COOH
4. Separation- Separation-Detection
4.1. Thin-Layer Chromatography
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with a 5-min extraction at pH 4 and a temperature of 35◦C, which accommodates the need for absence of organic solvents (47).
A relatively new solid-phase extraction method used a molec-ularly imprinted polymer as the sorbent to determine quercetin in red wine (48). The recovery was over 98% when using methanol containing 15% acetic acid or acetonitrile containing 10% aque-ous triethylamine, as eluent. The molecularly imprinted polymer was proved to be highly selective for the target analyte enhanc-ing, at the same time, the intensity of the quercetin and reducing the complexity of the chromatographic trace. Another molecu-larly imprinted polymer was evaluated toward six phenolic acids extracting selectively the analytes from Melissa officinalis (49).
Matrix solid-phase dispersion is an alternative for sample preparation workable for liquid and semi-liquid samples. Sample extraction and cleanup are carried out simultaneously with, gen-erally, good recoveries and precision. Matrix solid-phase disper-sion is frequently used to determine pesticides in, e.g., foods, but application to flavonoid analysis was reported only recently. For the determination of isoflavone aglycones and glycosides in Radix astragali, this extractive cleanup step was compared to Soxhlet and ultrasonic extraction with respect to the extraction capacity (50). For the aglycones, matrix solid-phase dispersion yielded the best extraction efficiency but for the glycosides Soxhlet proved to be more efficient.
4.
Occurrence and Analysis of Phenolic Compounds 75
UV light at 350–365 nm or 250–260 nm or with densitometry, at the same wavelengths.
Quantification, generally, is not the main goal of TLC stud-ies. However, densitometry is used in several studies in this direc-tion. Kaempferol and quercetin were determined in an extract of Ginko biloba leaves by scanning the HPTLC silica plates in the reflectance mode at 254 nm (53).
Two-dimensional TLC on cyanopropyl-bonded silica was used to separate eight flavonoids and three phenolic acids in Flos sambuci L. (54). The first dimension relied on a normal-phase separation for which seven binary eluents were tested, and the sec-ond one provided a reversed-phase separation, studied by using three binary eluents. From amongst the 21 combinations, the three best ones all contained n-hexane in the first, and water in the second dimension. More than 12 spots were discerned and 9 flavonoids and 3 phenolic acids were identified in the Flos sambuci L. extract.
Special attention is to be drawn on the native fluorescence of flavonoids. Their fluorescence properties were examined apply-ing TLC separation with fluorodensitometric detection (55). The native fluorescence of 14 flavone and 26 flavonol type com-pounds was enhanced by their in situ reaction on the plate with 2-aminoethyl ester of diphenylboric acid.
4.2. Gas
Chromatography
The high separation capacity and compatibility, with various kinds of detectors, renders gas chromatography (GC) a technique to be taken into consideration seriously, even if the additional stage of analytes derivatization has to be included into the analytical procedure. Gas chromatography is an important chromatographic technique employed for the analysis of phenolic compounds. In particular, when combined with mass spectrometry it offers high sensitivity and selectivity.
Preparation of samples for GC may include the removal of lipids or proteins, depending on the nature of sample, liberation of phenolics from ester and glycosidic bonds by alkali, acid, and enzymatic hydrolysis.
The significant concern with phenolic compounds is that they are not directly amenable to analysis by gas chromatography.
In addition to sample extraction, isolation, and cleanup, anal-ysis requires a chemical modification step, often referred to as derivatization. Nonetheless, Christov et al. described flame ion-ization and electron capture as detection methods in the analysis of underivatized acids (56). Earlier work with derivatized pheno-lics was typically performed with flame ionization detection but mass spectrometry (MS) has become widespread. Most of the GC–MS work is performed in the electron ionization mode, with the ionization voltage set to a standard 70 eV. The spectra are collected up to m/z 650 in scanning mode.
