Study of flavonoids in aqueous spinach extract using positive electrospray ionisation tandem quadrupole mass spectrometry

The presence of three flavonols (quercetin, kaempferol, myricetin) and two flavones (apigenin, luteolin) were investigated in the extract of fresh spinach leaves. Aqueous spinach extracts were prepared with a leaf/water ratio of 1:2 at 80 °C for 30 min stirring. Ferric ammonium sulphate method was used for measuring total polyphenols in the extracts and expressed as catechins and tannic acid equivalents. The flavonoids glycosides in the extract were hydrolysed to their aglycons with 1.2 M HCl in boiling 50% water methanol solution. The resulting aglycons were identified and quantified by a C18 reverse phase high-performance liquid chromatography (RP-HPLC). Furthermore, the results were confirmed by HPLC coupled to an electrospray ionisation tandem triple-quadrupole mass spectrometer ESI-MS performing low-energy collision induced dissociation (CID-MS/MS) in the collision cell (HPLC-ESI-MS/MS). Analyses were made in the multiple reaction monitory (MRM) mode. Results showed that total polyphenols contents in fresh spinach leaves were 270 mg kg⁻¹ and 390 mg kg⁻¹ as tannic acid and catechin equivalents, respectively, in which, major flavonoids aglycons were apigenin (170 mg kg⁻¹), quercetin (50 mg kg⁻¹) and kaempferol (30 mg kg⁻¹).     

HPLC–MS identification and quantification of flavonol glycosides in 28 wild and cultivated berry species

Berries and red fruits are rich dietary sources of polyphenols with reported health benefits. More than 50 different flavonols (glycosides of quercetin, myricetin, kaempferol, isorhamnetin, syringetin and laricitrin) have been detected and quantified with HPLC–MSⁿ in fruits of blueberry, bilberry, cranberry, lingonberry, eastern shadbush, Japanese wineberry, black mulberry, chokeberry, red, black and white currants, jostaberry, red and white gooseberry, hardy kiwifruit, goji berry, rowan, dog rose, Chinese and midland hawthorn, wild and cultivated species of blackberry, raspberry, strawberry and elderberry. The phenolic constituents and contents varied considerably among the analyzed berry species. Elderberry contained the highest amount of total flavonols (450–568 mg kg⁻¹ FW), followed by berry species, containing more than 200 mg kg⁻¹ FW of total: chokeberry (267 mg kg⁻¹), eastern shadbush (261 mg kg⁻¹), wild grown blackberry (260 mg kg⁻¹), rowanberry (232 mg kg⁻¹), american cranberry (213 mg kg⁻¹) and blackcurrants (204 mg kg⁻¹). Strawberry (10.5 mg kg⁻¹) and white currants (4.5 mg kg⁻¹) contained the lowest amount of total flavonols. Quercetins represent the highest percentage (46–100%) among flavonols in most analyzed berries. In wild strawberry and gooseberry the prevailing flavonols belong to the group of isorhamnetins (50–62%) and kaempferols, which represent the major part of flavonols in currants (49–66%). Myricetin glycosides could only be detected in chokeberry, rowanberry and species from the Grossulariaceae, and Adoxaceae family and Vaccinium genus. Wild strawberry and blackberry contained from 3- to 5-fold higher total flavonols than the cultivated one.     

Flavonols (kaempeferol,quercetin, myricetin) contents of selected fruits,vegetables and medicinal plants

The concentrations of flavonols (kaempeferol, quercetin, myricetin) were determined in 22 plant materials (9 vegetables, 5 fruits, and 8 medicinal plant organs). The materials were extracted with acidified methanol (methanol/HCl, 100:1, v/v) and analyzed by reverse phase high-performance liquid chromatographic (RP-HPLC) with UV detection. The total flavonols contents varied significantly (P < 0.05) among vegetables, fruits and medicinal plant organs ranged from 0 to 1720.5, 459.9 to 3575.4, and 2.42 to 6125.6 mg kg⁻¹ of dry matter, respectively. Among vegetables, spinach and cauliflower exhibited the highest amounts of flavonols (1720.5 and 1603.9 mg kg⁻¹, respectively), however, no flavonols were detected in garlic. Within fruits, highest level of flavonols was observed in strawberry (3575.4 mg kg⁻¹), whereas, the lowest in apple fruit (459.9 mg kg⁻¹). Of the medicinal plant organs, moringa and aloe vera leaves contained the highest contents of flavonols (6125.6 and 1636.04 mg kg⁻¹), respectively, whereas, lowest was present in barks (2.42–274.07 mg kg⁻¹). Overall, leafy green vegetables, soft fruits and medicinal plant leaves exhibited higher levels of flavonols.     

