Systematic identification and characterization of anthocyanins by HPLC-ESI-MS/MS in common foods in the United States: fruits and berries

Anthocyanins were systematically identified and characterized by HPLC-ESI-MS/MS coupled with diode array detection in common fruits from U.S. food markets and other commercial sources. Of the 25 different fruits that were screened, 14 fruits were found to contain anthocyanins; the number of anthocyanins varied from 2 in peaches and nectarines to 31 in Concord grape. The individual anthocyanins were identified by comparing their mass spectral data and retention times with those of standards and published data. In all of the samples analyzed, only 6 common anthocyanidins, delphinidin, cyanidin, pelargonidin, petunidin, peonidin and malvidin, were found. In addition to the well-known major anthocyanins, a number of minor anthocyanins were identified for the first time. Some possible guidelines that help to identify anthocyanins in foods with complex anthocyanin composition were deduced and discussed. For the first time, this paper presents complete anthocyanin HPLC profiles and MS spectral data of common fruits using the same uniform experimental conditions.     

Concentrations of anthocyanins in common foods in the United States and estimation of normal consumption

Anthocyanins (ACNs) are water-soluble plant pigments that have important functions in plant physiology as well as possible health effects. Over 100 common foods were screened for ACNs, and 24 of them were found to contain ACNs. Concentrations of total ACNs varied considerably from 0.7 to 1480 mg/100 g of fresh weight in gooseberry ('Careless' variety) and chokeberry, respectively. Not only does the concentration vary, but the specific anthocyanins present in foods are also quite different. Only six common aglycones, delphinidin, cyanidin, petunidin, pelargonidin, peonidin, and malvidin, were found in all of these foods. However, their sugar moieties and acylation patterns varied from food to food. Results from this study will add to the available data for the USDA Nutrient Database of flavonoids. On the basis of the concentration data and updated food intake data from NHANES 2001-2002, the daily intake of ACNs is estimated to be 12.5 mg/day/person in the United States. Of the different aglycones, cyanidin, delphinidin, and malvidin were estimated to contribute 45, 21, and 15%, respectively, of the total ACN intake. Nonacylated contributed 77% compared to 23% from acylated ACNs.     

Bound fumonisin B1: analysis of fumonisin-B1 glyco and amino acid conjugates by liquid chromatography-electrospray ionization-tandem mass spectrometry

To study the formation of fumonisin artifacts and the binding of fumonisins to matrix components (e.g., saccharides and proteins) in thermal-treated food, model experiments were performed. Fumonisin B(1) and hydrolyzed fumonisin B(1) were incubated with alpha-d-glucose and sucrose (mono- and disaccharide models), with methyl alpha-d-glucopyranoside (starch model), and with the amino acid derivatives N-alpha-acetyl-l-lysine methyl ester and BOC-l-cysteine methyl ester (protein models). The reaction products formed were analyzed by liquid chromatography-electrospray ionization-tandem mass spectrometry. The incubation of d-glucose with fumonisin B(1) or hydrolyzed fumonisin B(1) resulted in the formation of Amadori rearrangement products. Whereas conjugates were found following the reaction of sucrose, methyl alpha-d-glucopyranoside, and the amino acid derivatives with fumonisin B(1), the heating with hydrolyzed fumonisin B(1) yielded no artifacts. For structural determination, the stable reaction product formed by heating of methyl alpha-d-glucopyranoside (as starch model) with fumonisin B(1) was purified and identified by nuclear magnetic resonance spectroscopy as the diester of the fumonisin tricarballylic acid side chains with methyl alpha-d-glucopyranoside. These model experiments demonstrate that fumonisins are able to bind to polysaccharides and proteins via their two tricarballylic acid side chains.     

