5. AOACSPDSMethods-2018AwardsV3

15

1472 O fitserova & N erkar : J ournal of AOAC I nternational V ol . 99, N o . 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.

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.

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 (50  μ g/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. 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, E. Sample Preparation and Extraction

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 0.5 0.5 0.5 0.5 0.5 0.5

25 25 25 25 25 25 25

40 25 10

10 10 10 10 10 10 10

1.250 0.500 0.374 0.250 0.125 0.050

F. Safety

7.48

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.

5

2.5

1