HPLC-PDA-MS/MS of anthocyanins and carotenoids from dovyalis and tamarillo fruits

Anthocyanins and carotenoids are natural pigments responsible for the color of vegetables and fruits, and they are also bioactive compounds, both demonstrating important biological, therapeutic, and preventative properties. Considering the biodiversity of edible fruits, high performance liquid chromatography coupled to photodiode array and mass spectrometry detectors (HPLC-PDA-MS) was used to establish the composition of carotenoids and anthocyanins from dovyalis and tamarillo fruits. Ten anthocyanins and 26 carotenoids were found in dovyalis, whereas tamarillo showed 3 anthocyanins and 17 carotenoids. Higher contents of anthocyanins and carotenoids were found in dovyalis, 42.0 and 6.6 mg/100 g, respectively, as compared to tamarillo fruits with 8.5 and 4.4 mg/100 g, respectively. Although these fruits belong to different families, delphinidin 3-rutinoside and beta-cryptoxanthin were found to be, respectively, the major anthocyanin and carotenoid in both fruits.     

Identification and quantification of seed carotenoids in selected wheat species

Selected primitive and modern wheat species were evaluated on the basis of their carotenoid composition and effects of the genotype and environment on lutein using spectrometry and liquid chromatography. Carotenoids in the wheat extracts were identified and confirmed on the basis of their UV/vis and mass spectra compared with those of authentic standards. The protonated molecule (M + 1)+ at m/z 569 was the predominant ion for zeaxanthin compared to the fragment ion at m/z 551 for lutein. A similar carotenoid profile was obtained for the wheat species investigated, but significant differences were observed in the concentration of carotenoids. Einkorn (Triticum monococcum) exhibited the highest level of all-trans-lutein, averaging 7.41 microg/g with small amounts of all-trans-zeaxanthin, cis-lutein isomers, and beta-carotene. Durum, Kamut, and Khorasan (Triticum turgidum) had intermediate levels of lutein (5.41-5.77 microg/g), while common bread or pastry wheat (Triticum aestivum) had the lowest content (2.01-2.11 microg/g). Lutein in einkorn appeared to be influenced significantly by environmental growing conditions.     

Detection of phloridzin in strawberries (Fragaria x ananassa Duch.) by HPLC-PDA-MS/MS and NMR spectroscopy

The phenolic profile of strawberry fruits (Fragaria x ananassa Duch., Rosaceae) was investigated by high-performance liquid chromatography with photodiode array detection. A peak displaying retention time and UV spectral data identical to those of phloridzin (phloretin 2'-O-beta-d-glucoside), a dihydrochalcone glucoside so far considered characteristic of apples, was monitored. For further characterization, crude extracts of strawberries were purified on polyamide, and the target compound was isolated by preparative and analytical HPLC. Structure elucidation was performed on the basis of APCI- and ESI-MS in the negative ion mode as well as by 1D and 2D NMR spectroscopy using authentic phloridzin for comparison. The d-configuration of the sugar moiety was established by HPLC analysis of the corresponding acyclic 1-deoxy-1-(N-acetyl-alpha-methylbenzylamino)alditol acetate. Apart from its chemotaxonomic relevance, this first report on the occurrence of phloridzin in strawberries is of particular interest for the authenticity control of strawberry products such as juices, jams, and fruit preparations since phloridzin has so far been used for the detection of fraudulent admixtures.     

Identification and quantification of caffeoylquinic acids and flavonoids from artichoke (Cynara scolymus L.) heads, juice, and pomace by HPLC-DAD-ESI/MS(n)

A method for the identification and quantification of phenolic compounds from artichoke (Cynara scolymus L.) heads, juice, and pomace by HPLC with diode array and mass spectrometric detection was developed. Among the 22 major compounds, 11 caffeoylquinic acids and 8 flavonoids were detected. Quantification of individual compounds was carried out by external calibration. Apigenin 7-O-glucuronide was found to be the major flavonoid in all samples investigated. 1,5-Di-O-caffeoylquinic acid represented the major hydroxycinnamic acid, with 3890 mg/kg in artichoke heads and 3269 mg/kg in the pomace, whereas in the juice 1,3-di-O-caffeoylquinic acid (cynarin) was predominant, due to the isomerization during processing. Total phenolic contents of approximately 12 g/kg on a dry matter basis revealed that artichoke pomace is a promising source of phenolic compounds that might be recovered and used as natural antioxidants or functional food ingredients.     

