Pressurized liquid extraction of isoflavones from soybeans

Isoflavone derivatives from freeze-dried soybeans were extracted by pressurized liquid extraction (PLE) and determined by reverse-phase high performance liquid chromatography (HPLC) with both photo diode array and mass spectrometry (MS) detection. Both real and spiked samples were used in the development of the method.Several extraction solvents (methanol (MeOH) and ethanol (EtOH), 30-80% in water and water), temperatures (60-200 °C), pressures (100-200 atm), as well as the sample size (0.5-0.05 g) and cycle length (5-10 min) were studied for the optimization of the extraction protocol. The optimized extraction conditions for quantitative recoveries were: 0.1 g of sample, 100 °C, three (7 min) static extraction cycles and ethanol 70% as extracting solvent. The stability of the isoflavones during the PLE was also determined. Under PLE conditions, degradation of malonyl glucoside forms of the isoflavones takes place using temperatures higher than 100 °C whereas degradation of glucosides takes place above 150 °C. Using the optimized protocol, isoflavones can be extracted from freeze-dried soybeans without degradation.     

Fast analysis of isoflavones by high-performance liquid chromatography using a column packed with fused-core particles

The recent development of fused-core technology in HPLC columns is enabling faster and highly efficient separations. This technology was evaluated for the development of an fast analysis method for the most relevant soy isoflavones. A step-by-step strategy was used to optimize temperature (25-50 °C), flow rate (1.2-2.7 mL/min), mobile phase composition and equilibration time (1-5 min). Optimized conditions provided a method for the separation of all isoflavones in less than 5.8 min and total analysis time (sample-to-sample) of 11.5 min. Evaluation of chromatographic performance revealed excellent reproducibility, resolution, selectivity, peak symmetry and low limits of detection and quantification levels. The use of a fused-core column allows highly efficient, sensitive, accurate and reproducible determination of isoflavones with an outstanding sample throughout and resolution. The developed method was validated with different soy samples with a total isoflavone concentration ranging from 1941.53 to 2460.84 μg g⁻¹ with the predominant isoflavones being isoflavone glucosides and malonyl derivatives.     

Microwave assisted extraction of soy isoflavones

A fast and reliable analytical method using microwave assisted extraction has been developed. Several extraction solvents (methanol (MeOH) and ethanol (EtOH), 30-70% in water and water), temperatures (50-150 °C), extraction solvent volume, as well as the sample size (1.0-0.1 g) and extraction time (5-30 min) were studied for the optimization of the extraction protocol. The optimized extraction conditions for quantitative recoveries were: 0.5 g of sample, 50 °C, 20 min and 50% ethanol as extracting solvent. No degradation of the isoflavones was observed using the developed extraction protocol and a high reproducibility was achieved (>95%).     

Fast analysis of soy isoflavones by high-performance liquid chromatography with monolithic columns

A fast method using high-performance liquid chromatography based on two monolithic columns has been developed for the simultaneous determination of isoflavones extracted from soybeans and derived foods. The 12 main isoflavones were resolved in 10 min in two coupled monolithic columns working at 35 °C using a elution gradient of acidified water (0.1% acetic acid) and methanol (0.1% acetic acid) at a flow rate of 5 mL min⁻¹. Retention time and relative area standard deviations were below 1% for all isoflavones. The method developed was successfully applied to several soy food samples and spiked samples. Total isoflavone concentration in sampled soy foods ranged from 34.28 mg L⁻¹ to 4.29 mg g⁻¹.     

