COOH
4. Separation- Separation-Detection
4.4. Capillary Electrophoretic and
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recent study on the flavonoid constituents of a red clover extract, stopped-flow LC-NMR and stand-alone NMR were used to iden-tify structural isomers that could not be distinguished on the basis of MS/MS information (86). A recently developed cryoflow NMR probe exhibits detectability about fourfold better than with conventional probes or, alternatively, the scan time is 16-fold shorter for the same amount of sample. The probe has been applied for the analysis of an oregano extract where five flavonoids were identified using an LC–UV–solid-phase extraction–NMR–
MS setup (87).
Other less common means of detection, coupled to LC, have been through refractive index and evaporative light scattering detection. The latter offers freedom from some of the limitations of spectroscopic detection because it is not limited to compounds that contain UV-absorbing chromophores and it is immune to mobile-phase variations and gradient baseline shifting. Although being less sensitive than those previously described, both of the detectors have been successfully used: the HPLC-refractive index system in the quantification of (3,3,4,4,5,7-hexahydroxyflavan) in unripe banana pulp (88) and the HPLC-evaporative light scattering detection in the determination of Radix Astragali flavonoids (89).
4.4. Capillary
Occurrence and Analysis of Phenolic Compounds 83
separation of flavonoid-7-O-glycosides in the presence of sodium dodecyl sulfate under neutral conditions (pH 7.1) proved to be the optimum.
Huang et al. compared the microemulsion electrokinetic chromatography with MEKC methods for the analysis of phe-nolic compounds (95). A higher voltage and a higher column temperature improved the separation efficiency, without any noticeable reduction in resolution, for microemulsion electroki-netic chromatography, although they caused a poor resolution for the MEKC system.
Other authors used an amperometric system based on the end-column wall-jet configuration, in which the working elec-trode is placed at the outlet of the separation capillary (96). This configuration allows the use of normal size (i.e.,>100-m diam-eter) working electrodes without introducing significant post-capillary zone broadening which could jeopardize separation effi-ciency.
A handful of papers discuss the use of CE–MS for the deter-mination of flavonoids and phenolic acids (97). In the CE–ESI–
MS study by Lafont et al., a standard mixture of eight phenolic compounds was analyzed (98). With selective-ion monitoring–
MS the authors were able to identify all eight compounds based on their retention times and characteristic fragment ions ([M−H]−, loss of CO, CO2, and CH3) and obtained LODs of 0.1–40g/L. Huck et al. developed a CE method for the analysis of a flavonoid mixture consisting of 5-methoxyflavone, biochanin A, hesperetin, and naringenin obtained from plant extracts (99).
carrier electrolyte based on ammonium acetate and a bare fused silica capillary suitable for coupling to a quadrupole MS were selected. Optimization of parameteric values for both CE and MS provided detection limits comparable to UV detection and con-clusive proof-of-application was produced by determining narin-genin in a phytomedicine prepared from five different herbal drugs (Fig. 5.6). The authors concluded that the selectivity of UV detection was much too low to enable reliable quantitative analysis in this complex sample. Although CE–MS is not a tech-nique that will replace others, it can be made into a complemen-tary analytical tool for the analysis of phenolics.
4.5.
Spectrophotometric Methods
The development of a satisfactory ultraviolet spectrophotomet-ric assay is a rather cumbersome and difficult task and highly dependent on the material to be analyzed. In addition, the char-acteristics of absorption spectra of phenolics are affected by the nature of the solvent and the pH of the solution (100). Visible spectroscopic techniques are often used for the identification of isolated phenolic compounds, particularly flavonoids to iden-tify the presence of groups of predominant phenolic compounds (101, 102). The Folin–Denis assay is the first and most widely used rapid reaction procedure for the quantification of total
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Fig. 5.6. CE–ESI–MS of naringenin in a phytomedicine prepared from five different herbal drugs: (a) total ion current (TIC) and (b) single-ion monitoring trace (SIM). Capil-lary, fused silica, 60 cm, 50m I.D.; buffer, 40 mM ammonium acetate (pH 9.5), 15%
acetonitrile (v/v); detection, negative mode (Reprinted with permission from Huck et al.
(99)).
phenolics in plant materials (103). The method relies on the reduction of phosphomolybdic–phosphotungstic acid (Folin–
Denis) reagent to a blue colored complex in an alkaline solution.
The generated phosphomolybdic–phosphotungstic–phenol com-plex gives an easily detected absorbance at 760 nm. The Folin–
Ciocalteu assay is also used for the determination of the total content of plant food phenolics (104, 105). Neither of the above-mentioned is specific as they detect all phenolic groups; besides, they suffer from interference from reducing agents such as ascor-bic acid.
The vanillin method is specific for flavan-3-ols, dihydrochal-cones, and proanthocyanidins which have a single bond at the 2,3-position and possess free meta-hydroxy groups on the B-ring (106). This is the reason that this assay is mainly recognized as a useful method for the detection and quantification of proantho-cyanidins in plant materials.
The complexation of phenolics with Al(III) has been reported for the development of spectrophotometric methods to deter-mine of total caffeic acid, total flavonoids, and total tannins at 425 nm (107, 108). The method is based on the formation of a complex between the aluminum ion, Al(III), and the carbonyl and hydroxyl groups of the flavonoid.
Occurrence and Analysis of Phenolic Compounds 85
For the quantification of flavanones and dihydroflavonols, the spectrophotometric method based on the interaction of these compounds with 2,4-dinitrophenylhydrazine in acidic media (sul-furic acid) to form colored phenylhydrazones has been reported (109). The absorbance was measured at 486 nm.
Quantification of anthocyanins takes advantage of their char-acteristic behavior in acidic media. The analytical procedure for quantification of anthocyanins was first developed by Sondheimer and Kertesz (110) and modified by Swain and Hillis (111) who suggested expressing the concentration of pigments in terms of the change in the absorbance at λmax between pH 3.5 and pH≤ 1.0.
Finally, Schulz et al. (112) used a near-infrared reflectance spectroscopic method for prediction of polyphenols in the leaves of green tea (Camelia sinensis L) while Edelmann et al. (113) developed a rapid method of discrimination of Austrian red wines based on mid-infrared spectroscopy of phenolic extracts of wine.