PhD: Study of the polyphenolic profile and antioxidant activity of selected apple cultivars from Macedonia with spectrophotometry, liquid chromatography and mass spectrometry

Domesticated apple (Malus domestica Borkh.) contains wide array of polyphenolic compounds belonging to flavonoids and nonflavonoids. In this study, systematic evaluation of the polyphenolic content of 21 commercial, domesticated and autochthonous apple cultivars from Republic of Macedonia harvested during two years (2014 and 2015) was performed with spectrophotometric and HPLC-DAD-HESI-MSn methods. For determination of total polyphenol compounds, total flavonoids, anthocyanins and flavan-3-ols, and their antioxidant activity with DPPH, ABTS and FRAP, spectrophotometric methods were used, and for identification and quantification of individual polyphenolic compounds a HPLC-DAD-HESI-MSn method was optimized and validated. Additionally, efficiency of acetone, water, methanol and methanol/water mixture (90:10, V/V) as solvent was tested and compared. The latter was chosen as the most efficient extraction solvent. Results obtained with this extraction mixture showed significant differences in the content of total polyphenols, flavonoids, and flavan-3-ols between the commercial and autochthonous cultivars. Also, diversity between lyophilized pulp and peel and air dried leaves from the same fruit was observed (peel of Tetovka and Livadarka contained 15.63 mg/g GAE and 14.85 mg/g GAE, respectively, and the pulpof Pasalma, Livadarka and Tetovka contained 12.55 mg/g GAE, 11.45 mg/g GAE and 11.22 mg/g GAE, respectively). In addition, a strategy for systematic qualitative and quantitative analysis of the individual polyphenolic compounds was developed using an ion trap mass spectrometer with heated electrospray ionization (HPLC-HESI-MS). Polyphenols were separated on a reversed phase UHPLC column with optimized gradient consisting of 1% formic acid in water and 1% formic acid in methanol within 20 minutes. For identification purposes, screening was performed consisting of: full scan, single ion monitoring and data-dependent scan for the aglycone/glycoside pairs with the expected aglycones as targets. Then, five MS experiments were tested on sixteen reference polyphenolic compounds to optimize the ion trap for quantification. A segmented MS tune method, where each class of polyphenolic compounds was quantified employing its own optimized tune against a suitable/available standard, was found as most satisfactory. According to their retention times, UV-Vis maxima and fragmentation patterns, phenolic acids (hydroxycinnamic and hydroxybenzoic acids), flavones, flavonols, flavanols, dihydrochalcones and their derivatives were identified and quantified in the apple samples. Validation of the UHPLC-HESI-MSn method after extraction of the polyphenols from apple peel, flesh and leaves showed that analytical characteristics of the developed MS method were comparable to the UV-DAD with regards to recovery, precision and robustness, and were superior for its linear range, sensitivity and selectivity.