ABBOTTNUTRITIONDIVISION
TITLE
PAGENO.
Determination of TransVitaminK
1
byHPLC andFluorescenceDetection
5 of 14
Since vitaminK
1
is light sensitive, all samples and standardsmust be prepared, handled, and stored in the
dark or under yellow shielded lighting (see apparatus) unless otherwise stated. If the standards and
samplesmust be transported through or into an areawithout yellow shielded lighting, theymust be
wrapped tightly in foil.
1.
HPLCAnalyticalColumnMobilePhase
Add~900mLof iso-octane to a 1000mLvolumetric flask. Add 0.4-0.5mLof isopropanol to the
volumetric flask and dilute to volumewith iso-octane
.
NOTE: The isopropanol concentration in themobile phase can be adjusted slightlyuntil adequate
resolution of cis and trans vitaminK
1
from other peaks present in some samples is achieved. See
example chromatograms inAppendixA.
2.
PostColumnElectrolyte Solution
Weigh 1.37 (±0.02) grams zinc chloride and 0.420 (±0.05) grams sodium acetate anhydrous into a 1000
mLvolumetric flask containing~ 900mL reagent alcohol. Add 300 (±10) µL concentrated acetic acid
and dilute to volumewith reagent alcohol. Add a stir bar and stir solution for ~ 15minutes or until all
salts are dissolved.
3.
VitaminK
1
(Phytonadione) StockStandardSolution
Weigh approximately0.0550± 0.0055 grams vitaminK
1
(phytonadione) into a 250mLvolumetric vlask
and record theweight to at least the nearest 0.00001 gram. Dissolve vitaminK oil in iso-octane. Dilute
to volumewith isooctane andmixwell. The stock standard is good for 6months if stored in a tightly
stoppered container under refrigeration (2 to 8
C), protected from light.
4.
VitaminK
1
(Phytonadione) Intermediate I StandardSolution
Pipet 1.0mLof vitaminK
1
stock standard into a 100mLvolumetric flask. Bring to volumewith
isooctane andmixwell. Discard remaining Intermediate I solution at the end of the day. Prepare a new
Intermediate I standard from stock each time newworking standards aremade.
4.
VitaminK
1
(Phytonadione) Intermediate II StandardSolution
Pipet 10.0mLof vitaminK
1
Intermediate I Standard into a 50mLvolumetric flask. Bring to volume
with isooctane andmixwell. Discard remaining Intermediate II solution at the end of the day. Prepare
a new Intermediate II standard from stock each time newworking standards aremade.
5.
VitaminK
1
(Phytonadione)WorkingStandardSolutions
Pipet 3.0, 6.0, and 10.0mLs of Intermediate II standard into 100mLvolumetric flasks. Dilute each
standard to volumewith isooctane.Mixwell.With a clean glass Pasteur pipette, transfer a portion of
eachworking standard into a labeled autosampler vial and cap the vial. (Do not pour the standards
from the volumetric flasks into the vials.)Working standards stored in tightly stoppered flasks at room
temperature or under refrigeration and protected from the light are good for 3months. Theworking
standard expiration date cannot be extended beyond the stock standard expiration date.
E.
PROCEDURE
VitK-02
FORWORKINGGROUP/ERPUSEONLY
DONOTDISTRIBUTE
