Kinetics method for the quantitation of anthocyanidins, flavonols, and flavones in foods.

Flavonoids are important dietary constituents owing to their health-promoting properties. As a result, simplified analytic techniques are required for the population of databases with food values so that associations between dietary intake and disease risk/incidence can be established. The current research provides a simplified sample preparation procedure for the accurate estimation of food anthocyanidins, flavones, and flavonols as aglycons. Traditionally, flavonoid aglycons have been formed by acidic hydrolysis. However, some flavonoid aglycons are slowly degraded by acid. A procedure has been developed whereby anthocyanidins and flavonols are deglycosylated with HCl in 50% aqueous methanol and the resulting aglycons subsequently quantified by application of pseudo-first-order kinetics to their degradation. Flavones are also deglycosolated under similar conditions but, at appropriate temperatures, their aglycons are stable in acid, so kinetics were not required for the quantitation of this subclass of flavonoids. Catechins and flavanones were rapidly degraded under the hydrolytic conditions used in these studies.     

UHPLC-PDA-ESI/HRMS/MS(n) analysis of anthocyanins, flavonol glycosides, and hydroxycinnamic acid derivatives in red mustard greens (Brassica juncea Coss variety)

An UHPLC-PDA-ESI/HRMS/MS(n) profiling method was used for a comprehensive study of the phenolic components of red mustard greens ( Brassica juncea Coss variety) and identified 67 anthocyanins, 102 flavonol glycosides, and 40 hydroxycinnamic acid derivatives. The glycosylation patterns of the flavonoids were assigned on the basis of direct comparison of the parent flavonoid glycosides with reference compounds. The putative identifications were obtained from tandem mass data analysis and confirmed by the retention time, elution order, and UV-vis and high-resolution mass spectra. Further identifications were made by comparing the UHPLC-PDA-ESI/HRMS/MS(n) data with those of reference compounds in the polyphenol database and in the literature. Twenty-seven acylated cyanidin 3-sophoroside-5-diglucosides, 24 acylated cyanidin 3-sophoroside-5-glucosides, 3 acylated cyanidin triglucoside-5-glucosides, 37 flavonol glycosides, and 10 hydroxycinnamic acid derivatives were detected for the first time in brassica vegetables. At least 50 of them are reported for the first time in any plant materials.     

Discriminating between the six isomers of dicaffeoylquinic acid by LC-MS(n)

The fragmentation behavior of all six dicaffeoylquinic acids (diCQA) has been investigated using LC-MS(4). It is possible to discriminate between each of the isomers including those for which commercial standards are not available. For diCQA, the ease of removal of the caffeoyl residue during fragmentation is 1 approximately = 5 > 3 > 4. The distinctive fragmentation observed for the little-studied 1,4-dicaffeoylquinic acid involves elimination of the C1 caffeoyl residue, repeated dehydrations leading to the aromatization of the quinic acid moiety, and its decarboxylation. It is suggested that this process is initiated by the C1 carboxyl protonating the C5 hydroxyl in the inverted chair conformer, followed by its protonating the C1 caffeoyl residue in the favored chair conformation. The fragmentation of 1-caffeoylquinic acid is indistinguishable from that of the commercially available 5-caffeoylquinic acid, but these two isomers can be distinguished easily by their facile chromatographic resolution on reversed phase packings. The hierarchical key previously developed for characterizing chlorogenic acids has been extended to accommodate 1-caffeoylquinic acid and the 1-acyl dicaffeoylquinic acids.     

