Based on our results, it would be best to use fresh flowers and blend or homogenize the flowers to extract the highest levels of phenolic compounds.Before analyzing samples, method parameters were evaluated to find the optimal equilibrium time, temperature, and the extraction time for headspace VOCs. The optimal parameters for flower samples were determined to be 40 °C, 20 min equilibration , and 30 min extraction with a SPME fiber. The optimized conditions for the tea samples were 40 °C, 30 min equilibration, and 30 min extraction. The profiles of volatile compounds were evaluated in fresh whole, dry ground, and teas made from whole fresh and dry flowers by HS-SPME/GC-MS, and relative levels of identified volatile compounds were calculated. The fresh flowers were evaluated as whole flowers because homogenizing them can cause oxidation and create artifacts in the volatile headspace profiles. This led to higher variability in the relative peak areas of compounds, but it is believed to be truer to the real headspace VOC profile as compared to a homogenized fresh flower sample. Overall, 25 compounds in the fresh whole flowers, compounds in the dry ground flowers, and 18 compounds in the tea preparations were identified. Table 1S contains the m/z ion of the base peak and the average match factor for each of the compounds identified in the headspace of samples. In the headspace of fresh flower, the most concentrated compounds were pentadecane > methyl eugenol > cis-3-hexenyl acetate > α-farnesene > and cis-3-hexenyl-α- methylbutyrate .

The contribution of each compound’s odor to the overall aroma of these elderflowers cannot be determined from the concentration alone, vertical vegetable tower as each compound has its own odor activity and threshold. However, it can be useful to know the characteristic odors of these compounds as a way to understand what comprised the general aroma. These compounds are described to have odors such as waxy; clove, spice; fresh, green, sweet, fruity, apple, pear, melon;wood, sweet; ; fruity, sweet, minty, fresh, and green apple, respectively. In addition to pentadecane, several straight chain hydrocarbons were also present, which may be released from the cuticle of the petal or peduncle of the flower. These include 1-pentadecene, heptadecane, 8- heptadecene and 6,9-heptadcadiene. Flowers also contain 4.6% methyl salicylate a compound with a sweet, minty odor80 that is frequently used as an analgesic in liniments to relieve pain. The profile of headspace VOCs in elderflowers of S. nigra ssp. cerulea differ significantly from the European elderflowers as linalool oxides and other derivatives predominate in the European flowers, and are absent in the present study. Furthermore, the present study indicates a unique headspace VOC profile in the blue elderflowers because pentadecane and methyl eugenol have been identified as major contributors to the headspace VOC profile. Pentadecane has been identified at trace levels in some European elderflower extracts, however methyl eugenol has not been identified in European elderflowers. Methyl eugenol, which has a clove-like aroma, appears to be unique to the S. nigra ssp. cerulea elderflower, and could be a unique volatile marker for this subspecies. Tea made with fresh whole elderflowers presented a slightly different headspace VOC profile as compared to fresh flowers.

Although methyl eugenol was still a prominent compound in the headspace , there were also two ketones in relatively high concentrations: 2,2,6-trimethyl-4H-1,3-dioxin-4-one and 4-methyl-2-heptanone . Aroma descriptors were not found for these compounds. In addition, two aldehydes including heptanal and nonanal were also present in the headspace of the tea but not the fresh flowers. Heptanal odor is described to be fresh, fatty, green, and herbal,whereas nonanal is described to have waxy, rose, orange peel or fatty notes.80 Methyl salicylate comprised only 1.27 ± 0.29% of the headspace volatiles in the tea made from fresh flowers. In the headspace of dry elderflowers, the most concentrated compounds were -3-hexen-1-ol > 1-penten-3-ol > 3-methyl-butanal > heptanal, > isocyanato-methane . In general, the dry flowers contain a wider range of volatiles than the fresh flowers, including more aldehydes, alcohols, alkanes, and other hydrocarbons. Other notable volatiles identified include methyl salicylate , dihydroedulan , which is a driver of typical elderberry aroma,63 and linalyl acetate which is the only linalool derivate identified any preparations of the elderflowers of S. nigra ssp. cerulea, unlike European elderflowers which typically have high concentrations of linalool derivatives. In tea prepared from dried flowers, the headspace aroma less complex than the dried flowers, but many of the aldehydes were still identified, including nonanal , heptanal , and hexanal . Levels of methyl salicylate were about two-fold higher that fresh flower tea . In addition, methyl eugenol, an important compound in fresh flowers that has not been identified in European elderflowers was also present in low quantities , meaning aqueous products that use dry flowers may have a unique aroma profile as compared to the European elderflower that are used in virtually all commercial elderflower products.

