Comparison of microemulsion electrokinetic chromatography and micellar electrokinetic chromatography methods for the analysis of phenolic compounds

In this study, microemulsion electrokinetic chromatography (MEEKC) and micellar electrokinetic chromatography (MEKC) were compared for their abilities to separate and detect thirteen phenolic compounds (syringic acid, p-coumaric acid, vanillic acid, caffeic acid, gallic acid, 3,4-dihydroxybenzoic acid, 4-hydroxybenzoic acid, (+)-catechin, (-)-epigallocatechin, (-)-epicatechin gallate, (-)-epigallocatechin gallate, (-)-epicatechin, and (-)-gallocatechin), and two other ingredients (caffeine and theophylline) in teas and grapes. Separation of phenolic compounds was improved by changing the SDS concentration for MEEKC, but the SDS concentration rarely affected the resolution for MEKC. Organic modifier (acetonitrile or methanol) was found to markedly influence the resolution and selectivity for both MEEKC and MEKC systems. In addition, a higher voltage and a higher column temperature improved the separation efficiency without any noticeable reduction in resolution for MEEKC whereas they caused a poor resolution for the MEKC system. Although separations with baseline resolution were achieved by the optimized MEEKC and MEKC methods, the separation selectivity resulting from the proposed MEEKC method was completely different from that of MEKC.     

Further knowledge on barley (Hordeum vulgare L.) leaves O-glycosyl-C-glycosyl flavones by liquid chromatography-UV diode-array detection-electrospray ionisation mass spectrometry

Thirty-seven flavonoids and a hydroxycynnamic acid have been characterized in barley leaves (Hordeum vulgare L.) by liquid chromatography-UV diode-array coupled to ion trap mass spectrometry with electrospray ionisation interface (negative mode). Their structures have been determined by the study of the ion mass fragmentation which characterizes C-glycosyl flavones and O-glycosyl-C-glycosyl flavones, and differentiates di-O-glycosyl flavones from O-diglycosyl-flavones. The majority of them are described for the first time in barley. Saponarin (isovitexin-7-O-glucoside), lutonarin (isoorientin-7-O-glucoside) and isoscoparin-7-O-glucoside derivatives constitute the major part of the detected compounds. Some acylated derivatives are also described, namely, 7-O-[6-acyl]-glucoside and -7-O-[6-acyl]-glc-4'-glucoside of isovitexin, isoorientin and isoscoparin.     

Analysis of some cytokinins in coconut (Cocos nucifera L.) water by micellar electrokinetic capillary chromatography after solid-phase extraction

Micellar electrokinetic capillary chromatography (MECC) was developed for the separation of cytokinins including trans-zeatin, trans-zeatin-O-glucoside, dihydrozeatin, dihydrozeatin-O-glucoside, meta-topolin riboside, N6-isopentenyladenine and N6-benzylaminopurine. Under the optimum conditions, i.e. a combination of 10 mM phosphate and 10 mM borate as the running buffer containing 50 mM sodium dodecyl sulphate at pH 10.4, the separation of seven cytokinin standards was accomplished within 11 min. The C18 solid-phase extraction (SPE) method was used to pre-concentrate the putative cytokinins present in the coconut water. Following which, the eluate was further purified using mixed mode Oasis MCX SPE columns and this additional step helps to reduce matrix interference during MECC. After the two solid-phase extraction steps, the optimized MECC method was able to screen for certain cytokinins (zeatin-O-glucoside and dihydrozeatin-O-glucoside) present in coconut water. After this screening, the presence of zeatin-O-glucoside and dihydrozeatin-O-glucoside in coconut water was further confirmed by independent high-performance liquid chromatography and liquid chromatography-mass spectrometry experiments.     

