Characterisation of selenium compounds in rye seedling biomass using <Superscript>75</Superscript>Se-labelling/SDS-PAGE separation/γ-scintillation counting,and HPLC-ICP-MS analysis of a range of enzymatic digests

In the present study, selenium-enriched plant biomass was investigated to evaluate the ability of rye seedlings to take up, and assimilate, inorganic selenium. Two different analytical approaches were used. Electrophoretic separation (SDS-PAGE) of proteins extracted from ⁷⁵Se-labelled biomass was used to investigate the biotransformation of selenite into organic forms of the element. Ion-pair chromatography coupled with ICP-MS detection was chosen for the analysis of selenium species, enzymatically extracted from the plant biomass. The results of three enzymatic hydrolysis procedures and three sequential enzymatic extractions procedures are compared. The most effective single extraction was proteolysis (using protease type XIV), giving an overall extraction efficiency of 48%. However, for combinations of enzymes, the most effective was cellulase (Trichoderma viride) followed by sequential extraction of the solid pellet using protease type XIV, giving an extraction efficiency of 70%. The complementary data from the electrophoretic fractionation of proteins, and the HPLC separation of Se-species in the proteolytic digests, reveal the existence of large number of selenium-containing compounds in the rye seedling plant biomass. The results showed the complete biotransformation of inorganic selenium into organic forms during germination of the rye seedlings. HPLC-ICP-MS analysis of extracts from the plant biomass did not show the presence of selenate or selenite. At the time of this study, the lack of suitable organic-MS facilities meant that it was not possible to characterise them fully. However, the data does show that a combination of different enzymes, rather than just the commonly-used protease, should be considered when developing an extraction strategy for selenium in different food types to those already reported in the literature.     

Antioxidant capacity and antibacterial activity from Annona cherimola phytochemicals by ultrasound-assisted extraction and its comparison to conventional methods

In recent years, the food, pharmacy, and cosmetic industries have focused on the search of natural compounds with antimicrobial and antioxidant properties; commonly, these compounds are obtained from Kingdom plantae. The aim of the present work is comparing antibacterial and antioxidant capacity of Annona cherimola Mill leaves, using different extraction methods. The ultrasound assisted extraction technique (UAE) was compared with conventional techniques: Soxhlet and maceration. Water and ethanol were used as solvents for leaves extractions performed with these three methods. The main acetogenins reported in Annona cherimola Mill and Annona muricata L. species were simulated using the functional hybrid B3LYP and to confirm its presence, analysis of the compound composition was performed using FT-IR, UV–Vis and HPLC. Total phenolics (TP) and flavonoids (TF) were determined by spectroscopy techniques and novel Differential Pulse Voltammetry (DPV) electrochemical technique. Total Antioxidant Capacity (TAC) of the extracts was measured, using the DPPH, FRAP and CUPRAC techniques. The highest antioxidant content was found in the Soxhlet water extracts; even so, the UAE technique presented an attractive alternative due to considerable reduction in extraction time, which was greater than 99%, and possible selectivity in compounds extraction. Finally, antibacterial activity of the extracts was evaluated, obtaining the best results against gram-positive bacteria using UAE water extract. In this way, the UAE technique presents an excellent extraction option due to the considerable reduction in time and energy, as well as the increase in antibacterial activity.     

HPLC-ICP-MS for a comparative study on the extraction approaches for arsenic speciation in terrestrial plant, Ceratophyllum demersum

For the determination of arsenic compounds in terrestrial plant samples, a crucial step is the efficient extraction of arsenic from the solid plant matrix. However, the use of methanol-water extraction often resulted in low extraction efficiencies of less than 50%. In this study, eight solid-liquid extraction procedures (mainly based on mechanical mixing and sonication) were evaluated for the recovery of arsenic species from a submerged freshwater plant, coontail (Ceratophyllum demersum), collected in Moira River, Ontario, Canada. Speciation of As in the extracts was carried out with both anion-, and cation-exchange HPLC with sector-field inductively coupled plasma mass spectrometric (SF-ICP-MS) detection. The results obtained depended critically on the extraction solvents used in different extraction procedures. Extraction with methanol-water led only to 9%–44% recoveries of As. A high extraction yield (approximately 82%) was obtained by water extraction. Alkaline hydrolysis also resulted in high extraction efficiencies (86%–98%), but severe oxidation of As(III) to As(V) was observed. A protease enzymatic extraction led to a recovery of 48%. Approximately 0.5% of the total As in the plant sample was lipid-soluble. It was found that the extraction of inorganic arsenic species was suppressed by the presence of methanol in the extraction solvents, while high content of methanol in the extraction solvents was effective for the extraction of organic arsenic species. Therefore, it is recommended to perform the extraction both with water alone and with methanol-water (9+1, v/v), in order to obtain the complete As species profile in terrestrial plants.     

