ABBOTTNUTRITIONDIVISION
TITLE
PAGENO.
Determination of TransVitaminK
1
byHPLC andFluorescenceDetection
3 of 14
3.
This normal phaseHPLCmethodwith post column reduction and fluorescence detection allows for the
quantitativemeasurement of trans vitaminK
1
in infant andmedical nutritional products. The sample
preparation procedure for products involves vortexingproduct sampleswithmethanol to precipitate
proteins and free the lipids for extraction bydisruptingmicelles. Isooctane is used to extract vitaminK
1
and laboratorywater is added to enhance the separation ofmethanol and isooctane. After vitaminK
1
is
extracted into the isooctane layer, it is injected onto a silicaHPLC column. Cis and trans vitaminK
1
are
separatedwith an isooctane/isopropanolmobile phase. The column eluant ismixedwith a dilute
solution of zinc chloride, sodium acetate, and acetic acid in ethanolwhich allows vitaminK
1
to be
reduced to a fluorescent derivative in a zinc reductor. The resulting fluorescent compound is then
detectedwith a fluorescence detector setwith an excitationwavelength of 245 nm and an emission
wavelength of 440 nm.
C.
APPARATUS
1.
Instrumentation
a)
HPLCFluorescenceDetector; ShimadzuRF-20Axs or equivalent
b)
HPLC Isocratic pump (formobile phase): ShimadzuLC-20AT or equivalent
c)
HPLC Isocratic pump (for post column solution): ShimadzuLC-20ATor equivalent
d)
HPLC autosampler
e)
Analytical Column: Silica 150 x 3.0mm, 3micron, 60A silica or equivalent
f)
HPLC autosampler vials, clear or amber glass vialswithTeflon/silicon/Teflon caps
g)
Data collection system
h)
High pressuremixing tee
i)
Post-columnReductor: 20 x 4mm stainless steel,Waters P/NWAT084550 or equivalent
2. General LaboratoryEquipment/Facilities
b) Analytical Balance;minimumweighing capacityof at least 0.00001 grams
c) Beakers; assorted sizes
f) Centrifuge
h) CentrifugeTubes: Corex, 50mL
i) CentrifugeTubeCaps: Teflon lined caps
j) Centrifuge tube holder
l) Flasks, Volumetric: assorted sizes
m) GasCylinder SafetyHolders
n) GasTankRegulator, helium, deliverypressure range 5-180 psi
cylinder pressure gauge 0-4000 psi
o) Graduated cylinder; assorted sizes
VitK-02
FORWORKINGGROUP/ERPUSEONLY
DONOTDISTRIBUTE