Studies on phytoestrogenic and nonphytoestrogenic compounds in Trifolium incarnatum L. and other clover species using pressurized liquid extraction and high performance column liquid chromatography with photodiode-array and fluorescence detection

HPLC coupled with photodiode array (PDA) and fluorescence (FL) detectors has been used for the identification and determination of phytoestrogenic (isoflavones and coumestrol) and nonphytoestrogenic (flavones) compounds in hydrolyzed and nonhydrolyzed extracts obtained from aerial parts of Trifolium incarnatum L. and related clover species. The effective isolation technique of pressurized liquid extraction (PLE) was used. Various types of extraction solvents, i.e., ethanol, n-propanol, n-butanol, and water solutions (75%, v/v) of methanol or ethanol at changeable or constant temperatures were tested, taking into account the chemical character of isolated compounds. Higher PLE efficiency in relation to isoflavone aglycones was found for ethanol. Predominant isoflavone glycosides determined in clover samples were sissotrin and ononin, reaching levels above 1.4% dry weight (wt) in T. medium. The presence of flavone compounds (apigenin and luteolin) in aerial parts of T. incarnatum, occurring in amounts exceeding 0.4% dry wt, documented chemotaxonomic distinction of this species from other clovers examined. Additionally, within the group of phytoestrogenic isoflavones, only biochanin A and formononetin derivatives were identified in above-ground parts of T. incarnatum. The application of simultaneous PDA and FL detection enabled unambiguous confirmation of the lack of a strong phytoestrogen, coumestrol, in all hydrolyzed and nonhydrolyzed clover extracts.     

Simultaneous analysis of different classes of phytohormones in coconut (Cocos nucifera L.) water using high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry after solid-phase extraction

Coconut (Cocos nucifera L.) water, which contains many uncharacterized phytohormones is extensively used as a growth promoting supplement in plant tissue culture. In this paper, a high-performance liquid chromatography (HPLC) method was developed for the simultaneous determination of various classes phytohormones, including indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), abscisic acid (ABA), gibberellic acid (GA), zeatin (Z), N⁶-benzyladenine (BA), α-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) in young coconut water (CW). The analysis was carried out using a reverse-phase HPLC gradient elution, with an aqueous mobile phase (containing 0.1% formic acid, pH adjusted to 3.2 with triethylamine (TEA)) modified by methanol, and solute detection made at 265 nm wavelength. The method was validated for specificity, quantification, accuracy and precision. After preconcentration of putative endogenous phytohormones in CW using C₁₈ solid-phase extraction (SPE) cartridges, the HPLC method was able to screen for putative endogenous phytohormones present in CW. Finally, the identities of the putative phytohormones present in CW were further confirmed using independent liquid chromatography–tandem mass spectrometry (LC–MS/MS) equipped with an electrospray ionization (ESI) interface.     

Impacts of PEG-6000 pretreatment for barley (Hordeum vulgare L.) seeds on the effect of their mature embryo in vitro culture and primary investigation on its physiological mechanism

In this paper, we studied polyetheneglycol (PEG) pretreatment effect on the mature embryo culture in vitro by using barley (Hordeum vulgare L.) seeds. Meanwhile, we analyzed and assayed its mineral element and endogenous hormone level. The experimental results were as follows: (1) PEG-6000 imbibition could obviously slow down the water timecourse absorbed by barley seeds; (2) 10% PEG-6000 treatment of barley seeds for 3 h had a positive effect on germination in vitro and callus induction of the barley seed mature embryos; (3) 10% PEG-6000 treatment inhibited soluble leakage from the seeds; (4) N leakage was mainly from the endosperms, Mn2+ leakage from embryos; (5) PEG-6000 treatment changed greatly the hormone level (ABA, IAA, GAs), which influenced the percentage of plantlets from the mature embryo callus. The results can provide some clues to scientific sowing of crop seeds, pretreatment for the purpose of uniform seedlings, and the explant response quality in plant tissue culture.     

Purification of the total steroidal saponins from fenugreek seeds (Trigonella foenum-graecum L.) using aqueous two-phase system and determination of diosgenin content using micellar electrokinetic chromatography method

The total steroidal saponins, particularly its major steroidal sapogenin (diosgenin), are the main active principles of fenugreek seed extract. In this study, an ethanol-salt aqueous two-phase system (ATPS) was explored for the purification of the total steroidal saponins, and the process conditions were optimized by response surface methodology (RSM). Under the optimized conditions, the RSM predicted recovery of the total steroidal saponins in the top phase of ATPS was 97.9%, which agreed with the average experimental recovery (98.3 ± 4.2% (n = 6)). Moreover, a rapid micellar electrokinetic chromatography (MEKC) method was developed for the determination of diosgenin from extracts. The diosgenin content in the ATPS top phase extract was 3-fold higher than that in crude extract, suggesting this ATPS having a great potential for purification pharmacological active ingredients from fenugreek seeds.     