Qualitative characterization of benzoxazinoid derivatives in whole grain rye and wheat by LC-MS metabolite profiling

Benzoxazinoids are metabolites occurring in a restricted group of plant species including crops such as rye, wheat, and maize. Focus on the analysis of benzoxazinoid metabolites has typically been due to their importance to plant biochemistry and physiology as highly bioactive molecules that plants use as alleochemicals to defend themselves against predators and infections. However, the potential dietary contribution of these compounds has not been addressed. This study conducted a detailed qualitative characterization of benzoxazinoid metabolites present in the whole grain rye and processed fractions of rye bran, and their presence was also detected in whole grain wheat samples. Several novel benzoxazinoid metabolites of the hydroxamic acids (2,4-dihydroxy-1,4-benzoxazin-3-one, DIBOA; 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one, DIMBOA), lactams (2-hydroxy-1,4-benzoxazin-3-one, HBOA), and benzoxazolinones (1,3-benzoxazol-2-one, BOA) were identified, including double-hexose derivatives of DIBOA, DIMBOA, and HBOA. This paper presents an important addition to the information on the phytochemical composition of rye and wheat grains, which deserves attention in the discussion of the potential health-promoting effects of these grains.     

Characterization of phytoecdysteroid glycosides in Meadowfoam (Limnanthes alba) seed meal by positive and negative ion LC-MS/MS

Meadowfoam ( Limnanthes alba) is an oilseed crop grown in western Oregon. The seed meal has potential value as a biopesticide due to glucosinolate degradation products and phytoecdysteroids, a group of polyhydroxylated triterpenoids with potent activities as arthropod molting hormones. Liquid chromatography in combination with tandem mass spectrometry operated in the precursor ion mode revealed the presence of four ecdysteroid glycosides in meadowfoam seed meal. The carbohydrate sequence and the identity of the ecdysteroid aglycones, ponasterone A and 20-hydroxyecdysone, were determined by product ion scanning. Ecdysteroids were detected in the negative ion mode as [M + formate] (-) ions, which yielded [M - H] (-) and alpha-cleavage fragments with retention of hydroxyl groups in MS/MS experiments (not seen in the positive ion mode), allowing the determination of the number of hydroxyl groups in the side chain and in the steroid ring system. MS/MS of glycoside ions ([MH] (+) or [M + formate] (-)) provided carbohydrate sequence information.     

Quantitative analysis of steroidal glycosides in different organs of Easter lily (Lilium longiflorum Thunb.) by LC-MS/MS

The bulbs of the Easter lily ( Lilium longiflorum Thunb.) are regularly consumed in Asia as both food and medicine, and the beautiful white flowers are appreciated worldwide as an attractive ornamental. The Easter lily is a rich source of steroidal glycosides, a group of compounds that may be responsible for some of the traditional medicinal uses of lilies. Since the appearance of recent reports on the role steroidal glycosides in animal and human health, there is increasing interest in the concentration of these natural products in plant-derived foods. A LC-MS/MS method performed in multiple reaction monitoring (MRM) mode was used for the quantitative analysis of two steroidal glycoalkaloids and three furostanol saponins, in the different organs of L. longiflorum. The highest concentrations of the total five steroidal glycosides were 12.02 ± 0.36, 10.09 ± 0.23, and 9.36 ± 0.27 mg/g dry weight in flower buds, lower stems, and leaves, respectively. The highest concentrations of the two steroidal glycoalkaloids were 8.49 ± 0.3, 6.91 ± 0.22, and 5.83 ± 0.15 mg/g dry weight in flower buds, leaves, and bulbs, respectively. In contrast, the highest concentrations of the three furostanol saponins were 4.87 ± 0.13, 4.37 ± 0.07, and 3.53 ± 0.06 mg/g dry weight in lower stems, fleshy roots, and flower buds, respectively. The steroidal glycoalkaloids were detected in higher concentrations as compared to the furostanol saponins in all of the plant organs except the roots. The ratio of the steroidal glycoalkaloids to furostanol saponins was higher in the plant organs exposed to light and decreased in proportion from the aboveground organs to the underground organs. Additionally, histological staining of bulb scales revealed differential furostanol accumulation in the basal plate, bulb scale epidermal cells, and vascular bundles, with little or no staining in the mesophyll of the bulb scale. An understanding of the distribution of steroidal glycosides in the different organs of L. longiflorum is the first step in developing insight into the role these compounds play in plant biology and chemical ecology and aids in the development of extraction and purification methodologies for food, health, and industrial applications. In the present study, (22R,25R)-spirosol-5-en-3β-yl O-α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl-(1→4)-β-d-glucopyranoside, (22R,25R)-spirosol-5-en-3β-yl O-α-l-rhamnopyranosyl-(1→2)-[6-O-acetyl-β-d-glucopyranosyl-(1→4)]-β-d-glucopyranoside, (25R)-26-O-(β-d-glucopyranosyl)furost-5-ene-3β,22α,26-triol 3-O-α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl-(1→4)-β-d-glucopyranoside, (25R)-26-O-(β-d-glucopyranosyl)furost-5-ene-3β,22α,26-triol 3-O-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranosyl-(1→3)-β-d-glucopyranoside, and (25R)-26-O-(β-d-glucopyranosyl)furost-5-ene-3β,22α,26-triol 3-O-α-l-rhamnopyranosyl-(1→2)-α-l-xylopyranosyl-(1→3)-β-d-glucopyranoside were quantified in the different organs of L. longiflorum for the first time.     

