Flavonols play an important role as antioxidants; for example, they protect ascorbic acid from autoxidation in juices and which can lead to juice discoloration. Although flavonoids are abundant in fruit, and fruits or beverages can be a significant source of dietary flavonoids, levels will vary depending on the varieties, environmental conditions, soil, and climatic factors. Berries are a good source of quercetin and its derivatives , whereas the most abundant dietary flavanone glycoside is hesperetin-7-O-rutinoside present in citrus fruits. Peterson et al. reported that the most prevalent dietary flavanone aglycones are naringenin, hesperetin, isosakuranetin, and eriodictyol. ,e same authors demonstrate that a citrus fruit is also a primary source of narirutin, eriocitrin, didymin, neohesperidin, naringin, hesperidin, neoeriocitrin, and poncirin. ,e ratio of these compounds to each other can vary. For example, narirutin and naringin were detected in grapefruit in high ratios, while the levels of hesperidin and narirutin in oranges and eriocitrin in lemons were even higher. In addition, some flavanone glycosides such as 7-rutinoside are tasteless, in contrast to neohesperidin , naringin, and hesperetin which have an intense bitter taste isolated from bitter oranges and grapefruit. Apigenin is another key flavone found in fruits, vegetables, spices, and herbs and is abundant in grapefruit, beverages, some vegetables, and herbal plants such as chamomile. Isoflavones are present in plants in the glycosylated forms but are converted to aglycone forms through the action of intestinal microflora. Isoflavones are detected commonly in legumes such as green beans, fava, and soybeans, and among them, container size for raspberries genistein -4H-1-benzopyran-4- one and daidzein -4H-1- benzopyran-4-one are the two major forms of dietary isoflavones and are consumed in soy products.

Fermented soy products also contain an additional seven isoflavone aglycones in significant levels. Due to the structural similarities to human hormone estrogen, isoflavones have potent estrogenic properties. Anthocyanins are another important class of flanovids that are colorful water-soluble glycosides and acylglycosides of anthocyanidins. 3-O-glycosides or 3,5-di-O-glycosides of malvidin, delphinidin, pelargonidin, cyanidin, petunidin, and peonidin are known as the most common natural anthocyanins and are classified based on the number and position of hydroxyl and methoxy groups. Anthocyanins are responsible for the brilliant colors of various plant parts including flowers and leaves and especially fruits having red, blue, purple colors, particularly strawberries, blueberries, black currants, cherries, raspberries, and red and purple grapes. Anthocyanidins are also responsible for the color of red wines. ,eir color based upon the degree of methylation and with pH is discrete from other phenolics by the range of colors each forms. Color differences of anthocyanins depend on the substitutions of the B ring, the pattern of glycosylation, and the degree and nature of esterification of the sugars with aliphatic or aromatic acids, and also on the pH, temperature, type of solvent, and the presence of copigments. Berries are a good source of anthocyanins, and 100 g of berries can provide up to 500 mg of anthocyanins. Flavan-3-ols have been previously reported as an antioxidant, chemopreventive, and immunoregulation agents. Procyanidins exist in a wide range of foods and often exist in foods in a range of galloylated forms. Most widely used techniques for phenolics are HPLC , LC/MS, GC , GC/MS, UV-Vis spectrophotometry, mass spectroscopy, electrochemical, and fluorometric methods. Liquid chromatography mass spectrometry is used to determine phenolics in both APCI and ESI techniques, ABTS+ and DPPH.