Identification of flavonoids in Hakmeitau beans (Vigna sinensis) by high-performance liquid chromatography-electrospray mass spectrometry (LC-ESI/MS)

Liquid chromatography coupled with electrospray mass spectrometry (LC-ESI/MS) with positive and negative ion detection was used for the identification of flavonoids in Hakmeitau beans, a black seed cultivar of cowpea (Vigna sinensis). Gradient elution with water and acetonitrile, both containing 2% formic acid, was employed in chromatographic separation. The peaks were identified by comparison of the retention times and the UV-vis spectroscopic and mass spectrometric data with authentic standards and/or literature data. The identified flavonoids included six anthocyanins (cyanidin 3-O-galactoside, cyanidin 3-O-glucoside, delphinidin 3-O-glucoside, malvidin 3-O-glucoside, peonidin 3-O-glucoside, and petunidin 3-O-glucoside) and four flavonol/flavonol glycosides (kaempferol 3-O-glucoside, quercetin, quercetin 3-O-glucoside, and quercetin 3-O-6' '-acetylglucoside). The tentatively identified flavonoids included two anthocyanins (malvidin 3-O-acetylglucoside and peonidin 3-O-malonylglucoside) and three flavonol glycosides (myricetin-3-O-glucoside, quercetin 7-O-glucoside, and quercetin-3-O-diglucoside). These flavonoids are present in seed coats, and the contents of anthocyanins and flavonol glycosides were 20.7 and 2.0 mg/g, respectively.     

Identification of procyanidins and anthocyanins in blueberries and cranberries (Vaccinium spp.) using high-performance liquid chromatography/mass spectrometry

Blueberries and cranberries were analyzed for procyanidins using normal-phase HPLC/MS. Monomers, identified as (+)-catechin and (-)-epicatechin, and a series of oligomers were detected in blueberries, and MS data confirmed that the oligomers consisted of (epi)catechin units that were exclusively singly linked (B-type). The procyanidin "fingerprints" were similar for Tifblue and Rubel but higher than that for lowbush blueberries. In whole cranberries, (-)-epicatechin was present, along with a complex series of oligomers. Both A-type (contained only one double linkage per oligomer) and B-type oligomers were present. Two commercial cranberry juices exhibited similar procyanidin profiles, except that one contained increased quantities. There were processing effects on the procyanidin content of cranberry extract and juices when compared to those of the unprocessed fruits. Monomer, dimers, and A-type trimers were the primary procyanidins, with only trace levels of the B-type trimers and A-type tetramers and with an absence of the higher oligomers in cranberry extract and juices.     

Liquid chromatography-electrospray ionization-tandem mass spectrometry method for the determination of epoxidized soybean oil in food products

Epoxidized soybean oil (ESBO) is widely used as a plasticizer and stabilizer in such polymers [poly(vinyl chloride) in particular] commonly adopted for manufacturing of gaskets of the lids for glass jars and plastic films for food packaging. Human exposure to ESBO and its derivatives is likely to occur over a lifetime with a significant variation according to life stage. A reversed phase liquid chromatography interfaced with electrospray ion trap tandem mass spectrometry method for the determination of ESBO in foods was developed. A simple sample treatment procedure entailing the use of an extraction step with dichloromethane without any further cleanup was proved. Chromatographic separation was performed using two C18 columns with an aqueous acetic acid-acetone-acetonitrile mixture as the mobile phase under gradient conditions. The method was validated in terms of detection limits (4 mg kg(-1)), quantitation limits, linearity (established over 2 orders of magnitude), recovery (good mean recoveries, higher than 90% for all of the signals detected), precision (RSD% < 8), and trueness. The applicability of the method to the determination of ESBO in different food matrices (in particular those rich in edible oil) was demonstrated, and the performances were compared to those reachable by the commonly well-known gas chromatography-mass spectrometry procedure.     

Determination of trinexapac in wheat by liquid chromatography-electrospray ionization tandem mass spectrometry

A quantitative and confirmatory method for the analysis of trinexapac (free acid metabolite of trinexapac-ethyl) in wheat is described. Residues were extracted from wheat with acetonitrile in aqueous phosphate buffer (pH 7) overnight. The extract was directly injected into the HPLC system. Chromatographic separation was achieved on an octadecylsilica column, and detection was performed by negative ion electrospray ionization tandem mass spectrometry. The precursor ion of trinexapac [M - H](-) at m/z 223 was subjected to collisional fragmentation with argon to yield two intense diagnostic product ions at m/z 135 and 179, respectively. Accuracy and specificity for routine analysis of trinexapac were demonstrated. The validated concentration range was 10-200 microg/kg based on a 0.10 g/mL wheat sample extract. Recoveries were within the range of 71-94%, with associated relative standard deviations better than 10%. The limit of detection for trinexapac in wheat was estimated at 5 microg/kg. The method has been applied to a survey of 100 samples of wheat. In 46% of the samples analyzed, a quantifiable amount of trinexapac was detected, ranging from 10 to 110 microg/kg. It has been demonstrated that analyses of trinexapac accurately reflect the total amount of residues of the plant growth regulator, trinexapac-ethyl, in the wheat samples following field application. No residues of the parent compound, trinexapac-ethyl, in wheat were detected.     

