Flavanones HPLC separation

It has been stated that the extraction, hydrolysis, and RP-HPLC separation method is specific and sensitive for the analysis of flavonols, flavones and flavanons. The data can be used for the estimation of the daily intake of these compounds [187]. 

Fig. 12 HPLC separation of flavanone glycosides in grapefruit juice and orange juice.

Fig. 13 HPLC separation of flavanone glycosides and polymethoxylated flavones (PMFs) in orange juice spiked with didymin and PMFs.

An HPLC separation method with diode array detector and mass spectrometric (MS) detection equipped with atmospheric pressure ionization (API) was developed to determine flavone, flavonol, and flavanone in various vegetables, including green bean, broccoli, brussels sprouts, celery, kale, leek, onion, parsley, pepper (green, yellow, and red), and tomato (118). The flavonoids were analyzed as aglycones after acid hydrolysis. The extraction and acid hydrolysis conditions are based on previous work by Hertog et al. (119). Quercetin is the overall major flavonol, followed by kaempferol. The flavones, apigenin and luteolin, were found only in limited foods,... 

Various analytical methods exist for flavonoids. These range from TLC to CE. With the introduction of hyphenated HPLC techniques, the analytical potential has been dramatically extended. Gas chromatography (GC) is generally impractical, due to the low volatility of many flavonoid compounds and the necessity of preparing derivatives. However, Schmidt et al. ° have reported the separation of flavones, flavonols, flavanones, and chalcones (with frequent substitution by methyl groups) by GC. 

For the HPLC of flavanone glycosides in citrus, isocratic elution is often preferred because of the simplicity of the major flavanone glycosides occurring in citrus, and the flavanone glycosides are also present in citrus juice at fairly high levels. Furthermore, isocratic separations require no re-equilibration time between analyses and therefore are sometimes faster when only a few components are to be analyzed. 

Fig. 2 High-performance liquid chromatographic profile of a standard solution at 280, 320, and 350 nm. Separation was achieved with an analytical HPLC unit (Gilson), using a reversed-phase Shepherisorb ODS2 (25.0 x 0.46 cm 5 pm particle size) column. The solvent system used was a gradient of water/formic acid (19 1) (A) and methanol (B). The gradient was as follows 0 min, 30% B 15 min, 30% B 20 min, 40% B 30 min, 45% B 50 min, 60% B 53 min, 100% B and 55 min, 100% B. Detection was accomplished with a DAD. 1—Gallic acid (hydroxybenzoic acid) 2—caffeic acid (hydroxycinnamic acid) 3—mangiferin (xanthone) 4—ferulic acid (hydroxycinnamic acid) 5—eriodictiol (flavanone) 6—hemiarin (coumarine) and 7—quercetin (flavonol).

Only one major compound (SF-X) was present in the estrogenically active fractions, and this was isolated using semipreparative HPLC. The principles of separation are the same as for qualitative HPLC (see Section 25.4.1.2), with the difference that higher amounts of material can be loaded onto the ODS-column (Alltech, Econosil, Cig lOp, 250x22mm). For the identification of SF-x, a combination of spectroscopic techniques was used. Electrospray ionization in the mass spectrometer (HPllOO LC/MSD, Hewlett-Packard) with positive ionization mode gave a pseudo-molecular ion with m/z=439. H-NMR, C-NMR, DEPT, HMQC, and COSY spectra were recorded on a Varian-300 (300MHz) spectrometer. Analysis of the COSY spectram showed the presence of a lavandulyl (5-methyl-2-isopropenyl-hex-4-enyl) side chain. From the HMQC spectrum and a DEPT experiment, it appeared that SF-x possesses a disubstituted flavanone skeleton. 

HPLC is the method of choice for the separation of complex mixtures containing non-volatile compounds such as various flavonoids in extracts prepared from different samples. A survey of literatures revealed that most researchers have used Cjg-reversed stationary phases, which proved to be superior to the normal phase technique. The reversed phases are suitable for separating flavonoids in a wide range of polarities, as Vande Casteele et al. have demonstrated the separation of 141 flavonoids from polar triglycosides to relatively non-polar polymetoxy-lated aglycones belonging to the classes of flavones, flavonols, flavanones, dihydroflavonols, chalcones, and dihydrochalcones. 

HPLC-UV-NMR is a powerful technique for the identification and characterization of flavonoids. However, there are drawbacks, as NMR remains rather insensitive because of the need for solvent suppression, which has restricted the observable NMR range. Recently, two major research developments in HPLC-UV-NMR are post-column solid-phase extraction (HPLC-UV-SPE-NMR) and combination of HPLC-UV-SPE with capillary separations and NMR detection [89]. A post-column treatment of analyte focusing and multiple trapping through a SPE has solved the problem of sensitivity and solvent suppression. The separation and elucidation of three C-methylated flavanones and five dihydrochalcones from Myrica gale seeds have been achieved by HPLC-DAD-SPE-NMR and... 

The resolved polysubstituted flavanones have two hydroxyl groups in positions 5 and 7 with the exception of the 7-methoxy substituted sakuranetin. However, this last compound has a—OH group in position 4, which could justify the different behavior on MCTA with respect to the not resolved pinocembrin-7-methylether. A comparable behavior was observed on MCTA columns for polysubstituted flavanones [24], In particular, the comparison of separation factors (a) obtained on MCTA plates with those on MCTA columns, using similar water-alcohol mixtures as mobile phases, evidenced lower a-values on TLC (ranging from 0.4 to 0.8) with respect to those observed on HPLC, except for sakuranetin for which similar a-values (1.22) were obtained by both techniques [3]. 

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