Single-drop microextraction followed by in-syringe derivatization and gas chromatography-mass spectrometric detection for determination of organic acids in fruits and fruit juices

A simple analytical procedure based on single-drop microextraction combined with in-syringe derivatization and GC-MS was developed for determination of some phenolic acids in fruits and fruit juices. Cinnamic acid, o-coumaric acid, caffeic acid, and p-hydroxybenzoic acid were used as model compounds. The analytes were extracted from a 3-mL sample solution using 2.5 microL of hexyl acetate. The extracted phenolic acids were derivatized inside the syringe barrel using 0.7 microL of N,O-bis(trimethylsilyl)acetamide before injection into the GC-MS. The influence of derivatization conditions on the yield of in-syringe silylation was studied. Experimental SDME parameters such as selection of organic solvent, solvent volume, extraction time, extraction temperature, pH, and ionic strength of the solution on the extraction performance were studied. The method provided fairly good precision for all compounds (2.4-11.9%). Detection limits were found to be between 0.6 and 164 ng/mL within an extraction time of 20 min in the GC-MS full scan mode.     

A general analytical strategy for the characterization of phenolic compounds in fruit juices by high-performance liquid chromatography with diode array detection coupled to electrospray ionization and triple quadrupole mass spectrometry

In the present study, a methodology based on liquid chromatography with diode array detection (HPLC/DAD) coupled to an electrospray ionization (ESI) interface and a triple quadrupole mass spectrometer for the simultaneous identification of phenolic compounds in fruit juices has been developed. 72 available phenolic compound standards from diverse families present in fruits have been studied in order to analyze their fragmentation pattern. As a result, a general strategy for the characterization of unknown phenolic compounds in fruit juices was designed: (i) taking into account its UV–visible spectrum and elution order, assign the unknown polyphenol to a polyphenol class, (ii) identify the quasi-molecular ion using positive and negative MS spectra, being supported by adducts generated with solvent or sodium and molecular complexes, (iii) determinate the pattern of glycosylation in positive mode using ESI(+)-CID MS/MS product ion scan experiments, selecting the quasi-molecular ion as precursor ion, and finally, (iv) study the identity of the aglycone through ESI(+)-CID MS/MS product ion spectra from the protonated aglycone, [Y₀]⁺. This strategy was successfully employed for the characterization of known and unknown phenolic compounds in juices from 17 different fruits.     

Use of high-performance liquid chromatography with diode array detection coupled to electrospray-Qq-time-of-flight mass spectrometry for the direct characterization of the phenolic fraction in organic commercial juices

We have developed a direct method for the qualitative analysis of polyphenols in commercial organic fruit juices. The juices were diluted with water (50/50), filtered and directly injected. The analysis of phenolic compounds was carried out by reversed-phase high-performance liquid chromatography (RP-HPLC) coupled to photodiode array detection (DAD) and electrospray ionisation-Qq-time-of-flight mass spectrometry (ESI-Qq-TOF-MS). A unique gradient program has been optimized for the separation of several phenolic classes and the analysis time was only 5 min. The fruit juice samples were successfully analysed in positive and negative ionisation modes. In positive mode the anthocyanins were identified whereas the vast majority of polyphenols were identified using the negative ionisation mode. The sensitivity, together with mass accuracy and true isotopic pattern of the Qq-TOF-MS, allowed the identification of the phenolic compounds. Moreover, the advantage of the proposed method is the combined search of MS and MS/MS spectra, which improves the identification of compounds considerably, reducing ambiguities and false positive hits. Therefore the total fragmentation of the compound ion leading to the aglycone ion or other fragments was corroborated by MS–MS. The method was successfully employed to characterize diverse phenolic families in commercially available organic juices from four different fruits and consequently could be used in the future for the quantification purposes to compare different content of polyphenols in juices.     

