Flavonoids detection systems

Negative atmospheric pressure chemical ionization (APC) low-energy collision activation mss spectrometry has also been employed for the characterization of flavonoids in extracts of fresh herbs. Besides the separation, quantitative determination and identification of flavonoids, the objective of the study was the comparison of the efficacy of the various detection systems in the analysis of flavonoids in herb extracts. Freeze-dried herbs (0.5g of chives, cress, dill, lovage, mint, oregano, parsley, rosemary, tarragon and thyme) were ground and extracted with 20 ml of 62.5 per cent aqueous methanol. After sedimentation the suspension was filtered and used for HPLC analyses. Separations were carried out in an... 

As regards the sensitivity of the MS detection in HPLC analysis of flavonoids, this technique has proved to be the most sensitive as compared to UV and fluorescence detection. A very comprehensive comparison of the four detection systems—UV, fluorescence, and two MS systems [atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI)]—for the determination of the previously identified 3, 4, 5 -trimethoxyflavone is presented in Table 1. Fluorescence detection is 10 times more sensitive than UV detection, whereas MS detection is 50 times more sensitive than UV detection and 5 times more sensitive than fluorescence detection. 

Electrochemical detection is very sensitive for the compounds that can be oxidized or reduced at low-voltage potentials. Therefore, it could also be applied in the HPLC analysis of phenolic acids that are present in natural samples at very low concentrations. With the recent advances in electrochemical detection, multi-electrode array detection is becoming a powerful tool for detecting phenolic acids and flavonoids in a wide range of samples. The multi-channel coulometric detection system may serve as a highly sensitive way for the overall characterization of antioxidants the coulometric efficiency of each element of the array allows a complete voltametiic resolution of analytes as a function of their reaction (redox) potential. Some peaks may be resolved by the detector, even if they are unresolved when they leave the HPLC column. ... 

Uquid chromatography (HPLC) is estabUshed as the most convenient method which enables separation and identification of flavonoids using various detection sys-tems. As for the quantitative analysis, much data have been published in the last few years confirming the suitability of this technique for simultaneous determination of flavonoid compounds in various samples, which gives an insight into the distribution of flavonoids in the studied material. HPLC methods are developed for qualitative and quantitative analyses of flavonoids in fiuits and beverages, wine, honey, propolis, and, especially, in various plant materials " " using different detection systems, from which UV diode array detectors are settled as the most suitable for these compounds and the most accessible as well. 

Some examples of more flavonoid analyses by HPLC have been selected and detailed as presented in Tables 69.3-69.5. These examples are divided into plant (Table 69.3), food (Table 69.4), and biological (Table 69.5) samples. Notably, these examples are mainly from work published from 2008 onward, except for three papers in 2006 and 2007. These examples have been the selected ones due to the research article details including good representation(s) of chromatogram(s) to assist other researchers to easily validate their studies. These examples show the application of this chapter s aforementioned types of extractions, separations (in terms of column chemistry, dimensions), and the detection systems. 

The most common detection systems used to quantify these compounds are U V absorbance, fluorescence, and MS detection. MS is the detection system that is gaining major attention in the most recent reports [59], especially MS/MS (tandem mass spectrometry) systems, since they are the most important technique for the identification of flavonoids and the structural characterization of unknown member of this class of compounds [60]. 

Flame ionization detectors (FID), 171,361-372 applications of TLC-FID systems, 369 coated rod systems, 366-369 tubular systems, 365 TLC-FID systems, 362-369 coated rod systems, 364-369 tubular systems, 362-364 Flame diermionic ionization detector (flll>), 369 Flat-bed chromatography, 3 Flavonoids, 717-722 distribution, 717 practical experiments, 719-722 structure, 717 TLC of, 718-719 Fluorescence, 939 measurement of, 208-210 for quantitative determination in TLC, 281-287 Fluorgenic detection Q>esticide detection system), 810-811 Folic acid, 1051 Food additives, 803 Food dyes, 1006-1014 Foodstuff dyes, 1004... 

After TMS derivatization, flavonoids are injected onto a nonpolar capillary column in the split or splitless modes, and separated with a lineal 30-90 min temperature program up to 320°C. MS detector with positive electrospray ionization (ESI) and a temperature source of up to 250°C is the most common detection system used for the identification of flavonoids in the selected-ion monitoring (SIM) mode (Table 3.6). The molecular ion, [M-l-H], and fragment ions formed by the loss of CH3 or CO, as well as retro-Diels-Alder (RDA) reaction are typically used for identiflcation. 

The detection systems that are described in this section can be coupled to the different instruments where separation of flavonoids is performed. 

Although UV-Vis detector is the most widespread detection system, fluorescence or electrochemical detectors have also proven adequate for flavonoid analysis. However, the lack of active fluorescent or electrochemical groups in many flavonoids may affect the accuracy of these detection methods. MS is a powerful, highly sensitivity technique. Its use is spreading among food scientists because of its effectiveness in the identification of flavonoids and their applicability for quantitative analysis. Common sources of error in flavonoid analysis may derive from nonlinearity of the detector, which should be checked since many detectors are linear over only one or two decades. In addition, the different response factors of different flavonoids should be taken into account since detectors usually are not equally sensitive to aU components of a mixture. 

The primary limitation associated with GC/MS is the need for derivatization. Derivatization introduces additional complexity to the system and is not 100% efficient. Inefficient reactions result in the presence of multiple derivatized forms of the same compound. For example, we can detect three different derivatization products of the amino acid asparagine (mw = 132) in M. truncatula roots (Fig.3.4). These include asparagine, N,0-TMS (mw = 276), asparagine, N,N,0-TMS (mw = 348), and asparagine, N,N,N,0-TMS (mw = 420). Inefficiency of the derivation reactions also limits the lower concentration range of analytes that can be profiled. Finally, derivatization is not capable of achieving volatility for all compounds, such as many of the flavonoid glycosides. If derivatization is successful and the analyte is... 

A new poly(7-oxobomene-5,6-dicarboxylic acid-Wod -norbomene)-coa(cd silica has been synthesized and applied for the separation of flavonoids in model systems and in the extracts of onion, elder flower blossom, lime blossom, St. John s Wort and red wine. Separation was performed in a (150 X 4 mm i.d. particle size 7 /rm) column at room temperature. Flavonoids (quercitrin, myricetin, quercetin, kaempferol and acacetin) were separated with gradient elution water-ACN (20 mmol TFA) from 78 22 to 70 30 v/v in 3min. The flow rate was 2 ml/min. The separation of the standard mixture is shown in Fig. 2.51. It has been stated that the method is rapid, accurate and the MS detection makes possible the reliable identification of flavonoids [153],... 

Solvent systems used for thin layer chromatography were 1) n-butanol acetic acidiwater (4 1 5 upper phase), 2) acetic acid water (15 85), 3) ethyl acetate pyridine water (12 5 4), and 4) chloroform acetic acid water (50 45 5). Silica gel plates were used for chromatography of flavonoid aglycones and cellulose plates for all other components. Aluminum chloride was used for detection (under long UV light) of flavonoids, aniline phthalate for sugars, ninhydrin for amino acids and iodine for other components. Cellulose thick layer plates were developed with solvents 1 or 2. 

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