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The most active form of sulfur in biomolecules is the thiol group, present in a number of biologically active compounds. Here we present a comprehensive study of thiol analysis using flow injection analysis/HPLC with electrochemical detection. The effect of different potentials of working electrodes, of organic solvent contents in the mobile phase, and of isocratic and gradient elution on simultaneous determination of thiol compounds (cysteine, cystine, N-acetylcysteine, homocysteine, reduced and oxidised glutathione, desglycinephytochelatin, and phytochelatins) are described and discussed. These thiol compounds were well separated and detected under optimised HPLC-electrochemical detection conditions (mobile phase: 80 mM trifluoroacetic acid and methanol with a gradient profile starting at 97:3 (TFA:methanol), kept constant for the first 8 min, then decreasing to 85:15 during one minute, kept constant for 8 min, and finally increasing linearly up to 97:3 from 17 to 18 min; the flow rate was 0.8 mL/min, column and detector temperature 25 degrees C, and the electrode potential 900 mV). We were able to determine tens of femtomoles (3 S/N) of the thiols per injection (5 microL), except for phytochelatin5 whose detection limit was 2.1 pmole. This technique was consequently used for simultaneous determination of compounds of interest in biological samples (maize tissue and human blood serum).

An ultrafast HPLC/UV-vis DAD method working at 254 nm was applied for the determination of isoflavone aglycons and glycosides (genistin, genistein, daidzein, daidzin, glycitin, glycitein, ononin, formononetin, sissotrin, and biochanin A) in roots, stems, leaves, and soy pods of soy plants and in soybeans of five varieties (Korada, Quito, Rita, OAC Erin, and OAC Vison). An Atlantis dC18 ultrafast RP chromatographic column (20 mm x 2.1 mm, 3 microm particle size) was applied for separation of the isoflavone aglycons and glycosides. A flow rate of the mobile phase (0.1% (v/v) acetic acid, pH 3.75-solvent A and methanol-solvent B) was 0.35 mL min(-1), and the column temperature was 36 degrees C. A linear gradient profile from 13 up to 22% B (v/v) from zero to 2.5 min, up to 30% B to 3.21 min, up to 35% B to 4 min, up to 40% B to 4.5 min, up to 50% B to 5.14 min, and followed by negative gradient up to 13% B to 7.71 min was used. The absolute limits of detection per sample injection (5 microL) were the highest for biochanin A (166.2 fmol) and the lowest for genistin (17.0 fmol), respectively. An accelerated solvent extraction (ASE) in combination with sonication was applied for isolation of biologically active compounds. A solid-phase extraction procedure was used to purify the extracts in the case of analysis of soy plants parts. The recoveries of 96-106% were obtained for the different concentrations of the isoflavone aglycons and glycosides and the different matrixes (overall RSDs 2-9%). The highest isoflavone concentrations were found in roots (12.5 microg g(-1) dry weight), while the amounts were about 3-1100 microg g(-1) fresh weight in different varieties of soybeans.