The elderflower teas evaluated herein were prepared without the addition of added ingredients that are commonly used in the preparation of elderflower syrups, tonics, and beverages. Sugar, lemon, citric acid or vinegar, and preservatives like sodium benzoate are common ingredients used in these products but can impact the headspace VOC profiles. Elderflower syrups are a cooked product, and the time and temperature at which they are processed will influence the headspace VOC profile. Elderflower syrups evaluated via dynamic headspace sampling and GC/MS, show that cis-rose oxide, hotrienol, linalool, -3-hexenol, cis-linalool oxide and trans-rose oxide are some of the predominant volatiles. Of these, the only compound also identified in the present study is -3-hexenol, which was present at 3.51 ± 0.41% in fresh flowers and 18.60 ± 0.67% in dry flowers. Heptanal and nonanal were also identified in the syrup, and levels ranged from 15.5 to 80.7 ng mL-1 and 13.1 to 33.2 ng mL-1 , respectively.35 These compounds represent < 1-3% of the total concentration of VOCs and are significantly lower as compared with the levels identified in the present study. In another study of elderflower syrup with European elderflowers, a variety of process parameters, such as extraction temperature, time, and syrup composition, were evaluated for their impacts on VOCs profiles. 81 While the concentration of volatile compounds was dependent upon how the syrup was made, -rose oxide, linalool, -3-hexen-1-ol, -rose oxide, 1,1,6-trimethyl,1,2-dihydronaphthalene, -linalool oxide, and 2- and 3-methyl-1-butanol were some of the most concentrated compounds identified. In comparison to our results, -3- hexen-1-ol was identified to be the isomer present, and 3-methyl-1-butanol was present in dry ground flowers but only at 3.61 ± 0.13%. Not surprisingly, the volatile profiles from European elderflower syrups do not correspond well with the blue elderflower teas. However, future studies evaluating syrup made from blue elderflowers, vertical farm tower could provide better insight into how the aroma,flavor and biological potential differs from syrups made from European subspecies. Studies could also include the American elderflower, which has not been evaluated for its headspace VOC profile before and results could further differentiate these subspecies. The results from this study present the first information on the phenolic and VOC composition of the blue elderflower, the subspecies native to the western region of North America. The phenolic profile elucidated unique characteristics of this subspecies compared to the other more widely used subspecies, S. nigra ssp. nigra and S. nigra ssp. canadensis, namely that IR was the predominant phenolic compound measured. A novel phenolic compound, 5-hydroxypyrogallol hexoside, was also identified in the blue elderflowers. Furthermore, the headspace VOC profile of the fresh and dry flowers as well as teas made from both types of flowers showed distinctive aroma profiles, highlighting that how elderflowers are processed post-harvest will impact the volatile compounds present and their relative concentration. Methyl eugenol and 5-HPG hexoside were identified for the first time in elderflowers. Further sensory evaluation would help determine if consumers differentiate between products using various subspecies of elderflower.Elderberry is a small fruit commonly used in food, beverages, and in supplements. Most products available use the European subspecies which has established cultivars and growing regions, and for which there is knowledge on composition. The blue elderberry is native to the western regions of North America, grows in a wide range of climates and soil types, and is notable due to a white bloom caused by yeast that make the dark purple berries appear light blue. In California, in addition to growing wild, typically in riparian environments, blue elderberry is grown in hedgerows to improve biodiversity near other crops. 

