ALN-01

B rown et al .: J ournal of AOAC I nternational V ol . 97, N o . 5, 2014  1325

( c )  Analytical column .—Phenomenex (Torrance, CA) Kinetex 2.6 µm C 18 , 100 × 4.60 mm id, 2.6 µm particle size. ( d )  Column temperature .—30°C. ( e )  Detector conditions .—Monitor at 357 nm (4 nm bandwidth, no reference). ( f )  Flow rate .—1.850 mL/min. ( g )  Injection volume .—15 µL with methanol wash. ( h )  Run time .—18 min. ( i )  Isocratic elution conditions .— See Table 1. The calculation used to determine aloin concentration is as follows: Concentration, mg/mL: C = (A – B)/D where A = peak area (mAU × seconds), B = intercept of the calibration curve, and D = slope of the calibration curve. To quantify the individual aloins on a % (w/w) basis, the following calculation is used: Aloin, % (w/w) = [(C)(FV)(D)(100%)]/W where C = concentration (µg/mL) from linear regression analysis, FV = the final volume (mL) of the sample preparation, D = the dilution factor of the sample preparation, and W = the sample weight (mg). For the validation study, the following equations were used for evaluating precision: × 100 where SD(r) = population SD (σ/ n , where σ = sum of squares and n = number of replicates). PRSD r (RSD r calculated, %): PRSD r = 2C –0.15 where C = the concentration of the analyte expressed as a mass fraction. HorRat value: HorRat = RSD r PRSD r Within-day precision average and SDs are of four data points within a day, whereas within-laboratory precision average and SDs are 12 data points over 3 days (separate batches on 3 days). Calculations RSD r (found, %): RSD r = SD(r) mean ( a )  Liquid and juice samples .—( 1 ) Highly concentrated (>50 µg/mL) or viscous samples–mix thoroughly. Dilute 1:1 (v/v) with 0.1% acetic acid in methanol. Centrifuge at 5000 rpm for 3 min. ( 2 ) Filter supernatants and low concentrated juices through 0.2 µm PTFE filters into HPLC vials for analysis. ( b )  Extraction of powders and capsules .—( 1 ) The total capsule weight was determined by weighing 20 capsules. Combine and homogenize the contents of 20 capsules into a conical tube. ( 2 ) Weigh 100.0 ± 1.0 mg A. vera powder into a 15 mL conical centrifuge tube. Add 1.0 mL of 0.1% acetic acid in water and sonicate for 5 min three times, vortexing for 30 s between intervals. Preparation of Test Samples

( 3 ) Centrifuge at 5000 rpm for 3 min. ( 4 ) The supernatant is then filtered through 0.2 µm PTFE filter to an HPLC vial. ( 5 ) Highly concentrated (>50 µg/mL) or viscous samples.— Extract 100.0 mg Aloe powder with 1.0 mL 0.1% acetic acid in methanol first, gently vortex, and then add 1.0 mL 0.1% acetic acid in water. Sonicate for 5 min three times; vortex for 30 s between intervals. Centrifuge at 5000 rpm (4500 × g ) for 3 min prior to filtering with 0.2 µm PTFE filter into an HPLC vial. If samples are not analyzed immediately after preparation, they are refrigerated at 4°C prior to analysis to prevent degradation of aloins for 72 h. ( a )  Aloin A calibration curve .—Because the HPLC method uses an isocratic elution, only aloinAis required as the calibration standard for both aloins. A seven point calibration curve is generated from the 100 µg/mL stock solution as described below. ( 1 )  Linearity 1 (50 µg/mL) .—500 µL aloin A stock solution was pipetted into a 2.0 mL HPLC vial and diluted with 500 µL methanol and mixed well. ( 2 )  Linearity 2 (25 µg/mL).— 250 µL aloin A stock solution was diluted with 750 µL methanol and mixed well. ( 3 )  Linearity 3 (10 µg/mL).— 100 µL aloin A stock solution was diluted with 900 µL methanol and mixed well. ( 4 )  Linearity 4 (5 µg/mL) .—100 µL Linearity 1 was diluted with 900 µL methanol and mixed well. ( 5 )  Linearity 5 (1 µg/mL) .—100 µL Linearity 3 was diluted with 900 µL methanol and mixed well. ( 6 )  Linearity 6 (0.5 µg/mL) .—100 µL Linearity 4 was diluted with 900 µL methanol and mixed well. ( 7 )  Linearity 7 (0.3 µg/mL).— 300 µL Linearity 5 was diluted with 700 µL methanol and mixed well. ( b )  QC sample .—Amixed standard of 25 µg/mL aloin A and B is used for QC. Prepare the QC standard by combining 1.0 mL 100 µg/mL aloin A stock solution and 1 mL 100 µg/mL aloin B stock solution. Dilute the aloin A and B mixture with 2.0 mL of 0.1% acetic acid in water. Table 1. The isocratic elution conditions for the separation of aloin A and aloin B with column cleanup and post time re-equilibration Time, min Mobile Phase B, % 0.0 17 8.0 17 12.0 100 13.0 100 14.0 17 18.0 17 Preparation of Calibration and Quality Control Solutions

Single-Laboratory Validation (SLV) Parameters

This method was validated according to the guidelines of AOAC INTERNATIONAL SLV criteria (18). ( a )  Selectivity .—The selectivity of themethodwas established by injecting each of the aloins to show chromatographic resolution between analytes.