Compounds were identified based on retention time and spectral comparisons with standards. Information about the linear equations and lower limits of detection and quantitation can be found in Table S1 in the supplementary material. The LLOD was calculated as 3.3 times the standard deviation of the y-intercept of the curve divided the slope, while the LLOQ was calculated as 10 times those values.Several peaks appeared in the HPLC chromatograms that could not be identified using the above parameters. Chromatographic eluents of these peaks were collected individually and dried under vacuum. These extracts were reconstituted with mobile phase A, and 5 µL were injected into the HPLC- QTOF-MS/MS for accurate mass analysis . A Poroshell 120 EC-C18 column was used at 35 °C. Mobile phase A was 1% formic acid in distilled water, and mobile phase B was 1% formic acid in acetonitrile. The gradient used was 0 min 3% B, 30 min 50% B, 31-32 min 95% B, 33-38 min 3% B. The mass spectrometer was used in negative mode, and the mass range for MS was 100 to 1000 m/z while the range for MS/MS was 20-700 m/z. Collision energies at 10, 20, and 40 V were applied. The drying gas was set to a flow of 12 L/min at 250 °C, while the sheath gas was set to 11 L/min at 350 °C. The nebulizer was set to 40 psig, the capillary voltage was 3500 V, the nozzle was set to 500 V, vertical gardening in greenhouse and the fragmentor was set to 100 V. Data was analyzed using Agilent MassHunter Workstation Qualitative Analysis 10.0 .
Tentative identification was achieved by comparing the mass to charge ratio of the precursor and fragment ions to online libraries of compounds as well as using formula generation for the peaks in the spectra.The composition of blue elderberries is presented for the first time, which is key to understanding how this subspecies of Sambucus nigra compares to commercialized elderberry subspecies, S. nigra ssp. nigra and S. nigra ssp. canadensis. These data help to establish the blue elderberry grown in hedgerows in California as a viable source of berries and bio-active compounds. Data for the compositional assays is presented for the 2018 and 2019 harvest years as the average of all shrubs sampled in Table 2. The average moisture for the blue elderberries was 79.5 ± 1.5% in 2018 and 79.5 ± 1.6% in 2019, which is very similar to the levels found in wild elderberries in Spain 95. The average soluble solids found in blue elderberry ranged from 11.94 ± 2.08 to 14.95 ± 1.02 g per 100 g FW in 2018 and from 12.64 ± 1.86 to 17.09 ± 1.60 g per 100 g FW in 2019. These values are slightly higher than the soluble solids found in S. nigra ssp. cerulea grown in Slovenia29 and American elderberries grown in Ohio52. Compared to European and American elderberries evaluated in other studies, blue elderberries have similar levels of soluble solids 8,18,29,49,50,95. In the present study, the overall average content of soluble solids was significantly different between years, as blue elderberries harvested in 2019 had significantly higher average soluble solids than the elderberries harvested in 2018 . The pH in the blue elderberry ranged from 3.44 to 3.86 in 2018 and from 3.46 to 3.79 in 2019, with no significant difference found between harvest years.
These values are slightly lower than the values found in European elderberry, which ranged from 3.9 ± 0.06 to 4.1 ± 0.04 with an average pH of 3.9 ± 0.2, and American elderberry, which ranged from 3.9 ± 0.04 to 4.5 ± 0.03 with an average pH of 4.2 ± 0.2 49 Another evaluation of pH in American elderberries had a range of 4.5 ± 0.08 to 4.9 ±0.12,higher than those found in the blue elderberry.52 The higher sugar and lower pH levels in blue elderberry could potentially impact taste and performance in food and beverages as compared with the European and American species. The average titratable acidity in blue elderberries ranged from 0.45 ± 0.08 to 0.77 ± 0.03 g citric acid per 100 g FW in 2018 and from 0.54 ± 0.06 to 0.77 ± 0.11 g citric acid per 100 g FW in 2019 with no significant difference found between harvest years. These values are lower than the total acids found by Mikulic-Petkovsek et al. 29 in S. nigra ssp. cerulea , but they are similar to the levels found in European elderberry 8,18,49,50 .Anthocyanins are a class of phenolics that contribute red, purple, and blue hues to fruits and vegetables, act as attractants for pollinators, and are potent antioxidants. European and American elderberries are well-known for containing high levels of anthocyanins 8,18,49. The anthocyanin content of elderberries strongly correlates to the antioxidant potential of the fruit, which may confer health-promoting properties 50,89, which is one reason why elderberries are used in supplements and value-added products. Elderberry is also used as a source of natural food colorants due to the levels of anthocyanins35. Understanding the levels of anthocyanins in the blueelderberry grown in hedgerows is critical towards establishing this native fruit as an additional and more sustainable elderberry. TMA was variable between hedgerows in both years of harvest, with relative standard deviation values between 16% and 30%, yet there was not a significant difference in the overall average TMA between 2018 and 2019 . Furthermore, most hedgerows were not significantly different from the other hedgerows harvested that year despite significant differences in TMA values found between farms in both years .