Pharmacokinetic study of nobiletin and tangeretin in rat serum by high-performance liquid chromatography-electrospray ionization-mass spectrometry

Nobiletin (NOB) and tangeretin (TAN), two of the main polymethoxylated flavones (PMFs) in citrus, influence a number of key biological pathways in mammalian cells. Although the impacts of NOB and TAN on glucose homeostasis and cholesterol regulation have been investigated in human clinical trials, much information is still lacking about the metabolism and oral bioavailability of these compounds in animals. In this study, NOB and TAN were administered to rats by gavage and intraperitoneal (ip) injection, and the blood serum concentrations of these compounds and their main metabolites were monitored by high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS). In addition to the administered compounds, two metabolites of TAN and eight metabolites of NOB were detected and measured over 24 h. With identical oral doses, nearly 10-fold higher absorption of NOB occurred compared to TAN. For both compounds, maximum levels of glucuronidated metabolites occurred in the blood serum at later time points (～5-8 h) compared to the earlier T(max) values for NOB and TAN. In most cases the glucuronides occurred at substantially higher concentrations than the aglycone metabolites. Low levels of NOB and TAN and their metabolites were detectable in rat blood serum even at 24 h after treatment.     

Identification and quantification of epsilon-(gamma-glutamyl)lysine in digests of enzymatically cross-linked leguminous proteins by high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS)

A rapid and convenient method for the precise quantification of epsilon-(gamma-glutamyl)lysine isopeptide in lyophilized proteolytic digests of cross-linked plant protein samples was developed. The isopeptide was baseline-separated from three other isomers containing lysyl and glutamyl residues by reverse-phase high-performance liquid chromatography after exhaustive proteolytic digestion of the samples cross-linked by a microbial transglutaminase (MTG). Highly selective detection was performed by electrospray mass spectrometry in MS/MS mode. Demonstrating the applicability of the suggested analytical procedure, enzymatic cross-linking of protein isolates from soy [Glycine max (L.) Merr.], pea [Pisum sativum L.], and the sweet lupin species Lupinus albus L. and Lupinus angustifolius L. was investigated after incubation with 0.01 g of MTG/100 g of protein for 0-240 min at 40 degrees C. The liquid chromatography-mass spectrometry (LC-MS) method was successfully applied to monitor the kinetics of epsilon-(gamma-glutamyl)lysine isopeptide formation. Since the calculated initial levels of epsilon-(gamma-glutamyl)lysine in the genuine leguminous protein isolates were between 40 and 77 micromol/100 g, an isopeptide detection limit of 0.5 microg/mL, corresponding to approximately 50 micromol/100 g of protein, was shown to suffice for quantifying the cross-linking rate enzymatically induced by MTG. Concentrations of epsilon-(gamma-glutamyl)lysine in the texturized proteins ranged from 100 to 500 micromol/100 g of protein.     

Determination of oleandrin in tissues and biological fluids by liquid chromatography-electrospray tandem mass spectrometry

A rapid LC-MS/MS method, using a triple-quadrupole/linear ion trap mass spectrometer, was developed for the quantitative determination of oleandrin in serum, urine, and tissue samples. Oleandrin, the major cardiac glycoside of oleander (Nerium oleander L.), was extracted from serum and urine samples with methylene chloride and from tissues with acetonitrile. The tissue extracts were cleaned up using Florisil solid-phase extraction columns. Six replicate fortifications of serum and urine at 0.001 microg/g (1 ppb) oleandrin gave average recoveries of 97% with 5% CV (relative standard deviation) and 107% with 7% CV, respectively. Six replicate fortifications of liver at 0.005 microg/g (5 ppb) oleandrin gave average recoveries of 98% with 6% CV. This is the first report of a positive mass spectrometric identification and quantitation of oleandrin in tissue samples from oleander intoxication cases. The sensitivity and specificity of the LC-MS/MS analysis enables it to be the method of choice for toxicological investigations of oleander poisoning.     

