ABBOTTNUTRITIONDIVISION
TITLE
PAGENO.
Determination of TransVitaminK
1
byHPLC andFluorescenceDetection
7 of 14
isooctane layer. (A good separation of solvent layers can usuallybe achieved byvortexing
samples for approximately10minutes at 800 relative centrifugal force.)
6) Remove samples from the centrifuge and inspect the samples to verify that the isooctane and
laboratorywater/methanol layers are separated.With a glass pipette carefully rinse down the
upperwalls of the centrifuge tubewith a portion of the isooctane layer. If the layers become
mixed together, centrifuge the sample again. Pipet a portion of the clear isooctane layer into a
labeled autosampler vial and cap the vial.
2. HPLCAnalysis
a) InstrumentalOperatingConditions
1) Initial Instrument Conditions
HPLCAnalytical ColumnPumpPressureLimit:
400 bar
HPLCAnalytical ColumnPump FlowRate
0.4mL/min
RunTime
20.00minutes
InjectionVolume
20-50 uL
Post ColumnPump FlowRate
0.1mL/min
Post ColumnPumpPressureLimit
400 bar
Fluorescence detector settings
ExcitationWavelength
245 nm
EmissionWavelength
440 nm
Adjust gain and sensitivity settings so that the standard curve iswithin the range
of the detector.
b) Instrument Startup
Pack the post column reductorwith zinc. Degas themobile phase and post column electrolyte
solutions bybubblinghelium through them for at least 15minutes before connecting the post
column zinc reductor. Allow the column and post column reductor to equilibratewithmobile
phase flowing at 0.4mL/min and post column electrolyte solution flowing at 0.1ml/min for at
least 1/2 hour prior to the first injection. Bubble heliumVERYSLOWLY through themobile
phase and post column electrolyte solution continuously throughout the entire run. Allow the
fluorescence detector lamp towarm up½ hour prior to the first injection.
NOTE: When themobile phase and post column electrolyte solution are continuously sparged
with helium throughout a run, it is not necessary to pack the post column reductorwith zinc at the
beginningof every run.
c) HPLC of Standards andSamples
Inject themost concentrated standard (approximately40µg/L) onto the column and observe the
response on the fluorescence detector. If necessary, adjust the detector gain and sensitivity settings
so that the standard curve iswithin the range of the detector. Once the detector settings have been
determined, inject themost concentrated standard 3-4 times and note the peak areas. If the system
VitK-02
FORWORKINGGROUP/ERPUSEONLY
DONOTDISTRIBUTE
