*2.6. HPLC*/*ESI-MS Analysis of Polyphenolics*

An analytical Hewlett Packard 1100 series HPLC instrument (Palo Alto, CA, USA) equipped with an autosampler, binary HPLC pump, and UV/Vis detector was used. For HPLC/MS analysis, the HPLC apparatus was interfaced to a Bruker model Esquire-LC/MS ion trap mass spectrometer (Billerica, MA, USA). Mass spectral data were collected with the Bruker software (Bruker Co., DataAnalysis version 4.0, Billerica, MA, USA), which also controlled the instrument and collected the signal at 520 nm. Typical conditions for mass spectral analysis conducted in positive-ion electrospray mode for anthocyanins and negative-ion electrospray mode for flavonols included a capillary voltage of 4000 V, a nebulizing pressure of 30.0 psi, a drying gas flow of 9.0 mL/min, and a temperature of 300 ◦C. Data were collected in full scan mode over a mass range of *m*/*z* 50−1000 at 1.0 s per cycle. Characteristic ions (*m*/*z*) were used for peak assignment. For compounds where chemical standards were commercially available, retention times were also used to confirm the identification of components.

#### *2.7. Analysis of Percent Polymeric Color*

Percent polymeric color (% PC) of extracts was determined using the spectrophotometric assay of Giusti and Wrolstad (2001) [33]. Sample extracts were diluted with water in order to have an absorbance reading between 0.5 and 1.0 at 512 nm when evaluated by an 8452A Diode Array Spectrophotometer (Hewlett Packard, Palo Alto, CA, USA). For analysis, 0.2 mL of 0.90 M potassium metabisulfite was added to 2.8 mL diluted sample (bisulfite bleached sample) and 0.2 mL of DI water was added to 2.8 mL diluted sample (non-bleached, control sample). After equilibrating for 15 min, but not more than 1 h, samples were evaluated at λ = 700 nm, 512 nm, and 420 nm. Color density was calculated using the control sample according to the following formula:

$$\text{Color Density} = [(\text{A}\_{420\,\text{nm}} - \text{A}\_{700\,\text{nm}}) + (\text{A}\_{512\,\text{nm}} - \text{A}\_{700\,\text{nm}})] \times \text{Dilution Factor} \tag{1}$$

Polymeric color was determined using the bisulfite-bleached sample using the following formula:

$$\text{PolynomialColor} = \left[ (\text{A}\_{420\,\text{nm}} - \text{A}\_{700\,\text{nm}}) + (\text{A}\_{512\,\text{nm}} - \text{A}\_{700\,\text{nm}}) \right] \times \text{Dilution Factor} \tag{2}$$

Percent polymeric color was calculated using the formula:

$$\% \text{ Polymemic color} = \text{(polymemic color/color density)} \times 100\tag{3}$$

### *2.8. Statistical Analysis*

The effect of storage time (0, 2, 4, 6, and 8 weeks) on anthocyanins, flavonols, chlorogenic acid, and % polymeric color in each blueberry product was evaluated using the Fit Model platform of JMP (JMP Pro, version 15, SAS Institute, Cary, NC, USA)), and the percent retention of each compound after 8 weeks of storage was calculated using the fit model equation. The effect of storage temperature on phenolic compounds stability was not evaluated in this study due to the length of time (4 months) the WBB powder was stored between processing the products in Experiment 1 (products stored at 21 ◦C) and Experiment 2 (products stored at 4.4 ◦C). During this four-month storage time, the powder stored at 15.5 ◦C presumably absorbed moisture evident by powder clumping, resulting in different amounts of polyphenolics in the products immediately after processing. Principal component analysis (PCA) was performed with the total and individual anthocyanins variables, using the Multivariate platform in JMP, on the mean value (*n* = 3) of each sample per time point and using the correlation method. Correlations among total anthocyanins and percent polymeric color were determined by pairwise correlations method in the multivariate platform of JMP.

#### **3. Results**