Unlike the commercialized European elderberry subspecies, the blue elderberry has not yet been bred for specific cultivars/genotypes and is not used in commercial production of elderberry products. Due to the environmental benefits of the blue elderberry, like improved water, soil, and air quality in addition to food and shelter for pollinators, there is rising interest in utilizing the fruit for commercial applications; especially as a juice, in beverages and supplements. At the same time, elderberry-based products have increased in popularity as consumers look for natural ways to support their immune system. A better understanding of how thermal processing influences key chemical components of this fruit may help to increase the use of this native and sustainable plant in value-added products in the US. Numerous important crop plants contain cyanogenic glycosides including cassava, sorghum, lima beans, the pits of Prunus species as well as elderberry. Cyanogenic glycosides are nitrile‐containing plant secondary defense compounds that can present a potential toxicity risk to consumers yet cyanogenic glycosides themselves are not toxic. However, they can release hydrogen cyanide when the plant material is either damaged, chewed or digested through the action of endogenous β-glucosidase and/or gutmicroorganisms. Hydrolysis results in the production of sugar and a cyanohydrin that spontaneously decomposes to HCN and a ketone or aldehyde . Exposure to HCN can lead to rapid pulse, headache, vomiting, diarrhea, convulsions, and in extreme cases, death. Chronic consumption of CNGs, particularly in regions that rely on a cyanogenic staple food but have other nutritional inadequacies, can also cause complications and may lead to diseases such as konzo or tropical ataxic neuropathy. While individual metabolism of CNGs can vary significantly and is impacted by the structure of the CNG, the acute lethal oral dose of cyanide in humans is 0.5 to 3.5 mg/kg body weight. To make foods safe, cyanogenic glycosides are frequently hydrolyzed through processes including grinding, pounding, boiling, steaming or during soaking or fermentation in water. Thermal treatments have also been used to effectively decrease levels of cyanogenic glycosides in foods. Commercial elderberry juice is thermally processed to inactivate enzymes, improve microbiological stability, and to degrade the potentially toxic cyanogenic glycosides found in this berry which include amygdalin, dhurrin, linamarin, and sambunigrin .The range and content of cyanogenic glycosides in the blue elderberry have not yet been identified. However, there was an occurrence of people getting sick after consuming raw blue elderberry juice at a at a religious gathering in Monterey County, California in 1983. Therefore understanding levels of cyanogenic glycosides in this subspecies is critical if it is going to be considered for use in commercial products. In European elderberry, sambunigrin is the main CNG in all parts of the plant, with the highest levels in the leaves and the lowest levels in the ripe berries . Sambunigrin is a diastereoisomer of prunasin, containing L-glucose instead of D-glucose. In the American subspecies, amygdalin, dhurrin, linamarin, and sambunigrin were identified in the skin, seed, and juice of the berry as well as in the stem. Juice of the American elderberry contained the lowest levels of CNGs relative to the skin, seeds and stems, averaging about 4 µg g- 1 , while the stems had the highest concentrations in the two cultivars evaluated. A recent study demonstrated that the total phenolic content of the blue elderberry is similar to the European and American subspecies, however this subspecies has significantly lower levels of anthocyanins. The dominant phenolic compounds identified in the blue elderberry include the flavonols rutin and isorhamnetin glucoside, the phenolic acid chlorogenic acid, a novel phenolic compound tentatively identified as 5-hydroxypyrogallol hexoside , and the anthocyanins cyanidin 3-glucoside and cyanidin 3-sambubioside . Anthocyanins are responsible for the brilliant blue-purple color of elderberries. At the same time, anthocyanidins are desired by consumers for their potential health promoting bioactivity.