Comparison of pressurised fluid and ultrasonic extraction methods for analysis of plant antioxidants and their antioxidant capacity

The analytical method based on the high-performance liquid chromatography coupled with UV detection (HPLC/UV) for determination of selected antioxidants (i.e., esculetin, scopoletin, 7-hydroxycoumarine, rutin, xanthotoxin, 5-methoxypsoralen and quercetin) in plant material was developed. Pressurised fluid extraction (PFE) and ultrasonic extraction (USE) methods for the isolation of these compounds from ten real plant samples were used. Both extraction methods were optimised and compared to each other. For the proposed HPLC/UV method the LOQ values (limit of quantification) in the range from 22.7 (xanthotoxin) to 97.2 ng mL⁻¹ (rutin) were obtained. For all extracts the antioxidant capacity based on the reduction of free 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) was also determined. Results ranged from 82.04 to 94.43% of DPPH radical inhibition for PFE method and from 76.01 to 89.94% in the case of USE method.     

Multi-walled carbon nanotube-based dispersive solid phase extraction with following back-extraction for HPLC/UV determination of Rosmarinic acid in lemon balm and Rosemary plant samples

In this paper, dispersive solid phase extraction with following back-extraction and HPLC/UV determination of Rosmarinic acid have been investigated. Multi-walled carbon nanotubes were used as sorbent in the suggested method. The effectiveness of some parameters such as extraction time, sorbent dosage, pH and ionic strength of the aqueous solution on Rosmarinic acid adsorption were studied. The extraction time of 5.0 min, adsorbent weight of 5.0 mg, and pH of 2.5 were obtained as the optimum experimental parameters. Adsorption of Rosmarinic acid molecules was almost independent from ionic strength. For back-extraction purpose a basic solution containing methanol was brought into contact with sorbent to desorb Rosmarinic acid molecules from sorbent containing adsorbed Rosmarinic acid molecules. Linear range of HPLC method for Rosmarinic acid quantification was 1.80–21.62 mg/L (R² = 0.998). The relative standard deviation, limit of detection, and limit of quantification for the suggested method were 0.81%, 0.68 mg/L, and 2.86 mg/L, respectively. The proposed method was applied for extraction and HPLC/UV determination of Rosmarinic acid in Lemon balm and Rosemary plant samples. Comparison between obtained chromatograms before adsorption and after desorption processes showed that suggested extraction/back-extraction process has good selectivity and efficiency for separation of Rosmarinic acid molecules from interfering molecules existing in the plant extracts. The proposed method is efficient, low cost, fast, and simple for separation, extraction, and determination of Rosmarinic acid.     

Anthocyanin Monitoring in Four Red Grape Skin Extract Varieties Using RP‐HPLC‐ED

Abstract

Anthocyanin compounds, cyanin, peonidin, myrtillin, malvin, kuromanin, oenin, petunidin and pelargonidin, present in four varieties of red grape skin extracts were quantified by reversed‐phase high‐performance liquid chromatography with electrochemical detection (RP‐HPLC‐ED). Several extraction procedures were evaluated for anthocyanin extraction from red grape skins of varieties of the Dão wine demarcated region in Portugal. The developed method was successfully applied to the analysis of the anthocyanins in skins extracts of red grapes during the 2005 harvest campaign, and monitoring these compounds can give great insight toward choosing the best time for the grape harvest for achieving a better wine quality.     

Development and validation of a reversed‐phase high‐performance liquid chromatographic method with solid‐phase extraction for the quantification of hydrochlorothiazide in ex vivo permeation studies

Hydrochlorothiazide (HCT) is a diuretic used to treat hypertension. In order to study its intestinal permeation behavior applying an ex vivo methodology, a rapid, sensitive and selective reversed‐phase liquid chromatography (RP‐HPLC) method coupled with UV detection (RP‐HPLC UV) was developed for the analysis of HCT in TC199 culture medium used as mucosal and serosal solutions in the everted rat intestinal sac model. Also, analytical procedures for the quantification of HCT by RP‐HPLC with UV detection required a sample preparation step by solid‐phase extraction. The method was validated in the concentration range of 8.05 × 10⁻⁷ to 3.22 × 10⁻⁵ m for HCT. Chromatographic parameters, namely carry‐over, lower limit of quantification (1.4491 × 10⁻⁷ m ), limit of detection (3.8325 × 10⁻⁸ m ), selectivity, inter‐ and intraday precision and extraction recovery, were determined and found to be adequate for the intended purposes. The validated method was successfully used for permeability assays across rat intestinal epithelium applying the ex vivo everted rat gut sac methodology to study the permeation behavior of HCT.     