Identification and quantification of polyphenols in carob fruits (Ceratonia siliqua L.) and derived products by HPLC-UV-ESI/MSn

The polyphenolic patterns of carob pods (Ceratonia siliqua L.) and derived products were identified and quantified using high-performance liquid chromatography-UV absorption-electrospray ion trap mass spectrometry after pressurized liquid extraction and solid-phase extraction. In carob fiber, 41 individual phenolic compounds could be identified. In addition, spectrophotometric quantification using the Folin-Ciocalteu and vanillin assays was performed, and the antioxidative activity was determined as the 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity. Carob pods contain 448 mg/kg extractable polyphenols comprising gallic acid, hydrolyzable and condensed tannins, flavonol-glycosides, and traces of isoflavonoids. Among the products investigated, carob fiber, a carob pod preparation rich in insoluble dietary fiber (total polyphenol content = 4142 mg/kg), shows the highest concentrations in flavonol-glycosides and hydrolyzable tannins, whereas roasted carob products contain the highest levels of gallic acid. The production process seems to have an important influence on the polyphenolic patterns and quantities in carob products.     

Resonance Raman quantification of nutritionally important carotenoids in fruits, vegetables, and their juices in comparison to high-pressure liquid chromatography analysis

A rapid nondestructive estimation of carotenoid levels in intact fruits and vegetables and their juices could have great value when selecting nutritionally valuable crops for further propagation and commercial use. Carotenoid levels of a variety of agricultural products and juices were measured using resonance Raman spectroscopy and compared to levels determined by extraction and high-pressure liquid chromatography. A strong correlation was observed between the two methods when evaluating juices and when comparing different strains of intact tomatoes at the same stage of ripening.     

Identification and quantification of flavonol glycosides in almond seedcoats using MALDI-TOF MS

Interest in the molecular composition of almonds is growing, due to their popularity in a wide variety of food formulations. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a powerful new technique that can be used to rapidly identify and quantify possible bioactive compounds in these popular tree nuts. Four flavonol glycosides were identified in almond seedcoats for the first time: isorhamnetin rutinoside, isorhamnetin glucoside, kaempferol rutinoside, and kaempferol glucoside. A MALDI-TOF MS methodology was developed using rutin (quercetin-3-rutinoside) as an internal standard to quantitatively determine each of the four flavonol glycosides. Results of MALDI-TOF MS analysis were verified by high performance liquid chromatography.     

Sources of antioxidant activity in Australian native fruits. Identification and quantification of anthocyanins

Selected native Australian fruits, muntries (Kunzea pomifera F. Muell., Myrtaceae), Tasmanian pepper berry (Tasmanian lanceolata R. Br., Winteraceae), Illawarra plum (Podocarpus elatus R. Br. ex Endl., Podocarpaceae), Burdekin plum (Pleiogynium timorense DC. Leenh, Anacardiaceae), Cedar Bay cherry (Eugenia carissoides F. Muell., Myrtaceae), Davidson's plum (Davidsonia pruriens F. Muell. var. pruriens, Davidsoniaceae), and Molucca raspberry (Rubus moluccanus var. austropacificus van Royen, Rosaceae), were evaluated as sources of antioxidants by 2,2-diphenyl-1-picrylhydrazyl and ferric reducing antioxidant power assays and compared with blueberry (Vaccinum spp. cv. Biloxi). The total reducing capacity of five fruits was 3.5-5.4-fold higher than that of blueberry, and the radical scavenging activities of muntries and Burdekin plum were 1.5- and 2.6-fold higher, respectively. The total phenolic level by Folin-Ciocalteu assay highly correlated with the antioxidant activity. Therefore, systematic research was undertaken to identify and characterize phenolic complexes. In the present study we report on the levels and composition of anthocyanins. The HPLC-DAD and HPLC/ESI-MS-MS (ESI = electrospray ionization) analyses revealed simple anthocyanin profiles of one to four individual pigments, with cyanidin as the dominating type. This is the first evaluation of selected native Australian fruits aiming at their utilization for the development of novel functional food products.     

Characterization and quantification of phenolic compounds in olive oils by solid-phase extraction, HPLC-DAD, and HPLC-MS/MS

A simple and reproducible method for qualitative and quantitative analysis of phenolic compounds in virgin olive oils by solid-phase extraction (SPE), high performance liquid chromatography with diode array detector (HPLC-DAD), and HPLC-mass spectrometry (MS) in tandem mode was developed. The polar fraction was obtained from samples of three different virgin olive oils. Detection and quantification were performed at 280, 240, and 320 nm. For identification purposes, HPLC-MS/MS was equipped with turbo ion spray source in the negative-ion mode. Twenty compounds of twenty-three detected and quantified were characterized. The method showed satisfactory linearity (r > 0.99), good recovery, satisfactory precision, and appropriate limits of detection (LOD) and quantification (LOQ).     