Separation and determination of flavonoids and other phenolic compounds in cranberry juice by high-performance liquid chromatography

A HPLC method was developed for the separation and determination of flavonoid and phenolic antioxidants in cranberry juices. Free flavonoid and phenolic compounds were fractionated into neutral and acidic groups by means of a solid-phase extraction method, followed by subsequent HPLC separations. Combined flavonoids and phenolics were hydrolyzed by acid before HPLC analysis. This developed method provides a fast and high resolution of individual flavonoid and phenolic compounds. In cranberry fruit, flavonoids and phenolic acids exist predominantly in combined forms, such as glycosides and esters. A total of 400 mg of total flavonoids and phenolic compounds/l of sample was found in a freshly squeezed cranberry juice, which was distributed as about 44% of phenolic acids and 56% of flavonoids. Benzoic acid was the major phenolic compound. Major flavonoids in the freshly squeezed cranberry juice were quercetin and myricetin.     

Comparative Study of the Chemical Compositions and Antioxidant Activities of Fresh Juices from Romanian Cucurbitaceae Varieties

Cucurbitaceae is a family of health-promoting plants due to their compounds with beneficial effects. The aim of this study was to analyze, for the first time, the chemical composition, the antioxidant activity and the metal chelating properties of fruit juices obtained from four different species of the Cucurbitaceae family cultivated in Romania, namely Momordica charantia, Cucumis metuliferus, Benincasa hispida and Trichosanthes cucumerina. The samples of juice were analyzed by high-performance liquid chromatography (HPLC) and all the four species displayed high levels of the two triterpenes, oleanolic and ursolic acids, and also in phenolic compounds, including catechin, (−)-epicatechin and gallic acid. The juices demonstrated significant antioxidant activity against the free radical 2,2-diphenyl-1-picrylhydrazyl (ranging from 20 to 95%,), a good iron binding ability (ranging from 7.45 ± 0.28% to 86.95 ± 0.97%) and also promising antioxidant potential against the ABTS radical (ranging from 4.97 to 32.60 μETx/mL juice). Our findings raise interesting questions for further research on Cucurbitaceae fruit juices and, consequently, their very good antioxidant potential suggests these fruits should be further explored for their protective effect against oxidative damage. This is the first time the chemical composition and antioxidant activities of fruit juices from these four Romanian Cucurbitaceae varieties have been investigated.     

Determination of phenolic compounds in wines by novel matrix solid-phase dispersion extraction and gas chromatography/mass spectrometry

A novel matrix solid-phase dispersion (MSPD) extraction method was developed to extract simultaneously 23 phenolic compounds from wine samples prior to determination by gas chromatography with mass spectrometric detection in the selected ion monitoring mode. Different parameters of the MSPD technique such as dispersant solid-phase, eluting solvent, and sample ionic strength and pH were optimized. The optimized MSPD procedure requires a small volume of wine (1 mL), commercial silica gel (1.5 g) as dispersant solid-phase and a small volume of ethyl acetate (5 mL) as eluting solvent. Under these conditions, the extraction of the studied compounds was almost complete (mean values of recoveries between 87 and 109%) in a short time (15 min). Moreover, satisfactory standard deviations of repeatability (RSD<9% in most cases), linear regression coefficients (r(2)>0.993) and detection limits (<8 microg/L) confirm the usefulness of the methodology for routine monitoring of the concentration of individual phenolic antioxidants in wines. Application was illustrated by analysis of different wine samples.     

Application of deep eutectic solvents for the extraction of phenolic compounds from extra-virgin olive oil

A HPLC–DAD/ESI–MS method has been developed and validated for the analysis of the most representative phenolic compounds in extra-virgin olive oil (EVOO) samples using a green extraction approach based on deep eutectic solvents (DESs) at room temperature. We examined ten DESs based on choline chloride and betaine in combination with different hydrogen bond donors comprising six alcohols, two organic acids, and one urea. Five phenolic compounds, belonging to the classes of secoiridoids and phenolic alcohols, were selected for the evaluation of extraction efficiency. A betaine-based DES with glycerol (molar ratio 1:2) was found to be the most effective for extracting phenolic compounds as compared to a conventional solvent. The optimization of the extraction method involved the study of the quantity of water to be added to the DES and evaluation of the sample-to-solvent ratio optimal condition. Thirty percent of water added to DES and sample to solvent ratio 1:1 (w/v) were selected as the best conditions. The chromatographic method was validated by studying LOD, LOQ, intraday and interday retention time precision, and linearity range. Recovery values obtained spiking seed oil sample aliquots with standard compounds at 5 and 100 μg/g concentration were in the range between 75.2% and 98.7%.     

