ABBOTTNUTRITIONDIVISION
TITLE
PAGENO.
Determination of TransVitaminK
1
byHPLC andFluorescenceDetection
10 of 14
b) TransVitaminK
1
StandardConcentration
C
1t
=C
1
xT
a
C
2t
=C
2
xT
a
C
3t
=C
3
xT
a
Where :
C
1t
, C
2t
, andC
3t
are the concentrations inmicrograms/liter of trans vitaminK
1
in the
working standardsC
1
, C
2
, andC
3
respectively.
4. PreparationOf TheStandardCurve
For eachworking standard concentration, average the peak areas from each two consecutive sets of
standards (This is known as bracketing the standards at the beginning and the end of each set of
samples). Prepare a standard curve byperforming a linear least squares (regression) on the
concentration versus averaged peak areas. A standard curve should have a correlation coefficient of
0.999 or better to be acceptable. If the correlation coefficient is less than 0.999, replace the zinc in the
post-column reductor and/ormake new standards, and repeat the analysis.
5. CalculationOf TransVitaminK
1
InASample
S
C
P
S
R
C
C
c
c
p
0.10
1000
1000 0.10
Where:
C
p
is the concentration of trans vitaminK
1
inmicrograms/kilogram;
10.0 is the dilution volume of the sample inmilliliters;
1000 is the conversion factor for grams to kilograms;
R is the final dilutionweight of a product reconstitution (if necessary);
1000 is the conversion factor formilliliters to liters;
S is the sample size in grams;
P is theweight of product that is reconstituted (if necessary); and
C
c
is the concentration of trans vitaminK
1
in the injected sample determined from the
standard curve inmicrograms/liter.
6. CalculationOf TransVitaminK
1
InTheControl Sample
a) Calculate the trans vitaminK
1
concentration of the control sample using the equation inF.
Calculations, step 5. For each sample set, the control resultmust bewithin 3 standard deviations of
the controlmean.
7. ExampleCalculationOf TransVitaminK
1
InASample
a) StandardConcentration
Assume 0.05527 grams of vitaminK
1
stock standardwereweighed.
1) Concentration of StockStandard
C
s
=Wx 4000=0.05527 x 4000=221.08mg/L
2) Concentration of Intermediate I Standard
VitK-02
FORWORKINGGROUP/ERPUSEONLY
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