Sample preparation and extraction methods varied widely based on the nature of the sample matrix of the fruit or vegetable and based on the chemical structures of the phenolic compounds being extracted. As most samples contain a mixture of simple and complex polyphenolic compounds, such as phenolic acids, flavonoids, anthocyanins, and proanthocyanins, it is critical to choose a suitable method for sample preparation and extraction. Proteins, carbohydrates, lipids, or other elements may play negative effect to extraction of phenolics. In addition, it is not always possible to extract fresh samples, and special preparation techniques such as lyophilization, nitrogen pulverization, or drying may be needed. Particle size of extracted material and solvent-to-solute ratios need to be considered. As seen in Figure 1, there are many reliable qualitative and quantitative methods available for the measurement and characterization of the phenolic content in different natural products. Moreover, the success of these techniques will depend on the most effective sample preparation and extraction methods. Extraction efficiency is greatly influenced by solvent choice and composition and plays a critical role in the extraction yield of phenolics from fruits and vegetables. Generally, for the extraction of phenolics, water, acetone, ethyl acetate, alcohols , and their various percentages of mixtures are used. In addition to the solvent type extraction conditions, parameters such as temperature and duration also influence the yield of phenolics.Khoddami et al. previously reported that recovery of phenolics varied from one sample to another sample. It is also reported that acid- or base-catalyzed hydrolysis is also an important consideration for the stability of the phenolics in extracts. Davidov-Pardo and Marn-Arroyo reported that the extraction pH plays an important role in the extraction efficiency of phenolic compounds, and the same authors implied that catechins and their isomers are detected more efficiently in alkaline conditions as compared with acidic ones. Extraction of phenolic compounds are commonly done using either liquid-liquid or solid-liquid extraction technique. However, liquid-liquid extraction has some disadvantages because of using costly and potentially toxic solvents. For this reason, improved extraction methods such as solid-phase microextraction and solid-phase extraction techniques are used to extract phenolics from liquid samples.

In general, inexpensive and simple methods such as soxhlet, reflux, and maceration processes are the more conventional procedures used to recover phenolics from solid samples. In addition, ultrasound-assisted extraction , microwave-assisted extraction , ultrasound microwave-assisted extraction , supercritical fluid extraction , subcritical water extraction , and high hydrostatic pressure processing are the methods that help us to shorten extraction times and decrease the release of toxic pollutants through reducing organic solvent consumption and are relatively simple to perform. Pulsed electric field is also another extraction technique that can be applied at room temperature conditions and performed in a matter of seconds requiring low energy to increase cell membrane breakdown in mass transfer which were applied previously in several fruits such as strawberry and grapes.,e analysis of phenolic acids and flavonoids by liquid and/or gas chromatography techniques is the most widely and commonly applied methods for the quantification of phenolics in fruits and vegetables. In addition, spectrophotometric assays are used as nonspecific methods used for evaluating the levels of phenolics in many fruits and vegetables.Although, fruits differ in the quantity and types of phenolic antioxidants, degree of conjugation, and composition of sugar, total phenolic compounds can be estimated in fruits using the reagent proposed by Otto Folin and Vintila Ciocalteu and recently modified by Li et al.. ,is Folin–Ciocalteu method is robust, highly reproducible , convenient, and fast, requiring only a UV spectrophotometer. ,e method is typically standardized with either gallic acid, rutin, or a combination of pinocembrin/galangin. ,e Method is based on a reaction of the chemical reagent with phenolic electron transfer. ,e phenolic compounds are oxidized to phenolates by the reagent at alkaline pH in a saturated solution of sodium carbonate resulting in a blue molybdenum-tungsten complex and can be measured at 765 nm. ,e absorbance of each sample can be compared with those obtained from the standard curve, and the obtained data are expressed as µmol gallic acid equivalents per gram of fresh or dry matter. Because the reaction is quantitative and presumable, the analysis of a mixture of phenols can be recalculated based on any other standard. ,e assay comprises of monophenols and provides predictable reactions based on the phenols and provides measuring of all compounds readily oxidizable under the reaction conditions.Recently, chromatographic techniques such as highperformance liquid chromatography , HPLC electrospray ionization mass spectrometry , big plastic pots gaschromatography-mass spectrometry , capillary electrophoresis , and near-infrared spectroscopy techniques are developed for identification, separation, and quantification of phenolics. Phenolic content of plant materials can be measured and identified using HPLC employing different stationary phase-solvent combinations and various detectors. HPLC relies on comparisons of unknown compounds with standard reference compounds to make both qualitative and quantitative analytical measurements. Columns can be selected to impart specific separations based on the stationary phase type and the size and structure of the packing materials to which the stationary phase is bound to [52].

