ABBOTTNUTRITIONDIVISION
TITLE
PAGENO.
Determination of TransVitaminK
1
byHPLC andFluorescenceDetection
9 of 14
Where:
C
II
is the concentration of Intermediate II standard solution inmicrograms/liter;
10.0 is the volume of the Intermediate I standard solution aliquot inmilliliters; and
50 is the dilution volume inmilliliters.
d) Concentration ofWorkingStandards
240
03.0
100
0.3
1
W
C
C
C
II
II
480
06.0
100
0.6
2
W
C
C
C
II
II
800
10.0
100
0.10
3
W C
C
C
II
II
Where:
C
1
, C
2
, andC
3
are the concentrations of theworking standard solutions inmicrograms/liter;
3.0, 6.0, and 10.0 are the volumes of the intermediate standard solution inmilliliters; and
100 is the dilution volume inmilliliters.
2.
Measurement Of PeakArea (SeeSystemSuitabilityCheckList, SectionG)
The peak area ismeasuredwith a data system. Before calculating concentrations, review all
chromatograms tomake sure that cis and trans vitaminK
1
are baseline separated and that there are no
interferingpeaks. Trans vitaminK
1
concentrations cannot be calculated for any sampleswith
interferingpeaks or poor separationbetween the cis and trans isomers. Check the integration of the cis
and trans vitaminK
1
peaks. Cis vitaminK
1
elutes 1 to 3minutes before trans vitaminK
1
depending on
the analytical column used. If the peak areas of the same standards injected before and after a set of
samples have changed bymore than7%, the systemwas not equilibrated and the data is not acceptable.
3. CalculationOf TransVitaminK
1
StandardConcentration
a) Calculation of TransVitaminK
1
Fraction in theStandard
T=
A
2
A
1
+A
2
Where:
T is the trans vitaminK
1
fraction;
A
1
is the peak area of cis vitaminK
1
; and
A
2
is the peak area of trans vitaminK
1
.
The trans vitaminK
1
fraction is calculated for all standards and the calculated trans vitaminK
1
fraction for all standards is averaged together (T
a
) and used to calculate the trans vitaminK
1
concentration for all standard curves.
VitK-02
FORWORKINGGROUP/ERPUSEONLY
DONOTDISTRIBUTE