Solid Phase Derivatizations in HPLC: Borohydride/Silica Reductions for Carbonyl Compounds

Abstract

Sodium borohydride adsorbed onto silica gel has now been utilized for on-line, pre-and post-analytical column chemical derivatizations via reductions of various organic carbonyl compounds. These on-line reactions have been performed using normal phase HPLC conditions, involving conventional silica gel packings, organic mobile phases, and commercially available HPLC equipment and instrumentation. This approach for on-line HPLC derivatizations has been evaluated for a large number of organic carbonyl compounds, at a variety of temperatures for aldehydes and ketones. The overall rates of such carbonyl reductions via sodium borohydride/silica are sufficiently different as a function of temperature of the reaction to permit for compound/class identifications. Analyte identification can be both qualitative and quantitative, even wherein an analyte co-elutes with a non-carbonyl compound. In-house prepared borohydride/silica gel reactors can be characterized via standard iodine titration procedures along with inductively coupled plasma (ICP) elemental boron analysis. These normal phase derivatization approaches have been applied to certain standard vitamins, and to cinnamaldehyde found in a commercial spice product. Such approaches to on-line HPLC derivatizations     

High Performance Liquid Chromatographic Determination of Phenylenediamines in Aqueous Environmental Samples

Abstract

The use of high performance liquid chromatography (HPLC) for the determination of phenylenediamines has been studied. Detection limits using both ultraviolet (UV) and electrochemical (EC) detectors have been determined and EC is superior in most cases. Chromatographic conditions and sample preparation procedures are described for many phenylenediamines of environmental significance.     

High-Performance Liquid Chromatography of Domoic Acid,a Marine Neurotoxin,with Application to Shellfish and Plankton

Abstract

A recent outbreak of poisoning resulting from the consumption of cultured blue mussels (Mytilus edulis L.) from a localized area in Eastern Canada has been attributed to the presence of domoic acid (1), a relatively rare neurotoxic amino acid, previously found only in some algae of the family Rhodomelaceae. Studies on aqueous extracts of shellfish tissue indicated that the toxin and several of its isomers could be separated (and isolated in sufficient amounts for subsequent structural identification) by reversed-phase high-performance liquid chromatography (HPLC) with ultraviolet (UV) diode array detection (DAD). Aqueous acetonitrile containing 0.1% v/v trifluoroacetic acid was used as mobile phase. As the retention time and characteristic UV absorption spectrum of 1 (λ_max = 242 nm) permit unequivocal identification, the HPLC-DAD procedure was refined with a microbore column to provide a rapid (5 min), sensitive (0.3 ng detection limit) and reproducible assay method for the determination of 1 in shellfish tissue. Extraction was accomplished by boiling homogenized shellfish tissue for 5 min with distilled water. Extracts were taken through an octadecylsilica solid phase extraction clean-up prior to HPLC. This method has been applied to a variety of shellfish and phytoplankton samples.

BRIEF

Reversed-phase HPLC with ultraviolet diode array detection was used to analyze shellfish tissue and phytoplankton extracts for domoic acid. A rapid (5 min) and sensitive (0.3 ng detection limit) assay is presented.     

Analysis of phenolic compounds in spruce needle extracts using an UV-VIS-diode array detector

Summary High-performance liquid chromatography (HPLC) coupled with ultraviolet-visible spectroscopy, using a photodiode-array detector, was applied to the investigation of plant extracts for polyphenolic compounds. Simultaneous detection at different wavelengths and measurement of the UV spectrum of each separated compound during the elution allows an easy and rapid identification of the derivatives of benzoic acid, o-coumaric acid, p-hydroxyacetophenone, stilbenes and flavonoide compounds as well as several catechins. However, as some of these compounds have closely related structures, a characterization by their UV-spectra is insufficient. Hydrolysis with hydrochloric acid and enzymes results in the formation of their monomeric non glycosidated compounds as well as in an increase of the peak area assigned to the monomeric compounds. This technique was applied for the phenol analysis of purified spruce needle extracts of picea abies species.