Gas chromatographic-mass spectrometric fragmentation study of flavonoids as their trimethylsilyl derivatives: analysis of flavonoids, sugars, carboxylic and amino acids in model systems and in citrus fruits

The fragmentation patterns and quantitation possibilities of three anthocyanidins (pelargonidin, cyanidin, malvidin), one flavonol (quercetin), two flavones (apigenin, luteolin) and two flavanones (naringenin, hesperetin) have been investigated as trimethylsilyl and as trimethylsilyl (oxime) derivatives by gas chromatography-mass spectrometry. Results proved that anthocyanidins and flavanones form trimethylsilyl (oximes), while flavonol and flavones provide simple trimethylsilyl derivatives. In all cases, characteristic fragments of high masses are formed proper for quantitation purposes. Hydrolysis conditions for naringin, hesperidin and rutin have been optimized, resulting in the quantitative release of naringenin, hesperetin and quercetin together with their corresponding saccharides. These basic studies made possible the identification and quantification of the flavonoid, carboxylic-/amino acid and sugar constituents of citrus fruit juices and albedos, without any extraction/enrichment procedure. In total 33 compounds have been determined in hydrolyzed samples, such as 2 flavonoids (naringenin and hesperetin), 6 phenolic acids (trimethoxybenzoic, 4-hydroxybenzoic, vanillic, quinic, chlorogenic and rosmarinic acids), 3 aliphatic carboxylic acids (levulinic, malic, citric acids), phosphoric acid, 4 amino acids (aspartic, glutamic acids, alanine, proline), 9 monosaccharides (xylose, arabinose, rhamnose, fucose, fructose, galactose, glucose, galacturonic acid, sedoheptulose), inositol, sugarphosphate, 5 disaccharides and tocopherol. Measurements were carried out as the trimethylsilyl (oxime) ether/ester derivatives of constituents, in the concentration range of 2 x 10(-3) to 49.9%. Identification level of samples varied between 26.4 and 77.5%, expressed in dry matter content of juices and albedos.     

Optimization of a new mobile phase to know the complex and real polyphenolic composition: towards a total phenolic index using high-performance liquid chromatography

An HPLC method is reported for the separation and quantification of five major polyphenolic groups found in fruits and related products: single ring phenolic acids (hydroxybenzoic acid and hydroxycinnamic acid derivatives), flavan-3-ols, flavonols, anthocyanins, and dihydrochalcones. A binary mobile phase consisting of 6% acetic acid in 2 mM sodium acetate aqueous solution (v/v, final pH 2.55) (solvent A) and acetonitrile (solvent B) was used. The use of sodium acetate was new and key to the near baseline separation of 25 phenolics commonly found in fruits. A photodiode array detector was used and data were collected at four wavelengths (280, 320, 360, and 520 nm). This method was sensitive and gave good separation of polyphenolics in apple, cherry, strawberry, blackberry, grape, apple juice, and a processing by-product. The improved separation has led to better understanding of the polyphenolic profiles of these fruits. Individual as well as total phenolic content was obtained, and the latter was close to and correlated well with that obtained by the Folin-Ciocalteu method (FC). The HPLC data can be used as a total phenolic index (TPI) for quantification of fruit phenolics, which is advantageous over the FC because it has more information on individual compounds.     