European elderberry (Sambucus nigra L.) rich in sugars,organic acids,anthocyanins and selected polyphenols

Sugars and organic acids in the fruit of two cultivars and three selections of black elderberry (Sambucus nigra L.): ‘Haschberg’, ‘Rubini’, ‘Selection 13’, ‘Selection 14’ and ‘Selection 25’ were quantified. The anthocyanin as well as quercetin profiles of this plant material were also established by the use of HPLC/MS. Significant differences in the concentration of sugars and organic acids were detected between the widely spread cultivar ‘Haschberg’ and all other cultivars/selections; ‘Haschberg’ was the richest in organic acids (6.38 g kg⁻¹ FW), and it contained the least sugar (68.5 g kg⁻¹ FW). The following major cyanidin based anthocyanins were identified in the fruit of black elderberry: cyanidin 3-sambubioside-5-glucoside, cyanidin 3,5-diglucoside, cyanidin 3-sambubioside, cyanidin 3-glucoside and cyanidin 3-rutinoside. The most abundant anthocyanin in elderberry fruit was cyanidin 3-sambubioside, which accounted for more than half of all anthocyanins identified in the berries. The ‘Rubini’ cultivar had the highest amount of the anthocyanins identified (1265 mg/100 g FW) and the lowest amount was measured in berries of the ‘Selection 14’ (603 mg/100 g FW). The ‘Haschberg’ cultivar contained a relatively low amount of anthocyanins in ripe berries (737 mg/100 g FW). From the quercetin group, quercetin, quercetin 3-rutinoside and quercetin 3-glucoside were identified; the latter prevailing in black elderberry fruit. The cultivar with the highest amount of total quercetins was ‘Selection 25’ (73.4 mg/100 g FW), while the ‘Haschberg’ cultivar contained average amounts of quercetins (61.3 mg/100 g FW). The chemical composition of the ‘Haschberg’ cultivar, the most commonly planted, conforms to the standards for sugars, anthocyanins and quercetins and exceeds them in the content levels of organic acids, the most important parameter in fruit processing.     

Extraction and on-line concentration of flavonoids in Brassica oleracea by capillary electrophoresis using large volume sample stacking

Flavonoids are bioactive compounds found in plants. Studies indicate consumption of food containing these compounds may reduce the incidences of cancer and cardiovascular diseases. In broccoli, the flavonoids are present at variable concentrations and so far have mainly been determined using high performance liquid chromatography (HPLC). This paper describes a rapid capillary electrophoresis method, involving large volume sample stacking (LVSS), suitable for the analysis of flavonoids in broccoli. Following acid hydrolysis, the two key flavonoids (kaempferol and quercetin) in a broccoli extract were concentrated on-line by LVSS prior to separation by capillary zone electrophoresis (CZE). Using an optimised method, the extract was injected for 50 s into a 50 μm (internal diameter) × 85 cm (total length) capillary followed by stacking/matrix removal at −5 kV for 83 s. The two analytes were then separated in less than 8 min by CZE using a 10 mM sodium borate buffer (pH 8.40) and a separation voltage of +30 kV at 30 °C. A linear relationship in the range 1–20 ppm was observed for the method (r² = 0.9991–0.9995) with detection limits of 0.9 and 0.6 mg/kg of broccoli for kaempferol and quercetin, respectively. This method demonstrated good repeatability for the standard and extract with relative standard deviations of less than 5% for both peak area and migration time measured over five different days (n = 5). The method was successfully applied to quantitatively determine kaempferol and quercetin contents in a commercial broccoli sample as 11.8 and 14.6 mg/kg fresh weight, respectively. This result was validated by HPLC analysis and is within the ranges reported in the literature.     

