Capillary electrophoresis fingerprinting and spectrophotometric determination of antioxidant potential for classification of Mentha products.

In this work aqueous infusions from ten Mentha herbal samples (four different Mentha species and six hybrids of Mentha x piperita) and 20 different peppermint teas were screened by capillary electrophoresis with UV detection. The fingerprint separation was accomplished in a 25 mM borate background electrolyte with 10% methanol at pH 9.3. The total polyphenolic content in the extracts was determined spectrophotometrically at 765 nm by a Folin-Ciocalteu phenol assay. Total antioxidant activity was determined by scavenging of 2,2-diphenyl-1-picrylhydrazyl radical at 515 nm. The peak areas of 12 dominant peaks from CE analysis, present in all samples, and the value of total polyphenolic content and total antioxidant activity obtained by spectrophotometry was combined into a single data matrix and principal component analysis was applied. The obtained principal component analysis model resulted in distinct clusters of Mentha and peppermint tea samples distinguishing the samples according to their potential protective antioxidant effect. Principal component analysis, using a non-targeted approach with no need for compound identification, was found as a new promising tool for the screening of herbal tea products.

Mercury speciation by CE: an update.

This review provides an update on mercury speciation by CE. It includes a brief discussion on physicochemical properties, toxicity and transformation pathways of mercury species (i.e. methyl-, ethyl-, phenyl- and inorganic mercury) and outlines recent trends in Hg speciation by CE. CE is presented as a complementary technique to chromatographic separation techniques, especially in cases when speed, high efficiency and low sample volumes are required. The development of suitable sample preconcentration/isolation (sample stacking, ion exchange, liquid-liquid-liquid extraction, dual-cloud point extraction) to achieve low LODs for analysis of trace concentrations of mercury species in real samples is emphasized. Hyphenation of CE to element specific detectors (i.e. electrothermal atomic absorption spectrometry, atomic fluorescence spectrometry, inductively coupled plasma-optical emission spectrometry, inductively coupled plasma-mass spectrometry) is discussed as well as a potential of CE in interaction studies that may provide useful information on interaction of various Hg species with selected bio-macromolecules.

Enhancing sensitivity of liquid chromatographic/ion-trap tandem mass spectrometric determination of estrogens by on-line pre-column derivatization.

A rapid and sensitive liquid chromatographic/ion-trap mass spectrometric method has been developed for the simultaneous determination of estriol, alpha-, beta-estradiol, 17alpha-ethynylestradiol and estrone. The substances were extracted from river water samples (typically 250 ml) and cleaned-up using two-stage solid-phase extraction (SPE). 12-(Difluoro-1,3,5-triazinyl)-benz[f]isoindolo-[1,2b][1,3]benzothiazolidine was applied as a derivatizing agent. The recovery for each compounds ranged from 75 to 88% and the repeatability represented as RSDs ranged from 5.6 to 8.4%. Limits of detection (LOD, 3 x S/N) were 67-285 pg/l.

Capillary electrochromatography of inorganic cations in open tubular columns with a controllable capacity multilayered stationary phase architecture.

In this paper capillary electrochromatography of alkali and alkaline-earth metal cations in open tubular capillary columns is described. Capillary columns are prepared by coating fused silica capillaries of 75 microm I.D. with poly(butadiene-maleic acid) copolymer (PBMA) in multiple layers. Thermally initiated radical polymerization is used to crosslink the stationary phase. Capillary columns with different number of stationary phase layers can be prepared and allow for the adjustment of separation selectivity in the electrochromatographic mode. Fast and sensitive separations of common inorganic cations are achieved in less than 6 min in a 60 cm capillary column with on-column capacitively coupled contactless conductivity detector. Limits of detection (S/N=3) for the determination of alkali and alkaline-earth metal cations range from 0.3 to 2.5 microM and repeatability is better than 0.5, 4.5 and 6.1% for migration times, peak heights and peak areas, respectively.

Current trends in isolation, separation, determination and identification of isoflavones: a review.

