THE EFFECT OF FREEZING AND EXTRACTION SOLVENT ON THE TOTAL POLYPHENOL CONTENT MEASUREMENT IN WHOLE 'ROYAL GALA' APPLE FRUIT

The Folin-Cioccalteau (FC) procedure is commonly used to measure the total polyphenol content (TPP) of fruit. There is at present no agreement on the most effective solvent for this assay, however, the most widely reported extractants are acetone or methanol based. There is also limited knowledge on the effect of freezing the fruit on the extraction efficiency of the solvent used for the assay. The aim of this study was to examine the effects of freezing on the extraction efficiencies of a variety of solvents by measuring the TPP values for ‘Royal Gala’ apples stored at -20°C for up to six months. The polyphenol composition was determined concurrently on each of the extracts by ultra-high pressure liquid chromatography-photodiode array-ion trap mass spectrometry (UHPLC-PDA-MSⁿ). Four different extractants were evaluated: acetone-water 50-50% (A50); acetone 70%, water 29.5% and acetic acid 0.5% (AWA); methanol-water 90-10% (M90); and methanol-water 50-50% (pH 2.0), followed by acetone-water 70-30% (M50A70). TPP values were determined in fruit frozen for 1, 15, 30, 90 or 180 days and were compared with values for fresh apples. A50 provided the highest TPP values (120 mg GAE/100 g FW) in fresh apples, followed by AWA (110 mg GAE/100 g FW), M90 (84 mg GAE/100 g FW) and M50A70 (81 mg GAE/100 g FW). There was a significant interaction between extractant type and freezing time. TPP values decreased over time with A50 (10-20%), remained constant with AWA and M90 and increased with M50A70 (10-25%). AWA provided the most consistent TPP values throughout the experiment. If differences were detected, AWA gave the highest values while M90 the lowest when the polyphenol levels were determined by UHPLC-PDA-MS. This experiment suggests that AWA is the optimal extractant for measuring TPP values in frozen apple fruit after storage for up to 6 months and is also suitable for identifying selected polyphenols in fruit by UHPLC-PDA-MSⁿ.