Development of new efficient method for isolation of phenolics from sea algae prior to their rapid resolution liquid chromatographic-tandem mass spectrometric determination.

The extraction of phenolic compounds from 4 different sea algae samples, three brown algae (Cystoseira abies-marina, C. abies-marina grinded under cryogenic conditions with liquid nitrogen, Undaria pinnatifida and Sargassum muticum) and one red algae (Chondrus crispus) via solid phase extraction using micro-elution solid-phase extraction (µ-SPE) plate method was studied. Prior to µ-SPE, 50mg of algae with 80% methanol mixture was extracted in hyphenated series by various extraction techniques, such as pressurized liquid extraction and Ika Ultra-Turrax® Tube Drive, in combination with ultrasound assisted extraction. The µ-SPE plate technique reduced the time of sample pre-treatment thanks to higher sensitivity and pre-concentration effect. Selected groups of benzoic acid derivatives (p-hydroxybenzoic, protocatechuic, gallic, vanillic, and syringic acids), hydroxybenzaldehydes (4-hydroxybenzaldehyde, and 3,4-dihydroxybenzaldehyde), and cinnamic acid derivatives (p-coumaric, caffeic, ferulic, sinapic, and chlorogenic acids) were determined using rapid resolution liquid chromatography coupled to mass spectrometry detection with negative ion electrospray ionization (RRLC-ESI-MS) using multiple reactions monitoring. LOQs of measured samples varied in the range 0.23-1.68ng/mL and LODs in the range 0.07-0.52ng/mL. The applied method allowed a simultaneous determination of phenolics (i.e. free, esters soluble in methanol, glycosides, and esters insoluble in methanol) in less than 5min (including alkaline or acidic hydrolysis of raw extracts) from sea algae extracts.

Influence of glutamic acid enantiomers on C-mineralization.

Seasonal dynamics in the mineralization of glutamic acid enantiomers in soils from selected ecosystems was determined and subjected to a range of treatments: ambient x elevated CO2 level and meadow x dense x thinned forest environment. Mineralization of glutamic acid was determined by incubation of the soil with 2 mg L- or D-glutamic acid g(-1) of dry soil to induce the maximum respiration rate. Mineralization of glutamic acid enantiomers in soils fluctuates over the course of a vegetation season, following a similar trend across a range of ecosystems. Mineralization is affected by environmental changes and management practices, including elevated CO2 level and thinning intensity. L-glutamic acid metabolism is more dependent on soil type as compared to metabolism of its D-enantiomer. The results support the hypothesis that the slower rate of D- compared to L- amino acid mineralization is due to different roles in anabolism and catabolism of the soil microbial community.

Natural occurrence of enantiomers of organic compounds versus phytoremediations: should research on phytoremediations be revisited? A mini-review.

Decontamination of polluted soils using plants is based on the ability of plant species (including transgenic plants) to enhance bioavailability of pollutants in the rhizosphere and support growth of pollutant-degrading microorganisms via root exudation and plant species-specific composition of the exudates. In this work, we review current knowledge of enantiomers of low-molecular-weight (LMW) organic compounds with emphasis on their use in phytoremediation. Many research studies have been performed to search for plants suitable for decontamination of polluted soils. Nevertheless, the natural occurrence of L- versus D-enantiomers of dominant compounds of plant root exudates which play different roles in the complexation of heavy metals, chemoattraction, and support of pollutant-degrading microorganisms were not included in these studies. D-enantiomers of aliphatic organic acids and amino acids or L-enantiomers of carbohydrates occur in high concentrations in root exudates of some plant species, especially under stress, and are less stimulatory for plants to extract heavy metals or for rhizosphere microflora to degrade pollutants compared with L-enantiomers (organic acids and amino acids) or D-carbohydrates. Determining the ratio of L- versus D-enantiomers of organic compounds as a criterion of plant suitability for decontamination of polluted soils and development of other types of bioremediation technologies need to be subjects of future research.

Significance of the natural occurrence of L- versus D-pipecolic acid: a review.

Pipecolic acid naturally occurs in microorganisms, plants, and animals, where it plays many roles, including the interactions between these organisms, and is a key constituent of many natural and synthetic bioactive molecules. This article provides a review of current knowledge on the natural occurrence of pipecolic acid and the known and potential significance of its L- and D-enantiomers in different scientific disciplines. Knowledge gaps with perspectives for future research identified within this article include the roles of the L- versus the D-enantiomer of pipecolic acid in plant resistance, nutrient acquisition, and decontamination of polluted soils, as well as rhizosphere ecology and medical issues.

