5. AOACSPDSMethods-2018AwardsV3

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1448 K oshy et al .: J ournal of AOAC I nternational V ol . 99, N o . 6, 2016

(peak area/concentration) at each concentration examined. Over the full range of concentrations examined, r 2 varied from 0.76 for withanolide B to 0.99 for withanoside IV and V. A plot of the residuals by concentration revealed a convex pattern with significant residuals as high as 78%. The pattern indicates a lack of linearity and suggests that alternative regression analyses (e.g., weighted regression or polynomial regression) should be evaluated for a better fit to the data over the large concentration range. Alternatively, the concentration range can be narrowed to allow for a better approximation of linearity. Using the latter approach, the data for each analyte were analyzed over a narrower concentration range until linearity was more closely approximated. As can be seen in the nonrandom residual plots of Figures 4–9, even over a narrower concentration range, the data are not strictly linear. The residuals, however, are much smaller and tolerable at ≤6.25%. In the linear regression plots, all r 2 are >0.999 and the y -intercept has been greatly reduced in most cases. Over the narrower concentration ranges, the response factors, defined as mean peak area divided by concentration, vary by <5% from the mean response factor. The ranges for each analyte that approximate linearity are as follows: withanoside IV 20–330 μg/mL, withanoside V 19–300 μg/mL, withaferin A 23–184 μg/mL, 12-deoxywithastramonolide 22–178 μg/mL, withanolide A 22–173 μg/mL, and withanolide B 21–169 μg/mL. For routine analyses, the method includes three concentrations of standards within the linear range.

Results and Discussion

Specificity

A representative chromatogram of the mixed standard with UV detection is shown in Figure 1. The peaks are well- resolved and the retention times are very similar to injections of the individual standards (data not shown). Figures 2 and 3 show chromatograms of a methanolic extract sample and a raw material sample using a UV detector and a PDA detector. Although more peaks are observed in the samples compared with the mixed standard, the six withanolide peaks are still well- resolved and the retention times and relative retention times of the six withanolide peaks in the samples are very similar to those in the mixed standard (Table 6). Furthermore, the PDAspectra of the withanolide peaks in the samples are identical to the spectra of the corresponding standards, and the peak purities based on PDA spectral analysis are >0.99 in the standards and samples (data not shown). These results indicate no matrix interference in the LC method. The relative retention times of the analyte peaks were 0.70 for withanoside IV, 0.89 for withanoside V, 0.92 for withaferin A, 0.96 for 12-deoxywithastramonolide, 1.0 for withanolide A, and 1.15 for withanolide B. These relative retention times agree with the values from the United States Pharmacopeia LC method as reported in SMPR 2015.007 (1).

Linearity

Precision

The mean peak areas of the linearity standards were plotted against concentration and are presented in Figures 4–9. Table 7 summarizes the data. After performing linear regression, linearity was assessed by determining goodness-of-fit (square of the correlation coefficient, r 2 ), by examining residuals over the concentration range, and by determining the response factor

Peak areas and retention times.— Repeatability precision of standards in methanol based on the measurement of the peak area is presented in Table 7 and retention time in Table 8. The RSDs from five replicate injections at each concentration were all <1%. When a sample, WS/06Lot10, was analyzed

Figure 1. Representative chromatogram of mixed standard solution.