Solid-phase extraction of soy isoflavones

An automated method using solid-phase extraction (SPE) for the concentration and clean-up of soy isoflavone extracts is proposed in this work. Using a standardized sample (0.1 g of a freeze dried soybean extract/25 mL of water); eight SPE cartridges with a wide range of sorbents (C18, divinylbenzene and modified divinylbenzene) from different suppliers were evaluated and compared. A large variation on SPE cartridges performance was observed, especially regarding retention and breakthrough volume of isoflavones during sample load and washing steps. The most effective cartridges were the divinylbenzene based cartridges, especially Strata X (from Phenomenex) and HLB oasis (from Waters). Using Strata X cartridges, several extraction parameters, such as sample loading flow (5-15 mL min(-1)), extracting solvent volume (2-6 mL of methanol), pH of the extracting solvent and the necessity of drying the sorbent before elution, were evaluated to provide a fast, specific, quantitative and reproducible SPE method. The optimized method consists of conditioning the cartridge with 10 mL of methanol and 10 mL of water (10 mL min(-1)), loading 25 mL of the standardized extract onto the cartridges (5 mL min(-1)), washing the cartridge with 10 mL of water (10 mL min(-1)) and finally eluting with 4 mL of methanol (10 mL min(-1)). Mean isoflavones recovery was 99.37% and mean intra- and inter-day reproducibility was higher than 98%. The developed sample clean-up/concentration (6.25:1) method takes less than 10 min and can be used in the analysis of isoflavones from soy extracts.     

Ultrasound-assisted extraction of soy isoflavones

Efficiency in extracting four isoflavone derivatives (daidzin, glycitin, genistin and malonyl genistin) from freeze-dried ground soybeans was compared for mix-stirring extraction and ultrasound-assisted extraction, using different solvents and extraction temperatures with both. The efficiency of the extraction of soy isoflavones was improved by ultrasound but was dependent on the solvent employed. Optimization of the ratios of sample quantity to solvent volume and length of extraction time was also performed. Isoflavones can be quantitatively extracted from soybeans with 50% ethanol at 60 degrees C using ultrasound-assisted extraction in 20 min.     

Hyphenated technique for the extraction and determination of isoflavones in algae: Ultrasound-assisted supercritical fluid extraction followed by fast chromatography with tandem mass spectrometry

New hyphenated technique for the extraction and determination of isoflavones in sea and freshwater algae and cyanobacteria was developed. The method consists of sonication sample pretreatment, extraction by supercritical CO₂ modified by 3% (v/v) of MeOH/H₂O mixture (9:1, v/v) at 35 MPa and 40 °C for 60 min, fast chromatography analysis by the means of Agilent 1200 Series Rapid Resolution and MS/MS determination. Agilent 1200 Series RRLC was used with Zorbax SB-CN chromatographic column (100 mm × 2.1 mm, particle size 3.5 μm), 3 μl injection volume, mobile phase consisting of 0.2% (v/v) acetic acid in water (solvent A) and acetonitrile (solvent B) and used with linear gradient (30% B at 0 min, from 0 min to 3 min up to 50% B, from 3 to 6 min up to 80% B and from 6 to 10 min down to 30% B). The flow-rate was 0.4 mL/min, column oven temperature 35 °C. MS detector Agilent Technologies 6460 Triple quadrupole LC/MS with Agilent Jet Stream was used in a negative ESI mode under following conditions: gas temperature 350 °C, gas flow 13 L/min, nebulizer gas pressure 50 psi, sheath gas temperature 400 °C, sheath gas flow 12 L/min, capillary voltage was 4 kV. Samples were analysed in the multiple reaction monitoring (MRM) mode. Eight isoflavone compounds were found for the first time in seven real samples of sea algae and in three control samples of freshwater algae and cyanobacteria. Usual optimisation study of extraction parameters was performed. Pressure and temperature optima for algae matrix are different from those obtained sooner for other matrices for most of the analytes, but the results of modifier optimisation study are in good accordance with those obtained sooner for spiked samples and red clover matrix. It seems that matrix has very small or no effect on the modifier selection. Two different approaches of sonication pretreatment were tested: sonication bath and the thorn instrument. In longer extraction time experiments, thorn sonication was more efficient and recovery of following supercritical fluid extraction was higher.     

Pressurized liquid extraction versus other extraction techniques in micropreparative isolation of pharmacologically active isoflavones from Trifolium L. species

As a new sample preparation technique, pressurized liquid extraction (PLE), in combination with reversed-phase liquid chromatography (RP-LC) and photodiode-array (PDA) detection were used for the isolation and determination of phytoestrogenic isoflavones in hydrolyzed extracts obtained from aerial parts of five Trifolium L. (clover) species. To optimize the effectiveness of PLE procedure, variable extraction parameters: methanol and acetone (or their 75% aqueous solutions), as extraction solvents, temperatures (75, 100 and 125 °C) and the changeable number of static extraction cycles were tested. Additionally, two other micropreparative techniques: ultrasound-assisted extraction (UAE), and conventional solvent extraction (CSE), under optimized conditions, were also used and compared. Optimum extraction efficiency, expressed in the highest yield of biochanin A, formononetin, daidzein and genistein from plant material, with PLE, using methanol-water (75:25, v/v) as an extraction solvent, an oven temperature of 125 °C and three 5-min static extraction cycles, was obtained.     