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Analyse phenolischer Fichtennadelinhaltsstoffe unter Verwendung eines UV-VIS-Dioden-Array-Detektors

     

Comparison of gas and high performance liquid chromatography with selective detection for determination of triazine herbicides and their degradation products extracted ultrasonically from soil

GC and HPLC with selective detectors were compared for simultaneous determination of triazine herbicides simazine, atrazine, propazine, terbuthylazine, cyanazine, ametryn, prometryn, and atraton, and of their dealkylated degradation products in soil. The compounds were ultrasonically extracted from spiked agricultural soil samples (organic matter content < 5%) with a 2:1 acetone:n‐hexane mixture. High efficiency of GC capillary column and high selectivity of the thermionic sensitive detector (TSD) and ion trap detector (ITD) made it possible to directly analyse uncleaned soil extracts and determine all 12 compounds in one run. In reversed‐phase HPLC with diode‐array detector (DAD), the co‐elution of soil matrix components interfered with the determination of methylthiotriazines and terbuthylazine. The recovery of triazine compounds, determined by GC‐TSD, from a silty sand soil (organic matter content 1.82%, pH 6.22) spiked at levels of 15–600 ng g^–1, were 70–90% (RSD 9–19%), except for deisopropylatraton (38%). GC‐TSD analysis with detection limits of 5–15 ng g^–1 for chloro‐ and methylthiotriazines and 30 ng g^–1 for methoxytriazines was more sensitive than GC‐MS(ITD). GC analysis with electron capture detection was sensitive for some chlorotriazines, but a reliable compound quantification in complex chromatograms of uncleaned soil of extracts was not possible. For all compounds save didealkylatrazine, HPLC‐DAD was at least two times less sensitive than GC‐TSD. Soil/sediment organic matter, clay and silt content, and pH were identified as matrix characteristics which might affect ultrasonic extraction recovery of a particular compound.     

Modelling and Optimization of Ultrasound-Assisted Extraction of Phenolic Compounds from Black Quinoa by Response Surface Methodology

Phenolic compounds are currently the most investigated class of functional components in quinoa. However, great variability in their content emerged, because of differences in sample intrinsic and extrinsic characteristics; processing-induced factors; as well as extraction procedures applied. This study aimed to optimize phenolic compound extraction conditions in black quinoa seeds by Response Surface Methodology. An ultrasound-assisted extraction was performed with two different mixtures; and the effect of time; temperature; and sample-to-solvent ratio on total phenolic content (TPC) was investigated. Data were fitted to a second-order polynomial model. Multiple regression analysis and analysis of variance were used to determine the fitness of the model and optimal conditions for TPC. Three-dimensional surface plots were generated from the mathematical models. TPC at optimal conditions was 280.25 ± 3.94 mg of Gallic Acid Equivalent (GAE) 100 g⁻¹ dm upon extraction with aqueous methanol/acetone, and 236.37 ± 5.26 mg GAE 100 g⁻¹ dm with aqueous ethanol mixture. The phenolic profile of extracts obtained at optimal conditions was also investigated by HPLC. The two extracting procedures did not show different specificities for phenolic compounds but differed in the extraction yield.     

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.     

Extraction of antioxidants from plants using ultrasonic methods and their antioxidant capacity

The analytical method based on the HPLC coupled with UV detection (HPLC‐UV) for the determination of selected antioxidants (i.e. esculetin, scopoletin, 7‐hydroxycoumarine, rutin, xanthotoxin, 5‐methoxypsoralen and quercetin) in plant material was developed. Two ultrasonic extraction methods for the isolation of these compounds from the plants such as Mentha longifolia L., Mentha spicata L., Ruta graveolens L., Achyllea millefolium L., Plantago lanceolata L. and Coriandrum sativum L. were used. Both of these methods, i.e. ultrasonic probe and ultrasonic bath, were optimised and compared to each other. For the proposed HPLC‐UV method LOQ values in the range from 22.7 (xanthotoxin) up to 97.2 ng/mL (rutin) were obtained. For all extracts the antioxidant capacity based on the reduction of free 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) radical was also determined. Obtained results ranged from 10.11 up to 73.50% of DPPH radical inhibition.     