Identification and quantification of betalains from the fruits of 10 mexican prickly pear cultivars by high-performance liquid chromatography and electrospray ionization mass spectrometry

Qualitative and quantitative analyses of betalain pigments in 10 cultivars/lines of prickly pear (Opuntia spp.) fruit grown in Mexico were conducted with reverse phase high-performance liquid chromatography-diode array detection (HPLC-DAD) coupled with electrospray mass spectrometry (ESI-MS). Betacyanins and betaxanthins were identified by comparison with the UV/vis and mass spectrometric characteristics as well as the retention times of semisynthesized reference betaxanthins. Data revealed that the ratio and concentration of betalain pigments are responsible for the color in the different cultivars, showing the highest betalains content in the fruit of purple colored Camuesa (O. robusta Wendl.) (8.1 mg/g dry fruit), which is comparable to that found in red beet Beta vulgaris L. ssp. Var. Pablo) (8.6 mg/g dry tissue). Yellow betalains were absent in Reyna (O. alba-carpa) prickly pear cultivar. A total of 24 known/unknown betalains were present in the prickly pear fruit samples studied, including 18 betaxanthins and 6 betacyanins. Our results indicate that prickly pear cultivars can be considered as a potential source of yellow and red natural colorants.     

Identification and quantification of carotenoids including geometrical isomers in fruit and vegetable juices by liquid chromatography with ultraviolet-diode array detection

A method was established for the identification and quantification of carotenoids including geometrical isomers in fruit and vegetable juices by liquid chromatography with an ultraviolet-diode array detector, using a C(18) Vydac 201TP54 column. The mobile phase used was the ternary methanol mixture (0.1 M ammonium acetate), tert-butyl methyl ether and water, in a concentration gradient, and a temperature gradient was applied. Retinol palmitate was added as an internal standard. An extraction process (ethanol/hexane, 4:3, v/v) was performed, followed by saponification with diethyl ether/methanolic KOH (0.1%, w/v, BHT) (1:1, v/v) for 0.5 h at room temperature. Seventeen different (cis and trans) carotenoids were identified by UV-vis spectra and retention times in HPLC in the juices analyzed. The analytic parameters show that the method proposed is sensitive, reliable, accurate, and reproducible.     

Structural profiling and quantification of sphingomyelin in human breast milk by HPLC-MS/MS

The sphingolipid composition of food as well as of physiological samples has received considerable interest due to their positive biological activities. This study quantified the total amount of sphingomyelin (SM) in 20 human breast milk samples from healthy volunteers and determined the structures of SM by detailed mass spectrometric studies in combination with enzymatic cleavage. The quantification of SM was performed by hydrophilic interaction liquid chromatography coupled to electrospray ionization-tandem mass spectrometry (HILIC-HPLC-ESI-MS/MS) measuring the characteristic fragment ion of the phosphorylcholine group at m/z 184.2 and by using hexanoylsphingomyelin (C6-SM) and heptadecanoylsphingomyelin (C17-SM) as internal standards. The structures of SM species were identified after enzymatic cleavage with alkaline sphingomyelinase (SMase) to the corresponding ceramides. Structure elucidation of the sphingoid base and fatty acid backbone was performed by reversed-phase HPLC-ESI-MS/MS. The method includes the sphingoid bases dihydrosphingosine (d18:0), sphingosine (d18:1(Δ4)), 4,8-sphingadienine (d18:2(Δ4,8)), 4-hydroxysphinganine (phytosphingosine (t18:0)), and 4-hydroxy-8-sphingenine (t18:1(Δ8)) and fatty acids with even-numbered carbon atoms (C12-C26) as well as their (poly)unsaturated and monohydroxylated analogues. The total amount of SM in human breast milk varied from 3.87 to 9.07 mg/100 g fresh weight. Sphingosine (d18:1) was the predominant sphingoid base, with 83.6 ± 3.5% in human breast milk, followed by 4,8-sphingadienine (d18:2) (7.2 ± 1.9%) and 4-hydroxysphinganine (t18:0) (5.7 ± 0.7%). The main SM species contained sphingosine and palmitic acid (14.9 ± 2.2%), stearic acid (12.7 ± 1.5%), docosanoic acid (16.2 ± 3.6%), and tetracosenoic acid (15.0 ± 3.1%). Interestingly, the fatty acid composition of SM species in this study differs from the total fatty acids in human breast milk, and the fatty acids are not consistently distributed among the different sphingoid bases.     