Lipophilic and hydrophilic antioxidant capacities of common foods in the United States

Both lipophilic and hydrophilic antioxidant capacities were determined using the oxygen radical absorbance capacity (ORAC(FL)) assay with fluorescein as the fluorescent probe and 2,2'-azobis(2-amidinopropane) dihydrochloride as a peroxyl radical generator on over 100 different kinds of foods, including fruits, vegetables, nuts, dried fruits, spices, cereals, infant, and other foods. Most of the foods were collected from four different regions and during two different seasons in U.S. markets. Total phenolics of each sample were also measured using the Folin-Ciocalteu reagent. Hydrophilic ORAC(FL) values (H-ORAC(FL)) ranged from 0.87 to 2641 micromol of Trolox equivalents (TE)/g among all of the foods, whereas lipophilic ORAC(FL) values (L-ORAC(FL)) ranged from 0.07 to 1611 micromol of TE/g. Generally, L-ORAC(FL) values were <10% of the H-ORAC(FL) values except for a very few samples. Total antioxidant capacity was calculated by combining L-ORAC(FL) and H-ORAC(FL). Differences of ORAC(FL) values in fruits and vegetables from different seasons and regions were relatively large for some foods but could not be analyzed in detail because of the sampling scheme. Two different processing methods, cooking and peeling, were used on selected foods to evaluate the impact of processing on ORAC(FL). The data demonstrated that processing can have significant effects on ORAC(FL). Considering all of the foods analyzed, the relationship between TP and H-ORAC(FL) showed a very weak correlation. Total hydrophilic and lipophilic antioxidant capacity intakes were calculated to be 5558 and 166 micromol of TE/day, respectively, on the basis of data from the USDA Continuing Survey of Food Intakes by Individuals (1994-1996).     

Analysis of eight capsaicinoids in peppers and pepper-containing foods by high-performance liquid chromatography and liquid chromatography-mass spectrometry

Diverse procedures have been reported for the isolation and analysis of secondary metabolites called capsaicinoids, pungent compounds in the fruit of the Capsicum (Solanaceae) plant. To further improve the usefulness of high-performance liquid chromatography (HPLC), studies were carried out on the analysis of extracts containing up to eight of the following capsaicinoids: capsaicin, dihydrocapsaicin, homocapsaicin-I, homocapsaicin-II, homodihydrocapsaicin-I, homodihydrocapsaicin-II, nonivamide, and nordihydrocapsaicin. HPLC was optimized by defining effects on retention times of (a) the composition of the mobile phase (acetonitrile/0.5% formic acid in H2O), (b) the length of the Inertsil column, and (c) the capacity values (k) of the column packing. Identification was based on retention times and mass spectra of individual peaks. Quantification was based on the UV response at 280 nm in HPLC and recoveries from spiked samples. The method (limit of detection of approximately 15-30 ng) was successfully used to quantify capsaicinoid levels of parts of the pepper fruit (pericarp, placenta, seeds, and in the top, middle, and base parts of whole peppers) in 17 species of peppers and in 23 pepper-containing foods. The results demonstrate the usefulness of the method for the analysis of capsaicinoids ranging from approximately 0.5 to 3600 microg of capsaicin equiv/g of product. The water content of 12 fresh peppers ranged from 80.8 to 92.7%. The described freeze-drying, extraction, and analysis methods should be useful for assessing the distribution of capsaicinoids in the foods and in defining the roles of these biologically active compounds in the plant, the diet, and medicine.     

Identification of anthocyanins in berries by narrow-bore high-performance liquid chromatography with electrospray ionization detection

Qualitative determination of anthocyanins in extracts of red fruits by narrow-bore HPLC/ESI-MS was carried out. This method was used to investigate anthocyanin contents of black bilberry (Vaccinium myrtillus L.), blackberry (Rubus sp.), and mulberry (Morus nigra). An ultraviolet diode array and a mass spectrometer with ESI source were used for detection. Anthocyanin identifications were made by using retention time data and UV-vis and mass spectra and comparing them with those of commercially available standard compounds. The method allowed the identification of fourteen anthocyanins in black bilberry extract, six anthocyanins in blackberry extract, and five anthocyanins in mulberry extract.     