Inula viscosa (L.) Aiton leaves and flower buds: Effect of extraction solvent/technique on their antioxidant ability,antimicrobial properties and phenolic profile

Abstract

This study was designed to establish the most effective solvent/technique for extracting antioxidant phytoconstituents from leaves and flower buds of Inula viscosa (L.) Aiton (Asteraceae) grown wild in Morocco. Maceration and hot extraction with methanol or water and Soxhlet ethanol extraction were utilized. The antioxidant potential was evaluated in vitro by DPPH, reducing power, and ferrous ions chelating activity assays. I. viscosa leaf and flower bud extracts displayed the strongest effect in the DPPH test, being the Soxhlet ethanol the most active ones (IC₅₀ = 54.24?±?0.21?μg/mL and 39.77?±?0.23?μg/mL); thus, they were selected for further investigations. The antimicrobial efficacy of the Soxhlet ethanol extracts against ATCC and food isolates strains was assayed; the leaf extract showed the best activity, and Candida albicans was the most sensitive strain (MIC = 125?µg/mL). The extracts resulted non-toxic against Artemia salina. Among the phenolics characterised by HPLC-PDA-ESI-MS, hispidulin hexoside, patuletin and spinacetin were identified for the first time.     

Quality evaluation of Rhizoma Belamcandae (Belamcanda chinensis (L.) DC.) by using high-performance liquid chromatography coupled with diode array detector and mass spectrometry

A high-performance liquid chromatography coupled with diode array detector and mass spectrometry (HPLC-DAD-MS) method was developed to evaluate the quality of Rhizoma Belamcandae (Belamcanda chinensis (L.) DC.) through establishing chromatographic fingerprint and simultaneous determination of seven phenolic compounds. The analysis was achieved on an Alltima C(18) analytical column (250 mm x 4.6 mm i.d. 5 microm) using linear gradient elution of acetonitrile-0.1% trifluoroacetic acid. The correlation coefficients of similarity were determined from the HPLC fingerprints, and they shared a close similarity. By using an online APCI-MS/MS, twenty phenols were identified. In addition, seven of these phenols including mangiferin, 7-O-methylmangiferin, tectoridin, resveratrol, tectorigenin, irigenin and irisflorentin were quantified by the validated HPLC-DAD method. These phenols are considered to be major constituents in Rhizoma Belamcandae, and are generally regarded as the index for quality assessment of this herb. This developed method by having a combination of chromatographic fingerprint and quantification analysis could be applied to the quality control of Rhizoma Belamcandae.     

Extract with high 1,1-diphenyl-2-picrylhydrazyl (DPPH) inhibitory capability from pericarp and seed of mangosteen (Garcinia mangostana L.) using microwave-assisted extraction (MAE) two-phase solvent technique

The human body needs compounds that are antioxidants to prevent oxidative stress. Some parts of the mangosteen fruit (Garcinia mangostana L.) have been known as sources of bioactive compounds that have antioxidant properties. The pericarp and seeds of mangosteen were extracted using the MAE method to produce the extract with the greatest antioxidant activity. There are two types of solvent mixtures used in the extraction process: single-phase and two-phase solvents. The solvents used were ethanol (EtOH), ethyl acetate (EtOAc), isopropyl alcohol (IPA), and water. First, utilizing dried mangosteen pericarp powder as the raw material, a study was undertaken to determine the ideal operating conditions for the MAE process. A one-factor-at-a-time approach was used to find the best operating conditions. A mixture of solvents with varied ratios (mL/mL), extraction temperature (°C), extraction time (min), and solid to solvent ratio (g/mL) were applied as independent variables. Then, dried mangosteen seed powder extraction was carried out based on the best-operating conditions previously achieved. The DPPH scavenging activity, total phenolic content (TPC) value, and α-mangostin content of the two extracts were compared. It was discovered that the mangosteen pericarp extract showed higher antioxidant activity (IC₅₀ DPPH = 9.40 µg/mL) than the mangosteen seed extract (IC₅₀ DPPH = 37.54 µg/mL), even slightly better than ascorbic acid (IC₅₀ DPPH = 10.47 µg/mL). The best extract was produced from the bottom phase of two-phase solvent system (EtOAc:EtOH:Water 2:1:2), with an MAE temperature of 50 °C, a time of 4 min, and a solid-to-solvent ratio of 1:16. The TPC value of the best extract is 903.54 mgGAE/g extract, with a yield of 16.53 % and an α-mangostin concentration of 0.11 %.     