Enhanced extraction of polychlorinated organic compounds from soil samples by fluidized-bed extraction (FBE)

Summary The extraction and determination of polychlorinated organic compounds, like hexachlorobenzene, pentachlorobenzene, octachlorostyrene and polychlorinated biphenyls in soils and solid wastes continues to be a subject for study. In this work Soxhlet extraction and a new extraction technique, fluidized-bed extraction, have been compared. The extraction of polychlorinated organic compounds by this technique has been optimized using experiemental design procedures. The variation of the number of extraction cycles, composition of extraction solvent (mixtures ofn-hexane-acetone) and the holding time after reaching the heating temperature were considering as experimental variables to generate a surface response design. Gas chromatography with mass spectrometric detection was used to determine levels of the analytes in the extracts. Extraction and analysis of a certified reference material (BCR CRM 392) showed the applicability of the method.     

Rapid isocratic HPLC analysis of caffeic acid derivatives from <Emphasis Type="Italic">Echinacea purpurea</Emphasis> cultivated in Iran

Cichoric acid and caftaric acid are the main phenolic compounds in Echinacea purpurea tops. The level of these phenolic compounds in E. purpurea extracts is affected by different factors such as seasonal variations, drying methods, extraction methods, and growing location of the plant. HPLC analysis of caffeic acid derivatives in extracts of Echinacea purpurea (Cultivar) aerial parts, produced by boiling water extraction and ethanol-water extraction methods, showed various levels of the derivatives. Our findings revealed that the Iranian cultivated E. purpurea had a high level of cichoric acid (3.5–5.7 %). Caftaric acid was also the main phenolic compound in E. purpurea tops (3.1–4.5 %). After 2 h of boiling water extraction, the level of cichoric acid was 5.7 %, whereas the level of this acid in 60:40 ethanol-water extraction did not exceed 3.9 %. Published in Khimiya Prirodnykh Soedinenii, No. 2, pp. 150–152, March–April, 2008.     

Liquid Chromatographic Determination of 4-Amino-N-(2,6-dimethylphenyl)-benzamide in Rat Serum and Urine

Abstract

The compound 4-amino-N-(2,6-dimethylphenyl)-benzamide has shown potential as a new anticonvulsant. A method for the liquid chromatographic determination of serum and urine concentrations of the compound and its N-acetylated metabolite was developed for pharmacokinetic studies. Quantitation was achieved via UV detection at 275 nm following isocratic reversed phase (C₁₈) separation using a ternary solvent system of water:acetonitrile:acetic acid (60:39:1) at a flow rate of 1.5 mL/min. The compounds were isolated from a 50 μL sample of serum using solid phase extraction with prior protein precipitation. The compounds and internal standard were eluted from the extraction column with acetonitrile. Isolation from urine was achieved similarly with the exclusion of protein precipitation. The assay procedure is useful for the determination of concentrations of parent compound from 0.68 to 204.6 μg/mL.     

Differential Measurement of Cortisol and Cortisone in Human Saliva by HPLC with Uv Detection

Abstract

Both cortisol and its dehydro metabolite cortisone are present in normal human saliva. A method for differential Measurement of both compounds in 1 ml samples of saliva by HPLC/UV is described. the method uses an extraction column having a cyclodextrin bonded phase to retain the compounds of interest while allowing elution of interfering compounds. A steroid-bearing fraction is eluted from the cyclodextrin column, dried, reconstituted in a weak mobile phase, and injected on a reversed phase HPLC/UV system provided with an injector-mounted reversed phase extraction column. Samples containing corticosteroid concentrations as low as 0.5 ng/ml can be effectively analyzed by this method.     

Application of HPLC and Dual Wavelength Detection to the Analysis of Phenolic Compounds in Apples

Abstract

High-performance liquid chromatography, coupled with a programmable UV/visible detector, was applied to the identification and quantification of the phenolic compounds of apple tissue. The identity and purity of unknown components eluted from the HPLC column were established by comparing retention times and absorbance ratios with those of authentic compounds. In Golden Delicious apples, components with retention times agreeing with those of authentic catechin, epicatechin, chlorogenic acid, protocatechuic acid, p-hydroxybenzoic acid, p-coumaric acid, ferulic acid, and sinapic acid were found. However, comparison of absorbance ratios with those of authentic compounds suggested that many of the components that eluted in this system as single, symmetrical peaks were mixtures rather than individual compounds. This method helped to eliminate inaccurate identifications or faulty assumptions about homogeneity of HPLC “peaks”, errors that can occur when traditional HPLC methods that rely solely on detection at a single wavelength are employed. The method should have general applicability to plant extracts.