Use of solid-supported liquid-liquid extraction in the analysis of polyphenols in wine

Solid-supported liquid-liquid extraction (SS-LLE) was compared to liquid-liquid extraction (LLE) for the analysis of phenolic compounds in wine. Diatomaceous earth commercial cartridges were evaluated together with "in-house" made cartridges for the wine phenolic extraction. Statistical treatment, analysis of variance ANOVA-single factor, was used to compare the extraction yields obtained by these methods, and for the majority of the studied compounds, significantly higher yields were obtained by the SS-LLE methodology using the "in-house" prepared cartridges. This is an environmentally friendly low-cost sample preparation method which proved to be reproducible (RSD<5% for the most compounds) and yielding high recoveries (80-100%) for the compounds studied.     

Experimental design for extraction and quantification of phenolic compounds and organic acids in white "Vinho Verde" grapes

An experimental design was applied for the optimization of extraction and clean-up processes of phenolic compounds and organic acids from white “Vinho Verde” grapes. The developed analytical method consisted in two steps: first a solid-liquid extraction of both phenolic compounds and organic acids and then a clean-up step using solid-phase extraction (SPE). Afterwards, phenolic compounds and organic acids were determined by high-performance liquid chromatography (HPLC) coupled to a diode array detector (DAD) and HPLC-UV, respectively. Plackett-Burman design was carried out to select the significant experimental parameters affecting both the extraction and the clean-up steps. The identified and quantified phenolic compounds were: quercetin-3-O-glucoside, quercetin-3-O-rutinoside, kaempferol-3-O-rutinoside, isorhamnetin-3-O-glucoside, quercetin, kaempferol and epicatechin. The determined organic acids were oxalic, citric, tartaric, malic, shikimic and fumaric acids. The obtained results showed that the most important variables were the temperature (40 °C) and the solvent (acid water at pH 2 with 5% methanol) for the extraction step and the type of sorbent (C18 non end-capped) for the clean-up step.     

Determination of phenolic acids in fruit juices by isocratic column liquid chromatography

A simple and rapid analytical method of five phenolic acids, gallic, chlorogenic, caffeic, ellagic and ferulic acid, which are naturally occurring bioactives, were determined in fruit juices by isocratic LC using photodiode array UV detection. The sample was pre-treated by solid-phase extraction (a combination of Sep-Pak Plus tC18 and Bond Elut PSA).     

Determination of Phenolic Compounds and the Antioxidant Capacity of Ximenia parviflora Benth. var. parviflora (Olacaceae) Fruit by High-Performance Liquid Chromatography with Diode Array Detection

The total phenolic and flavonoid content, phenolic composition, and in vitro antioxidant capacity of ethanolic extracts of Ximenia parviflora Benth. var. parviflora fruits collected at Zinaparo, Michoacan (in central Mexico) were determined. Fruit extracts present a high scavenging activity of 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azino-bis[3-ethylbenzothiazoline-6-sulfonic acid] radicals (71.49?±?0.11% and 85.00?±?1.29% inhibition, respectively). The four phenolic compounds identified in fruit extracts by high-performance liquid chromatography with diode array detection were gallic acid, chlorogenic acid, caffeic acid, and quercetin. X. parviflora fruits may be used as a starting material for the extraction of high value antioxidant phenolic compounds with potential applications in the pharmaceutical and dietary supplement industries.     

Anthocyanins as antimicrobial agents of natural plant origin

Anthocyanins are particularly abundant in different fruits, especially in berries. The beneficial effects of these compounds for human health have been known from at least the 16th century. Despite the great number of papers devoted to the different biological effects exerted by anthocyanins only a limited number of studies is focused on the antimicrobial activity of these compounds. Anthocyanin content of berry fruits varies from 7.5 mg/100 mg fresh fruit in redcurrant (Ribes rubum) up to 460 mg/100 g fresh fruit in chokeberry (Aronia melanocarpa). After consumption, anthocyanins are intensively metabolized, mainly in the intestines and liver. Glucorination, methylation and sulfation are the most typical metabolic reactions. Antimicrobial activity of crude extracts of plant phenolic compounds against human pathogens has been intensively studied to characterize and develop new healthy food ingredients as well as medical and pharmaceutical products. However, there is very little information available about the antimicrobial activity of the pure anthocyanins. In the last part of this review we present the collection of papers describing the anthocyanin profiles of different fruits (mainly berries) and the antimicrobial properties of the identified compounds. Generally, anthocyanins are active against different microbes, however Gram-positive bacteria usually are more susceptible to the anthocyanin action than Gram-negative ones. Mechanisms underlying anthocyanin activity include both membrane and intracellular interactions of these compounds. Antimicrobial activity of berries and other anthocyanin-containing fruits is likely to be caused by multiple mechanisms and synergies because they contain various compounds including anthocyanins, weak organic acids, phenolic acids, and their mixtures of different chemical forms. Therefore, the antimicrobial effect of chemically complex compounds has to be critically analyzed.     