An on-line method for pressurized hot water extraction and enzymatic hydrolysis of quercetin glucosides from onions

A novel environmentally sound continuous-flow hot water extraction and enzymatic hydrolysis method for determination of quercetin in onion raw materials was successfully constructed using a stepwise optimization approach. In the first step, enzymatic hydrolysis of quercetin-3,4′-diglucoside to quercetin was optimized using a three level central composite design considering temperature (75–95 °C), pH (3–6) and volume concentration of ethanol (5–15%). The enzyme used was a thermostable β-glucosidase variant (termed TnBgl1A_N221S/P342L) covalently immobilized on either of two acrylic support-materials (Eupergit^® C 250L or monolithic cryogel). Optimal reaction conditions were irrespective of support 84 °C, 5% ethanol and pH 5.5, and at these conditions, no significant loss of enzyme activity was observed during 72 h of use. In a second step, hot water extractions from chopped yellow onions, run at the optimal temperature for hydrolysis, were optimized in a two level design with respect to pH (2.6 and 5.5), ethanol concentration (0 and 5%) and flow rate (1 and 3 mL min⁻¹) Obtained results showed that the total quercetin extraction yield was 1.7 times higher using a flow rate of 3 mL min⁻¹ (extraction time 90 min), compared to a flow rate of 1 mL min⁻¹ (extraction time 240 min). Presence of 5% ethanol was favorable for the extraction yield, while a further decrease in pH was not, not even for the extraction step alone. Finally, the complete continuous flow method (84 °C, 5% ethanol, pH 5.5, 3 mL min⁻¹) was used to extract quercetin from yellow, red and shallot onions and resulted in higher or similar yield (e.g. 8.4 ± 0.7 μmol g⁻¹ fresh weight yellow onion) compared to a conventional batch extraction method using methanol as extraction solvent.     

Ultrasonic assisted headspace single drop micro-extraction and gas chromatography with nitrogen-phosphorus detector for determination of organophosphorus pesticides in soil

This work describes optimization of headspace single drop micro-extraction for extraction of five organophosphorus pesticides; thionazin, sulfotep, dimethoate, disulfoton and parathion in soil. Ultrasound has also been used successfully to improve and accelerate the extraction of the analytes from the sample. The optimized extraction performance was obtained when the experimental parameters were set as follows; 3.0 μL of octanol as extraction solvent, high ionic strength (20% sodium chloride), 1:1 (w/v) sample dilution with water, extraction temperature at 60 °C for 30 min; applying ultrasound and without any pH adjustment. The optimized method was linear over the calibration range (5–200 and 10–300 for different analytes) with limits of detection of 0.1–2.0 ng g⁻¹. The enrichment factor for OPPs was 1.4–12.7 and the method was also reproducible with the relative standard deviations (RSD%) of 2.1–6.9%.     

Headspace solvent microextraction: a very rapid method for identification of volatile components of Iranian Pimpinella anisum seed

At the present study, a new and rapid headspace solvent microextraction (HSME), for the extraction and pre-concentration of the volatile components of plant sample into a microdrop was applied. The extraction occurred by suspending a microliter drop of the solvent from the tip of a microsyringe to the headspace of a ripen and powdered dry fruit sample (Iranian Pimpinella anisum seed) in a sealed vial for a preset extraction time, then the microdrop was retracted back into the microsyringe and injected directly into a GC injection port. The chemical composition of the HSME extracts were confirmed according to their retention indexes and mass spectra (EI, 70 eV); and quantitative analysis was performed by GC-FID.Parameters such as the nature of the extracting solvent, particle size of the sample, temperatures of the microdrop and sample, volume of sample and the extraction time were studied and optimized, and the method's performance was evaluated. The optimized conditions were: sample particle size, 1 mm; sample volume, 5 ml (in a 15 ml vial); sample temperature, 60 °C; microsyringe needle temperature, 0 °C; and extraction time, 10 min. Finally, accordingly, the percentage of trans-anethole (the major compound of P. anisum) and the relative standard deviation for extraction and determination of trans-anethole (seven-replicated analysis) were determined to be 90% and 3.9%, respectively.     