Isoflavones are natural substances which elicit a number of physiological effects in living organisms. The substances are synthesized in plant tissues as protective agents against biotic stress (i. e. bacterial infection). Isoflavones are also an important dietary constituent in human nutrition. Modern trends in studies of isoflavones in plant materials and foodstuffs and procedures for chemical analyses of isoflavones in human body fluids and plant tissues are discussed in this review. Highly effective extraction and purification techniques, i. e. solid-phase extraction, accelerated-solvent extraction, and Soxhlet extraction, are presented. Latest procedures in chromatographic separation of isoflavones that apply different types of sorbents are described. Immunochemical analysis, electrochemical sensing of isoflavones, and spectrometric and other analytical techniques and their applications are also mentioned. Special attention is paid to the highly selective and sensitive technique of mass spectrometry and its application for identification of isoflavones and their glucosides in plants. Studies of interactions of isoflavones with cell receptors and a number of biologically active substances such as DNA and proteins are described. The review does not intend to give a complete overview of the topics considered but rather to present modern and most recent methods used in studies of isoflavones.

Gravity-flow open tubular cation chromatography.

We describe ion chromatography (IC) on open tubular cation exchange columns with a controllable capacity multilayered stationary phase architecture. The columns of relatively large bore (75 microm id) are fabricated by coating fused-silica capillaries with multiple layers of poly(butadiene-maleic acid) (PBMA) copolymer and crosslinking the deposited layers by thermally initiated radical polymerisation. Column capacity increases in a predictable manner with increase in the number of successively coated layers. Gravity flow with a modest head (< 2 m) can provide the desired separations within a reasonable period. We provide a minimalist configuration where no suppression is used, the sample is injected hydrodynamically as in CE, and detection is accomplished by an inexpensive homebuilt contactless conductivity detector or a capacitance to voltage digital converter. A 1 m long 75 microm bore column coated with two layers of PBMA allows gravity-flow open tubular IC to separate four alkali cations in < 10 min with a 1 mM tartaric acid (TA) eluent. Simultaneous separation of alkali and alkaline earth metal cations can be accomplished in less than 25 min using 1.75 mM pyridinedicarboxylic acid as an eluent. Contactless conductometric detection (C(4)D) allows LODs down to 150 nmol/L, corresponding to 30 fmol injections. Analysis of real water samples is demonstrated.

Supercritical fluid extraction of amino acids from birch (Betula pendula Roth) leaves and their liquid chromatographic determination with fluorimetric detection.

A method for supercritical fluid extraction (SFE) of amino acids was adapted and optimal experimental conditions were selected for a matrix consisting of dry leaves. The matrix-dependent SFE method uses a mixture of MeOH-H(2)O-acetonitrile (10:10:1 v/v/v) as a modifier (0.5 mL in situ, 300 muL on-line) at 70 degrees C and 40 MPa and no HCl is needed as an entrainer. The amino acids were quantified using high-performance liquid chromatography with fluorimetric detection (HPLC/FLD) after gradient elution on Zorbax Eclipse AAA columns (4.6x150 mm, 3.5 mum) with aqueous Na(2)HPO(4 )buffer of pH 7.8 and ACN-MeOH-water as a mobile phase. In comparison with Soxhlet extraction, SFE gave higher recovery and selectivity, but it required longer extraction time (90 min) and it was more labor-intensive (clean-up step after the pre-concentration). Both methods should be used separately or in combination according to the matrix, number of samples, and levels of ballast compounds.

Combined isolation and purification procedures prior to the high-performance liquid chromatographic-ion-trap tandem mass spectrometric determination of estrogens and their conjugates in river sediments.

A fast and sensitive analytical procedure has been developed for the simultaneous separation and determination of alpha-estradiol, beta-estradiol, estriol, estrone and ethynylestradiol and their sulfate, glucuronide and acetate conjugates in river sediments. The procedure includes a microwave-assisted extraction (MAE) with aqueous methanol (25:75, v/v) at 100 degrees C in 10 min, a clean-up on Oasis WAX cartridge and a high-performance liquid chromatography-ion-trap tandem mass spectrometry (HPLC-IT-MS/MS) with electrospray ionization. The recovery for each compounds ranged from 83 to 107% and the repeatability represented as RSDs ranged from 4.9 to 9.6%. The limits of detection (LODs) were down to 1 ng g(-1). The analytical performance of the method was evaluated using determination of free and conjugated estrogens in river sediments.