Combined microwave-assisted isolation and solid-phase purification procedures prior to the chromatographic determination of phenolic compounds in plant materials

A fast, sensitive and selective procedure employing a combination of microwave-assisted extraction (MAE) and solid phase extraction (SPE) was applied prior to liquid chromatographic identification and quantification of phenolic compounds in plant materials. MAE has been tested and optimized for the isolation of phenolic acids (gallic, protocatechuic, p-hydroxybenzoic, chlorogenic, vanilic, caffeic, syringic, p-coumaric, ferulic, sinapic, benzoic, m-coumaric, o-coumaric, rosmarinic, cinnamic acids) and 3,4-dihydroxybenzaldehyde, syringaldehyde, p-hydroxybenzaldehyde, and vanillin in various plants. The effects of experimental conditions on MAE efficiency, such as solvent composition, temperature, extraction time, have been studied. The extraction efficiencies were compared with those obtained by computer-controlled, two-step Soxhlet-like extractions. Plant extracts were purified and phenolic compounds were pre-concentrated using SPE on polymeric RP-105 SPE sorbent prior to HPLC analysis. Chromatographic separation was carried out on a Hypersil BDS C₁₈ column using a mobile phase consisted of 0.3% (v/v) acetic acid in water (solvent A) and methanol (solvent B) at flow rate 0.6 ml min⁻¹ and column temperature 30 °C with gradient elution.     

Determination of pesticides in fruit and fruit juices by chromatographic methods. An overview

In order to combat a variety of pests, pesticides are widely used in fruits. Several extraction procedures (liquid extraction, single drop microextraction, microwave-assisted extraction, pressurized liquid extraction, supercritical fluid extraction, solid-phase extraction, solid-phase microextraction, matrix solid-phase dispersion, and stir bar sorptive extraction) have been reported to determine pesticide residues in fruits and fruit juices. The significant change in recent years is the introduction of the Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) methods in these matrices analysis. A combination of techniques reported the use of new extraction methods and chromatography to provide better quantitative recoveries at low levels. The use of mass spectrometric detectors in combination with liquid and gas chromatography has played a vital role to solve many problems related to food safety. The main attention in this review is on the achievements that have been possible because of the progress in extraction methods and the latest advances and novelties in mass spectrometry, and how these progresses have influenced the best control of food, allowing for an increase in the food safety and quality standards.     

Thin-layer chromatography applied in quality assessment of beverages derived from fruits

ABSTRACT

The analysis of food products is important for the assessment of food quality and authenticity, the control of a technological process, the determination of nutritional values, and the detection of compounds which could exert beneficial or toxic effects on human health. Techniques which are usually chosen for these purposes must provide accurate and reliable results, being relatively simple and inexpensive to perform. The aim of this paper is to give a brief overview of the thin-layer chromatography methods for the analysis of different compounds (polyphenols, dyes, carboxylic acids, biogenic amines, and vitamin C), used for quality assessment and authentication of non-fermented or fermented beverages derived from fruits, namely fruit juices and wines.     

