A standard addition method to assay the concentration of biologically interesting polyphenols in grape berries by reversed-phase HPLC

A reversed-phase HPLC method which allows the simultaneous assay of (+)- catechin, (-)-epicatechin, trans-resveratrol, quercetin and quercetin glycosides in grape berries is described. Kromasil 100 served as stationary phase and a gradient of acetic acid, water and methanol was used. The analytical run requires 42 min for complete sample elution. Satisfactory peak resolution was achieved following a novel extraction process and direct injection of a 20 microL sample. The method was used for the analyses of eighteen samples. Linearities were in the range of 0.98 to 0.999 regression coefficient, for all phenolics, while detection limits ranged from 30 microg mL(-1) for trans-resveratrol to 1.5 mg mL(-1) for (+)-catechin. Recoveries ranged from 95.1 to 98.7% while the method provided good precision, with standard deviations between 3.5 and 6.1%, n=5.     

Standardisation in reversed-phase HPLC

25 Years after Kovats proposed the use of a retention index scale to standardise the reporting of gas-liquid chromatographic separations there has been a growth of interest in methods for the standardisation of high-performance liquid chromatography. A number of systems have been suggested based on internal standards or retention indexes. These methods can also provide a basis for the classification of columns and eluents.     

Study of polyphenols in grape berries by reversed-phase high-performance liquid chromatography

Several polyphenols have been tested in grape berries from Spain. The flavonoid content is important because of the pharmacological properties of these compounds, whereas resveratrol has been proved to be an antifungal, antiinflammatory and an anticarcinogenic compound. A reversed-phase HPLC method has been developed and applied to determine resveratrol, quercetine, quercitrine and rutine content in several grape berries samples in a single analysis. Covering the grapes with a preservative paper yields a healthier product, but one which has a lower polyphenol content than unprotected grapes.     

Analysis of nucleotides,nucleosides, nucleobases in cells by ion-pair reversed-phase HPLC

Summary HPLC methods for the separation of nucleotides, nucleosides and nucleobases by ion-pair reversed-phase are reviewed. The advantages of these are discussed versus anion-exchange and reversed phase separations. Extraction procedures for nucleotide determinations from cells and tissues are pointed out in detail. Extracts from red blood cells, Ehrlich ascites tumour cells, hepatocytes, intestine are used for determination of nucleotide concentrations by the methods described.     

Solvent-strength selectivity in reversed-phase HPLC

Summary Solvent-strength selectivity refers to the variation of band spacing by changing the %-organic in the mobile phase (ion-pair or reversed-phase HPLC). A review of the literature has been combined with new experimental data to illustrate the general potential of this approach for HPLC optimization. It appears that most samples exhibit significant changes in band spacing method development based on solvent-strength optimization plus computer simulation (DryLab software) are given for illustration. For relatively simple mixtures (10 or fewer components), it appears that solvent-strength optimization compares favorably with other methods such as mapping the organic-solvent selectivity of methanol, acetonitrile, tetrahydrofuran, and water.     

Facile discrimination of aldose enantiomers by reversed-phase HPLC

One-pot reactions of aldoses with L-cysteine methyl ester and o-tolyl isothiocyanate yielded methyl 2-(polyhydroxyalkyl)-3-(o-tolylthiocarbamoyl)-thiazolidine-4(R)-carboxylates. Direct HPLC analysis of the reaction mixture and UV detection at 250 nm discriminated D- and L-enantiomers of aldoses. The reaction was applied to the determination of absolute configuration the sugar residues of an aroma precursor.     

Determination of triacylglycerols of manketti oil by reversed-phase HPLC

Using reversed-phase HPLC with refractometric detection, we determined the composition of triacylglycerols (TAGs) of manketti oil and calculated its fatty acid composition. It was shown that the oil consists of octadecatrienoic (34.6 ± 0.8 mol %), linoleic (35.4 ± 0.8 mol %), oleic (14.6 ± 0.4 mol %), palmitic (7.2 ± 0.3 mol %), and stearic acids. Using spectrophotometric detection, for the oils of centrathus and jacaranda seeds, it was demonstrated that octadecatrienoic acid is presented mainly by α-eleostearic acid and small impurities of β-eleostearic (2.8 mol %) and jacarandic (0.3 mol %) acids. The results of the quantitative determination of TAGs, obtained by refractometric detection (with corrections of the peak areas to the calculated values of the refractive indices of TAGs), are consistent with the data obtained using spectrophotometric detection.     

Determination of phenolic compounds in medicinal herbs by reversed-phase HPLC

An HPLC procedure is proposed for the determination of 12 phenolic compounds in plant tissues by reversed-phase HPLC with gradient elution and UV detection. The influence of pH, composition of the mobile phase, concentration of the organic modifier, and temperature on the separation of gallic, protocatechuic, trans-ferulic, trans-caffeic acid, rutin, quercetin, dihydroquercetin, and (_)-epicatechin for 30 min is studied. The lower limit of quantification of phenolic compounds is 1–2.5 μg/L. The procedure was applied to the determination of phenolic compounds in aqueous extracts of Hypericum perforatum; its sample was found to contain protocatechuic acid, (_)-epicatechin, and also rutin.     

