Flavanones HPLC

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]. 

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. 

The evaluation criteria applied during database development highlighted a lack of acceptable anthocyanin food content literature. Values were often presented as percentage of the total anthocyanin content." " In addition, test samples were frequently gathered from noncommercial sources, such as horticultural research stations.Moreover, analytical procedures often employed spectral pH differential methodology rather than HPLC to estimate anthocyanin content. Consequently, although there is a substantial amount of characterization information with crude estimates of total anthocyanin content (T able 4.8), anthocyanins had to be excluded from the final database. Similarly, the flavanone eriodictyol was also excluded from the final database due to a lack of rigorously analyzed quantitative information. 

Chalcones are comparatively rare in foods. Naringenin chalcone is present in tomato skin and may be present in juice, paste and ketchup. Acid hydrolysis, commonly applied prior to HPLC, converts the chalcone to the corresponding flavanone (naringenin), which is naturally present only in trace amounts (2-15 mg/kg) in the tomato [23],... 

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. 

Table 5 Isocratic HPLC Analysis for Flavanone Glycosides HPLC condition...

Table 7 Gradient HPLC for the Simultaneous Analysis of Flavanone Glycosides and Polymethoxylated Flavones...

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,... 

Table 9.2 Selected HPLC analysis methods for eitrus flavanones. 

The current efforts in flavanone analysis are directed toward enhanced sensitivity, especially in samples such as human plasma. In addition, advancements in HPLC and LC-MS techniques are another area of focused research. Another area of active research in flavonoid analysis is the capillary electrophoresis/electrochromatography of flavonoids. During the past decade a growing number of publications have addressed the efficient use of capillary electrophoresis. However, there are a limited number of reports on the use of capillary electrochromatography in the analysis of flavanones. This technique needs further consideration to fully elucidate its potential in flavanone analysis. 

Cautela, D. Laratta, B. Santelli, F. Trifiro, A. Servillo, L. Castaldo, D. 2008. Estimating Bergamot juice adulteration of lemon juice by high-performance liquid chromatography (HPLC) analysis of flavanone glycosides. J. Agric. Food Chem. 56 5407-5414. 

Castillo, J. Benavente-Garcia, O. Del Rio, J.A. Study and optimization of Citrus flavanone and flavones elucidation hy reverse phase HPLC with several mobile phases Influence of the stmctural characteristics. J. Liq. Chromatogr. 1994, 17 (7), 1497-1523. 

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).

In Fig. 6 a characteristic HPLC chromatogram of lemon juice phenolics is shown. The main phenolics in lemon juice are flavanones and C-glucosylflavones. The small peaks at the beginning of the chromatogram correspond to hydroxycinnamic acid derivatives, which are minor constituents in lemon juice. 

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. 

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