Quantitation of Mycotoxins in Food and Feed from Burkina Faso and Mozambique Using a Modern LC-MS/MS Multitoxin Method

In this study an LC-MS/MS multitoxin method covering a total of 247 fungal and bacterial metabolites was applied to the analysis of different foods and feedstuffs from Burkina Faso and Mozambique. Overall, 63 metabolites were determined in 122 samples of mainly maize and groundnuts and a few samples of sorghum, millet, rice, wheat, soy, dried fruits, other processed foods and animal feeds. Aflatoxin B(1) was observed more frequently in maize (Burkina Faso, 50% incidence, median = 23.6 μg/kg; Mozambique, 46% incidence, median = 69.9 μg/kg) than in groundnuts (Burkina Faso, 22% incidence, median = 10.5 μg/kg; Mozambique, 14% incidence, median = 3.4 μg/kg). Fumonisin B(1) concentrations in maize were higher in Mozambique (92% incidence, median = 869 μg/kg) than in Burkina Faso (81% incidence, median = 269 μg/kg). In addition, ochratoxin A, zearalenone, deoxynivalenol, nivalenol, and other less reported mycotoxins such as citrinin, alternariol, cyclopiazonic acid, sterigmatocystin, moniliformin, beauvericin, and enniatins were detected. Up to 28 toxic fungal metabolites were quantitated in a single sample, emphasizing the great variety of mycotoxin coexposure. Most mycotoxins have not been reported before in either country.     

Phenolics of Arbutus unedo L. (Ericaceae) fruits: identification of anthocyanins and gallic acid derivatives

Arbutus unedo L., the strawberry tree (Ericaceae family), is an evergreen shrub or small tree, typical of the Mediterranean fringe and climate. The aim of the present study was to evaluate the profile of the phenolic constituents of A. unedo fruits. Seven compounds were purified by Sephadex LH-20 column chromatography of the MeOH extract followed by HPLC and were characterized as arbutin, beta-D-glucogalline, gallic acid 4-O-beta-D-glucopyranoside, 3-O-galloylquinic acid, 5-O-galloylquinic acid, 3-O-galloylshikimic acid, and 5-O-galloylshikimic acid, by means of NMR and ESI-MS analyses. Moreover, LC-PDA-MS analysis of the red pigment of A. unedo fruits revealed the presence of three anthocyanins recognized as cyanidin 3-O-beta-D-galactopyranoside, delphinidin 3-O-beta-D-glucopyranoside, and cyanidin 3-O-beta-D-arabinopyranoside. These pigments were also quantified.     

