ABBOTTNUTRITIONDIVISION
TITLE
PAGENO.
Determination of TransVitaminK
1
byHPLC andFluorescenceDetection
8 of 14
is equilibrated, theRSD of the standard peak areas should be<2% and the peak areas should not
steadily increase or decrease bymore than 4% from the first injection to the third or fourth
injection. If theRSD>2%, locate the source of the imprecision and correct it before beginning the
sample analysis. If peak areas steadily increase or decrease bymore than 4%, the system is not
equilibrated andmust be allowed to equilibrate longer. Once the system has reached equilibrium
and theRSD is ≤2%, inject a set of standards, the control sample, unknown samples, and another
set of standards. Every set of unknown samplesmust be bracketed by standards and the first
sample in each setmust be the control sample.
3. CALCULATIONS
The vitaminK
1
concentrations of samples analyzed on theHPLC system are determined by
comparison of peak areas from samples of knownweightwith the peak areas of standards of known
concentration. Because the cis and trans vitaminK
1
retention timesmay shift slightlyduring a run,
peak areasmust be used to quantitate trans vitaminK
1
.
1.
Calculationof theStandardConcentrations
a) Concentration of StockStandard
4000
250 .0
1000
W
W
C
s
Where:
C
s
is the concentration of total (cis and trans) vitaminK
1
stock solution inmilligrams/liter;
W is theweight of vitaminK
1
standard in grams;
1000 is the conversion factor for grams tomilligrams; and
0.2500 is the dilution volume in liters.
b) Concentration of Intermediate I Standard
000 ,40
00.10
100
1000 0.1
W
C
C
C
s
s
I
Where:
C
I
is the concentration of Intermediate I standard solution inmicrograms/liter;
1.0 is the volume of the stock standard solution aliquot inmilliliters;
1000 is the conversion factor formilligrams tomicrograms; and
100 is the dilution volume inmilliliters.
b)
Concentration of Intermediate II Standard
000 ,8
2000 .0
50
0.10
W
C
C
C
I
I
II
VitK-02
FORWORKINGGROUP/ERPUSEONLY
DONOTDISTRIBUTE
