Phenolic compounds HPLC analysis

Most papers dealing with phenolic acid HPLC analysis in herbs describe only simple liquid extraction without the hydrolysis step. Acetone, methanol, or alcoholic-water or acetone-water mixtures are applied. Very rarely, pure water is used as the extraction solvent. " It was found that the extraction recoveries for water extracts are often lower in comparison to alcoholic-water mixtures, especially when the simultaneous separation of polar and less polar phenolic acids has been performed. Sometimes, the control of pH can improve the recovery. If necessary, n-hexane, chloroform, diethyl ether, benzene-acetone, petroleum ether, or other less polar solvents are recommended for removing interfering compounds. The extraction is usually performed by refluxing the samples for a specific time in a Soxhlet apparatus, with simple mechanical or magnetic stirring of the sample with the extraction solvent, or by plant sample maceration. The application of an ultrasonic bath for the liquid extraction has also become popular in recent years. The hydrolysis steps have also been recommended for medicinal species preparation, especially when other phenolic compounds are also analyzed simultaneously with phenolic acids in herbs. 

Knowledge of the identity of phenolic compounds in food facilitates the analysis and discussion of potential antioxidant effects. Thus studies of phenolic compounds as antioxidants in food should usually by accompanied by the identification and quantification of the phenols. Reversed-phase HPLC combined with UV-VIS or electrochemical detection is the most common method for quantification of individual flavonoids and phenolic acids in foods (Merken and Beecher, 2000 Mattila and Kumpulainen, 2002), whereas HPLC combined with mass spectrometry has been used for identification of phenolic compounds (Justesen et al, 1998). Normal-phase HPLC combined with mass spectrometry has been used to identify monomeric and dimeric proanthocyanidins (Lazarus et al, 1999). Flavonoids are usually quantified as aglycones by HPLC, and samples containing flavonoid glycosides are therefore hydrolysed before analysis (Nuutila et al, 2002). 

Paganga, G. et al.. The polyphenolic content of fruit and vegetables and their antioxidant activities what does a serving constitute Free Radical Res., 30, 153, 1999. Maatta, K.R. et al.. High-performance liquid chromatography (HPLC) analysis of phenolic compounds in berries with diode array and electrospray ionization mass spectrometric (MS) detection Rihes species, J. Agric. Food Chem., 51, 6736, 2003. 

Ibern-Gomez M, Andres-Lacueva C, Lamuela-Raventos RM and Waterhouse AL. 2002. Rapid HPLC analysis of phenolic compounds in red wines. Am J Enol Vitic 53(3) 218—221. 

Tomas-Barberan FA, Gil MI, Cremin P, Waterhouse AL, Hess-Pierce B and Kader AA. 2001. HPLC-DAD-ESIMS analysis of phenolic compounds in nectarines, peaches, and plums. J Agric Food Chem. 49(10) 4748 t760. 

Among the numerous applications of SPE are separations of phenolic acids and flavonoids from wines and fruit juices. Sep-Pak Cig cartridges have been used for the fractionation of flavonol glycosides and phenolic compounds from cranberry juice into neutral and acidic parts before HPLC analysis. Antimutagenic flavonoids were identified in aqueous extracts of dry spinach after removal of lipophilic compounds by SPE. ... 

Stalmach A, Mullen W, Nagai C, Crozier A. On-line HPLC analysis of the antioxidant activity of phenolic compounds in brewed, paper-filtered coffee. Brazilian Journal of Plant Physiology. 2006 18(1) 253-262. 

In a polyphenolic extract, anthocyanins can interfere with other polyphenolics such as pro-cyanidins during HPLC analysis and hence should be removed prior to analysis. Anthocyanins from crude polyphenolic extracts can be removed as described in Basic Protocol 2. The ethyl acetate used for elution of phenolic compounds other than anthocyanins is removed using a rotary evaporator at 20°C. The non-anthocyanin polyphenolics are dissolved in deionized distilled water and the pH is adjusted to 7.0 with NaOH as described in Alternate Protocol 2 or the method developed by Oszmianski and Lee (1990a). In the latter method, polyphenolics were fractionated into three groups neutral fraction A (flavanols and other polar phenolics), neutral fraction B (flavonols), and acidic phenolics. Polyphenolic extracts were adjusted to pH 7.0 with NaOH... 

An accurate sample weight before extraction and the amount of final extract after sample cleanup should be known for an accurate quantification of phenolic compounds extracted from plant materials by HPLC analysis. The characteristic wavelengths for detection of polyphenolics can be selected at the discretion of the experimenter. The solvent gradients described in the Basic and Alternate Protocols can be modified for better resolution. 

Many excellent discussions of natural occurrence, structure, characterization, and analysis of phenolic compounds are available in the literature, and a series of books devoted to flavonoid chemistry has also been published. Detailed discussions on various chromatographic modes, including HPLC, GC, column chromatography (CC), capillary electrophoresis (CE), PC, and TLC, of simple phenolics and polyphenols are also presented in the recent book, Handbook of Food Analysis, volume 1, edited by Nollet (1). Due to their diversity and the chemical complexity of phenolic compounds, this chapter is limited to phenolic compounds that are considered to be important to foods and the food industry. 

The variety of detection modes available for HPLC analysis that provide additional information about the eluent as it exits the column greatly facilitates unknown characterization. The majority of analytical methods for phenolic compounds includes HPLC with spectrophotometric-based detection techniques (UV absorption, fluorescence, photo diode array—PDA) as well as HPLC with electrochemical detection. 

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