Simultaneous determination of multiple phytohormones in plant extracts by liquid chromatography-electrospray tandem mass spectrometry

A rapid multiresidue method to quantify three different classes of plant hormones has been developed. The reduced concentrations of these metabolites in real samples with complex matrixes require sensitive techniques for their quantification in small amounts of plant tissue. The method described combines high-performance liquid chromatography with electrospray ionization tandem mass spectrometry. Deuterium-labeled standards were added prior to sample extraction to achieve an accurate quantification of abscisic acid, indole-3-acetic acid, and jasmonic acid in a single run. A simple method of extraction and purification involving only centrifugation, a partition against diethyl ether, and filtration was developed and the analytical method validated in four different plant tissues, citrus leaves, papaya roots, barley seedlings, and barley immature embryos. This method represents a clear advantage because it extensively reduces sample preparation and total time for routine analysis of phytohormones in real plant samples.     

Direct determination of paclobutrazol residues in pear samples by liquid chromatography-electrospray tandem mass spectrometry

A rapid and sensitive liquid chromatography/electrospray ionization/tandem mass spectrometry (LC-ESI-MS-MS) method has been developed for the determination of the plant growth regulator paclobutrazol in pear samples. Extraction was performed with methanol by using a high-speed blender Ultra-Turrax, and 10 microL of pear extract was directly injected in the LC-ESI-MS-MS system without any previous sample treatment. The highest sensitivity of the method was achieved under MS-MS conditions obtaining a limit of detection of 0.7 microg/kg and a quantification limit of 5 microg/kg, with a run time of only 5.5 min. Recoveries for paclobutrazol from spiked pear samples at 0.005, 0.05, and 0.5 mg/kg were around 82-102% with relative standard deviations between 2 and 7%. The method was applied to real treated and untreated samples of pears, using quality control samples as a evaluation of the method reliability. Two MS-MS transitions were selected, one for quantification (294 > 70) and the other for confirmation of the analyte (296 > 70). All the experiments were performed in compliance with good laboratory practices.     

Determination of capsaicin and dihydrocapsaicin in capsicum fruits by liquid chromatography-electrospray/time-of-flight mass spectrometry

A simple, highly selective, sensitive, and reproducible liquid chromatography-electrospray ionization/time-of-flight mass spectrometry method has been developed for the direct and simultaneous determination of capsaicin and dihydrocapsaicin in Capsicum fruit extracts. Capsaicin and dihydrocapsaicin are the two major members of the so-called capsaicinoid family, which includes other minor analogues, and usually account for at least 90% of the pungency trait in Capsicum fruits. Chromatographic separation of capsaicin and dihydrocapsaicin was achieved with a reversed-phase chromatography column, using a gradient of methanol and water. Quantification was done using as an internal standard (4,5-dimethoxybenzyl)-4-methyloctamide, a synthetic capsaicin analogue not found in nature. Analytes were base-peak resolved in less than 16 min, and limits of detection were 20 pmol for capsaicin and 4 pmol for dihydrocapsaicin. The intraday repeatability values were lower than 0.5 and 12% for retention time and peak area, respectively, whereas the interday repeatability values were lower than 0.6 and 14% for retention time and peak area, respectively. Analyte recoveries found were 86 and 93% for capsaicin and dihydrocapsaicin, respectively. The method developed has been applied to the identification and quantification of capsaicin and dihydrocapsaicin in fruit extracts from different Capsicum genotypes, and concentrations found ranged from 2 to 6639 mg kg(-1).     