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.

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.

GC/MS of terpenes in walnut-tree leaves after accelerated solvent extraction.

Terpenes, e. g. (+)-alpha-pinene, (-)-camphene, (-)-(-pinene, myrcene, R-(+)-limonene, eucalyptol, (+/-)-linalool, (-)-bornyl acetate, (-)-trans-caryophyllene, and alpha-humulene were determined in leaves of walnut trees from the Juglandaceae family (walnut tree, royal (J. regia L.), black (J. nigra L.), and Siebold (J. sieboldiana, var. Cordiformis Lam.) using gas chromatography with mass spectrometric detection. Terpenes were repeatedly (3 cycles, 5 min each) extracted from leaves of walnut trees by accelerated (pressurized) solvent extraction (ASE) 150 bar and 120 degrees C. The efficiency of ASE extraction was superior to that of steam distillation, solvent extraction according to Soxhlet, sonication, and extraction by agitation. Differences in relative concentrations and distribution of terpenes were studied in dependence on the species of walnut tree and on different locations.

Liquid chromatographic/electrospray mass spectrometric determination (LC/ESI-MS) of chelerythrine and dihydrochelerythrine in near-critical CO(2) extracts from real and spiked plasma samples.

Two polar benzo[c]phenanthridine alkaloids, chelerythrine (CHE) and dihydrochelerythrine (DHCHE), were extracted at 35 degrees C and 10MPa (15MPa for real samples) from real and spiked plasma samples with acceptable recoveries (95.1% and 81.0%, respectively) using near-critical CO(2) modified with aqueous (1:1, v/v) methanol. The alkaloids were quantified by a liquid chromatographic/electrospray mass spectrometric (LC/ESI-MS) method on a Zorbax SB-CN column (75mmx4.6mm, 3.5mum particle size) using methanol (organic phase) and 50mM ammonium formiate (aqueous phase) as a mobile phase. A linear gradient 0-1min, isocratic at 60% organic phase (v/v); from 1.0 to 7.0min, 60-71% organic phase (v/v); from 7.0 to 18.0min, 71-60% organic phase (v/v) was applied. The limit of detection was 1.22ng (3.50pmol) for CHE and 0.95ng (2.72pmol) for DHCHE per 1ml of the sample. The linearity of the calibration curves was satisfactory as indicated by coefficients of determination 0.9979 and 0.9995 for CHE and DHCHE, respectively. Repeatability and intermediate precision (average R.S.D.s) were 1.0-1.5%, accuracy was in the range 99.7-100.3%. Average recovery was 100.1% for both, standard solutions and spiked plasma extracts. Three samples of real rat plasma were extracted and analysed to test the method.

A hydrophilic interaction chromatography coupled to a mass spectrometry for the determination of glutathione in plant somatic embryos.

An electrospray ionization mass spectrometric (ESI-MS) determination of glutathione (GSH), a sulfur-containing tripeptide (gamma-Glu-Cys-Gly) with regulation and detoxication functions in metabolisms of most living organisms, from nanomolar to micromolar levels is described. A hydrophilic interaction chromatography (HILIC) with an isocratic elution using a mobile phase containing acetonitrile and an aqueous 0.00005% solution of trifluoroacetic acid (60/40%, v/v) was applied for the separation of GSH. The peptide detection was achieved in the presence of L-ascorbic acid which significantly enhanced the signal intensity of the molecular ion GSH [M+H]+ (m/z 308). The calibration curve was linear (R2=0.9995) in the concentration range from 2 nM to 10 microM with a detection limit (LOD, S/N=3) of 0.5 nM. The excellent detection limit, and the excellent selectivity and high reproducibility of this method enabled determination of GSH in a single plant somatic embryo of a Norway spruce (Picea abies). The average amount of GSH in the single somatic embryos (n=18) was 9 pmol per embryo. Owing to our results, it can be supposed that the proposed HILIC/ESI-MS analysis might be used for GSH determination in microscopic cell structures and in single cell analyses.

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.

Solid-phase extraction of 4(5)-methylimidazole (4MeI) and 2-acetyl-4(5)-(1,2,3,4-tetrahydroxybutyl)-imidazole (THI) from foods and beverages with subsequent liquid chromatographic-electrospray mass spectrometric quantification.

