AOACSPDSMethods-2017Awardsv2

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1472 Ofitserova & Nerkar: Journal of AOAC International Vol. 99, No. 6, 2016
(5) Green tea extract tablets.—Each tablet contained 500 mg
green tea standardized extract (175 mg epigallocatechin gallate),
calcium phosphate (47 mg calcium), stearic acid, modified
cellulose gum, and silica.
D. Preparation of Standard Solutions
(a) l-Theanine stock solution (500 μg/mL).—l-Theanine
stock solution was prepared by weighing 50 mg l-theanine in a
100 mL volumetric flask and diluting to volume with extraction
solution. The final concentration was corrected for purity stated
in the certificate of analysis. The solution was stored refrigerated
for up to 8 weeks.
(b) IS stock solution (500 μg/mL).—l-Norleucine stock
solution was prepared by weighing 50 mg l-norleucine in a
100 mL volumetric flask and diluting to volume with extraction
solution. The final concentration was corrected to the purity
stated in Certificate of Analysis. The solution was stored
refrigerated for up to 8 weeks.
(c) l-Theanine intermediate stock solution (50μg/mL).—
l-Theanine intermediate stock solution was prepared by
pipetting 2.5 mL l-theanine stock solution into a 25 mL
volumetric flask and diluting to volume with extraction solution.
(d) Mixed working calibration solutions.—Mixed working
calibration solutions were prepared by diluting stock solutions
of l-theanine and l-norleucine with extraction solution
according to Table 2016.10A. All working calibration solutions
were prepared on the day of analysis.
E. Sample Preparation and Extraction
Sample size and volume of extraction solution were chosen
based on sample availability, sample type, and expected
theanine concentration.
(a) For samples in tablet form.— At least 20 tablets were
finely ground and the resulting sample thoroughly mixed
before separating out the test portion. A 0.1–0.5 g portion was
accurately weighed in a 10 or 25 mL volumetric flask. IS stock
solution (500 μL) and extraction solution (20 mL) were added
to the 25 mL volumetric flask and mixed well. IS stock solution
(200 μL) and extraction solution (8 mL) were added to the
10 mL volumetric flask and mixed well. The flask was placed in
an ultrasonic water bath for 2 h. After cooling the flask to room
temperature, the solution was diluted to volume with extraction
solution and then mixed and transferred to a 50 mL centrifuge
tube. The extract was centrifuged for 20 min at 3800 rpm,
filtered through 0.45 μm syringe filter, and placed in an HPLC
autosampler vial for analysis.
(b) Forsamplesinpowderform.—Thesamplewasthoroughly
mixed before separating out the test portion. A 0.1–0.5 g portion
was accurately weighed in a 10 or 25 mL volumetric flask. IS
stock solution (500 μL) and extraction solution (20 mL) were
added to the 25 mL volumetric flask and mixed well. IS stock
solution (200 μL) and extraction solution (8 mL) were added
to the 10 mL volumetric flask and mixed well. The flask was
placed in an ultrasonic water bath for 2 h. After cooling the
flask to room temperature, the solution was diluted to volume
with extraction solution and then mixed and transferred to a
50 mL centrifuge tube. The extract was centrifuged for 20 min
at 3800 rpm, filtered through 0.45 μm syringe filter, and placed
in an HPLC autosampler vial for analysis.
(c) For samples in liquid form.—The sample was thoroughly
mixed before separating out the test portion. A 0.1–0.5 g portion
was accurately weighed in a 10 or 25 mL volumetric flask. IS
stock solution (500 μL) and extraction solution (20 mL) were
added to the 25 mL volumetric flask and mixed well. IS stock
solution (200 μL) and extraction solution (8 mL) were added
to the 10 mL volumetric flask and mixed well. The flask was
placed in an ultrasonic water bath for 2 h. After cooling the
flask to room temperature, the solution was diluted to volume
with extraction solution and then mixed and transferred to a
50 mL centrifuge tube. The extract was centrifuged for 20 min
at 3800 rpm, filtered through 0.45 μm syringe filter, and placed
in an HPLC autosampler vial for analysis.
(d) For softgels, gelcaps, or encapsulated dry supplement
samples.—The contents of at least 15 capsules were removed
and thoroughly mixed before separating out the test portion.
A 0.1–0.5 g portion was accurately weighed in a 10 or 25 mL
volumetric flask. IS stock solution (500 μL) and extraction
solution (20 mL) were added to the 25 mL volumetric flask and
mixed well. IS stock solution (200 μL) and extraction solution
(8 mL) were added to the 10 mL volumetric flask and mixed
well. The flask was placed in an ultrasonic water bath for 2 h.
After cooling the flask to room temperature, the solution was
diluted to volume with extraction solution and then mixed
and transferred to a 50 mL centrifuge tube. The extract was
centrifuged for 20 min at 3800 rpm, filtered through 0.45 μm
syringe filter, and placed in an HPLC autosampler vial for
analysis.
(e) For SRMs.—A 0.1 g sample was accurately weighed in
a 10 or 25 mL volumetric flask. IS stock solution (500 μL) and
extraction solution (20 mL) were added to the 25 mL volumetric
flask and mixed well. IS stock solution (200 μL) and extraction
solution (8 mL) were added to the 10 mL volumetric flask and
mixed well. The flask was placed in an ultrasonic water bath for
2 h. After cooling the flask to room temperature, the solution was
diluted to volume with extraction solution and then mixed and
transferred to a 50mLcentrifuge tube. The extract was centrifuged
for 20 min at 3800 rpm, filtered through 0.45 μm syringe filter,
and placed in an HPLC autosampler vial for analysis.
F. Safety
Postcolumn ninhydrin reagent (Trione) is sensitive to
oxidation, so reagent bottles were pressurized with nitrogen at
5 psi. Safety-coated bottles were used to hold the postcolumn
reagent.
Table 2016.10A. Preparation of mixed working calibration
solutions
Volume of
l-theanine
stock
solution, mL
Volume of
l-norleucine
stock solution,
mL
Total volume
of calibration
solution, mL
l-Theanine
concn, μg/mL
l-Norleucine
concn, μg/mL
2.00 0.5 25 40 10
1.250 0.5 25 25 10
0.500 0.5 25 10 10
0.374 0.5 25 7.48 10
0.250 0.5 25 5 10
0.125 0.5 25 2.5 10
0.050 0.5 25 1 10
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