AOAC 2019 First Action Methods

for 10 min at 15 ° C, and the supernatant was collected. The supernatant was taken to dryness by evaporation under vacuum at 35 ° C and solubilized in 10 mL of ethanol. A Kontes glass preparative column (2.5 cm I.D. x 25 cm L) filled with Sephadex LH-20 TM resin, previously swollen with water, to a height of 10 cm. Prior to loading the sample, the Sephadex LH-20 TM resin was eluted with water (200 mL) and equilibrated with ethanol (100 mL). Each of the ethanolic extracts (5 mL) were loaded onto a column and sequentially eluted with ethanol (150 mL), ethanol:methanol (1:1 v/v; 150 mL) to remove hydroxycinnamic acids, anthocyanins, flavonols. The column was then eluted with acetone (80% v/v; 200 mL) to remove the PAC. The acetone fraction was taken to dryness by roto-evaporation under vacuum at 35 ° C. The extract was solubilized in 1.5 mL of methanol prior to MALDI-TOF MS analysis. D. MALDI-TOF MS Analysis MALDI-TOF MS spectra were collected in positive reflectron mode using a Bruker UltraFlex® III MALDI TOF/TOF mass spectrometer (Billerica, MA, USA) equipped with a SmartBeam TM laser. Four factors were optimized to acquire high-resolution MALDI-TOF spectra of PAC, the selection of the matrix, the cationization agent, the pulsed ion extraction, and the laser power. The operating conditions for the optimized analysis were: ion source 1 (25.0 kV), ion source 2 (20.6kV), lens voltage (9.5 kV), reflector 1 (26.5 kV), reflector 2 (14.41 kV), pulsed ion extraction (130 ns), detector gain (2.64x; 1548V), preamplifier filter bandwidth (small), and digitizer sampling frequency (100 Hz). Spectra were the sum of different locations in each well, which accumulated a total of 2000 shots with deflection set at 900 Da. The 2,5-dihydrobenzoic acid (DHB; 1.30 M in methanol) was used as a matrix. The samples and matrix were deionized using strong cation resin tips PureSpeed IEX 1 mL/20 µ L (Rainin, Oakland, CA, USA). The deionized matrix was mixed with cesium trifluoroacetate (5 mM) in a 1:1 volume ratio. Each deionized sample was spotted on the stainless steel target (0.5 µ L), followed by addition of the matrix (0.6 µ L). Spectra were calibrated using bradykinin (1060.6 Da) and glucagon (3483.8 Da) as external standards. FlexControl, Flex Analysis (Bruker Daltonik GmbH, Bremen, Germany), and mMass (version 5.5.0) were used for data acquisition, data processing, and spectra analysis. E. Deconvolution of Isotope Patterns of A- to B-Type Interflavan Bonds in PAC Deconvolution of the overlapping isotope patterns of MALDI-TOF MS spectra in positive reflectron mode was used to determine the percentage of A- to B-type interflavan bonds in PAC at each DP. The absolute intensity ( ai) of each PAC peak in the positive reflectron mode MALDI-TOF MS data set was calculated using mMass software. Spectral data were excluded from deconvolution analysis when the signal-to- noise (S/N) ratio was lower than 3. The percentage of A- to B-type interflavan bonds in PAC was solved using the following algebraic matrix: − 1 × = where A -1 = inverse coefficient matrix of the relative intensity ( ri ) of the isotope patterns for each DP, b = constant matrix of ai from the spectra, and c = variable matrix of linear combinations that solve the simultaneous equation. The linear combinations obtained from − 1 × were divided by the sum of all possible iterations and multiplied by 100 to obtain the percentage of A- to B-type interflavan bonds.