Detector choice can also be manipulated to enhance detection and especially quantifi- cation. Phenolic compounds can easily be measured using UV-Vis, photodiode array detection , fluorometric detection , and electrochemical detection . Each stationary phase-detector combination will provide specific information on the phenolic composition of a sample. For example, UV detection can be used to measure benzoic acid at 246–262 nm, gallic acid at 271 nm, and 275 nm for syringic acid. Two different wavelengths 225–235 nm and 290– 330 nm can be used to measure cinnamic acids, but the common wavelength of 280 nm is issued for the general analysis of phenolics. However, many factors such as sample purification, column and detector types, solvents used as mobile phase and solvent purity, and their pH affect HPLC analysis of phenolics. It is previously reported that mixtures of water, methanol, acetonitrile, formic and acetic acids, and trifluoroacetic acid are used for mobile phase for phenolic compounds in reversed phase chromatography using octadecyl silica columns. Generally, among the HPLC detectors, UV-Vis and DAD detectors are more common compared to the fluorometric detection . Common stationary phases include C18 RP columns employing an acidified mobile phase and ammonium acetate buffers of organic solvents . Detection efficiency can be improved by using SPE cartridges composed of styrenedivinylbenzene to purify phenolic compounds from crude extracts prior to HPLC analysis. ,e wavelength selected for monitoring phenolics is an important criterion and generally ranges between 190 and 380 nm. Gradient elution is generally preferred rather than isocratic elution. Some of the authors previously reported that phenolics such as flavonones, flavonoids, and flavan-3-ols of plum, blueberry, raspberry, strawberry, orange, apple, and tea are possible to be measured by common HPLC techniques. In general, for identification and quantification of phenolics, individual stock solutions of each standard are prepared in methanol and stored at −20°C until analysis. ,e working standard mixture solutions are made by diluting the appropriate amount of each stock standard solution to obtain at least 5 calibration levels. Measurements of flavanols, hydroxycinnamates, flavonols, and anthocyanins of fruits can be detected at 280, 320, 360, and 520 nm by using HPLC. External standards are used to quantify the phenolic compounds. Stable isotopes can also be used to quantify phenolic compounds when HPLC-ESI/MS is being used as described below.HPLC-ESI/MS is used to increase the range of phenolic compounds detected in a sample and to improve sensitivity as compared with standard chromatographic methods. HPLC-ESI/MS is a robust and selective quantification method that is effective at measuring the complex array of phenolics typically found in fruits and vegetables. Mass spectrometry methods can be performed on a variety of instruments including electrospray ionization ion trap instruments, triple quadrupole instruments , and time-of flight instruments . ,e mass spectrometer is an analytical detector that gives both qualitative and quantitative measurements based on separation of ions by their m/z ratio and 0.01% correction. Mass spectrometry involved three stages: ionization, mass analysis, and detection of ions. Separation of phenolic compounds is best achieved in aqueous-organic extracts of foods with HPLC prior to MS analysis although GC can also be used. ,e most common solvent reduction and ionization technique is electrospray ionization . ,is can be performed using different voltages to create negative or positive pseudomolecular ions that can be accelerated into the mass analyzer. ,e mass analyzer separates ions based on the flight path as with a magnetic /electric field separation, time-of-flight in a filed free region, or by altering ion trajectories using quadrupole and ion trap mass analyzers. Detection is usually achieved with an ion multiplier tube. Triple quadrupole analyzers and ion trap analyzers are often used when higher sensitivity and specificity, or structural information is required for identification. Fidelity of MS measurements can be increased using MS/MS techniques. For example, a common technique is to create product ions through collision-activated dissociation of selected precursor ions in the collision cell of the triple quadrupole mass spectrometer , analyzing the fragment ions in the second analyzer of the instrument .