Influence of processing procedure on the quality of Radix Scrophulariae: A quantitative evaluation of the main compounds obtained by accelerated solvent extraction and high‐performance liquid chromatography

An improved high‐performance liquid chromatography with diode array detection combined with accelerated solvent extraction method was used to simultaneously determine six compounds in crude and processed Radix Scrophulariae samples. Accelerated solvent extraction parameters such as extraction solvent, temperature, number of cycles, and analysis procedure were systematically optimized. The results indicated that compared with crude Radix Scrophulariae samples, the processed samples had lower contents of harpagide and harpagoside but higher contents of catalpol, acteoside, angoroside C, and cinnamic acid. The established method was sufficiently rapid and reliable for the global quality evaluation of crude and processed herbal medicines.     

A multiresidue method for the analysis of pesticide residues in polished rice (Oryza sativa L.) using accelerated solvent extraction and gas chromatography and confirmation by mass spectrometry

An analytical procedure using accelerated solvent extraction and gas chromatography with an electron capture detector has been optimized to simultaneously determine the residue of two insecticides (diazinon and EPN) and one fungicide (isoprothiolane) in polished rice and was confirmed by GC-mass spectrometry. Several parameters, including temperature, pressure, solvent ratio, cell size and cell cycle, were thoroughly investigated to find the optimal extraction conditions. The average recoveries of the three pesticides were between 82.7 and 126.4% at spiking levels of 0.1 and 0.5 ppm. The relative standard deviations were less than 7% for all of the recovery tests. The optimum accelerated solvent extraction operating conditions were 100 degrees C, 1500 atm, acetone-n-hexane (20:80 v/v) as the extraction solvent, two cycles, and a cell size of 33 ml. The total extraction time was approximately 20 min. The optimized procedure has also been applied to the determination of diazinon, isoprothiolane and EPN in real rice samples. In conclusion, accelerated solvent extraction was used for the first time for the analysis of diazinon, isoprothiolane and EPN in polished rice and offers the possibility of a fast and simple process for obtaining a quantitative extraction of the studied pesticides.     

Ultra-performance liquid chromatography-Quadrupole time-of-flight tandem mass spectrometry-based metabolite profiling,quality evaluation,and marker analysis of Trachyspermum ammi (L.) Sprague by high-performance thin-layer chromatography

Trachyspermum ammi (L.) Sprague (Apiaceae), commonly known as “Ajwain” is distributed throughout India. Ajwain fruits contain fiber, carbohydrates, phenolic acids, flavonoids, and tannins. The fruits also yield a small amount of essential oil, with Thymol as the principal constituent. Ajwain has various pharmacological activities like anti-leishmanial, antimicrobial, cytotoxic, antispasmodic, nematocidal, and anthelmintic. The fruits are of high therapeutic value; thus, it becomes quite essential to evaluate the quality of Trachyspermum ammi (L.) Sprague to authenticate and ensure its therapeutic and nutritional properties. The ethyl acetate fraction of Trachyspermum ammi (L.) Sprague fruits exhibited the highest total phenolic and flavonoid content values of 149.55 ± 1.19 mg rutin equivalent and 682.85 ± 3.68 mg gallic acid equivalent, respectively. Metabolite profiling of the ethyl acetate fraction using ultra-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry analysis resulted in identifying 19 phytomolecules. A validated high-performance thin-layer chromatography method was developed to quantify standard phytomolecules in the ethyl acetate fraction. The highest and lowest percentages of phytomarker were found to be caffeic acid (5.51% ± 0.16% w/w) and gallic acid (1.29% ± 0.09% w/w), respectively. This validated rapid, accurate, and precise method for standardization of Trachyspermum ammi (L.) Sprague will be beneficial for its quality evaluation as well as the derived products.     

