5. AOACSPDSMethods-2018AwardsV3

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original test tube one more time using a 4 mL portion of the ethyl acetate–methanol (90 + 10) solution. Mix the sample on the vortex mixer for about 30 s. If necessary, centrifuge the sample at 2000 rpm for 5 min to separate layers. Transfer the top-most organic layer into the second test tube. ( 7 ) Evaporate the combined sample extract of the second test tube to dryness using an N 2 purge in a 50°C water bath. ( 8 ) Pipet 0.5 mL methanol–water (60 + 40) into the second test tube containing the dried sample residue. Mix the sample on a vortex mixer for 15 s. ( 9 ) Filter the sample through a 0.2 µm PVDF filter into a glass HPLC vial with an insert or a microvial. (b)  Finished product (powder and liquid form) .— ( 1 )  Powder product. —Accurately weigh about 0.5 g powder product into a 15 mL screw-cap test tube, pipet 1 mL purified water, and mix the sample well on the vortex mixer to dissolve the powder. ( 2 )  Liquid product .—Accurately weigh about 1 g liquid product into a 15 mL screw-cap test tube. ( 3 ) Pipet 1 mL reagent alcohol and 2 mL saturated sodium chloride solution into the test tube. Mix the sample well on the vortex mixer. ( 4 ) Pipet 4 mL ethyl acetate–methanol (90 + 10) solution into the test tube. ( 5 ) Cap the test tube and mix the sample on the vortex mixer for 60 s at maximum speed. ( 6 ) If necessary, centrifuge the sample at 2000 rpm for 5 min to separate layers. ( 7 ) Transfer the top-most organic layer into a second 15 mL screw-cap test tube. ( 8 ) Extract the sample from the original test tube one more time using a 4 mL portion of the ethyl acetate–methanol (90 + 10) solution. Mix the sample on a vortex mixer for 30 s. If necessary, centrifuge the sample at 2000 rpm for 5 min to separate layers. Transfer the top-most organic layer into the second test tube. ( 9 ) Evaporate the combined sample extract of the second test tube to dryness using an N 2 purge in a 50°C water bath. ( 10 ) Pipet 0.5 mL methanol–water (60 + 40) into the second test tube containing the dried sample residue. ( 11 ) Mix the sample on a vortex mixer for 15 s. ( 12 ) Filter the sample through a 0.2 µm PVDF filter into a glass HPLC vial with the insert or a microvial. (a)  In chromatographing the standards, the correlation coefficient (R) for the aloin A and B (and aloe-emodin, if necessary) curves should not be less than 0.998. (b)  Run a standard check (80 ppb standard) after every six sample injections and at the end of the run. The peak area of each check standard should be within 90–110% of the peak area of the working standard from the calibration curve. (c)  The theoretical plate count should not be less than 10000 for aloins A and B and aloe-emodin (if required). (d)  The tailing factor should not be more than 2.0 for aloins A and B and aloe-emodin (if required). H. System Suitability

E. Preparation of Extraction Solutions

(a)  Saturated sodium chloride solution.— Add 50 g sodium chloride to 100 mL freshly boiled purified water. (b)  Ethyl acetate–methanol (90 + 10) solution.— Combine 900 mL ethyl acetate with 100 mL methanol. Mix well. (c)  Methanol–water (60 + 40) solution.— Combine 600 mL methanol with 400 mL purified water. Mix well.

F. Preparation of Standards

(a)  Stock standard solutions .—( 1 )  Aloin A and B stock standard solution.— Accurately weigh about 5 mg each of aloin A and B reference standards into the same 50 mL volumetric flask. Dissolve and dilute to volume with methanol. Store the stock standard solution in a refrigerator [2–8°C, concentration of ~100 parts per million (ppm)]. ( 2 )  Aloe-emodin stock standard solution .—Accurately weigh about 5 mg aloe-emodin reference standard into a 50 mL volumetric flask. Dissolve and dilute to volume with methanol. Store the stock standard solution in a refrigerator (2–8°C, concentration of ~100 ppm). ( 3 )  Aloin A and B mid-standard solution .—Pipet 100 µL aloin A and B stock standard solution into a 10 mL volumetric flask and dilute to volume with methanol–water (60 + 40). Mix well (concentration of ~1 ppm). ( 4 )  Aloe-emodin mid-standard solution .—Pipet 100 µL aloe- emodin stock standard solution into a 10 mL volumetric flask and dilute to volume with methanol–water (60 + 40). Mix well (concentration of ~1 ppm). (b)  Working standard solution .—( 1 )  Standard [300 parts per billion (ppb)] .—Pipet 3 mL mid-standard solution into a 10 mL volumetric flask and dilute to volume with methanol– water (60 + 40). Mix well. ( 2 )  Standard (80 ppb) .—Pipet 2 mL mid-standard solution into a 25 mL volumetric flask and dilute to volume with methanol–water (60 + 40). Mix well. ( 3 )  Standard (20 ppb) .—Pipet 200 µL mid-standard solution into a 10 mL volumetric flask and dilute to volume with methanol–water (60 + 40). Mix well. (a)  Rawmaterial (powder and liquid form) .—( 1 ) Accurately weigh about 0.1 g powder raw material sample into a 15 mL screw-cap test tube; pipet 1 mL purified water and mix the sample well on a vortex mixer to dissolve the powder. For the liquid raw material, weigh ~1 g raw material sample into a 15 mL screw-cap test tube. ( 2 ) Pipet 1 mL reagent alcohol and 2 mL saturated sodium chloride solution into the test tube. Mix the sample well on the vortex mixer. ( 3 ) Pipet 4 mL ethyl acetate–methanol (90 + 10) solution into the test tube. ( 4 ) Cap the test tube and mix the sample on the vortex mixer for about 60 s at maximum speed. ( 5 ) If necessary, centrifuge the sample at 2000 rpm for 5 min to aid separation of layers. ( 6 ) Transfer the top-most organic layer into a second 15 mL screw-cap test tube. Extract the sample from the G. Preparation of Samples