Regarding the age of the elderberry shrub, hedgerows 2 and 14 had two of the three highest concentrations of TMA in 2019 . This suggests that blue elderberries can be harvested from plants as young as two years without a significant loss of TMA concentrations. TMA values for the blue elderberries are lower than those found in other elderberry subspecies. In European elderberries, TMA levels range from 170 ± 12 to 343 ± 11 with an average of 239 ± 94 mg CGE per 100 g FW 49. A study of American elderberry grown in Ohio showed a range from 354 ± 59 to 595 ± 26 mg CGE per 100 g FW. In the present study, bare root prerooted cuttings of American elderberries were planted, along with blue elderberries, on Farm 1 in 2018, and three shrubs were harvested in 2019. These American elderberries had an average TMA value of 263 ± 5.4 mg CGE per 100 g FW, which is more similar to what has been observed in other studies on this subspecies. This suggests it is a subspecies difference contributing to the lower anthocyanin concentration in the blue elderberry and not the difference in growing conditions. Compared to other berries, blue elderberries have similar levels of anthocyanins as raspberries, but lower levels than blueberries and blackberries . The lower concentration of anthocyanins in theblue elderberry may require adjustment of levels used in supplements, food and beverages for optimal performance or health benefit, or as natural coloring agents.In addition to anthocyanins, elderberries contain other phenolic compounds, such as flavonols and phenolic acids, which also contribute to the health promoting properties of elderberry. Phenolic compounds are responsible for organoleptic properties and can help protect foods against lipid oxidation. Therefore, TPC can be useful for making approximate comparisons, for example, greenhouse vertical farming between varieties of the same fruit, between similar fruits or in the evaluation of a processing step . It is important to note that the TPC assay is a non-selective assay and is easily impacted by extraction conditions and interfering substances, such as ascorbic acid and reducing sugars. Although there is no evidence that the beneficial effects of polyphenol-rich foods can be attributed to the TPC of a food, it can be a useful measure for making general comparisons with other studies in the literature which reported these values but should be supported by quantitative HPLC data. Herein, the range of TPC measured in the blue elderberries was from 514 ± 41 to 791 ± 34 mg GAE per 100 g FW in 2018 and from 459 ± 50 to 695 ± 41 mg GAE per 100 g FW in 2019 . TPC in the blue elderberries was significantly higher in 2018 than in 2019 . While there were significant differences found between the farms in both years , most hedgerows were not significantly different than most other hedgerows in the given year when evaluated together . Although the farms in this study were near each other and experience similar climates, there can still be differences in growing conditions for each hedgerow, such as water availability, which has been shown to influence the levels of phenolics in blueberries 101 and strawberries 102 . Hedgerows 2 and 14 were not significantly different from other hedgerows in 2019, indicating that the blue elderberries can be harvested earlyin the plant’s lifetime, which allows farmers to earn an early return on the investment of establishing hedgerows.
These comparisons show that blue elderberries from hedgerows are a rich source of phenolic compounds.Phenolic compounds were identified and quantified in the blue elderberry based upon retention time, absorbance spectra and authentic standards when available. Concentrations for samples from 2018 are presented in Table 4, while samples from 2019 are presented in Table 5. Two peaks with significant area were observed in the HPLC chromatograms at 6.96 min and 11.70 min that did not correlate to standards or library matching. Both compounds eluted between the retention time of gallic acid and protocatechuic acid. The first eluting compound had a maximum absorbance at 300 nm while the second compound had a maximum absorbance at 280 nm. These peaks were collected individually and further evaluated by accurate mass quadrupole time-of-flight tandem mass spectrometry . TOF acquires mass spectral data by pulsing ions entering the flight tube in an orthogonal beam, therefore full spectra are collected. The data captured is accurate enough to determine the elemental composition therefore allowing identification without standards. The two compounds were tentatively identified using high mass accuracy as 5-hydroxypyrogallol hexoside, a tetrahydroxybenzene , and protocatechuic acid dihexoside . Accurate mass was especially helpful since commercial standards for these compounds are not available. 5- HPG hexoside was identified by its fragmentation pattern , showing a precursor ion [MH]- at m/z 303.0723 and product ion [M-hexose-H]- at m/z 141.0186 . This compound was one of the most abundant phenolic compounds in the blue elderberry. While no evidence of5-HPG glycoside was found in the literature, the aglycone has shown to have a high radical scavenging activity compared to other simple phenols.Like other elderberry species, rutin was the predominant flavonol and overall had the highest concentration of any of the flavonols measured, with an average of 57.01 ± 17.42 mg per 100 g FW in 2018 and 51.89 ± 25.53 mg per 100 g FW in 2019. These values fall within the range of what has been found in European elderberry. Other flavonols identified include isoquercetin , kaempferol-3-rutinoside, and isorhamnetin-3-rutinoside, which was also a major phenolic compound in the berry. Isorhamnetin- 3-rutinoside averaged 28.30 ± 14.03 mg per 100 g FW in 2018 and 24.71 ± 14.83 mg per 100 g FW in 2019, which is higher than what has been found in other subspecies. Overall, the blue elderberry analyzed in the present study has much higher levels of total flavonols as compared to European elderberry. In the American elderberry, the main flavonols are rutin followed by isorhamnetin-3-rutinoside whereas in European elderberries, the main flavonols are rutin followed by isoquercetin. In blue elderberry grown in Slovenia, rutin and isoquercetin were the two predominant flavonols, though the total flavonols in found for the subspecies was similar to the levels found in this study 59 . The predominant anthocyanin present in the blue elderberry is cyanidin-3-sambubioside, like the European subspecies. The average concentration in 2018 was 32.70 ± 10.18 mg per 100 g FW and 29.66 ± 16.81 mg per 100 g FW in 2019.