A rapid and selective high-performance liquid chromatographic (HPLC) method is developed for the separation and determination of caffeine, theobromine, and theophylline. The chromatography is performed on a Zorbax Eclipse XDB-C8 column (4.6x150 mm i.d., 5-microm particle size) at 25 degrees C, with a mobile phase of water-THF (0.1% THF in water, pH 8)-acetonitrile (90:10, v/v). The flow rate is 0.8 mL/min, and detection is by UV at 273 nm. This method permits the simultaneous determination of caffeine, theobromine, and theophylline in food, drinks, and herbal products with detection limits of 0.07-0.2 mg/L and recoveries of 100.20-100.42%. Correlation coefficients, for the calibration curves in the linear range of 0.2-100 mg/L, are greater than 0.9999 for all compounds. The within- and between-day precision is determined for both retention times and peak area. The data suggests that the proposed HPLC method can be used for routine quality control of food, drinks, and herbal products.

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A simple and rapid HPLC method for determination of chlorogenic acid (5-O-caffeoylquinic acid) in mate tea extracts was developed and validated. The chromatography used isocratic elution with a mobile phase of aqueous 1.5% acetic acid-methanol (85:15, v/v). The flow rate was 0.8 mL/min and detection by UV at 325 nm. The method showed good selectivity, accuracy, repeatability and robustness, with detection limit of 0.26 mg/L and recovery of 97.76%. The developed method was applied for the determination of chlorogenic acid in mate tea extracts obtained by ethanol extraction and liquid carbon dioxide extraction with ethanol as co-solvent. Different ethanol concentrations were used (40, 50 and 60%, v/v) and liquid CO 2 extraction was performed at different pressures (50 and 100 bar) and constant temperature (27 ± 1 °C). Significant influence of extraction methods, conditions and solvent polarity on chlorogenic acid content, antioxidant activity and total phenolic and flavonoid content of mate tea extracts was established. The most efficient extraction solvent was liquid CO 2 with aqueous ethanol (40%) as co-solvent using an extraction pressure of 100 bar.

Considering the growing interest for mushrooms and the demand search of natural antioxidants sources, the aim of this study was to investigate the antioxidant properties of two edible widely used Boletus species, Boletus edulis, and Boletus auranticus, collected from Istra region in Croatia in late summer 2007. To evaluate the antioxidant properties and content of antioxidant compounds, scavenging capacity on DPPH˙, OH˙, and O 2˙− radicals, reducing power and capacity to inhibit lipid peroxidation has been investigated. It is determined that content of total phenols (41.82± 0.08 mg gallic acid equivalent per gram of dry extract) was higher for B. edulis. Using high performance liquid chromatography/ diode array detector analysis, the main antioxidant compound, variegatic acid, has been detected and quantified. 1,1-Diphenyl-2-picryl-hydrazyl-hydrate assay was used as a preliminary free radical-scavenging evaluation. By this assay, it has been found that B. edulis dry mushroom extract exhibits 50% of inhibition value at the extract concentration of 0.016±0.0003 mg/ml. The extracts were capable of reducing iron(III) and, thus, are capable of donating electrons. Using electron paramagnetic resonance spintrapping and spin-probing techniques, activity against relevant reactive species,˙OH and O 2˙− radical, was analyzed for both mushroom extracts. Both investigated extracts are determined as good inhibitors for˙OH radical reduction, and both exhibited significant capacity for scavenging O 2˙− radical and for that could help to prevent or meliorate oxidative damage. Only B. edulis extract prevents lipid peroxidation. Investigated mushroom extracts could represent easily accessible natural antioxidant resource. AbbreviationsLDL Low density proteins DPPH 1,1-Diphenyl-2-picryl-hydrazyl-hydrate BHA Butylated hydroxyanisole 7-DS 2-(5-Carboxypentyl)-2-undecyl-4, 4-dimethyloxazolidine-3-oxylstearate DEPMPO 5-Diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide GAE Gallic acid equivalent CE Catechin equivalent RI Relative inhibition LC/MS Liquid chromatography/mass spectrometry HPLC/ DAD High performance liquid chromatography/ diode array detector EPR Electron paramagnetic resonance TP Total phenolic content TF Total flavonoid content UV/VIS Ultraviolet/visible spectroscopy IC 50 50% of inhibition value RSC radical scavenging capacity

A study of the chemical composition of essential oil and CO 2 extracts of the middle and upper leaf positions of the oriental tobacco Prilep was performed using GC and GC-MS. The major constituents were: in the essential oil, neophytadiene and solanone; in CO 2 extract I, neophytadiene; and in CO 2 extract II, nicotine, nonacosane and solanesol. The antimicrobial activity of the essential oil and CO 2 extracts were investigated against the microorganisms: Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. The results showed that oil from the middle leaves showed greater activity than the oil of the upper leaves. Both leaf oils mainly showed greater activity than CO 2 extracts.