HPLC flavonoids

Tomas-Barberan, F. A., Martos, I., Ferreres, F., Radovic, B. S., and Anklam, E. (2001). HPLC flavonoid profiles as markers for the botanical origin of European unifloral honeys. /. Sd. Food Agric. 81, 485-496. 

In many examples, with little regard of the sample type, the analyses of flavonoids are usually done in the reversed-phase HPLC mode using nonpolar Cig (in few cases, Cg) columns and polar mobile phase (mixed aqueous-organic solvents) due to their structural and physicochemical properties. Conversely, with more regard of the sample type, different extractions and sample pretreatments including Soxhlet, LLE, SPE, USAE, ASE, and SEE have been used prior to HPLC flavonoid analysis. 

As relatively few standard compounds are available from commercial or other sources, identification of flavonol glycosides has to be achieved by alternative means, for example UV-, H- and C-NMR spectroscopy. Therefore hydrolysing all glycosides to aglycones followed by HPLC determination offers a practical method for the quantitative determination of flavonoids in tea (Hertog et al, 1993a Wang and Helliwell, 2001). 

Knowledge of the identity of phenolic compounds in food facilitates the analysis and discussion of potential antioxidant effects. Thus studies of phenolic compounds as antioxidants in food should usually by accompanied by the identification and quantification of the phenols. Reversed-phase HPLC combined with UV-VIS or electrochemical detection is the most common method for quantification of individual flavonoids and phenolic acids in foods (Merken and Beecher, 2000 Mattila and Kumpulainen, 2002), whereas HPLC combined with mass spectrometry has been used for identification of phenolic compounds (Justesen et al, 1998). Normal-phase HPLC combined with mass spectrometry has been used to identify monomeric and dimeric proanthocyanidins (Lazarus et al, 1999). Flavonoids are usually quantified as aglycones by HPLC, and samples containing flavonoid glycosides are therefore hydrolysed before analysis (Nuutila et al, 2002). 

NUUTILA A M, KAMMioviRTA K and OKSMAN-CALDENTEY K-M (2002) Comparison of methods for the hydrolysis of flavonoids and phenolic acids from onion and spinach for HPLC-analysis, Food Chem, 76, 519-25. 

Just as with anthocyanin analysis, the advent of HPLC/mass spectrometer coupling made it possible to avoid acid hydrolysis for flavonoid identification. Maata... 

Ferreres, F., Tomas-Barberan, F. A., Soler, C., Garcia-Viguera, C., Ortiz, A., and Tomas-Lorente, F. (1994f). A simple extractive technique for honey flavonoid HPLC analysis. 

D. Cristea, I. Bareau and G.Vilarem, Identification and quantitative HPLC analysis of the main flavonoids present in weld (Reseda luteola L.), Dyes and Pigments, 57, 267 272 (2003). 

I. Surowiec, B. Szostek and M. Trojanowicz, HPLC MS of anthraquinoids, flavonoids, and their degradation products in analysis of natural dyes in archeological objects, J. Sep. Sci., 30, 2070 2079 (2007). 

Procedure Flavonoids are then further purified with 2 ml of methanolic HC1 (2 N), followed by centrifugation (2 min, 15 600 g), hydrolyzation of 150 il of suspension in an autoclave (15 min, 120 C). A reverse osmosis-Millipore UF Plus water purification system is used in high performance liquid chromatography (HPLC) with an autosampler. After injections of 5 pg of samples, the mobile phases flow at a rate of 1 ml/minute with isocratic elution in a column at 30 C. 

Pietta PG, Mauri PL, Manera E and Ceva PL. 1989. HPLC determination of the flavonoid glycosides from Betulae folium extracts. Chromatographia 28 311—312. 

The leaf flavonoids of the cruciferous species such as Camelina sativa, Crambe abyssinica, Crambe hispanica, Thlaspi arvense, Brassica napus and Sinapis alba were separated and identified with the combination of HPLC, TLC and paper chromatography. Llavonoid aglycones were extracted by cutting fresh three-week-old leaves in tiny pieces and boiled in 50 ml of 2 M HC1 for 45 min. 

The flavonoid content of the tinctures of Calendula officinalis L., Passiflora incarnata L and Silybum marianum (L.) Gaertn. was investigated by HPLC-DAD and HPLC-MS. The anti-inflammatory effect, and the beneficial influence to treat hepatic injuries, tension... 

The high sensitivity of hyphenated techniques such as HPLC-MS has also been exploited in the identification and structural studies of flavonoid glucosides [151] and the application of other hyphenated techniques such as LC-MS-MS and LC-NMR for the analysis of plant constituents has been discussed earlier [152],... 

RP-HPLC found application in the separation and identification of the main flavonoids in weld (.Reseda luteola L.). The aerial parts of the weld were dried, ground and extracted with various solvents and solvent mixtures such as methanol, ethanol, water, methanol-water... 

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... 

PRESENCE OF FLAVONOIDS IN SPECIES. SUBSPECIES AND VARIETIES OF LAVANDULA AND SABAUDIA BASED ON HPLC. PC AND ELECTROPHORESIS RESULTS. NUMBERS REFER TO FLAVONOIDS IN THE TEXT... 

Extract subjected to electrophoresis —, flavonoid not detected by HPLC , +, ++ relative amounts of flavonoids present (low, medium, or high UV absorbance of the flavonoid peak, respectively. Reprinted with permission from T. M. Upson et al. [155]. 

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