ALN-01

1326  B rown et al . : J ournal of AOAC I nternational V ol . 97, N o . 5, 2014 ( b )  Linearity .—Linearity was evaluated using a seven-point standard calibration curve of aloin A. Calibration curves were plotted and linear regression was used to determine the slope and y-intercept. Linearity was also confirmed visually by generating trendlines using a Microsoft Excel program (Microsoft, Mississauga, ON, Canada). 25 30 35

Aloin B

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( c )  LODand LOQ .—The LODand LOQ for the quantification of aloin A and aloin B were determined using the International Union of Pure and Applied Chemistry (IUPAC) method. This method, recommended byAOAC INTERNATIONAL, uses data sets from at least seven replicate injections of the blank matrix. The LOD is defined as the mean response plus 3x the SD, and the LOQ is the mean plus 10x the SD. ( d )  Precision (repeatability; precision study) .—Precision was evaluated by analyzing multiple replicates of each test sample. Four replicate preparations of each test material were prepared and analyzed on 3 separate days, thereby having 12 replicates of each test material prepared. The within-day, between-day, and total SDs for aloin A and aloin B for each of the nine test materials were calculated and consequently used to generate HorRat values by using the Horwitz formula for each of the test material to summarize the precision of the validation study (19). Acceptable HorRat values are 0.1 to 2.0. ( e )  Accuracy .—Accuracy of the method was assessed using both solid and liquid aloe test samples that were determined not to contain any detectable aloins. Stock solutions of known concentrations were spiked in both liquid and solid materials in triplicate at three levels that correspond to 10% (low), 100% (medium), and 200% (high) of aloin A and aloin B expected in typical aloin raw materials and finished products. For the solid matrix recovery study, a commercial spray-dried A. vera powder was spiked to expected concentrations of 4, 40, and 80 µg/mL. An A. vera inner fillet juice was spiked to expected concentrations of 1, 10, and 20 µg/mL for the liquid matrix. The samples were processed as per the Preparation of Test Samples section above, taking into account the volume of spike solution added. ( f )  Stability .—The stability of the aloin A and aloin B was determined by comparing the concentration of the reference standards either acidified with 0.1% acetic acid or nonacidified at room temperature over time. Each aloin was evaluated by storing the solutions at room temperature (18–22°C). Identical reference standards were aliquotted in two sets having four replicates each: one set was acidified and the other set nonacidified and analyzed at a given time (t) point (t = 0, 4, 8, 24, 48, and 240 h). Stability was determined by comparing the peak areas to time = 0 where the assumption of no degradation was made. Optimization Studies Prior to the method validation, the extraction and chromatography parameters were investigated through optimization studies. For the extraction optimization, parameters such as extraction solvent, the addition of acid, sonication time, and the effect of heat were explored. For the chromatography method, parameters such as analytical column length and type, mobile phase composition, gradient versus isocratic elution, peak resolution, and run time were factors that were Results and Discussion

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Figure 1. Figure 1. Chromatogram of an aloin A and aloin B reference standard.

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Figure 2.

examined to provide the best resolution and shortest run time for the separation of aloin A and aloin B. The optimization of the method was carried out using both aloin reference standards and A. vera test materials.

Method Validation Results–Performance Characteristics

Identification of aloin A and aloin B in the test materials was possible by comparing the retention times of the aloins from the reference standards. An isocratic elution was used for the analysis of aloin A and aloin B, where the retention order is aloin B followed by aloin A. Representative chromatograms of the reference standards and test material are displayed in Figures 1 and 2. Quantification of the analytes was carried out by a simple linear regression analysis using quadruplicate samples prepared on three separate days at seven concentration levels. The analytical range used for the following aloins are listed above in the section on Preparation of Calibration and QC Solutions . Table 2. The LOD and LOQ by the IUPAC method Analyte LOD, µg/mL LOQ, µg/mL Aloin A 0.09 0.23 Aloin B 0.09 0.21