Trace element content of Boletus tomentipes mushroom collected from Yunnan,China

The contents of As, Cd, Cr, Cu, Fe, Hg, Mg, Mn, Ni, Pb, and Zn in eleven fruiting bodies of Boletus tomentipes were determined. The results showed the values of the studied elements decreased in the order: Mg (208–279 mg kg⁻¹) > Fe (106–137 mg kg⁻¹) > Mn (29.5–46.8. mg kg⁻¹) > Zn (18.7–23.1 mg kg⁻¹), > Cu (11.4–15.8 mg kg⁻¹) > Cr (3.36–4.78 mg kg⁻¹) > Pb (1.38–3.88 mg kg⁻¹) > Ni (1.68–3.01 mg kg⁻¹) > Cd (0.16–0.32 mg kg⁻¹) > As (0.10–0.24 mg kg⁻¹) > Hg (<0.06 mg kg⁻¹).     

Influence of genotype,harvest time and plant part on polyphenolic composition of globe artichoke [Cynara cardunculus L. var. scolymus (L.) Fiori]

Globe artichoke is an ancient herbaceous plant native to the Mediterranean Basin. The edible part of the plant (head) is particularly rich in polyphenols, whose therapeutic properties are well documented. A field experiment was conducted in Sicily (south Italy) to examine the influence of genotype and harvest time on the polyphenol content and profile of different head parts. The concentrations of 19 phenolic compounds were determined by HPLC-DAD-ESI/MSⁿ analysis. It was observed that individual phenolic substances were preferentially accumulated in specific head parts and genotypes. Apigenin 7-O-glucuronide was found to be the major flavonoid, with 6298 mg kg⁻¹ DM in ‘Romanesco clone C3’ receptacle, whereas chlorogenic acid represented the main caffeoylquinic acid, reaching 14841 mg kg⁻¹ DM in the inner bracts of ‘Violetto di Sicilia’. Our findings prove also the influence of climatic conditions on the phenolic profile and thus suggest giving specific consideration to harvest time.     

Field incurred chlorpyrifos and 3,5,6-trichloro-2-pyridinol residues in fresh and processed vegetables

The effect of washing, peeling and cooking on residue levels of chlorpyrifos and 3,5,6-trichloro-2-pyridinol (TCP) in winter (spinach, cauliflower, potato) and summer vegetables (eggplant, tomato, okra) was determined. Analysis was carried out by capillary gas chromatography (DB-5MS capillary column) with mass selective detection. The samples were collected from trials conducted under controlled conditions as well as from the farmer’s field. In supervised field trials, the highest chlorpyrifos residue was found at raw stage in spinach (1.87 mg kg⁻¹) followed by okra (1.41 mg kg⁻¹) and eggplant (1.25 mg kg⁻¹). The lowest residue of chlorpyrifos was recorded in cauliflower (0.036 mg kg⁻¹). The chlorpyrifos residue reduced from 15 to 33% after washing, 65–85% post-peeling and cooking further lowered it from 12% to 48% in all the tested vegetables; while an increase in TCP concentration was observed during heat treatment. Out of 267 vegetable samples collected from the farmer’s field, 225 samples contained detectable residues representing 84% rate of contamination. About 6% of samples contained chlorpyrifos residues above maximum residue limits (MRLs). However, vegetable processing reduced the chlorpyrifos residue below the MRL.     

Determination of metal contaminations in sea foods from Marmara,Aegean and Mediterranean seas: Twelve fish species

The concentrations of cadmium, cobalt, chromium, copper, iron, manganese, nickel, lead and zinc were determined by ICP-AES in muscles and livers of 12 fish species sampled from the Marmara, Aegean and Mediterranean seas of Turkey. Iron showed the highest levels in examined tissues of all fish species. Following Fe, Zn generally showed the second highest levels. Metal concentrations in edible parts of fish species were 0.02–0.37 mg kg⁻¹ for cadmium, 0.04–0.41 mg kg⁻¹ for cobalt, 0.04–1.75 mg kg⁻¹ for chromium, 0.32–6.48  mg kg⁻¹ for copper, 7.46–40.1 mg kg⁻¹ for iron, 0.10–0.99  mg kg⁻¹ for manganese, 0.02–3.97 mg kg⁻¹ for nickel, 0.33–0.86 mg kg⁻¹ for lead, 4.49–11.2 mg kg⁻¹ for zinc, respectively. All metal concentrations in livers were higher than those in muscles. In some stations, cadmium and chromium concentrations in both muscles and livers, and lead levels in livers of the examined species were higher than permissible safety levels for human uses.     

