Investigating the quality of extraction and quantification of bioactive compounds in berries through liquid chromatography and multivariate curve resolution

Berries are a rich source of natural antioxidant compounds, which are essential to profile, as they add to their nutritional value. However, the complexity of the matrix and the structural diversity of these compounds pose challenges in extraction and chromatographic separation. By relying on multivariate curve resolution alternating least squares (MCR-ALS) ability to extract components from complex spectral mixtures, our study evaluates the contributions of various extraction techniques to interference, extractability, and quantifying different groups of overlapping compounds using liquid chromatography diode array detection (LC-DAD) data. Additionally, the combination of these methods extends its applicability to evaluate polyphenol degradation in stored berry smoothies, where evolving factor analysis (EFA) is also used to elucidate degradation products. Results indicate that among the extraction techniques, ultrasonication-assisted extraction employing 1% formic acid in methanol demonstrated superior extractability and selectivity for the different phenolic compound groups, compared with both pressurized liquid extraction and centrifugation of the fresh berry smoothie. Employing MCR-ALS on the LC-DAD data enabled reliable estimation of total amounts of compound classes with high spectral overlaps. Degradation studies revealed significant temperature-dependent effects on anthocyanins, with at least 50% degradation after 7 months of storage at room temperature, while refrigeration and freezing maintained fair stability for at least 12 months. The EFA model estimated phenolic derivatives as the main possible degradation products. These findings enhance the reliability of quantifying polyphenolic compounds and understanding their stability during the storage of berry products. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00216-024-05474-8.


Fig. S12:
Chromatographic peaks and spectral profile of compounds observed among samples stored at room temperature for 6 to 9 months.These peaks were not observed or were relatively low in the baseline sample...

Fig. S4 :
Fig. S4: Comparisons of chromatographic elution times of interfering compounds and analytes identified with ultra-high performance liquid chromatography and high resolution mass spectrometry.

Fig. S5 :
Fig. S5: Stability of anthocyanins monitored in berry smoothie drinks stored in the fridge.Error bars represent standard deviation for n=3.

Fig. S6 :
Fig. S6: Degradation of rate of cyanidin 3-O glucoside compared at three storage temperatures after fitting with first order reaction kinetics model.

Fig. S8 :
Fig. S8: Stability of flavonoids monitored in berry smoothie drinks stored in the fridge.Error bars represent standard deviation for n=3.

Fig. S9 :
Fig. S9: Stability of flavonoids monitored in berry smoothie drinks stored at room temperature.Error bars represent standard deviation for n=3.

Fig. S10 :
Fig. S10: pH stabilities of berry smoothie drink reported at different storage temperatures.Error bars represent standard deviation for n=3.

Table S1 :
Confirmation of identified analytes in berry smoothie samples using high resolution mass spectrometry (HRMS).All the mass shown are [M-H] -form of the compound.with exception of masses marked with * which represent [M+H] + obtained in positive mode.while ** shows M-2H aglycone form and tR is retention time.Some compounds shown here were not quantified as they were below limit of quantification in the diode array detector.

Table S2 :
Molar absorptivity of different compounds measured at different wavelengths.Maleic acid and tryptophan represent the organic acid and amino acids detected as interferences in this study, while other compounds represent different classes of polyphenols

Table S3 :
Interfering compounds of identified analytes in berry smoothie samples using high resolution mass spectrometry (HRMS).All the mass shown are [M-H] -form of the compound, with exception of masses marked with * which represent [M+H] + obtained in positive mode.

Table S4 :
Kinetic parameters for degradation of anthocyanins derived from first order fitting model.RT stand for room temperature.