Development of liquid phase microextraction method based on solidification of floated organic drop for extraction and preconcentration of organochlorine pesticides in water samples

A simple and efficient liquid-phase microextraction (LPME) in conjunction with gas chromatography-electron capture detector (GC-ECD) has been developed for extraction and determination of 11 organochlorine pesticides (OCPs) from water samples. In this technique a microdrop of 1-dodecanol containing pentachloronitrobenzene (internal standard) is delivered to the surface of an aqueous sample while being agitated by a stirring bar in the bulk of solution. Following completion of extraction, the sample vial was cooled by putting it into an ice bath for 5 min. Finally 2 μL of the drop was injected into the GC for analysis. Factors relevant to the extraction efficiency were studied and optimized. Under the optimized extraction conditions (extraction solvent: 1-dodecanol; extraction temperature: 65 °C; sodium chloride concentration: 0.25 M; microdrop and sample volumes: 8 μL and 20 mL respectively; the stirring rate: 750 rpm and the extraction time: 30 min), figures of merit of the proposed method were evaluated. The detection limits of the method were in the range of 7-19 ng L⁻¹ and the RSD% for analysis of 2 μg L⁻¹ of OCPs was below 7.2% (n = 5). A good linearity (r² ≥ 0.993) and a relatively broad dynamic linear range (25-2000 ng L⁻¹) were obtained. After 30 min of extraction, preconcentration factors were in the range of 708-1337 for different organochlorine pesticides and the relative errors ranged from −10.1 to 10.9%. Finally the proposed method was successfully utilized for preconcentration and determination of OCPs in different real samples.     

Optimization of dispersive liquid-liquid microextraction combined with gas chromatography for the analysis of nitroaromatic compounds in water

Dispersive liquid-liquid microextraction (DLLME) coupled with gas chromatography-flame ionization detector (GC-FID) was developed for preconcentration and determination of some nitroaromatic compounds in wastewater samples. The effects of different variables on the extraction efficiency were studied simultaneously using experimental design. The variables of interest in the DLLME process were extraction and disperser solvent volumes, salt effect, sample volume, extraction temperature and extraction time. A Plackett-Burman design was performed for screening of variables in order to determine the significant variables affecting the extraction efficiency. Then, the significant factors were optimized by using a central composite design (CCD) and the response surface equations were derived. The optimum experimental conditions found from this statistical evaluation included: sample volume, 9 mL; extraction solvent (CCl₄) volume, 20 μL; disperser solvent (methanol) volume, 0.75 mL; sodium chloride concentration, 3% (w/v); extraction temperature, 20 °C and extraction time, 2 min. Under the optimum conditions, the preconcentration factors were between 202 and 314. Limit of detections (LODs) ranged from 0.09 μg L⁻¹ (for 2-nitrotoluene) to 0.5 μg L⁻¹ (for 2,4-dinitrotoluene). Linear dynamic ranges (LDRs) of 0.5-300 and 1-400 μg L⁻¹ were obtained for mononitrotoluenes (MNTs) and dinitrotoluenes (DNTs), respectively. Performance of the present method was evaluated for extraction and determination of nitroaromatic compounds in wastewater samples in the range of microgram per liter and satisfactory results were obtained (RSDs < 10.1%).     