Fish and food safety: Determination of formaldehyde in 12 fish species by SPME extraction and GC–MS analysis

The formaldehyde (FA) content in different fish products was evaluated using a solid phase microextraction (SPME)-GC–MS method based on fiber derivatisation with pentafluorobenzyl-hydroxyl-amine hydrochloride. LOD and LOQ values of 17 and 28 μg kg⁻¹, respectively were calculated. Fish quality was assessed by the analysis of 12 species (sea-fish, freshwater-fish and crustaceans), revealing variable FA levels. Fresh, deep frozen, canned, boiled and roasted fish were analysed; cooking always produced a decrease in the analyte content. Fish belonging to the Gadidae family were the samples with the highest FA concentration (from 6.4 ± 1.2 mg kg⁻¹ to 293 ± 26 mg kg⁻¹), in four cases out of 14 exceeding the value of 60 mg kg⁻¹ proposed by the Italian Ministry of Health. Storage on ice was also investigated, showing moderate FA production also at temperature around 0 °C. FA contents lower than 22 mg kg⁻¹ were finally found in all the other samples.     

Metal content determination in four fish species from the Adriatic Sea

Concentrations of As, Cd, Cu, Hg and Pb were measured by atomic absorption spectrometry (AAS) in muscle tissues of four fish species: anchovy (Engraulis encrasicholus), mackerel (Scomber japonicus), red mullet (Mullus surmuletus) and picarel (Spicara smaris) from the Croatian waters of the Adriatic Sea during 2008 and 2009. Metal levels measured in anchovy were in the following ranges (mg kg⁻¹): As 0.01–54.8, Cd 0.001–0.02, Cu 0.001–6.29, Hg 0.001–0.52 and Pb 0.001–0.34 mg kg⁻¹. Metal ranges in red mullet were (mg kg⁻¹): As 0.01–70.9, Cd 0.002–0.85, Cu 0.001–57.3, Hg 0.001–2.07 and Pb 0.001–0.27 mg kg⁻¹. Metal level ranges measured in mackerel were (mg kg⁻¹): As 0.01–36.4, Cd 0.001–0.1, Cu 0.001–15.9, Hg 0.001–0.78 and Pb 0.002–0.24 mg kg⁻¹. In picarel, metal level ranges were (mg kg⁻¹): As 0.01–54.6, Cd 0.001–0.1, Cu 0.08–32.9, Hg 0.001–0.207 and Pb 0.001–0.46 mg kg⁻¹. Significant differences in metal concentrations were found among fish species. The results presented on metal contents in the examined species give an indication of the environmental conditions. Concentrations of Cd, Cu, Hg and Pb obtained were far below the established values by the European Community regulations. However, arsenic levels found in red mullet were higher than the recommended legal limits for human consumption and as such may present a human health issue.     

Flow injection analysis of nitrate-N determination in root vegetables: Study of the effects of cooking

Vegetables are the major vehicles for the entry of nitrate into the human system. Ever-increasing concern over nitrate toxicity has directed a number of countries to lay down maximum allowable threshold concentrations with regards to nitrate-N in vegetables. Fiji is an independent island nation, located in the southern Pacific Ocean, has a tropical oceanic climate and hence expected to have high nitrate-N levels in vegetables. Thus, the present study was devoted to establish a flow injection analysis (FIA) technique for nitrate-N determination in Fiji’s commonly consumed fresh and cooked root vegetables such as potato (Solanum tuberosum), dalo (Colocasia esculenta), sweet potato (Ipomoea batatas) and carrot (Daucus carota L.). Activated carbon extraction technique was applied to extract nitrate-N. FIA with colorimetric detection technique having linear dynamic range of determination 1.0–20.0 mg L⁻¹ and detection limit of 0.042 mg L⁻¹ (0.34 mg kg⁻¹), using sulphanilamide and N-(1-naphthyl)ethylenediamine dihydrochloride as colour reagents, was used to determine nitrate-N contents in selected fresh and cooked root vegetables. The samples throughput was 38 h⁻¹. The effects of various cooking (boiling, baking and frying) methods on nitrate-N contents in root vegetables have also been studied. The study shows that the nitrate content of fresh root vegetables ranges from 53.76–258.00 mg kg⁻¹ whereas boiling reduces nitrate content by 23.30–42.62%. The frying in soya bean oil elevates nitrate contents from 204.53–299.12% but after baking nitrate contents remains almost constant with slight increasing trend from 2.80–8.43%. A comparison of the nitrate obtained by standardised method and the nitrate contents in vegetables of other countries are also presented.     