Profiling the chlorogenic acids and other caffeic acid derivatives of herbal chrysanthemum by LC-MSn

Four samples of herbal chrysanthemum have been profiled qualitatively by LC-MS5 to identify their component chlorogenic acids and partially characterize other caffeic acid derivatives. The chlorogenic acids were minor components, and the four samples varied markedly in profile. Three p-coumaroylquinic acids, three feruloylquinic acids, four caffeoylquinic acids, six dicaffeoylquinic acids, and two tricaffeoylquinic acids were detected, 13 for the first time from this source. Partial characterization of minor components suggested the presence of five caffeoyl-hexose esters and caffeic acid-4-beta-d-glucose that have not previously been reported from this source, and eight caffeoylquinic acid glycosides and 16 dicaffeoylquinic acid glycosides that have not previously been reported in nature. Succinic acid-containing chlorogenic acids and chlorogenic acids based on epi-quinic acid, previously reported in Chrysanthemum spp., were not detected in these samples.     

Chemical synthesis of hydroxycinnamic acid glucosides and evaluation of their ability to stabilize natural colors via anthocyanin copigmentation

This work describes the chemical synthesis of O-aryl-beta-D-glucosides and 1-O-beta-D-glucosyl esters of hydroxycinnamic acids. In particular, O-aryl-beta-D-glucosides were efficiently prepared via a simple diastereoselective glycosylation procedure using phase transfer conditions. Despite the lability of its ester linkage, 1-O-beta-D-caffeoylglucose could also be obtained using a Lewis acid catalyzed glycosylation step and a set of protective groups that can be removed under neutral conditions. Hydroxycinnamic acid O-aryl-beta-D-glucosides were then quantitatively investigated for their affinity for the naturally occurring anthocyanin malvin (pigment). Formation of the pi-stacking molecular complexes (copigmentation) was characterized in terms of binding constants and enthalpy and entropy changes. The glucosyl moiety did not significantly alter these thermodynamic parameters, in line with a binding process solely involving the polyphenolic nuclei.     

HPLC-DAD/MS characterization of flavonoids and hydroxycinnamic derivatives in turnip tops (Brassica rapa L. Subsp. sylvestris L.)

Flavonoids and hydroxycinnamic derivatives of turnip tops (Brassica rapa L. subsp. sylvestris L.) were characterized for the first time in four samples from different origins. Turnip tops exhibit a high polyphenols content (ranging from 107 to 191 mg/100 g, fresh weight) and a good antiradical activity, determined with the DPPH* test. After a liquid-liquid extraction and fractionation procedures, most flavonoids (isorhamnetin, kaempferol, and quercetin glycosides) and hydroxycinnamic derivatives were identified by means of HPLC-DAD/MS techniques. Isorhamnetin glycosides were the main flavonoid derivatives, differing from that found in the vegetables belonging to the Brassica oleracea group.     

Structure determination and sensory analysis of bitter-tasting 4-vinylcatechol oligomers and their identification in roasted coffee by means of LC-MS/MS

Aimed at elucidating intense bitter-tasting molecules in coffee, various bean ingredients were thermally treated in model experiments and evaluated for their potential to produce bitter compounds. As caffeic acid was found to generate intense bitterness reminiscent of the bitter taste of a strongly roasted espresso-type coffee, the reaction products formed were screened for bitter compounds by means of taste dilution analysis, and the most bitter tastants were isolated and purified. LC-MS/MS as well as 1-D/2-D NMR experiments enabled the identification of 10 bitter compounds with rather low recognition threshold concentrations ranging between 23 and 178 micromol/L. These bitter compounds are the previously unreported 1,3-bis(3',4'-dihydroxyphenyl) butane, trans-1,3-bis(3',4'-dihydroxyphenyl)-1-butene, and eight multiply hydroxylated phenylindanes, among which five derivatives are reported for the first time. In addition, the occurrence of each of these bitter compounds in a coffee brew was verified by means of LC-MS/MS (ESI-) operating in the multiple reaction monitoring (MRM) mode. The structures of these bitter compounds show strong evidence that they are generated by oligomerization of 4-vinylcatechol released from caffeic acid moieties upon roasting.     

