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Jing et al.:
J
ournal of
AOAC I
nternational
V
ol.
98, N
o
. 5, 2015
1399
additives (recommended by column supplier). After verifying
equilibration of the UPLC system, inject the mid-level working
standard four times to verify system suitability. RSD of the
peak areas from these injections should be <5%. Once system
suitability has been established, inject working standards
(WS 1–3), followed by a reagent blank, control sample, and
samples. Reinject working standards approximately every 4 h
(e.g., enough time for 16 samples with analysis cycle time of
15 min).
H. System Suitability
(
a
) The RSD of the four standard injections to prove
equilibration prior to run must be <5%.
(
b
) Calibration curve residuals must be ≤4%. Samples
should be bracketed by two sets of such valid calibration curves.
(
c
) A suitable control sample is National Institute of
Standards and Technology Standard Reference Material (NIST
SRM) 1849a, reconstituted as a normal sample powder (each
packet contains about 10 g). A control sample must be run
concurrently with every sample set and a corresponding control
chart set up. The control chart RSD of the means of choline and
carnitine must be <4.0%.
(
d
) The method is valid for analytical solution concentrations
between 50% of WS1 and 10% above WS3.
I. Calculations
(
a
) For each of the three WS, the software plots each relative
response (analyte/internal standard) versus its corresponding
working standard concentration to obtain two separate
calibration curves for choline and carnitine (two data points for
each concentration, one from the beginning of the analysis and
one from the end). It applies a linear regression model to the
data and obtains an equation for the best-fit line.
(
b
) For each sample injected, the instrument measures the
response (analyte/internal standard) for choline and carnitine
and uses the linear regression equation to calculate the resulting
concentration in the analytical solution.
(
c
) The concentration in the analytical solution (ng/mL) is
multiplied by a dilution factor (DF) to project the results back to
the original sample, on a µg/g basis:
C
x
= C
s
× DF
(1)
where C
x
is the concentration of the analyte in the product (µg/g)
and C
s
is the concentration in the analytical solution measured
by the instrument (ng/mL).
Figure 1. Representative chromatograms of SRM 1849a (total analysis). Secondary transitions were monitored for both choline and
carnitine, including their internal standards (only the primary transitions are given in Table 2014.04D).
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