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There are a variety of reagents used to modify and gener-ate volatile derivatives via converting hydroxyl groups to ethers or esters. Prior to chromatography, phenolics are usually trans-formed to more volatile derivatives by methylation, conver-sion to trimethylsilyl derivatives, or derivatization with N- (tert-butyldimethylsilyl)-N-methyltrifluoroacetamide.Typically, in GC, flavonoids are hydrolyzed and converted into their trimethylsilyl derivatives, injected onto non-polar columns in the split or split-less mode and separated with a linear 30–90 min temperature program up to 300◦C. The reaction involves dissolving the dried sample in a base (e.g., pyridine or ethylamine), addition of the trimethylsilyl reagent, and then heating the reaction vial up to 70◦C, for 20–120 min. In an endeavor to speed up the silylation procedure, Chu et al. reasoned that the heat transfer was a slow process and devised a microwave derivatization procedure cutting the time to 30 s (57).
Huˇsek made use of ethyl and methyl chloroformate for the formation of ethyl and methyl esters, respectively (58). Dimethyl sulfoxide with methyl iodide in an alkaline medium is another alternative to methylation. However, methyl esters can lead to some confusion, as they are naturally occurring in some plant-based material. There are many advantages to generating the silylated derivatives instead of other derivatizating agents. Phe-nols and carboxylic acids are relatively reactive and are functional groups susceptible to silylation. Both functional groups (acids and phenols) are derivatized in the same step. Moreover, many of the minor products or artifacts have been well described and doc-umented, are extremely volatile, and do not interfere with the analysis (59).
Plasma levels of catechin and its metabolite 3 -O-methylcatechin have been determined by GC–MS of the trimethylsilyl derivatives, after consumption of red wine (60).
Glucuronide and sulfate conjugates were determined after enzy-matic hydrolysis. In conventional GC, it is very difficult to ana-lyze flavonoid glycosides even after derivatization. Pereira et al.
used high-temperature–high-resolution GC–MS for the glucoside hesperidin, with columns that can withstand temperatures up to 400◦C (61).
Phenolics of propolis were identified and quantitated sub-sequent to their derivatization with N,O-bis-(trimethylsilyl)-trifluoroacematide by flame ionization and MS detection (62, 63). Flavonoid content originating from the leaf extract of Ginkgo biloba in human urine (64) and in pharmaceutical preparations (65) has been determined in extracts immediately and subse-quent to hydrolysis. One of the methods utilized gas chromatog-raphy – negative ion chemical ionization mass spectrometry of the trimethysilyl derivatives of the flavonoids. Hydrolysis of urine samples resulted in markedly higher quercetin and kaempferol
Occurrence and Analysis of Phenolic Compounds 77
content due to the fact that these two aglycones are present in the urine as glucuronides.
An improved derivatization procedure, by Stalikas et al., pro-posed an in-vial derivatization–extraction method for the GC–
MS analysis of methylated flavonoids and phenolic acids in various herbs (38). Derivatization takes place under basic con-ditions so that the hydroxyl groups of the analytes are depro-tonated. The anionic nucleophiles are transferred to the organic phase as ion-pairs using a phase-transfer catalyst and are next sub-jected to reaction with methyl iodide (66). Polymer-bound tri-n-butylmethylphosphonium chloride proved to be the best cata-lyst. In the selective-ion monitoring mode, good separation was attained (Fig. 5.4) and the limits of detection of the phenolics in the extracts were 4–40 ng/mL.
Fig. 5.4. The GC–MS(SIM) chromatogram of aMentha spicata fortified extract after derivatization with methyl iodide and phase-transfer catalysis. Peak assignment: (1)p-hydroxy benzoic acid, (2) trans-cinnamic acid, (3) homovanillic acid, (4) vanillic acid, (5) 2-hydroxy cinnamic acid, (6) 4-hydroxy cinnamic acid, (7). syringic acid, (8) ferulic acid, (9) naringenin, (10) galangin, (11) kaempferol, (12) luteolin, and I.S. internal standard (Reprinted with permission from Stalikas et al.
(38)).