Total mercury and mercury species in birds and fish in an aquatic ecosystem in the Czech Republic.

Total mercury and mercury species (methylmercury-MeHg, inorganic mercury--Hg(2+)) were determined in the aquatic ecosystem Záhlinice (Czech Republic). Four tissues (muscle, intestines, liver and kidney) of three bird species--cormorant, great crested grebe and Eurasian buzzard, muscle tissues of common carp, grass carp, northern pike, goldfish, common tench, perch and rudd, aquatic plants (reed mace and common reed), sediments and water were analysed. Relative contents of MeHg (of total Hg) were in the range from 71% to 94% and from 15% up to 62% in the muscle and intestines and in liver, respectively, for all birds. Statistically significant differences were found between contents of MeHg in liver tissues of young and adult cormorant populations (F(4.60)=56.71, P<10(-5)). Relative contents of MeHg in muscle tissues of fishes were in the range from 65.1% to 87.9% of total Hg.

Non-edible parts of Solanum stramoniifolium Jacq. - a new potent source of bioactive extracts rich in phenolic compounds for functional foods.

Extracts prepared from leaves, roots, and stems of Solanum stramoniifolium Jacq. (Solanaceae) in 80% ethanol have been tested for their in vitro antioxidant, anti-inflammatory, antimicrobial, and cytotoxic activities with an aim to find new sources of substances for functional foods and food additives. The root extract revealed the highest antioxidant activity in all assays exceeding the trolox capacity, and was the only extract that inhibited nitric oxide production in mouse macrophage cells, showing also the capacity to suppress the growth of all tested human tumor cell lines (MCF-7, NCI-H460, HeLa and HepG2). The leaf extract showed the strongest antimicrobial activity inhibiting all tested clinical isolates. To the author's best knowledge it was the first time that all individual parts of this plant were tested for biological activity together with the phenolic compound characterization.

A liquid chromatographic-mass spectrometric evidence of dihydrosanguinarine as a first metabolite of sanguinarine transformation in rat.

Adult rats were orally administered with a single dose of sanguinarine (10 mg SA per 1 kg body weight) in 1.0 ml water. In the plasma and the liver, dihydrosanguinarine (DHSA) was identified as a SA metabolite by high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC/ESI-MS). Significantly higher levels of DHSA were found in both the plasma and the liver in comparison with those of SA. SA and DHSA were not detected in the urine. The formation of DHSA might be the first step of SA detoxification in the organism and its subsequent elimination in phase II reactions. Benz[c]acridine (BCA), in the literature cited SA metabolite, was found neither in urine nor in plasma and liver.

Determination of total content of phenolic compounds and their antioxidant activity in vegetables--evaluation of spectrophotometric methods.

This research studies in detail the contents of phenolic compounds determined by the Folin-Ciocalteu reagent and the antioxidant activities determined by the TEAC (Trolox equivalent antioxidant capacity), DPPH (using diphenyl-p-picrylhydrazyl radical), and FRAP (ferric reducing antioxidant power) methods, and their correlations for used standards with these methods (catechine, gallic acid, caffeic acid, ferulic acid, Trolox, ascorbic acid, and ferrous sulfate) and extracts from several species of commonly consumed vegetables were studied in detail. The comparison of absolute values of absorption coefficients for used standards and for individual methods allows one to choose optimal common standards for methods to be compared. The procedures applied for the same sets of the extracts using identical calibration procedures and common standards allowed better comparison of the results obtained by the TEAC, DPPH, and FRAP methods. The values of content of phenolic substances and total antioxidant activity of the sets of samples correlate very well for all used methods. The very high values of antioxidant activity were found in intensely colored vegetables (red cabbage, red onion, etc.), and the values were very low in watery vegetables such as potato, marrow, and cucumber.

Supercritical fluid extraction of bioavailable amino acids from soils and their liquid chromatographic determination with fluorometric detection.