Determination and quantification of phenolic compounds in methanolic extracts of Solanum ferrugineum (Solanaceae) fruits by HPLC-DAD and HPLC/ESI-MS/TOF

Solanum ferrugineum Jacq. is a wild species that in previous analysis reported a significant antioxidant capacity. The aim of our research was to determine total phenolic content (TPC) and total flavonoid content (TFC), and the phenolic composition by HPLC-DAD and HPLC/ESI-MS/TOF of methanolic extracts of S. ferrugineum fruits, collected from Paredones, Jiquilpan, and Fray Dominguez, Pajuacarán in the Mexican state of Michoacán. TPC and TFC were determined by the spectrophotometric Folin–Ciocalteu reagent and the AlCl₃ method, respectively. TPC in S. ferrugineum fruit [31.41?±?0.91?mg gallic acid equivalent/g dry tissue (DT)] was similar to those reported for Turkey berry (Solanum torvum Sw.) and eggplant (Solanum melongena L.) fruits. The TFC values of S. ferrugineum fruits (29.14?±?4.99?mg catechin equivalent /g DT) corresponded to 80.24% of the TPC. Eight phenolic compounds (PC) were identified by HPLC analysis. The main PC identified in S. ferrugineum fruits were chlorogenic acid, caffeic acid, p-coumaric acid, gallic acid, quercetin, and kaempferol. S. ferrugineum fruits could be used as a starting material for the extraction of high-value PC with potential applications.     

Choline Chloride–Lactic Acid-Based NADES As an Extraction Medium in a Response Surface Methodology-Optimized Method for the Extraction of Phenolic Compounds from Hazelnut Skin

Deep eutectic solvents (DESs) are promising green solvents for the extraction of compounds from food byproducts. Hazelnut (Corylus avellana L.) is one of the most commonly cultivated tree nuts worldwide. The skin represents one of the major byproducts of the hazelnut industry and accounts for 2.5% of the total hazelnut kernel weight. It is a rich source of phenolic compounds like flavan-3-ols, flavonols, dihydrochalcones, and phenolic acids. In this work, fifteen DESs based on choline chloride and betaine, with different compositions, were studied in order to test their phenolic compounds extraction efficiency through the determination of their total concentration via Folin–Ciocalteu assay. A qualitative analysis of extracted phenolic compounds was assessed by HPLC with UV and MS detection. Using the DES with the best extraction efficiency, a new ultrasound-assisted solid liquid extraction (UA-SLE) method was optimized though the response surface methodology (RSM), taking into account some extraction parameters. Efficient recovery of extracted phenolic compounds was achieved using a 35% water solution of choline chloride and lactic acid (molar ratio 1:2) as an extraction solvent, working at 80 °C and with a solid-to-solvent ratio of 1:25 gmL⁻¹. The optimized conditions made it possible to recover 39% more phenolic compounds compared to a classic organic solvent.     

Quantitative Analysis of Phenolic Acids in Extracts Obtained from the Fruits of Peucedanum alsaticum L. and Peucedanum cervaria (L.) Lap

Peucedanum alsaticum L. and Peucedanum cervaria (L.) Lap. are, in common with all species belonging to the Apiaceae family, rich in coumarins and essential oils. Phenolic acids also present in the plant are very important pharmacologically, because of their broad spectrum of biological activity. A simple high-performance liquid chromatographic method has been developed for separation and quantitative analysis of the major phenolic acids in extracts obtained from the fruits of P. alsaticum and P. cervaria. Soxhlet extraction, ultrasound extraction, and accelerated solvent extraction under different conditions were used to find the most efficient extraction conditions. Optimum chromatographic performance was obtained with a C₁₈ column and acetonitrile—1% (v/v) aqueous acetic acid as mobile phase. Ferulic, p-coumaric, caffeic, vanillic, syringic, p-hydroxybenzoic, protocatechuic, chlorogenic, and gallic acids were investigated in the fruits of the plants. For all calibration plots linearity was good (R ² > 0.9991) in the ranges tested. The highest yields of most of the phenolic acids were achieved by use of accelerated solvent extraction. The predominant phenolic acid in the fruits of both plants was chlorogenic acid. The amounts, which depended on the method of extraction, were approximately 146 ± 1.616 and 109.92 ± 3.405 mg per 100 g dry weight for P. cervaria and P. alsaticum, respectively.