ABBOTTNUTRITIONDIVISION
TITLE
PAGENO.
Determination of TransVitaminK
1
byHPLC andFluorescenceDetection
6 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. SamplePreparation
NOTE: If any liquid leaks or is displaced from the 50mL centrifuge tube at any time during the
sample pretreatment, the samplemust be prepared again beginningwith step 1.
To eliminate possible sources of contamination do not allow anyof the organic solvents or solutions
usedduring the sample preparation and analysis to come into contactwith plastics or rubber articles.
Solutions ofVitaminK
1
in isooctane are stable. If the isooctane extracts from prepared samples are
storedunder yellow lights in tightly capped centrifuge tubes, they can be reanalyzed for up to 7 days
after preparation.
All samples should be as uniform and representative of the product as possible. This should be
accomplished by thoroughlymixingor stirring the sample before sampling.
a) Product andControl Samples
1) Accuratelyweigh up to 4 grams of ready-to-feed products or powders diluted to ready-to-feed
concentrations into a 50mL centrifuge tube. For sampleweights that are less than 4 grams,
add enoughwater to the tubes so that the sampleweight plus the amount of addedwater
equals ~4.
2) Using a repeatingpipette, add 25 (±2.0)mLofmethanol to each sample just prior to
vortexingor stirring.Methanol should not be added tomore than two samples consecutively
without vortexingor stirring. Cap each centrifuge tube, vortex each sample at high speed for
at least 30 seconds, and allow samples to set undisturbed for at least 10minutes, but nomore
than 40minutes after vortexingwithmethanol, OR add amagnetic stir bar to each sample,
place each uncapped sample onto amagnetic stir plate, and stir each sample for at least 10
minutes, but notmore than40minutes at a spin-rate that causes a vortex to formwithin the
samplewithout any sample escaping from the tube.
3) Add 10 (±0.05)mLof isooctane to each samplewith a volumetric pipette. Add isooctane to
all samples before vortexingor stirring anyof the samples. Vortex each sample for at least 45
secondsOR stir each uncapped sample for at least 45 seconds at a spin-rate that causes a
vortex to formwithin the samplewithout any sample escaping from the tube.
4) Using a pipetter, add 5 (±1)mLof laboratorywater to each sample and vortex the sample for
at least 20 secondsOR stir each capped sample for at least 20 seconds at a spin-rate that
causes a vortex to formwithin the samplewithout any sample escaping from the tube.
Laboratorywater can be added to all the samples prior to vortexingor stirring, or each sample
can be vortexed or stirred after laboratorywater is added to it.
5) Centrifuge the samples until a clean separation of the isooctane and laboratory
water/methanol layers results. The isooctane layer should be a clear layer at the top of the
centrifuge tube and the laboratorywater/methanol layer should be a cloudy layer below the
VitK-02
FORWORKINGGROUP/ERPUSEONLY
DONOTDISTRIBUTE