Dispersive liquid-liquid microextraction preconcentration of palladium in water samples and determination by graphite furnace atomic absorption spectrometry

A new method for the determination of palladium was developed by dispersive liquid-liquid microextraction preconcentration and graphite furnace atomic absorption spectrometry detection. In the proposed approach, diethyldithiocarbamate (DDTC) was used as a chelating agent, and carbon tetrachloride and ethanol were selected as extraction and dispersive solvent. Some factors influencing the extraction efficiency of palladium and its subsequent determination, including extraction and dispersive solvent type and volume, pH of sample solution, concentration of the chelating agent and extraction time, were studied and optimized. Under the optimum conditions, the enrichment factor of this method for palladium reached at 156. The detection limit for palladium was 2.4 ng L⁻¹ (3σ), and the relative standard deviation (R.S.D.) was 4.3% (n = 7, c = 1.0 ng mL⁻¹). The method was successfully applied to the determination of trace amount of palladium in water samples.     

Improved methods for the extraction and analysis of isoflavones from soy-containing foods and nutritional supplements by reversed-phase high-performance liquid chromatography and liquid chromatography-mass spectrometry

An improved method for extraction and analysis of isoflavones from soy protein, soy foods and nutritional supplements is presented. The method uses acetonitrile extraction without acidification, with apigenin as internal standard. Samples extracted in acetonitrile-water are diluted to 50% acetonitrile and directly injected for gradient HPLC separation on a C18 reversed-phase column. This method saves significant time during sample preparation and improves accuracy and precision. Conventional and rapid HPLC analysis methods compatible with the extraction scheme were developed. During development of the methods, unexpected minor forms of malonyl and acetyl isoflavones were discovered in extracts of soy proteins and in pure isoflavone standard preparations. By LC-triple MS, these peaks have identical composition to the respective 6'-O-malonyl- and 6'-O-acetyl-isoflavones from which they form. These minor forms are believed to be malonyl and acetyl isoflavones where the site of attachment is a hydroxyl other than the 6'-OH of the glucose. These compounds can represent significant minor isoflavone components of foods, which contain high concentrations of malonyl or acetyl isoflavones.     

Headspace in-drop derivatization of carbonyl compounds for their analysis by high-performance liquid chromatography-diode array detection

A simple and rapid method has been reported for the determination of carbonyl compounds involving sample preparation by headspace single drop microextraction using 1-butanol as extraction solvent containing 2,4-dinitrophenylhydrazine for hydrazone formation, and direct transfer of the drop into the injector for high-performance liquid chromatography with diode array detection. An angle-cut polytetrafluoroethylene sleeve, 3 mm × 0.5 mm, was fixed at the tip of the syringe needle and this allowed the use of 7 μL drop of solvent drop for extraction and derivatization. The procedure has been optimized with respect to suitable solvent for headspace drop formation, drop volume, concentration of reagent, sample temperature, reaction time, and headspace-to-sample volume ratio. The method has been validated when rectilinear relationship was obtained between the amount of analyte and peak area ratio of hydrazones in the range 0.01-15 mg L⁻¹, the correlation coefficient over 0.996-0.999, and the limit of detection in the range 1.7-24.1 μg L⁻¹. Spiked real samples have been analyzed with adequate accuracy, and application has been demonstrated of the method for analysis of carbonyl compounds formed as oxidation products.     

Application of liquid-phase microextraction to the analysis of trihalomethanes in water

A liquid-phase microextraction method for the determination of trihalomethanes (THMs) including chloroform (CHCl₃), bromodichloromethane (CHBrCl₂), dibromochloromethane (CHBr₂Cl) and bromoform (CHBr₃) in water samples was developed, with analysis by gas chromatography-electron capture detection (GC-ECD). After the determination of the most suitable solvent and stirring rate for the extraction, several other parameters (solvent drop volume, extraction time and ionic strength of the sample) were optimized using a factorial design to obtain the most relevant variables. The optimized extraction conditions for 5 mL of sample volume in a 10 mL vial were as follows: n-hexane an organic solvent; a solvent drop volume of 2 μL; an extraction time of 5.0 min; a stirring rate of 600 rpm at 25 °C; sample ionic strength of 3 M sodium chloride. The linear range was 1-75 μg L⁻¹ for the studied THMs. The limits of detection (LODs) ranged from 0.23 μg L⁻¹ (for CHBr₂Cl) to 0.45 μg L⁻¹ (for CHCl₃). Recoveries of THMs from fortified distilled water were over 70% for a fortification level of 15 μg L⁻¹, and relative standard deviations of the recoveries were below 5%. Real samples collected from tap water and well water were successfully analyzed using the proposed method. The recovery of spiked water samples was from 73% to 78% with relative standard deviations below 7%.     