A new procedure with supercritical CO2 modified with 0.5 mL of water and 0.75 mL of 0.1 M HCl in situ and 0.75 mL of water on-line at 15 MPa and 50 degrees C for 45 min was applied for the extraction of bioavailable amino acids from soil samples. Total extraction time was 60 min, but more favorable conditions are even possible for selected groups of amino acids. All analytes were trapped into 20 mL of methanol with satisfactory recovery (94-104%) and determined using high-performance liquid chromatography with fluorometric detection on a Zorbax Eclipse column (4.6 x 75 mm, 3.5 microm) with Na2HPO4 and acetonitrile/methanol/water as a mobile phase. Linear calibration curves were obtained (r > 0.999 except 0.99823 for Ile) with lower limits of detection (S/N = 3) in the range from 1.54 pg (Gly) to 13.5 pg (Cy2) or from 18.6 fmol (Ser) to 64.8 fmol (Lys). Validation and repeatability data are also given. Comparable results were obtained with a robust, commonly used extraction method (0.5 M ammonium acetate, 60 min in shaker, followed by filtration and lyophilization). Limiting values of artificial release of amino acids were also determined for each soil sample to eliminate any false results to ensure that all extracted amino acids originate from soil solution and exchangeable bound positions of soil samples.

Determination of isoflavones in soy bits by fast column high-performance liquid chromatography coupled with UV-visible diode-array detection.

A fast determination of isoflavones (genistin, genistein, daidzein, daidzin, glycitin, glycitein, ononin, formononetin, sissotrin and biochanin A) by HPLC/UV-vis-DAD working at 254 nm is described. An Atlantis dC18 fast reversed-phase chromatographic column (20 mm x 2.1 mm, 3 microm particle size) was used at a flow rate 0.35 ml min(-1) of a mobile phase consisted from 0.1% (v/v) acetic acid (A) at pH 3.75 and methanol. (B). A linear gradient profile was used for separation at the column temperature 36 degrees C. Limits of detection (LODs for 3 S/N criterion) per sample injection (5 microl) ranged from 166.2 to 17.0 fmol (9.4-1.1 ng ml(-1) for biochanin A and genistin, respectively. The recoveries 96-106% were obtained for the different concentrations of the isoflavones (RSDs 2-8%). The pressurized liquid extraction/HPLC/UV-vis-DAD method was used for the determination of the isoflavones in soy bits (28-962 microg g(-1) dry weight). The proposed procedure is faster (ca. 8 min) without loosing its separation efficiency (up to 10 isoflavonoids can be determined) and sensitivity (tens to hundreds fmol).

Uptake of thallium from artificially and naturally contaminated soils into rape (Brassica napus L.).

An inductively coupled plasma mass spectrometry (ICP/MS) method was used for the evaluation of thallium transfer from naturally (pedogeochemically) and artificially contaminated soils into rape. Two sets of three different types of top soils (heavy, medium, and light) were used for pot experiments. The first set was collected from areas with high levels of pedogeochemical thallium (0.3, 1.5, and 3.3 mg kg(-1) DM). The second set of three soils with naturally low content of thallium was artificially contaminated with thallium sulfate to achieve five levels of contamination (0, 0.4, 2, 4, and 6 mg kg(-1) DM Tl). The soil samples and the samples of winter and spring rape (straw, seeds) from both sets were collected and analyzed. Plant and soil samples from fields were collected at 42 selected sites situated in South Bohemia and in Czech-Moravian Highlands where higher pedogeochemical content of thallium was expected. More intensive transport (better availability) of Tl was observed in the case of artificially contaminated soils. The physicochemical form and the total content of Tl in soil were found to be the main factors influencing its uptake by plants. The concentration of Tl in rapeseeds in the field samplings was mostly 45% of its content in the particular soil. Nevertheless the uptake of Tl from soils with naturally high pedogeochemical content can be high enough to seriously endanger food chains. These findings are very important because of the high toxicity of Tl and the absence of threshold limits for Tl in soils, agricultural products, feedstuffs, and foodstuffs in most countries including the Czech Republic.

Evaluation of isoflavone aglycon and glycoside distribution in soy plants and soybeans by fast column high-performance liquid chromatography coupled with a diode-array detector.

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.

Analysis of Organic Acids, Deacetyl Asperulosidic Acid and Polyphenolic Compounds as a Potential Tool for Characterization of Noni (Morinda citrifolia) Products.