Comparison of different solid phase extraction materials and techniques by application of multiresidue methods for the determination of pesticides in water by high-performance liquid chromatography (HPLC)

Solid phase extraction materials and techniques (C-18 EMPORE^® disks, polystyrenedivinylbenzene (SDB) EMPORE^® disks, C-18 BondElut cartridges and ENVI-Carb cartridges) are compared for the preconcentration of 33 basic/neutral and 10 acidic/phenolic pesticides and three metabolites in water. The efficiency of the different extraction procedures was investigated by application of appropriate multiresidue separation methods by reversed phase-high performance liquid chromatography with UV-diode-array detection. Calibrations were performed with multicomponent standard mixtures and recoveries, relative standard deviations and determination limits were calculated for comparing the described enrichment methods. Experiences made in practical application of the different techniques and materials were also considered for the final evaluation.     

Preparative separation of bioactive compounds from essential oil of Flaveria bidentis (L.) Kuntze using steam distillation extraction and one step high‐speed counter‐current chromatography

In order to utilize and control the invasive weed, bioactive compounds from essential oil of Flaveria bidentis (L.) Kuntze were studied. Steam distillation extraction and one step high‐speed counter‐current chromatography were applied to separate and purify the caryophyllene oxide, 7,11‐dimethyl‐3‐methylene‐1,6,10‐dodecatriene, and caryophyllene from essential oil of Flaveria bidentis (L.) Kuntze. The two‐phase solvent system containing n‐hexane/acetonitrile/ethanol (5:4:3, v/v/v) was selected for the one step separation mode according to the partition coefficient values (K) of the target compounds and the separation factor (α). The purity of each isolated fraction after a single high‐speed counter‐current chromatography run was determined by high performance liquid chromatography. A 3.2 mg of caryophyllene oxide at a purity of 92.6%, 10.4 mg of 7,11‐dimethyl‐3‐methylene‐1,6,10‐dodecatriene at a purity of 99.1% and 5.7 mg of caryophyllene at a purity of 98.8% were obtained from 200 mg essential oil of Flaveria bidentis (L.) Kuntze. The chemical structures of these components were identified by GC‐MS, ¹H‐NMR, and ¹³C‐NMR.     

Comparison of different solid phase extraction materials and techniques by application of multiresidue methods for the determination of pesticides in water by high-performance liquid chromatography (HPLC)

Solid phase extraction materials and techniques (C-18 EMPORE^® disks, polystyrenedivinylbenzene (SDB) EMPORE^® disks, C-18 BondElut cartridges and ENVI-Carb cartridges) are compared for the preconcentration of 33 basic/neutral and 10 acidic/phenolic pesticides and three metabolites in water. The efficiency of the different extraction procedures was investigated by application of appropriate multiresidue separation methods by reversed phase-high performance liquid chromatography with UV-diode-array detection. Calibrations were performed with multicomponent standard mixtures and recoveries, relative standard deviations and determination limits were calculated for comparing the described enrichment methods. Experiences made in practical application of the different techniques and materials were also considered for the final evaluation.     

Comparison of different extraction methods for the determination of α‐ and β‐thujone in sage (Salvia officinalis L.) herbal tea

Salvia officinalis L. (sage) is an important industrial plant used both for food and pharmaceutical purposes. The terpene fraction of this plant is responsible for many of its therapeutic and culinary properties. We used different extraction methods Tenax TA® purge and trap, headspace (HS) solid‐phase microextraction, HS sorptive extraction, and stir bar sorptive extraction to analyze the terpene fraction extracted from sage tea by GC–MS. Twenty compounds were identified, including α‐, β‐thujone, and several other oxygenated monoterpenes (1,8‐cineole, linalool, camphor, boneol, and bornyl acetate) and oxygenated sesquiterpenes (caryophyllene oxide, viridiflorol, humulene epoxide I, II, and III). Tenax TA® and HS sorptive extraction extracted a lower number of identified compounds, whereas HS solid‐phase microextraction allowed the complete extraction of volatiles with particular reference to α‐ and β‐thujone. The importance of the determination of thujones content in sage herbal tea is also discussed.     

