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1474
O
fitserova
& N
erkar
:
J
ournal of
AOAC I
nternational
V
ol
.
99, N
o
.
6, 2016
Table
2016.10C
. The effect of any specific factor was evaluated
by comparing the difference between the averages of two subsets
of four experiments with 2 ×SD, where SD represents the SD
between the replicates performed under the same conditions.
Dry green tea extract capsules were used for the ruggedness
trial and each experiment was performed in duplicate.
M. Calibration Curves
Sample analysis throughout the validation study was
performed using a seven-point working calibration curve
covering a range from 1 to 40 μg/mL (Table
2016.10A
).
Additionally, the linearity of the method was evaluated by
building extended calibration curves consisting of 13 mixed
calibration standards that ranged from 0.5 to 100 μg/mL.
Extended calibration curves were obtained on 6 separate
days by two different analysts using different lots of reagents.
Calibration standards for each curve were freshly prepared on
the day of analysis.
Results and Discussion
Validation Study
The single-laboratory validation (SLV) study was conducted
to compare performance characteristics of this method with
the characteristics of AOAC SMPR 2015.014, “
Standard
Method Performance Requirements
for Determination of
Catechins, Methyl Xanthines, Theaflavins, and Theanine in
Tea (
Camellia sinensis
) Dietary Ingredients and Supplements”
(8), which are listed in Table 1.
Matrixes
Eight matrixes were used in the validation study: five green
tea-containing dietary supplements and three NIST SRMs.
The dietary supplements included tablets, dry capsules,
liquid formulation, softgels, and gelcaps. According to label
claims, all dietary supplements contained green tea extract.
The liquid formulation contained up to 45% alcohol, whereas
the tablets and dry capsules contained calcium and magnesium
salts, as well as common inactive ingredients. The gelcaps
contained glycerin and the softgels contained fish oil, caffeine,
lecithin, glycerin, and several plant extracts. None of the dietary
supplements had label claims indicating theanine content.
NIST SRMs included SRM 3254
C. sinensis
(green tea)
leaves, SRM 3255
C. sinensis
(green tea) extract, and SRM 3256
green tea-containing solid oral dosage form. Only reference
(noncertified) mass fraction values for
l
-theanine were available
from NIST.
l
-Theanine reference values represented data from
a single laboratory using an LC-MS method.
Selectivity
Performing a postcolumn reaction with ninhydrin reagent is
specific for primary amino groups and allows for the selective
detection of amino acids in complex matrixes. Lithium cation-
exchange columns and lithium citrate buffers represent a
chromatographic system designed to separate free amino acids.
Only free amino acids and a very limited number of organic
amines are retained in a lithium cation-exchange column under
the analytical conditions used for this analysis, and therefore could
be detected after reaction with ninhydrin postcolumn reagent.
l
-Theanine peak identity was confirmed by comparing the
HPLC elution profiles of
l
-theanine standard solution with the
profiles of the samples by using two types of cation-exchange
columns and different sets of buffers as mobile phases. The
peaks of
l
-theanine and
l
-norleucine (IS) were fully resolved
from the other peaks present in the chromatograms with a
resolution R
S
≥1.5.
The chromatograms of dietary supplements analyzed during
the course of this study are shown in Figures 1–3.
Figure 1. Chromatogram of a green tea softgel sample. The concentration of theanine in the sample was 0.1432 mg/g.
Table 1. SMPR for the determination of
l
-theanine in tea
dietary ingredients and supplements (8)
Analytical range, ppm
10–100000
LOQ, ppm
≤ 5
Method performance parameters by range
10–50 ppm 51–500 ppm >500 ppm
Recovery, %
80–110
90–107
95–105
RSD
r
, %
≤7
≤5
≤5
RSD
R
, %
≤10
≤8
≤8
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