Effect of sprouting on physicochemical,antioxidant and flavonoid profile of onion varieties

This study investigated the impact of sprouting in four Indian onion varieties (Punjab White, Punjab Naroya, PRO‐6 and Commercial). Results showed significant (P < 0.05) increase in protein, crude fibre, ascorbic acid content, total phenol content, total flavonoid content, antioxidant activity, anthocyanin content and significant (P < 0.05) decrease in total carbohydrate, energy value and hardness due to sprouting in all the four varieties. HPLC analysis revealed irregular pattern of increase and decrease in flavonoid components. There was no particular trend observed within analysed flavonols due to sprouting in all the four varieties except with the maximum increase in quercetin (219.3–287.4 mg kg^?1), kaempferol (13.8–17 mg kg^?1), myricetin (34.6–40.9 mg kg^?1), quercetin‐3?‐glucoside (2.9–4.1 mg kg^?1) and quercetin‐4?‐glucoside (83–87.1 mg kg^?1) in case of PRO‐6 variety only. The present research work implied sprouting to be beneficial in onion as nutritional and functional potential of all the four onion varieties was improved by sprouting.     

Determination of glucosinolates and phenolic compounds in rocket salad by HPLC-DAD–MS: Evaluation of Eruca sativa Mill. and Diplotaxis tenuifolia L. genetic resources

The glucosinolate and phenolic profiles of 37 rocket salad accessions (32 Eruca sativa and 5 Diplotaxis tenuifolia) were obtained by liquid chromatography–mass spectrometry. Eleven desulpho-glucosinolates (DS-GLSs) were isolated and the glucosinolate profiles did not differ between the two species. Total DS-GLS content, expressed as sinigrin equivalents (SE) revealed a certain variability, ranging from 0.76 to 2.46 g kg⁻¹ d.w. but, again, the quantitative analysis did not discriminates Eruca from Diplotaxis. The polyphenol evaluation by HPLC-DAD–MS allowed the identification of two different classes of compounds in the two rocket salad species. Qualitative differences were observed between the polyphenol profiles at specific level: quercetin derivatives were the main phenolics of Diplotaxis, whereas kaempferol derivatives characterised Eruca samples. The contents of total flavonoids determined as rutin equivalents (RE) ranged from 4.68 to 31.39 g kg⁻¹ d.w. Kaempferol-3,4′-diglucoside (71.4–82.2%) and isorhamnetin-3,4′-di-glucoside (7.8–18.4%) were always isolated as first and second more abundant phenolic compounds in Eruca samples. No marker phenolic compounds were isolated in Diplotaxis samples.     

Elemental composition of cephalopods from Portuguese continental waters

Essential and contaminant elements concentrations were determined in the muscle tissue of octopus (Octopus vulgaris), squid (Loligo vulgaris) and cuttlefish (Sepia officinalis), caught off the Portuguese coast in 2004–2005. As expected, the largest concentrations found correspond to Cl, S, K, Na, P and Mg (average values between 629 mg (100 g)⁻¹, for Cl, and 435 mg kg⁻¹, for Mg, in octopus and squid, respectively). Above average concentrations of Zn, Cu, Fe and Sr were also found. The highest total Hg concentration was found in cuttlefish (0.36 mg kg⁻¹); however, this value did not exceed the recommended limit proposed by EU (0.5 mg kg⁻¹). Lead levels observed in all samples were always significantly lower than the EU limit (1.0 mg kg⁻¹). Regarding Cd, the 1.0 mg kg⁻¹ limit was only exceeded in two octopus samples. It may be concluded that the cephalopods studied do not constitute cause for concern, in terms of toxic elements, and could be safely used for daily intake of essential elements. Nevertheless, the squid contribution for elemental DI is minor in comparison to the other two species.     