Organic acids, deacetyl asperulosidic acid (DAA) and polyphenolic compounds in various noni (Morinda citrifolia L.) products (4 juices, 4 dry fruit powders and 2 capsules with dry fruit powder) were analyzed. Reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with a variable wavelength detector (VWD) and electrospray ionization time-of-flight mass spectrometer (ESI-TOF MS) was applied for simultaneous analysis of organic acids (malic, lactic, citric and succinic acid) and DAA. An RP-HPLC method with diode-array detector (DAD) was developed for the analysis of polyphenolic compound content (rutin, catechin, quercitrin, kaempferol, gallic acid, caffeic acid and p-coumaric acid). The developed methods can contribute to better characterization of available noni products that is required from the consumers. In our study, we discovered significant dissimilarities in the content of DAA, citric acid and several phenolic compounds in some samples.

Capillary electrophoretic studies of acid-base properties of sanguinarine and chelerythrine alkaloids.

Capillary zone electrophoresis with UV detection was used for determination of dissociation constants of alkaloids sanguinarine and chelerythrine. Despite the limited solubility of the uncharged forms of the alkaloids resulting in insufficient analytical signal at higher pH the reliable dissociation constants were obtained when acidified samples containing low amount of the alkaloid were injected into the capillary. The precipitation of the alkaloid in the capillary induced by injecting sample of low pH into the background electrolyte of higher pH does not affect the mobility of the alkaloid if its concentration injected exceeds the solubility only to a small extent. Dissociation constants (pK(R+)) of sanguinarine and chelerythrine calculated to 8.3 +/- 0.1 and 9.2 +/- 0.1, respectively, are relevant to Good buffers of ionic strength of 30 mM.

Application of a contactless conductometric detector for the simultaneous determination of small anions and cations by capillary electrophoresis with dual-opposite end injection.

A contactless conductometric detection (CCD) system for capillary electrophoresis (CE) with a flexible detection cell was applied for the simultaneous determination of small anions and/or cations in rain, surface and drainage water samples. The applied frequency, the amplitude of the input signal, the electrolyte conductivity and electrode distance were found to be the most significant factors affecting the detection sensitivity. 2-(N-Morpholino)ethanesulfonic acid/histidine-based (MES/His) electrolytes were used for direct conductivity detection of anions and cations, while ammonium acetate was selected for indirect conductivity determination of alkylammonium salts. For the simultaneous separation procedure, involving dual-opposite end injection, an electrolyte consisting of 20 mM MES/His, 1.5 mM 18-crown-6 and 20 microM cetyltrimethylammonium bromide provided baseline separation of 13 anions and cations in less than 6 min. The detection limits achieved were 7-30 micrograms/l for direct conductometric detection of various common inorganic cations and anions, excluding F- (62 micrograms/l) and H2PO4- (250 micrograms/l), and 35-178 micrograms/l for indirect conductometric detection of alkyl ammonium cations. The developed electrophoretic method with conductometric detection was compared to ion chromatography.

Determination of isoflavones in soybean food and human urine using liquid chromatography with electrochemical detection.

A highly sensitive high-performance liquid chromatographic method with electrochemical detection (HPLC-ED) was developed for the determination of isoflavones. Electrochemical behaviour of daidzein and genistein was studied on carbon paste electrode (CPE) by adsorptive transfer stripping square wave voltammetry. The obtained electrochemical results were used for the development of HPLC-ED method. Furthermore, isoflavones were separated on an Atlantis dC18 column using a mobile phase consisting of acetonitrile (solvent A) and 0.15M acetate buffer of pH 5.5 (solvent B) at a flow rate 0.4 mL/min. A linear gradient profile (solvent B) was at 0-2 min 87%; 22 min 60%; 27 min 50%; 31 min 45%; 47 min 87%. Full scan of multi-channel coulometric detection was tested and optimal potential at 450 mV was chosen for our purposes. Calibration curves were linear (daidzein R(2) = 0.9993 and genistein R(2) = 0.9987). The detection limit for daidzein/genistein was 480/394 pg/mL (1.8/1.5 nM) and per column 2.4/1.9 pg. Isoflavones extracted from soybean products (farina, meat, milk) by the accelerated solvent extraction (ASE) procedure and isoflavones present in human urine were determined by the HPLC-ED method.