Sample hydrolysis phenolic acids

RP-HPLC has been employed for the determination of flavonoids and other phenolic compounds in cranberry juice. The neutral and acidic analytes were preconcentrated octadecyl silica SPE cartridges conditioned with distilled water (neutral analytes) or with 0.01 M HC1 (acidic compounds). Hydrolysis of samples was carried out in aqueous methanol solution acidified with 6 M HC1 at 35°C for 16h. Chromatographic separation was performed in an ODS column (150 X 4.6mm i.d. particle size 5/.an). Solvents A and B were water-acetic acid (97 3, v/v) and methanol, respectively. The gradient started with 0 per cent B (flow rate, 0.9 ml/min), reached 10 per cent B in lQmin (flowrate, 1.0 ml/min) and increased to 70 per cent B in 40min (flowrate, 1.0 ml/min). Analytes were detected at 280 and 360 nm. Some typical chromatograms are presented in Fig. 2.71. The concentrations of flavonoids and phenolic acids are compiled in Table 2.69. It was stated that the SPE-HPLC procedure makes possible the simultaneous determination of phenolic compounds and flavonoids, therefore, it can be employed for the measurement of these classes of analytes in other fruit juices [188],... 

A reversed-phase HPLC procedure was proposed for the determination of seven phenolic acids in green coffee samples (144). The sample preparation technique involved extraction, alkaline hydrolysis, and liquid/liquid extraction. The chromatographic separation was achieved us-... 

To analyze free-form phenolic acids in fmit or vegetable juice, the sample preparation is straightforward and simple. The juice can be directly injected into an HPLC system after it is filtered to remove any insoluble particles. However, for a sample with a solid fraction containing both free and bound phenolic acids, the sample preparation is not as simple. A mechanical method is needed to physically break down the sample and release the free phenolic acids, which are blocked in the inner core of the sample matrix. Chemical (acid or alkaline) or enzymatic hydrolysis must be applied to break down linkages in the bound phenolic acids to release free phenolic acids. However, the recovery of total phenolic acids is significantly affected by hydrolysis and other extraction conditions. An intensive hydrolysis condition may increase the release rate of bound phenolic acids to free phenolic acids however, it can also cause degradation of some phenolic acids and lower... 

In general, the sample preparation and extraction steps of phenolic acid analysis are very critical to the final result. Solvent or solution composition, extraction temperature, extraction technology, acid, alkaline, or enzymatic hydrolysis, extraction time, and cleanup conditions are all factors that affect the recovery and profile of phenolic acids. Poor sample preparation and extraction result in unreliable outcomes, regardless of the precision of the chromatography quantification method. Table 3.1 lists various sample extraction methods for different types of samples. 

An HPLC-DAD method was developed for the separation and the determination of flavonoid and phenolic antioxidants in commercial and freshly prepared cranberry juice.Two sample preparation procedures were used with and without hydrolysis of the glycoside forms of flavonoids carried out by the addition of HCl in the step prior to solid-phase extraction (SPE). The flavonoid and phenolic compounds were then fractionated into neutral and acidic groups via a solid-phase extraction method (Sep-Pak Cig), followed by a RP HPLC separation with gradient elution with water-methanol-acetic acid and a detection at 280 and 360 nm. A comparison of the chromatograms obtained for extracts prepared with and without hydrolysis showed that flavonoids and phenolic acids exist predominantly in combined forms such as glycosides and esters. In a freshly squeezed cranberry juice, for instance, 400 mg of total flavonoids and phenolics per liter of sample was found, 56% of which were flavonoids. Quercetin was the main flavonoid in the hydrolyzed products, where it accounted for about 75% of the total flavonoids, while it was absent in the unhydrolyzed products. 

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. 

HS-SPME conditions for analysis of TFSV (temperature, sampling time, pH of solution) were reported by Fedrizzi et al., (2007b). Increase of the pH matrix at a value of 7, performed in order to reduce interference in the analysis due to presence of hexanoic acid, can reduce the 3-mercaptohexyl acetate hydrolysis. Unfortunately, the antioxidant protection operated from S02 is also reduced (due to the shift of equilibrium towards the HSOj form) as a consequence phenolic acids can be easily oxidized forming the correspondent quinones (Singleton, 1987 Murat et al., 2003). They can, in their turn, oxidize thiols (Rigaud et al., 1991), even if with an opportunely delayed time as evidenced by Blancard et al. 

Other plants (cereals, seeds, rye, nuts, barley, malt) can also be extracted with organic solvents or their mixtures with water, but, in many cases, less polar solvents are also used for the elimination of pigments, oils, non-polar and macromolecular compounds. Many of these assays have also included acidic and/or alkaline hydrolysis steps, column liquid clean-up procedures, or SPE. The simultaneous separation of phenolic acids and other phenolic compounds in these samples also complicates the preparation step. Very often, multi-step liquid extraction and sample clean-up assays are recommended. It is necessary to select extraction solvents according to the polarities of the analytes and the form of their bonding to the sample matrix. Very often, both free and bound phenolic acids are separated in plant samples, and hydrolyzed and non-hydrolyzed materials are analyzed separately. In some cases, two or more hydrolysis methods are applied for the analyses of cereals and nuts. ... 

The nature of the association between membrane and teichoic acid is unknown, and it is possible that these teichoic acids are chemically attached to other components of the cell. Samples obtained by extraction with phenol appear to have appreciably higher molecular weight than has the purified teichoic acid obtained by extraction with trichloroacetic acid, and it is likely that the prolonged, acid treatment used in earlier work may have caused hydrolysis of some of the phosphodiester linkages. It is noteworthy that this comment on earlier studies does not apply to ribitol teichoic acids. Detailed examination of preparations of membrane teichoic acid obtained by less drastic conditions is highly desirable, in order to confirm the supposed size of the naturally occurring polymers, as well as... 

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