16 / 63
O
fitserova
& N
erkar
:
J
ournal of
AOAC I
nternational
V
ol
.
99, N
o
.
6, 2016
1473
A 100 psi back-pressure regulator was installed on the
detector outlet line to prevent any liquid from boiling in the
postcolumn reactor.
G. HPLC Conditions
The equipment was connected in the following order: HPLC
pump, autosampler, guard column, analytical column, postcolumn
derivatization system, and UV-Vis detector. A Lithium cation-
exchange column and lithium-based buffer solutions were used
to separate
l
-theanine. The HPLC pump flow rate was set to
0.35 mL/min and the postcolumn reagent pump flow rate was set
to 0.3 mL/min. The column oven temperature was set to 37°C and
the postcolumn reactor temperature was set to 130°C. The HPLC
pump gradient conditions that were used for the analysis are listed
in Table
2016.10B
. The UV-Vis detector signal was monitored at
570 nm with the reference wavelength set at 630 nm. An injection
volume of 10 μL was used.
Before starting the analysis, the system was equilibrated
for at least 30 min until all temperatures and pressures were
stable. At least one reagent blank was injected to equilibrate
the column before injecting the working calibration solutions,
control samples, samples extracts, and reagent blank. A mid-
range calibration solution was run every 10 injections to confirm
the stability of the calibration curve.
H. System Suitability
(a)
Retention times for
l
-theanine in sample extracts and
calibration solutions were within 0.5 min.
(b)
Retention times for
l
-norleucine in sample extracts and
calibration solutions were within 0.5 min.
(c)
The correlation coefficient R
2
for the weighted linear
regression calibration curve was ≥0.9998.
(d)
Relative error for the back-calculated concentration for
the mid-range calibration standard was within ±4%.
I. Calculations
The response ratio for the calibration standards (Area
L-
theanine
/Area
IS
) vs its corresponding ratio for the concentrations
(Concn
L-theanine
/Concn
IS
) was plotted to obtain a weighted linear
regression calibration curve.
The concentration of
l
-theanine (μg/mL) in the sample
extracts was calculated by interpolating the calibration curve.
The amount of
l
-theanine in the sample was calculated by
using the following formula:
(
)
( )
( )
=
µ
×
×
Concn sample mg g
Concn extract
g
mL
Volume extract mL
Mass sample g 1000
J. Precision Testing
Each matrix was analyzed in triplicate over 4 days. Working
calibration solutions were prepared on each day of the analysis.
Repeatability precision was assessed by calculating s
r
and
RSD
r
(%) for same-day replicates measured under the same
conditions. To determine intermediate precision, the conditions
of analysis were intentionally varied by performing the analysis
on different days by two different analysts using different lots of
reagents and different calibration curves. In addition, samples
SRM 3254, SRM 3255, and SRM 3256 were analyzed using
two different HPLC systems.
The Grubbs’ outlier test for a 95% confidence interval was
applied to the results with no outliers detected.
K. Accuracy Testing
Method accuracy was evaluated by analyzing SRM 3254,
SRM 3255, and SRM 3256, as well as by conducting spike
recovery studies for seven matrixes.
SRMs were analyzed in triplicate over 4 days by two different
analysts using two different HPLC systems and different lots of
reagents and columns.
For spike recovery studies, each matrix was spiked at two
levels and samples analyzed in duplicate over 3 days by two
different analysts using different lots of reagents.
l
-Theanine
stock solution and
l
-theanine intermediate stock solution were
used to spike the samples. The overall mean for unspiked
samples determined during the course of the precision study
was used to calculate the recoveries.
L. Ruggedness Testing
The effect of seven factors (
see
Table
2016.10C
for a list) was
evaluated using the Youden ruggedness trial design (18). In each
experiment, the values of four factors were modified as shown in
Table 2016.10B. HPLC pump gradient conditions
Time, min
Li275, % Li750, % RG003, %
0
100
0
0
12
100
0
0
45
66
34
0
45.1
0
0
100
50
0
0
100
50.1
100
0
0
62
100
0
0
Table 2016.10C. Ruggedness trial experimental design
Factor
Value 1
Value 2
Formulation of ninhydrin
reagent
T100, 1-part
ninhydrin reagent (A)
T200, 2-part ninhydrin
reagent (a)
HPLC flow rate
0.35 mL/min (B)
0.38 mL/min (b)
Extraction volume
25 mL (C)
10 mL (c)
Analyst
Analyst 1 (D)
Analyst 2 (d)
Extraction time
2 h (E)
1.5 h (e)
Extraction solution
Li220
Lot 1 (F)
Lot 2 (f)
Reactor temperature
130 °C (G)
125 °C (g)
Experiment No.
Combination of factors
1
ABCDEFG
2
ABcDefg
3
AbCdEfg
4
AbcdeFG
5
aBCdeFG
6
aBcdEfG
7
abCDefG
8
abcDEFg
16