AOAC 2019 First Action Methods

minutes. Centrifuge tube at 1800 g for 10 minutes at 20°C. Dilute extracted solutions as necessary with dilution solution. (c) Cranberry juice concentrate. –Dilute juice as necessary with dilution solution. (d) 30% (w/v) PAC laboratory produced cranberry liquid. –SMPR 2017.003 calls for the evaluation of cranberry juice that contains >15% PAC (w/v). There are no juice products in the commercial market that meet this criterion. Thus, we created a 30% (w/v) PAC laboratory based juice using purified PAC isolated from cranberry fruit by methods previously described by Feliciano et al 2012 (3). Weigh 300 mg isolated cranberry PAC into a microfuge tube and add 1 mL water. Vortex tube and sonicate to ensure PAC is completely solubilized. Dilute juice as necessary with dilution solution.

F. Analysis

Transfer 70 µL of dilution solution (blank), standards, and samples into desired wells of a 96-well plate. Plate is read at an absorbance of 640 nm every 1 minute for 30 minutes at 25°C. Data reduction is calculated at maximum absorbance (t max ).

G. Calculations

The results are calculated from the ProA2 standard curve and the c-PAC standard curve by plotting the absorbance values on a linear y-axis and the concentration of PAC on a linear x-axis. The PAC concentration for powder samples (w/w) is calculated using the following equation: � � = − × where y is the maximum absorbance at 640 nm, A is the intercept, B is the slope, D is the dilution factor, V is the extraction volume (mL), and M is the sample mass (g). The PAC concentration for liquid samples (w/v) is calculated using the following equation: � � = − × where y is the maximum absorbance, A is the intercept, B is the slope, and D is the dilution factor.

H. Method Validation and Statistical Analysis

Validation of this method was conducted according to the ICH Harmonized Tripartite Guideline Validation of Analytical Procedures: Text and Methodology Q2(R1) (4), following the acceptability criteria AOAC SMPR.003 shown in Table 1. Statistical analysis was performed using RStudio (version 1.2.1335). The function lm() was used to create the regression model and the function plot() was used to evaluated the regression diagnostics plots.