Comparison of different extraction methods for the analysis of volatile secondary metabolites of Lippia alba (Mill.) N.E. Brown, grown in Colombia, and evaluation of its in vitro antioxidant activity

Hydrodistillation (HD), simultaneous distillation solvent extraction (SDE), microwave-assisted hydrodistillation (MWHD), and supercritical fluid (CO2) extraction (SFE) were employed to isolate volatile secondary metabolites from fresh leaves and stems of Colombian Lippia alba (Mill.) N.E. Brown. Kovàts indices, mass spectra or standard compounds were used to identify around 40 components in the various volatile fractions. Carvone (40-57%) was the most abundant component, followed by limonene (24-37%), bicyclosesquiphellandrene (5-22%), piperitenone (1-2%), piperitone (ca. 1.0%), and beta-bourbonene (0.6-1.5%), in the HD, SDE, MWHD, and SFE volatile fractions. Static headspace (S-HS), simultaneous purge and trap in solvent (CH2Cl2) (P&T), and headspace solid-phase microextraction (HS-SPME) were used to sample volatiles from fresh L. alba stems and leaves. The main components isolated from the headspace of the fresh plant material were limonene (27-77%), carvone (14-30%), piperitone (0.3-0.5%), piperitenone (ca. 0.4%), and beta-bourbonene (0.5-6.5%). The in vitro antioxidant activity of L. alba essential oil, obtained by hydrodistillation was evaluated by determination of hexanal, the main carbonyl compound released by linoleic acid subjected to peroxidation (1 mm Fe2+, 37 degrees C, 12 h), and by quantification of this acid as its methyl ester. Under the same conditions, L. alba HD-essential oil and Vitamin E exhibited similar antioxidant effects.     

Evaluation of a novel metal–organic framework as an adsorbent for the extraction of multiclass pesticides from coconut palm (Cocos nucifera L.): An analytical approach using matrix solid‐phase dispersion and liquid chromatography

We report the synthesis, characterization, and application of [Zn(1,4‐benzenedicarboxylate)(H₂O)₂]_n , Zn(1,4‐benzenedicarboxylate)_0.99(NH₂‐1,4‐benzenedicarboxylate)_0.01(H₂O)₂]_n , [Zn(1,4‐benzenedicarboxylate)_0.95(NH₂‐1,4‐benzenedicarboxylate)_0.05(H₂O)₂]_n , and [Zn(1,4‐benzenedicarboxylate)_0.9(NH₂‐1,4‐benzenedicarboxylate)_0.1(H₂O)₂]_n as sorbents for the extraction of multiclass pesticides from coconut palm. Liquid chromatography with ultraviolet diode array detection was used as the analysis technique, and the experiments were performed at one fortification level (0.1 μg/g). The recoveries were 47–67, 51–70, 58–72, and 64–76% for [Zn(1,4‐benzenedicarboxylate)(H₂O)₂]_n , Zn(1,4‐benzenedicarboxylate)_0.99(NH₂‐1,4‐benzenedicarboxylate)_0.01(H₂O)₂]_n , [Zn(1,4‐benzenedicarboxylate)_0.95(NH₂‐1,4‐benzenedicarboxylate)_0.05(H₂O)₂]_n , and [Zn(1,4‐benzenelate)_0.95(NH₂‐1,4‐benzenedicarboxylate)_0.05(H₂O)₂]_n , and [Zn(1,4‐benzenedicarboxylate)_0.9(NH₂‐1,4‐benzenedicarboxylate)_0.1(H₂O)₂]_n , respectively, with relative standard deviation ranging from 1 to 7% (n = 3). Detection and quantification limits were 0.01–0.05 and 0.05–0.2 μg/g, respectively, for the different pesticides studied. The method developed was linear over the range tested (0.01–10.0 μg/g) with r ² > 0.9991. A direct comparison of [Zn(1,4‐benzenedicarboxylate)_0.9(NH₂‐1,4‐benzenedicarboxylate)_0.1(H₂O)₂]_n with the commercially available neutral alumina showed that [Zn(1,4‐benzenedicarboxylate)_0.9(NH₂‐1,4‐benzenedicarboxylate)_0.1(H₂O)₂]_n was a similar extracting phase for the pesticides investigated.