Caffeic acid derivatives production by hairy root cultures of Echinacea purpurea

Inoculation of leaf explants of Echinacea purpurea (Moench) with Agrobacterium rhizogenes induced hairy roots with the capacity to produce biologically active caffeic acid derivatives (CADs), especially cichoric acid. The kinetics of growth, the uptake of macronutrients, and the accumulation of CADs were investigated in heterotrophically cultured hairy roots for a 50 day period. A maximum of 12.2 g L(-1) dry biomass was achieved in MS nutrients supplemented with 30 g L(-1) sucrose on day 40. The mathematical relationship between hairy root growth and conductivity was established during the exponential phase in Erlenmeyer flasks. HPLC analyses of methanolic (0.1% phosphoric acid; 70:30, v/v) extracts from hairy roots revealed the presence of important CADs: cichoric acid (19.21 mg g(-1) dry biomass), caftaric acid (3.56 mg g(-1) dry biomass), and chlorogenic acid (0.93 mg g(-1) dry biomass). These results demonstrate that biotechnological production of CADs in hairy roots of E. purpurea is possible. Furthermore, these hairy root cultures offer, for the very first time, an excellent biological model to study the biosynthetic pathway of medicinally important CADs.     

A quantitative stable-isotope LC-MS method for the determination of folic acid in fortified foods

A stable-isotope liquid chromatography-mass spectrometry (LC-MS) assay was developed for the quantitative determination of folic acid in fortified foods. Folic acid was extracted from food samples into a phosphate buffer, purified on a C-18 Sep-Pak cartridge, and analyzed by LC-MS in the negative ion mode using electrospray ionization. The analyte was quantified using (13)C(5)-folic acid as an internal standard. The coefficient of variation for the precision of the method was 5.6% based on the analysis of four sample replicates. The accuracy of the method was assessed using a standard method of addition of folic acid to a shredded whole-wheat cereal. The quantitative determination of folic acid in this matrix was linear over 1 order of magnitude having a concentration range of 2.4 to 24 microg/g of food (or 0.05 to 0.5 microg of analyte injected into the LC-MS). The overall quantitative efficiency of the method was evaluated using a standard reference material (infant formula SRM 1846). The method was applied to the determination of folic acid in several test samples (fortified breakfast cereals), and the values were in accord with the manufacturer's claim. This method advances a LC-MS technique for the determination of folic acid in fortified foods based on stable-isotope dilution methodology. The specificity of the technique and quantitative accuracy of the method in various food substrates suggests that the method may be adapted for routine analysis in other fortified foods.     

Monitoring of the fermentation media of citric acid by the trimethylsilyl derivatives of the organic acids formed

In this approach, a derivatization method is described for monitoring of organic acids in fermentation media without any separation step. The aqueous phase of fermentation media was evaporated and heated in a silylation reagent to form trimethylsilyl (TMS) derivatives. The silylated compounds are analyzed by 29Si nuclear magnetic resonance (29Si NMR) and gas chromatography-mass spectrometry (GC-MS). 29Si NMR can qualitatively monitor the components produced in the Krebs cycle. Quantification of these compounds is investigated by using selected ion monitoring mode of mass spectrometry. In this mode, mass to charge (m/z) values of their [M - 15]+ ions, which are 465, 275, 247, 221, 335, 251, and 313 of TMS derivatives of citric, alpha-ketoglutaric, succinic, fumaric, l-malic, oxaloacetic, and palmitic (as an internal standard), acids, respectively, are used. The limit of detection and the linear working range for derivatized citric acid were found to be 0.1 mg L(-1) and 10-3 x 10(4) mg L(-1). The relative standard deviation of the method for five replicates was 2.1%. The average recovery efficiency for citric acid added to culture media was approximately 97.2%. Quantitative results of GC-MS are compared with those obtained by an ultraviolet-visible method.     