A method for the simultaneous determination of 4(5)-methylimidazole (4MeI) and 2-acetyl-4(5)-(1,2,3,4-tetrahydroxybutyl)-imidazole (THI) was developed using SPE and HPLC/MS. Solid-phase extraction using SCX Disc cartridges was used for isolation of the analytes from liquid samples. The lower LOQwas 0.1 ng/mL for 4MeI and 0.2 ng/ mL for THI. The linearity of the calibration curves was satisfactory as indicated by correlation coefficients >0.999. The CV for the intra- and inter-day precision was <5% (n = 6); the accuracy was in the range 98-103%. The recovery was > or = 97 and > or = 98% for THI and 4MeI, respectively. The method was used to determine THI and 4MeI in beverages, coffee, caramel colours and other samples.

A two-step supercritical fluid extraction of polycyclic aromatic hydrocarbons from roadside soil samples.

A two-step procedure for the supercritical fluid extraction (SFE) of polycyclic aromatic hydrocarbons from soil samples was developed. The procedure consists of a static supercritical fluid treatment in a closed extraction cell at a high temperature (T=250 or 340degreesC for 20 min) and an SFE with a solvent trapping. During the static phase, the sample is exposed to a supercritical organic solvent (methanol, toluene, dichloromethane, ACN, acetone, and hexane). The solvent penetrates particles of the matrix to substitute strongly bonded molecules and dissolves the analytes in the supercritical phase. At ambient temperature, supercritical fluids became liquid and lost their solvation abilities. Most of the analytes condense on the surface of the particles or on the extraction cell walls without forming strong bonds or penetrating deep into the matrix. Thus, the pretreatment liberates the analytes and they behave similar to those in freshly spiked samples. The common SFE with toluene-modified CO2 as an extraction fluid follows the static phase. With the use of the most suitable extraction phases (toluene, ACN), the extraction efficiency of the combined procedure is much higher (approximately100%). The results of the combined procedure are compared to the SFE procedure of the same untreated sample (difference less than 5%) and to the Soxhlet extraction. The extracts were analyzed using a GC with the flame ionization detection.

Supercritical fluid extraction of isoflavones from biological samples with ultra-fast high-performance liquid chromatography/mass spectrometry.

An efficient method of modifier addition for supercritical fluid extraction (SFE) of polar isoflavones was developed and yielded extraordinarily high recoveries. To find the optimal extraction conditions, a temperature and pressure optimization and modifier impact study was performed in naturally contaminated and spiked samples. Ultra-fast high-performance liquid chromatography/mass spectrometry (HPLC/MS) was used for the determination of isoflavones on an Atlantis dC18 high-speed reversed phase chromatographic column (20 x 2.1 mm, 3 microm particle size). A newly elaborated supercritical fluid extraction (SFE) procedure allowed more accurate (< 5%) and precise (< 4-7%) determination of isoflavones in biological materials. The HPLC/MS method significantly reduced analysis time with simultaneous improvement of sensitivity and detection limits. The on-column limits of detection LOD (S/N = 3) for isoflavone glycosides (daidzin, genistin, glycitin, ononin, and sissotrin) were 1.3-3.6 fmol and 0.2-1.0 fmol for aglycones (daidzein, glycitein, genistein, formononetin, and biochanin A), respectively.

A solvent recirculation trapping device for supercritical fluid extraction of polyaromatic hydrocarbons.

A new device has been developed for the trapping of volatile pollutants in trapping solvents. The device allows solvent recirculation and cryogenic trapping of evaporated volatiles to minimize the stripping effect and any losses of volatile analytes. Due to solvent recirculation, the trapping solvent column height remains constant during the extraction without any need for replenishment. Also mass transfer conditions are favorable due to the flattened shape of bubbles of CO2 and the longer extraction time. The bubbles have higher interfacial area and they have to pass a three times longer distance in the solvent column. The device produces more concentrated extracts, reduces solvent consumption, and reduces or eliminates its evaporation to the environment. The cryotrapping part reduces losses of volatile analytes and the stripping effect. It also enables single-phase extraction into much smaller solvent volumes. Due to constant and favorable extraction conditions, the precision of the method was also greatly improved (RSDs decreased from 2.2 to 0.8%). As proved by a set of rapid spiked-sample extractions of highly volatile compounds at very high flow rates, the relative standard deviation of the experiments performed in the new device is 3.5 times lower.