The analysis of nitrate in environmental samples by reversed-phase HPLC

A method is presented for the analysis of nitrate in natural waters and waste water by high-performance liquid chromatography (HPLC) with a reversed-phase octadecyl column, aqueous phosphoric acid/dihydrogen phosphate mobile phase, and UV detector. The optimum nitrate concentration is 0.3 to 3 mg/L as N with linear detector response below 3 mg/L. The detection limit is 0.007 mg/L. Relative standard deviations in the optimum range are consistently less than one percent. Several potential interferences have been investigated; nitrite and organic chromophores are resolved from nitrate and do not interfere. Hexavalent chromium and sulfate are slight positive interferences, negligible at typical environmental concentrations. The method produces results in agreement with the accepted chromotropic acid method except in samples from eutrophic lakes, for which evidence is presented indicating that the chromotropic acid is inaccurate.     

Sensitive analysis of amino acids in injection liquor by reversed-phase HPLC

A convenient derivatization method of amino acids with l-fluoro-2,4-dimtrobenzene as reaction reagent and a separation system were described. The derivative amino acids were separated on a specific chemically bonded phase column with a simple linear gradient elution consisting of aqueous buffer and methanol. The eluate was detected by common ultraviolet absorption detector at 360 nm. The detection limits of amino acids were as low as 10 picomole. This method has been successfully applied to assay amino acid injection liquor used in hospital. It has good repro-ducibility and precision. The procedures avoid the requirements of particular derivative equipment and analyzer employed in conventional amino acid analysis.     

Behaviour of macromolecular RNA in reversed-phase HPLC

Large RNA molecules are retained on reversed-phase columns and can be eluted by increasing the concentration of acetonitrile. The retention behaviour depends on the base composition but not on the length of the chain, thereby providing a means for the separation of RNA molecules with different base composition. However, isocratic elution is not feasible due to the sudden changes in retention characteristics which are observed with slight variations in acetonitrile concentration. In order to interpret these results, a model has been developed called 'multiple-point interaction theory', based on the assumption that macromolecules are flexible and very large as compared to the hydrophobic phase. This model fits the experimental data and could be applied to all types of flexible macromolecules, especially proteins and nucleic acids, when they are chromatographed on reversed-phase columns. In the case of RNA, it has been observed that the retention time depends on ionic strength, pH, and temperature as expected for a true partition between two phases. Furthermore, an artifact called the 'polypeak phenomenon' appears at high flow rates which decreases the resolution of RNA. This polypeak phenomenon is controlled by the slope of the gradient d phi/dV.     

Simultaneous determination of dexamethasone and betamethasone in pharmaceuticals by reversed-phase HPLC

Chromatographia - An HPLC method for the simultaneous determination of the glucocorticoids betamethasone and dexamethasone is described. The method based on the separation of these compounds using...     

Direct determination of vanillylmandelic acid in human urine by reversed-phase HPLC

A reversed-phase HPLC method for the determination of epinephrine, norepinephrine, dopamine, and vanillylmandelic acid (VMA) has been developed. The concentration of VMA in the urine of hypertensive patients was measured by direct injection after centrifugation. The method is useful for the diagnosis of pheochromocytoma.     

A rapid method for the determination of benzylpenicillin in serum by reversed-phase HPLC

A simple procedure for the determination of benzylpenicillin in serum is described. The assay involves the extraction of the drug and the internal standard (phenoxymethylpenicillinic acid) from the sample into dichloromethane, using tetrabutylammonium hydrogen sulfate neutralized with NaOH and buffered with citrate as an ion-pairing reagent. RP-HPLC was performed on a Spherisorb 5 ODS column, eluting the drugs isocratically with 14% acetonitrile in 10 mM ammonium acetate buffer. Monitoring was by UV detection at 208 nm. Our results show that the method is accurate and reproducible, permitting quantification of serum levels of benzylpenicillin without interference from other drugs commonly used in therapy. Analytical recovery was greater than 79.2%.     

Generation of characteristic profiles of steroid hormones by reversed-phase HPLC

A method for the determination of steroid hormones by reversed-phase HPLC has been applied to the analysis of biological fluids from healthy individuals and patients with endocrine systems diseases. The obtained chromatographic profiles were processed by the method of principal component analysis and method of soft independent modeling of class analogy. The principle possibility of using the results of chemometric processing of the chromatographic profiles as an additional diagnostic criterion for the treatment of some endocrine pathologies has been shown.     

Microcomputer-assisted retention prediction system in reversed-phase HPLC

Summary The microcomputer-assisted retention prediction system in C₁₈ reversed-phase HPLC is described. The system is based on the use of the hydrophobic parameter and the correlation factor of alkylbenzenes and polycyclic aromatic hydrocarbons (PAHs) which are highly correlated to their retention in reversed-phase HPLC. The system is evaluated by comparing the retention data between measured and predicted values. One of the typical examples applied for PAH analysis in the extracts of diesel particulate matter shows the high potential of the system investigated.