ALN-01

1324  B rown et al . : J ournal of AOAC I nternational V ol . 97, N o . 5, 2014

( d )  Water (H 2

A validated method can also aid in future assessments regarding safety, use, and application of A. vera products.

O).—FW: 18.01 g/mol; CAS No. 7732-18-5,

HPLC grade (Fisher Scientific) or equivalent.

Solutions

Experimental

( a )  Reference standard diluent .—100% methanol. ( b )  Extraction solvent for high concentration test materials .—0.1% (v/v) acetic acid in methanol. ( c )  Extraction solvent for low concentration test materials .—0.1% (v/v) acetic acid in water. ( d )  Mobile phase A .—0.1% (v/v) acetic acid in water (filtered through a 0.2 µm nylon filter). ( e )  Mobile phase B .—0.1% (v/v) acetic acid in acetonitrile. Separate stock solutions of 100 µg/mL aloin A and aloin B are prepared by dissolving 2.83 mg aloin A in 25.0 mL reference standard diluent and 2.74 mg aloin B in 25.0 mL reference standard diluent. The stock solution of aloin A is then diluted to appropriate concentrations for external calibration. The aloin B stock solution is used as a QC sample. ( a )  Aloin A (C 21 H 22 O 9 ).—FW: 418.39 g/mol; CAS No. 8015- 61-0, purity 88.4%, acquired from Chromadex (Irvine, CA), Cat. No. 1625. ( b )  Aloin B (C 21 H 22 O 9 ).—FW: 418.39 g/mol; CAS No. 8015- 61-0, purity 91.3%, acquired from Chromadex, Cat. No. 1626. Reference standards are stored at 4°C until use. ( a )  Analytical balance .—Capable of reading ±0.01 mg (Mettler Toledo, Vancouver, BC, Canada) or equivalent. ( b )  HPLC vials .—Clear, volume 300 µL (Phenomenex, Torrance, CA). ( c )  Centrifuge .—Eppendorf 5810 table top centrifuge (VWR International) or equivalent. ( d )  Centrifuge tubes .—15 mL polypropylene (VWR International). ( e )  Micropipets .—Eppendorf Reference series 100, 200, and 1000 µL (Eppendorf, Mississauga, ON, Canada) or equivalent. ( f )  Solvent bottles .—100, 500, and 1000 mL. ( g )  Syringe filters .—Luer-lok ® type, 0.2 µm PTFE (VWR International). ( h )  Disposable syringes .—3 mL screw-type (VWR International). ( i )  Sonicating water bath .—Bransonic 3510 (VWR International) or equivalent. ( j )  Vortex mixer .—Thermo Scientific Maxi Mix1 (VWR International) or equivalent. Reference Standards Apparatus

Principle This method is used to detect and quantify aloin A and aloin B in A. vera leaf juice, dietary supplements, and finished products by HPLC with a diode array detector (DAD). This method has been summarized in the American Herbal Pharmacopeia (AHP) monograph for A. vera leaf, leaf juice, and inner leaf juice (17). The focus of the validation study is the diastereomers aloin A (barbaloin) and aloin B (isobarbaloin), although other anthraquinone-type compounds are known to exist in A. vera containing materials. These diastereomers are present in the latex of the plant, which is removed during processing of raw materials into juices and other finished products since recent studies have shown adverse effects when they are consumed in sufficient quantities. The validation consisted of nine test materials. ( a )  A. vera inner leaf dry juice powder A .—Obtained from Tampa Bay Analytical Research (Tampa Bay, FL). ( b )  A. vera inner leaf dry juice powder B .—Obtained from Tampa Bay Analytical Research. ( c )  A. vera nondecolorized leaf dry juice powder .—Obtained fromAloecorp (Ontario, Canada). ( d )  A. vera decolorized inner leaf dry juice powder .— Obtained fromAloecorp. ( e )  A. vera leaf juice powder capsule .—Obtained fromTampa Bay Analytical Research. ( f )  A. vera nondecolorized leaf juice .—Obtained from Aloecorp. ( g )  A. vera decolorized leaf juice .—Obtained fromAloecorp. ( h )  A. vera nondecolorized inner leaf juice .—Obtained from Aloecorp. ( i )  A. vera nondecolorized inner leaf juice concentrate .— Obtained fromAloecorp. All solid test samples were stored in a desiccator at room temperature, and liquid test samples were stored at 4°C. For capsules, to minimize variability due to nonuniformity between dosage units, contents of 20 capsules were combined and homogenized prior to analysis. ).—Formula weight (FW): 60.05 g/mol; CAS No. 64.19-7, ACS reagent grade (Fisher Scientific, Ottawa, ON, Canada) or equivalent. ( b )  Methanol (CH 4 O).—FW: 32.04 g/mol; CAS No. 67-65-1, HPLC grade (VWR International, Mississauga, ON, Canada) or equivalent. ( c )  Acetonitrile (C 2 H 3 N).—FW: 60.10 g/mol, CAS No. 75- 05-08, HPLC grade (VWR International) or equivalent. Test Samples Reagents and Supplies ( a )  Acetic acid, glacial (C 2 H 4 O 2

( k )  Volumetric flasks .—Class A; 2, 5, and 10 mL. ( l )  Volumetric pipets .—Class A; 1, 2, 4, and 8 mL.

HPLC Conditions

( a )  Instrument .—The HPLC system must be equipped with a DAD or UV detector, sample injection system, pump capable of delivering constant flow up to 400 bar, temperature-controlled column compartment, and computing data processor. ( b )  Autosampler temperature .—Room temperature.