A microwave-based extraction method for the determination of sugar and polyols: Application to the characterization of regular and peaberry coffees

The brewing properties of coffee products are defined by the chemical composition in the bean, including sugars and polyols. Some factors, such as coffee species and roasting, may affect the level of these compounds in the bean. A new analytical microwave-assisted extraction (MAE) method has been developed to extract sugars and polyols from the coffee bean. The studied extraction conditions for the MAE were temperature (30–80 °C), solvent composition (0–50% ethanol in water), and solvent-to-sample ratio (10:1–30:1 mL solvent per g sample). A Box-Behnken design was applied to study the effect of extraction variables, and subsequently, the influential variables were optimized by response surface methodology (RSM). In addition to the main effect of the solvent-to-sample ratio, all quadratic effects significantly influenced (p < 0.05) the recovery of sugars and polyols from the coffee beans. RSM suggested the optimized MAE conditions: temperature 52 °C, ethanol concentration in water 18.5%, and solvent-to-sample ratio 17:1. Under the optimum condition, a kinetics study confirmed that 15 min showed high precision and accuracy of the developed method. Ultimately, a real sample application of the developed MAE revealed that the new method successfully described the composition of sugars and polyols in regular and peaberry coffee beans. Additionally, the method also effectively characterized the green and roasted Arabica and Robusta coffee beans.     

Simple hollow fiber renewal liquid membrane extraction method for pre-concentration of Cd(II) in environmental samples and detection by Flame Atomic Absorption Spectrometry

A hollow fiber renewal liquid membrane (HFRLM) extraction method to determine cadmium (II) in water samples using Flame Atomic Absorption Spectrometry (FAAS) was developed. Ammonium O,O-diethyl dithiophosphate (DDTP) was used to complex cadmium (II) in an acid medium to obtain a neutral hydrophobic complex (ML₂). The organic solvent introduced to the sample extracts this complex from the aqueous solution and carries it over the poly(dimethylsiloxane) (PDMS) membrane, that had their walls previously filled with the same organic solvent. The organic solvent is solubilized inside the PDMS membrane, leading to a homogeneous phase. The complex strips the lumen of the membrane where, at higher pH, the complex Cd-DDTP is broken down and cadmium (II) is released into the stripping phase. EDTA was used to complex the cadmium (II), helping to trap the analyte in the stripping phase. A multivariate procedure was used to optimize the studied variables. The optimized variables were: sample (donor phase) pH 3.25, DDTP concentration 0.05% (m/v), stripping (acceptor phase) pH 8.75, EDTA concentration 1.5 × 10⁻² mol L⁻¹, extraction temperature 40 °C, extraction time 40 min, a solvent mixture N-butyl acetate and hexane (60/40%, v/v) with a volume of 100 μL, and addition of ammonium sulfate to saturate the sample. The sample volume used was 20 mL and the stripping volume was 165 μL. The analyte enrichment factor was 120, limit of detection (LOD) 1.3 μg L⁻¹, relative standard deviation (RSD) 5.5% and the working linear range 2-30 μg L⁻¹.     

Ligandless-dispersive liquid-liquid microextraction of trace amount of copper ions

In the present work, a new ligandless-dispersive liquid-liquid microextraction (LL-DLLME) method has been developed for preconcentration trace amounts of copper as a prior step to its determination by flame atomic absorption spectrometry. In the proposed approach 1,2-dicholorobenzene and ethanol were used as extraction and dispersive solvents, respectively. Some factors influencing on the extraction efficiency of copper and its subsequent determination were studied and optimized, such as the extraction and dispersive solvent type and volume, pH of sample solution, extraction time and salting out effect. Under the optimal conditions, the calibration curve was linear in the range of 1.0 ng mL⁻¹-0.6 μg mL⁻¹ of copper with R² = 0.9985. Detection limit was 0.5 ng mL⁻¹ in original solution (3S_b/m) and the relative standard deviation for seven replicate determination of 0.2 μg mL⁻¹ copper was ±1.4%. The proposed method has been applied for determination of copper in standard and water samples with satisfactory results.