Rapid determination of domoic acid in serum and urine by liquid chromatography-electrospray tandem mass spectrometry

A rapid, selective, and sensitive LC-MS/MS method was developed for the quantitative determination of domoic acid in serum and urine samples. Samples were prepared for analysis using an Oasis HLB SPE column. Determination was by a reversed phase HPLC using a mixture of methanol, acetonitrile, and water containing 1% acetic acid and an electrospray ionization (ESI) ion-trap mass spectrometer (Finnigan LCQ). The method was validated by analyzing five replicates each of negative control bovine serum or urine fortified with domoic acid at the 0.005 microg/g method detection limit (MDL) and at the 0.05 microg/g level. Recoveries ranged from 90 to 95% for fortifications at the MDL and from 92 to 98% for fortifications 10 times higher than the MDL. The diagnostic utility of the method was tested by analyzing samples from live animals showing clinical signs suggestive of domoic acid poisoning submitted to the veterinary toxicology laboratory.     

Development of analyses by high-performance liquid chromatography and liquid chromatography/tandem mass spectrometry of bilberry (Vaccinium myrtilus) anthocyanins in human plasma and urine

Anthocyanins are potent antioxidants that may possess chronic disease preventive properties. Here, rapid, reliable, and reproducible solid-phase extraction, high-performance liquid chromatography (HPLC), and mass spectrometry techniques are described for the isolation, separation, and identification of anthocyanins in human plasma and urine. Recoveries of cyanidin-3-glucoside (C3G) were 91% from water, 71% from plasma, and 81% from urine. Intra- and interday variations for C3G extraction were 9 and 9.1% in plasma and 7.1 and 9.1% in urine and were less than 15% for all anthocyanins from a standardized bilberry extract (mirtoselect). Analysis of mirtoselect by HPLC with UV detection produced spectra with 15 peaks compatible with anthocyanin components found in mirtoselect within a total run time of 15 min. Chromatographic analysis of human urine obtained after an oral dose of mirtoselect yielded 19 anthocyanin peaks. Mass spectrometric analysis employing multiple reaction monitoring suggests the presence of unchanged anthocyanins and anthocyanidin glucuronide metabolites.     

Identification of flavonol and xanthone glycosides from mango (Mangifera indica L. Cv. "Tommy Atkins") peels by high-performance liquid chromatography-electrospray ionization mass spectrometry

Flavonol O- and xanthone C-glycosides were extracted from mango (Mangifera indica L. cv. "Tommy Atkins") peels and characterized by high-performance liquid chromatography-electrospray ionization mass spectrometry. Among the fourteen compounds analyzed, seven quercetin O-glycosides, one kaempferol O-glycoside, and four xanthone C-glycosides were found. On the basis of their fragmentation pattern, the latter were identified as mangiferin and isomangiferin and their respective galloyl derivatives. A flavonol hexoside with m/z 477 was tentatively identified as a rhamnetin glycoside, which to the best of our knowledge, has not yet been reported in mango peels. The results obtained in the present study confirm that peels originating from mango fruit processing are a promising source of phenolic compounds that might be recovered and used as natural antioxidants or functional food ingredients.     

Determination of benzoxazinone derivatives in plants by combining pressurized liquid extraction-solid-phase extraction followed by liquid chromatography-electrospray mass spectrometry

A new analytical method based on the use of pressurized liquid extraction (PLE) followed by solid-phase extraction with LiChrolut RP C18 cartridges was evaluated for the sample preparation, extraction, and cleanup of eight naturally occurring benzoxazinone derivatives, 2-beta-D-glucopyranosyloxy-4-hydroxy-1,4-benzoxazin-3-one, 2-beta-D-glucopyranosyloxy-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one, 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA), 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one, 2-hydroxy-1,4-benzoxazin-3-one, 2-hydroxy-7-methoxy-1,4-benzoxazin-3-one, benzoxazolin-2-one, and 6-methoxybenzoxazolin-2-one in plant samples. Afterward, liquid chromatography-electrospray mass spectrometry, using the selected ion monitoring mode and internal standard (2-MeO-DIBOA, indoxyl-beta-D-glucoside, and quercetin-3-O-rutinoside) quantification method was performed. This paper demonstrates the effectiveness of the PLE method, in conjunction with sensitive and specific mass spectrometric detection, for the quantitative recovery of compounds of the benzoxazinone class from plants. The recoveries of the analytes ranged from 66 to 110% with coefficients of variation ranging from 1 to 14%. This method gave detection limits between 1 and 27 microg/g. The method was applied to foliage and roots of three different wheat cultivars, and the analytes were detected in the range of 11-3261 microg/g of dry weight.