Purification and identification of linoleic acid hydroperoxides generated by soybean seed lipoxygenases 2 and 3

It has been known that lipoxygenase (LOX) isozymes exhibit differences in product formation, but most product information to date is for LOX 1 among soybean (Glycine max) LOX isozymes. In this study, LOXs 2 and 3 were purified and used to generate hydroperoxide (HPOD) products in an in vitro system using linoleic acid as a substrate in the presence of either air or O2. The products were analyzed to determine their stereoisomeric characteristics. The control (no enzyme) showed only low levels of hydroperoxide production and no stereoisomeric specificity. LOX 2 shows high specificity in product formation, producing roughly 4 times more 13-HPOD than 9-HPOD, nearly all of which was 13-S(Z,E)-HPOD. LOX 3 produced more 9-HPOD than 13-HPOD at approximately a 2:1 ratio. No single stereoisomer was predominant among LOX 3 products. These results demonstrate that different isozymes of LOX have characteristic product profiles in in vitro reactions.     

Reactivity of ferulic acid and its derivatives toward hydrogen peroxide and peracetic acid.

The reactions of ferulic acid and its derivatives with hydrogen peroxide and peracetic acid in lignin-retaining bleaching conditions have been investigated to determine their susceptibility to oxidative degradation. The conjugated side chain of ferulic acid and its etherified or esterified derivative was shown to be fairly stable, especially to hydrogen peroxide. The major reaction was trans-cis isomerization that possibly involved a radical mechanism but did not cause bond cleavage. The peracetic acid reaction increased the rate of trans-cis isomerization and was also accompanied by a minor cleavage of the side chain. Esterification did not have a substantial effect on the reactivity of ferulic acid, but 4-O-etherification significantly stabilized it against these two oxidants. By contrast, aldehyde substitution tremendously enhanced the susceptibility of the cinnamyl side chain to oxidative degradation, as evidenced by an intensive degradation of coniferaldehyde.     

Determination of 4(5)-methylimidazole in soy sauce and other foods by LC-MS/MS after solid-phase extraction

A method for the determination of 4(5)-methylimidazole (4MeI) in naturally brewed soy sauce was developed for the first time using solid-phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). SPE on silica-based reversed-phase cartridges with heptafluorobutyric acid as an ion-pairing reagent was used for the efficient cleanup of 4MeI. A multimode ODS column was employed for the chromatographic separation. To subtract the matrix effect during LC-MS/MS analysis, a standard addition method was used. The levels of 4MeI found in naturally brewed soy sauce were extremely low (ranging from <0.002 to 0.023 μg/g), whereas those in soy sauces containing caramel color were generally high (ranging from 0.43 to 4.8 μg/g). The method proved to be useful for the analysis of 4MeI in other foods such as caramel colors, drinks, and Worcestershire sauce.     

Simultaneous identification and quantification of the sugar, sugar alcohol, and carboxylic acid contents of sour cherry, apple, and ber fruits, as their trimethylsilyl derivatives, by gas chromatography-mass spectrometry

Our gas chromatography-mass spectrometry method--developed for the simultaneous quantitation of mono-, di-, and trisaccharides, sugar alcohols, caboxylic and amino acids, measured as their trimethylsilyl-(oxime) ether/ester derivatives, from one solution by a single injection, prepared in the presence of the fruit matrix--has been extended/utilized for special purposes. The compositions of (i) freshly harvested and stored sour cherries (Prunus cerasus), (ii) apples obtained from organic and integrated productions (Malus domestica), and (iii) green and ripe bers (Zizyphus mauritiana L.) were compared. On the basis of earlier, basic researches (derivatization, quantitation, and fragmentation studies of authentic compounds), we demonstrate the reproducible quantitation of the main and minor constituents in a wide concentration range (approximately 1 x 10(-)(3) to >/=40%, in total up to < or =98%, calculated on dry matter basis of the fruit matrices). Reproducibility of quantitations, calculated on the basis of their total ion current values, provided an average reproducibility of 3.3 (sour cherries), 6.2 (apple), and 4.3 (ber) RSD %, respectively.