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first injection to the third or fourth injection. If the RSD is >2%,
locate the source of the imprecision and correct it before beginning
the sample analysis. If peak areas steadily increase or decrease by
more than 4%, the system is not equilibrated and must be allowed
to equilibrate longer. Once the system has reached equilibrium and
the RSD is ≤2%, inject a set of standards, unknown samples, and
another set of standards. Every set of unknown samples must be
bracketed by standards.
(
4
)
Instrument shut down
.—After analyzing a set of samples,
simultaneously turn off the flow on the mobile phase and
postcolumn electrolyte solution pumps. Remove the helium sparge
lines from the mobile phase and postcolumn electrolyte solution
and turn off the helium. Turn off the fluorescence detector lamp.
F. Calculations
The vitamin K
1
concentrations of samples analyzed on the HPLC
system are determined by comparison of peak areas from samples
of known weight with the peak areas of standards of known
concentration. Because the
cis
and
trans
vitamin K
1
retention
times may shift slightly during a run, peak areas must be used to
quantitate
trans
vitamin K
1.
(a)
Calculation of the standard concentrations:
(
) 1,000,000,000
(
)
1 2 3
1 2
3 4
C
W V V V
D D D D
s
= × × × ×
× × ×
where C
s
is the working standard concentration in µg/L; W is the
weight of standard in g; V
1
, V
2
, and V
3
are the aliquots of stock,
intermediate I, and intermediate standard II solutions, respectively,
in mL; 1,000,000,000 is the conversion factor from g/mL to mcg/L;
and D
1
, D
2
, D
3
, and D
4
are the dilution volumes of the stock,
intermediate I, intermediate II, and working standard solutions,
respectively, in mL.
(b)
Peak areas are measured with a data system. Before
calculating concentrations, review all chromatograms to make sure
that
cis
and
trans
vitamin K
1
are baseline separated and that there
are no interfering peaks.
Trans
vitamin K
1
concentrations cannot be
calculated for any samples with interfering peaks or poor separation
between the
cis
and
trans
isomers (
see
Figures
2015.09B–D
).
Check the integration of the
cis
and
trans
vitamin K
1
peaks.
Cis
vitamin K
1
elutes 1 to 3 min before
trans
vitamin K
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 by more
than 7%, the system was not equilibrated and the data are not
acceptable.
(c)
Calculation of
trans
vitamin K
1
standard concentration:
T
A
A A
(
)
2
1 2
= +
where T is the
trans
vitamin K
1
fraction, A
1
is the
cis
vitamin K
1
peak area, and A
2
is
trans
vitamin K
1
peak area. The
trans
vitamin
K
1
fraction is calculated for all standards, and the calculated
trans
vitamin K
1
fraction for all standards is averaged together (T
a
) and
used to calculate the
trans
vitamin K
1
concentration of all standards.
(d)
Trans
vitamin K
1
standard concentration:
C C T
ST
S a
= ×
where C
ST
is the concentration of
trans
vitamin K
1
in the working
standard C
S
in µg/L and C
s
is the working standard concentration
in µg/L.
(e)
Preparationof standard curves
.—For each working standard
concentration, average the peak areas from each two consecutive
sets of standards. Prepare a standard curve by performing a linear
least-squares regression on
trans
concentration versus averaged
peak areas. A standard curve must have an r
2
of 0.999 or better
to be acceptable.
(f)
Calculation of
trans
vitamin K
1
in a sample:
= × ×
×
10.0
C
C
R
S P
p
c
where C
p
is the concentration of
trans
vitamin K
1
in µg/kg, C
c
is the
concentration of
trans
vitamin K
1
in the injected sample determined
from the standard curve in µg/L, 10.0 is the dilution volume
of the sample in mL, R is the final dilution weight of a product
reconstitution in g (if necessary), S is the sample size in g, and P is
the weight of product that is reconstituted in g (if necessary).
References:
J. AOAC Int . 98 , 1382(2015)DOI: 10.5740/jaoacint.15-130
AOAC SMPR 2014.001
J. AOAC Int . 98 , 1036 (2015)DOI: 10.5740/jaoac.int.SMPR2014.001
Posted: October 12, 2015
Candidates for 2016 Method of the Year
108