Flavonoid hydrolysis

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... 

Identification of flavonoids Quantification of individual flavonoids depends heavily on the availability of standard references. Only a limited number of common flavonoids are commercially available as standards. Standard references for flavonoid glycosides are particularly difficult to find thus direct quantification of the native glycosides is nearly impossible. Analysis of the aglycones after acid or enzymatic hydrolysis is therefore common practice. When standard flavonoids are not available, or when unknown compounds are encountered in a particular fruit or vegetable, use of a DAD... 

Flavonoid aglycones (isolated by acid hydrolysis of the corresponding flavonoid glycosides)... 

Fig. 2.55. Gradient reversed-phase HPLC analysis of flavonoids in white onions (a) and celery (b). ODS column of 150 X 3.9mm i.d particle size 5pm. Mobile phase 20min gradient of 15-35 per cent acetonitrile in water adjusted to pH 2.5 with TFA. Fowrate lml/min. Upper and lower traces represent samples before and after hydrolysis, respectively. Detection wavelength 365 nm. IS = internal standard Qc = quercetin Ap = apigenin Lt = luteolin. Reprinted with permission from A. Crozier et al. [159],...

Because of the importance of soybean and soybean products in both human and animal nutrition their flavonoid content has been investigated many times. Thus, HPLC-UV and HPLC-MS have been applied for the determination of flavonoids and other phytochemicals in soybean extracts and in onion with and without hydrolysis, lg of onion was homogenized and mixed with 8 ml of methanol-water (8 2, v/v). After 2h the suspension was centrifuged at 4°C and the supernatant injected. Powdered soybean (500 mg) was defatted by 2 X 10 ml of hexane and further treated as the onion sample. Flavonoids were hydrolyzed by mixing 2 ml of extract with 2 ml of 2 M HC1 and heated to 130°C for 2 h. The solution was neutralized with 4 M of NaOH. Separation was performed in an ODS column (125 X 4.6 mm... 

RP-HPLC has been employed for the determination of flavonoids and other phenolic compounds in cranberry juice. The neutral and acidic analytes were preconcentrated octadecyl silica SPE cartridges conditioned with distilled water (neutral analytes) or with 0.01 M HC1 (acidic compounds). Hydrolysis of samples was carried out in aqueous methanol solution acidified with 6 M HC1 at 35°C for 16h. Chromatographic separation was performed in an ODS column (150 X 4.6mm i.d. particle size 5/.an). Solvents A and B were water-acetic acid (97 3, v/v) and methanol, respectively. The gradient started with 0 per cent B (flow rate, 0.9 ml/min), reached 10 per cent B in lQmin (flowrate, 1.0 ml/min) and increased to 70 per cent B in 40min (flowrate, 1.0 ml/min). Analytes were detected at 280 and 360 nm. Some typical chromatograms are presented in Fig. 2.71. The concentrations of flavonoids and phenolic acids are compiled in Table 2.69. It was stated that the SPE-HPLC procedure makes possible the simultaneous determination of phenolic compounds and flavonoids, therefore, it can be employed for the measurement of these classes of analytes in other fruit juices [188],... 

There have been many survey studies of citrus flavonoids, yet by no means has an exhaustive analysis been performed for all species and cultivars. Surveys often are initiated by hydrolysis of tissue extracts and identification of the flavonoid aglycones present in the tissues. This provides a basis for subsequent identification of the glycosylated compounds present... 

Sample extraction and hydrolysis details e.g., solvent extraction after freeze drying, with optimized acid or enzymatic hydrolysis Preparation of flavonoid standards and use of internal standards Chromatographic separation and detection method used, ideally RP-HPLC with UV or fluorescent detection Outline of quality assurance procedures employed... 

Bokkenheuser, V.D., Shackleton, C.H., and Winter, J., Hydrolysis of dietary flavonoid glycosides by strains of intestinal Bacteroides from humans, Biochem. J., 248, 953, 1987. 

It seems that overall percentage of absorption, determined by measuring plasma levels of flavonols after enzymatic hydrolysis, does not exceed 2-3% of the ingested dose. It is also likely that, as with other micronutrients, the existence of a steady-state concentration of these compounds could result in diminished absorption. Thus, it is conceivable that the major parts of these flavonoids are either degraded to phenolic acids in the large intestine or excreted in the faeces [72]. 

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