Chemical characterization of a traditional honey-based Sardinian product: Abbamele

The first chemical characterization of abbamele, a traditional honey decoction from Sardinia (Italy) is hereby reported. Water content (from 17.7% to 27.7%), electrical conductivity (from 0.19 to 0.81 mS cm⁻¹), pH (from 3.21 to 3.92), free acidity (from 26.1 to 87.6 meq kg⁻¹), invertase activity (from 0 to 1.02 U kg⁻¹), 5-(hydroxymethyl)-2-furaldehyde, HMF (from 881 to 4776 mg kg⁻¹), total polyphenols (from 188 to 984 mg kg⁻¹) and free amino acid contents of thirteen abbamele samples, from industrial and traditional producers, were obtained in an attempt to compare this traditional product with honey and to study the relationship between its main features and the production procedures. The long thermal treatment involved in the production of abbamele has been identified as the main cause of very low (or absent) invertase activity and free amino acid content as well as the very high content of HMF.     

Natural levels of ethyl formate in stored grains determined using an improved method of analysis

Ethyl formate was readily determined with a gas chromatograph (GC) equipped with a flame ionisation detector. Natural levels of ethyl formate in Australian wheat, barley, oats, and canola were analysed by GC, after extraction with ammonium nitrate solution. Background levels of ethyl formate were present in newly harvested and stored grain. The levels of ethyl formate (0.1-0.6 mg kg⁻¹) in grains varied with commodity, temperature, moisture and period of storage. The values ranged from 0.1 to 0.2 mg kg⁻¹ for newly harvested wheat, barley and oats, and 0.3-0.4 mg kg⁻¹ for newly harvested canola. Ethyl formate was present in grains at harvest, increased during the first 7 months of storage, and then began to decline, particularly at grain temperatures higher than 25°C and moisture contents higher than 12.5% (for wheat, barley and oats) and 6.5% (for canola). The natural levels of ethyl formate should be considered when establishing maximum residue limits.     

Selenium concentration in St. John’s wort (Hypericum perforatum L.) herb after foliar spraying of young plants under different UV-B radiation levels

St. John’s wort (Hypericum perforatum L.) was grown under different levels of UV-B radiation, with selenium (10 mg l⁻¹ Se applied by foliar spraying in the form of sodium selenate) or without foliar Se application. The different levels of UV-B radiation comprised an enhanced level simulating 17% ozone depletion, ambient level, and a reduced level of UV-B radiation. The concentration of Se in unsprayed plants was from 20 ng g⁻¹ to 120 ng g⁻¹. The concentration of Se in the organs of plants foliarly sprayed with Se ranged from 1000 ng g⁻¹ to 12,000 ng g⁻¹, the highest concentration being detected in plants grown under reduced levels of UV-B radiation. Foliar application of Se fertiliser is feasible and effective in St. John’s wort and results in Se-enriched nutritional supplements.     

Profiling and quantifying quercetin glucosides in onion (Allium cepa L.) varieties using capillary zone electrophoresis and high performance liquid chromatography

There is increasing evidence that flavonols demonstrate beneficial properties for human health. Quercetin is the major flavonol present in onion (Allium cepa cv) and is present predominantly as quercetin 3,4′-diglucoside and quercetin 4′-monoglucoside. These compounds are known to be potent free radical scavengers and antioxidants, and are considered to be protective against cardiovascular disease. Analysis for the presence of these compounds has therefore become more important. Robust capillary zone electrophoresis and high performance liquid chromatography procedures were developed for profiling and quantifying the levels of quercetin 3,4′-diglucoside and quercetin 4′-monoglucoside in 70% methanol/water extracts of six different onion varieties available in Victoria, Australia. Quercetin 3,4′-diglucoside, which is not commercially available as a reference standard, was isolated from freeze-dried onion powder by preparative high performance liquid chromatography and used to quantify the levels in the onion extracts. Significant differences in the levels and ratios of the two compounds were seen between red, brown and white onion varieties (e.g. ‘Redwing’; quercetin 3,4′-diglucoside 191 mg/100 g DW, quercetin 4′-monoglucoside 85 mg/100 g DW; ‘Cream Gold’, quercetin 3,4′-diglucoside 153 mg/100 g DW, quercetin 4′-monoglucoside 58 mg/100 g DW, ‘Spanish white’; quercetin 3,4′-diglucoside <1 mg/100 g DW, quercetin 4′-monoglucoside <1 mg/100 g DW).