Identification and structure elucidation of a p-phenoxybenzaldehyde in bamboo shoots by HPLC-ESI/MS/MS and NMR

In this study, a derivative of p-phenoxybenzaldehyde in bamboo shoots was investigated. Bamboo shoots were ground and extracted with water, and an aqueous suspension was purified by SPE using Oasis HLB cartridges. After the SPE procedure, the analytes were analyzed by HPLC with refractive index detection (HPLC-RI). In the HPLC-RI analysis for sucralose, a putative sucralose was detected. In the subsequent HPLC-PDA analysis, the suspicious peak showed a unique UV absorption spectrum with the maximum wavelength at 285 nm indicating the existence of an aromatic ring. The contents of the unknown compound in bamboo shoot products ranged from 0.01 to 0.15 mg/g. The identity of the unknown compound was further confirmed by HPLC-ESI/MS/MS. The molecular weight of the unknown compound was determined to be 244. The chemical structure of the unknown compound was elucidated on the basis of NMR spectroscopic analyses ((1)H, (13)C, DEPT, COSY, HMQC, and HMBC). Finally, the structure of the unknown compound was characterized as 4-(4-dihydroxymethylphenoxy)benzaldehyde.     

Detection and quantification of glycosylated flavonoid malonates in celery, Chinese celery, and celery seed by LC-DAD-ESI/MS

A screening method using LC-DAD-ESI/MS was applied to the analysis of flavonoids in celery, Chinese celery, and celery seeds (Apium graveolens L. and varieties). Fifteen flavonoid glycosides were detected in the three celery materials. They were identified as luteolin 7-O-apiosylglucoside, luteolin 7-O-glucoside, apigenin 7-O-apiosylglucoside, chrysoeriol 7-O-apiosylglucoside, chrysoeriol 7-O-glucoside, and more than 10 malonyl derivatives of these glycosides. The identification of the malonyl derivatives was confirmed by their conversion into glycosides upon heating and by comparison of some of the malonates with malonates that had previously been identified in red bell pepper and parsley. The concentrations of the glycosides and the malonyl glycosides in the three materials were estimated by comparison to aglycone standards. This is the first report of the presence of these glycosylated flavonoid malonates in celery.     

Analysis of methoxyfenozide residues in fruits, vegetables, and mint by liquid chromatography-tandem mass spectrometry (LC-MS/MS)

Methoxyfenozide [3-methoxy-2-methylbenzoic acid 2-(3,5-dimethylbenzoyl)-2-(1,1-dimethylethyl) hydrazide; RH-2485], in the formulation of INTREPID, was applied to various crops. Analysis of methoxyfenozide was accomplished by utilizing liquid-liquid extraction and partitioning, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Method validations for fruits, vegetables, and mint are reported. Methoxyfenozide mean recoveries ranged from 72 to 129% over three levels of fortification. The overall average of mean recoveries is 97 +/- 10%. The limit of quantitation for fruits, artichoke, cucumber, squash, and refined sugar was 0.010 ppm, with a detection limit of 0.005 ppm. For all other crops, the limit of quantitation was 0.050 ppm, with a detection limit of 0.025 ppm. No residues were found greater than the limit of quantitation in control samples. Residues above the limit of quantitation were found in all matrices except refined sugar. Foliage (bean, beet, pea, and radish) had greater residue levels of methoxyfenozide residue than their corresponding roots or pods. Other crop matrices contained <1.0 ppm of methoxyfenozide except artichoke, which had a mean of 1.10 ppm.     

Enzymatic treatment to improve extraction of capsaicinoids and carotenoids from chili (Capsicum annuum) fruits

Enzymatic treatments using noncommercial enzymes as a means to the improve the extraction of carotenoids and capsaicinoids from chili fruits are explored in this study. The results show that it is possible to obtain chili fruit powder with a higher concentration of both capsaicinoids and carotenoids than previously reported for similar processes. Furthermore, extraction yields above 96% for carotenoids and 85% for capsaicinoids as separate fractions can be achieved using a sequential and selective two-stage extraction. Evidence is presented demonstrating that the content and extraction yield depend directly on the extent of the enzymatic hydrolysis of chili cell walls, and higher yields are obtained when the sample is completely hydrolyzed. The enzymatic treatment described here is a promising alternative to current industrial practices, and it improves the extraction of carotenoids and capsaicinoids from chili fruits.     

