluble in methanol and ethanol. It is soluble in vegetable
oils and organic solvents such as pentane, hexane, iso-octane,
and 2-propanol. Vitamin K
1
has five ultraviolet absorption
maxima which are at 242, 248, 260, 269, and 325 nm and can
be reduced to a fluorescent hydroquinone. Vitamin K
1
is stable
to air, heat, oxidizing agents, and moisture, but its activity is
destroyed by light (especially UV radiation), reducing agents,
and alkalies (1).
Good sources of vitamin K
1
are alfalfa, cabbage, cauliflower,
green vegetables, tomatoes, cheese, dairy products, meat, egg
yolks, and canola and soy oil. Vitamin K
1
is also found in bacteria
and is synthesized in the intestinal tract by microorganisms.
Trans
vitamin K
1
is biologically active, while the
cis
form has
little if any activity (1).
At the September 2013 AOAC Annual Meeting, an AOAC
Stakeholder Panel on Infant Formula and Adult Nutritionals
(SPIFAN) working group developed
Standard Method
Performance Requirements
(SMPR
®
; 2) for
trans
vitamin K
1
and required separation of the
cis
and
trans
isomers
since
cis
vitamin K
1
has little if any biological activity. SPIFAN approved
AOAC SMPR 2014.001 at the March 2014 AOAC Mid-Year
Meeting. Subsequently, AOAC issued a call for methods.
In response to AOAC’s call for methods, a new vitamin K
1
method that combined the strengths of the two current AOAC
Official Vitamin K
1
methods,
992.27
and
999.15
, was developed
and validated. AOAC
992.27
uses liquid–liquid extraction in
separatory funnels, open column cleanup, normal phase (NP)
chromatography, and UV absorbance to extract, separate, and
quantitate
trans
vitamin K
1
(3). Although the AOAC
992.27
sample preparation procedure provides better recovery of
vitamin K
1
in more complex infant, pediatric, and adult
nutritional matrixes than
999.15
and the sample preparation
solvents are compatible with the NP chromatography, UV
detection is not very specific and the sample preparation
procedure is labor-intensive. AOAC
999.15
uses an enzyme
digestion and liquid–liquid extraction in glass tubes,
reversed-phase chromatography, and fluorescence detection
after postcolumn reduction with zinc to extract, separate, and
quantitate
trans
or total vitamin K
1
(4). Although AOAC
999.15
uses a more specific detection system and a simpler sample
preparation procedure, it will not separate
cis
and
trans
vitamin
K
1
if a C
18
column is used; sample extracts must be dried
down and the residue dissolved in a solvent compatible with
Determination of Vitamin K
1
in Infant, Pediatric, and Adult
Nutritionals by HPLC with Fluorescence Detection: Single-
Laboratory Validation, First Action 2015.09
Mary Bidlack, Linda D. Butler Thompson, Wesley A. Jacobs, and Karen J. Schimpf
1
Abbott Nutrition, 3300 Stelzer Rd, Columbus, OH 43219
91