Preparation of antioxidant enzymatic hydrolysates from alpha-lactalbumin and beta-lactoglobulin. Identification of active peptides by HPLC-MS/MS

We have investigated the antioxidant activity of hydrolysates from whey proteins bovine alpha-lactalbumin (alpha-La) and beta-lactoglobulin A (beta-Lg A) by commercial proteases (pepsin, trypsin, chymotrypsin, thermolysin, and Corolase PP). Corolase PP was the most appropriate enzyme to obtain antioxidant hydrolysates from alpha-La and beta-Lg A (ORAC-FL values of 2.315 and 2.151 micromol of Trolox equivalent/mg of protein, respectively). A total of 42 peptide fragments were identified by HPLC-MS/MS in the beta-Lg A hydrolysate by Corolase PP. One of the sequences (Trp-Tyr-Ser-Leu-Ala-Met-Ala-Ala-Ser-Asp-Ile) possessed radical scavenging (ORAC-FL value of 2.621 micromol of Trolox equivalent/micromol of peptide) higher than that of butylated hydroxyanisole (BHA). Our results suggest that whey protein hydrolysates could be suitable as natural ingredients in enhancing antioxidant properties of functional foods and in preventing oxidation reaction in food processing.     

Fast and versatile multiresidue method for the analysis of botanical insecticides on fruits and vegetables by HPLC/DAD/MS

A simple multiresidue method for screening analysis of 12 botanical insecticides used by organic farmers has been developed. The method involves a rapid and small-scale extraction procedure with acetonitrile. For all fruit and vegetable samples, there was no need for clean up. Rotenone, azadirachtin, ryanodines, and pyrethrins can be separated by high-performance liquid chromatography, quantified, and confirmed with a diode array detector (DAD) and atmospheric pressure chemical ionization mass spectrometry (APCI-MS) in the select ion-monitoring mode (SIM). The majority of pesticide recoveries for various fruits and vegetables were >70% in the concentration range from 0.01 to 5 mg/kg. The limit of quantitation for most of the pesticides was 0.01 mg/kg, with the majority of relative standard deviations (RSD) mostly below 10%.     

Automated strong cation exchange extraction of fatty acid esters of 3-(N-phenylamino)-1,2-propanediol from oil samples for routine quantification by HPLC-APCI/MS/MS.

Fatty acid esters of 3-(N-phenylamino)-1,2-propanediol are currently considered the best chemical markers of toxic oils related to the Spanish toxic oil syndrome. Recent research in this area has undertaken the exhaustive and quantitative characterization of these compounds in oils collected during the epidemic outbreak. Current methods developed in this laboratory are based on solid phase extraction (SPE) using SCX cartridges followed by HPLC-APCI/MS/MS quantification. To circumvent the long and tedious extraction procedure, the SPE protocol was adapted for automatic extraction and the problems derived from the use of the immiscible solvents required for the SCX extraction were solved. Linearity of the analytical method was found in the same range as for the manual method. Extraction recoveries were 87 and 75% for 2-hydroxy-3-(N-phenylamino)propyl linoleate and 2-(linoleyloxy)-3-(N-phenylamino)propyl linoleate, respectively, and the corresponding coefficients of variation were approximately 1%, greatly improving reproducibility over manual procedures.     

Isotopic labeling and LC-APCI-MS quantification for investigating absorption of carotenoids and phylloquinone from kale (Brassica oleracea)

The ability to study bioavailability of nutrients from foods is an important step in determining the health impact of those nutrients. This work describes a method for studying the bioavailability of nutrients from kale (Brassica oleracea var. Acephala) by labeling the nutrients with carbon-13, feeding the kale to an adult volunteer, and analyzing plasma samples for labeled nutrients. Results showed that conditions for producing atmospheric intrinsically labeled kale had no detrimental effect on plant growth. Lutein, beta-carotene, retinol, and phylloquinone were analyzed using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry. Analysis of plasma samples showed that labeled lutein peaked in plasma at 11 h (0.23 microM), beta-carotene peaked at 8 (0.058 microM) and 24 h (0.062 microM), retinol peaked at 24 h (0.10 microM), and phylloquinone peaked at 7 h (3.0 nM). This method of labeling kale with (13)C was successful for producing clearly defined kinetic curves for (13)C-lutein,(13)C-beta-carotene, (13)C-retinol, and (13)C-phylloquinone.