Determination of 2,4-D and Dicamba in food crops by MEKC

Summary The determination of 2,4-D (2,4-dichlorophenoxyacetic acid) and Dicamba (2-methoxy-3,6-dichlorobenzoic acid) residues in sugar cane, rice and corn was performed by a supercritical fluid extraction (SFE) method using CO₂/acetone as extraction mix and an SFE apparatus developed in our laboratory. The extracts were cleaned up after extraction by both liquid-liquid partition and a Florisil column. Micellar electrokinetic capillary chromatography (MEKC) coupled with ultraviolet on-column detection was used for the analysis of these pesticides. The detection limits were improved by the preparation of a special detection cell with an increased pathlength that gave detection limits of ca. 0.6 pg for 2,4-D and Dicamba. Our results demonstrated that capillary electrophoresis can be a powerful new analytical tool for pesticide residue analysis.     

Off-line SFE-CZE analysis of carbamates residues in tobacco samples

Summary Determination of carbamate residues in tobacco samples was carried out by solid-liquid extraction (SLE) and supercritical-fluid extraction (SFE) methods, both developed for this purpose. The clean-up step was carried out on SPE-Florisil cartridges and the extracts analysed by capillary zone electrophoresis (CZE) with UV detection.The results were compared and SFE using CO₂-acetone showed the best results in terms of recovery and generally higher extraction power. SFE in conjunction with CZE proved suitable for carbamate residue analysis in real tobacco samples.     

Supercritical fluid extraction of 2,4-D from soils using derivatization and ionic modifiers

Supercritical fluid extraction (SFE) with CO(2), a clean and rapid alternative to conventional organic solvent extraction techniques, was investigated for the extraction of 2,4-D from soils using a variety of pre-extraction soil treatments to enhance extraction recoveries. Initial experiments with silylation, ion-pairing, methyl esterification, and ionic displacement are reported. Methyl esterification and ionic displacement during SFE proved to be the most promising approaches for quantitative extraction. Although the SFE procedures were not fully optimized, comparison between SFE and a standard Soxhlet extraction method demonstrated the potential for improving analytical measurement for highly polar pesticides in soil by modifying SFE-CO(2) extraction with derivatizing reagents and ionic solutions.     

Identification of volatile compounds of Atractylode lancea Rhizoma using supercritical fluid extraction and GC–MS

Supercritical fluid was used to extract volatile components from the rhizoma of Atractylode lancea (A. lancea). An orthogonal array design (OAD), L₉ (3)⁴, was employed as a chemometric method for the optimization of the supercritical fluid extraction (SFE) of volatile compounds from the herbal medicine. Four parameters, namely, pressure, temperature, dynamic extraction time, and flow rate of CO₂, were studied and optimized by a three‐level OAD in which the interactions between the parameters were neglected. These compounds were identified according to their retention times and mass spectra by GC–MS. A total of 30 compounds of SFE extracts were identified. Atractylon (8.63%), hinesol (1.44%), β‐eudesmol (6.64%), elemol (0.42%), and atractydin (13.92%) were the major sesquiterpenes identified in A. lancea SFE extracts.     

Coupled solid phase extraction–supercritical fluid extraction–on-line gas chromatography of explosives from water

A method has been developed for the quantitative extraction of nitrotoluenes (2,3-dinitrotoluene, 2,4-dinitrotoluene and trinitrotolugene) from water using a Bakerbond^TM phenyl sorbent. The average solid phase extraction recoveries for spiked standards ranged from 80 to 95 percent for reagent water and 52 to 95 percent from well and surface water in the low ppb and ppt levels. After the nitrotoluenes had been trapped on the solid sorbent they were quantitatively eluted using SFE. Adding toluene to the extraction cell increased the rate of extraction, but did not improve analyte recovery versus unmodified CO₂. The extracts were analyzed off-line with GC–ECD using an internal standard. Extraction losses were due to analyte breakthrough, and not from poor SFE recoveries. This demonstrates that supercritical fluid extraction is a suitable elution technique for analytes trapped on solid phase extraction sorbents. Also, a method for the direct on-line coupling of SPE to GC, using SFE, has been developed and evaluated. Supercritical CO₂ is ideal for directly coupling SPE to GC, since carbon dioxide is a gas under ambient conditions. One potential problem of on-line SPE–SFE–GC is the presence of residual water trapped on the active sites of the Bakerbond¹³ phenyl sorbent. This problem was dealt with by using a split interface previously described by Hawthorne. From the results of this study, the relative standard deviation of the on-line SPE–SFE–GC interface was determined to be between 4 and 10 percent. In addition, there was no significant difference in the precision of the method with or without the use of an internal standard. A calibration curve was also constructed (r² = 0.995) from spiked controls, demonstrating that the method is quantitative.     

GC–MS Studies of Thiophenes in the Supercritical Fluid CO2 and Solvent Extracts of Tagetes patula L.

The intact plant parts and genetically modified hairy root clone #TpA6 of Tagetes patula were extracted with supercritical fluid CO₂ extraction (SFE) and a conventional solvent extraction. SFE optimization included the variation of fluid CO₂ pressure, dynamic time, and the addition of methanol modifier co-solvent. The four characteristic thiophene metabolites, 5-(3-buten-1-ynyl)-2,2′-bithienyl (BBT), 2,2′:5′,2″-terthiophene (α-T), 5-(4-acetoxy-1-butynyl)-2,2′-bithienyl (BBTOAc), and 5-(3,4-diacetoxy-1-butynyl)-2,2′-bithienyl [BBT(OAc)₂], were analysed by GC–MS. The proposed SFE method allowed the selective extraction of thiophenes in 60 min dynamic time with supercritical CO₂ without modifier co-solvent, at 30 MPa and 40 °C. The SFE and the reference solvent extraction yielded similar results. The SFE of intact roots and flowers yielded 717 ± 31.3 and 480 ± 26.6 μg g^?1 α-T, respectively, while the leaves did not contain considerable amounts of thiophenes. Remarkable amounts of BBT, BBTOAc, and BBT(OAc)₂ were characteristic of the SFE of hairy root cultures.     

GC–MS Studies of Thiophenes in the Supercritical Fluid CO2 and Solvent Extracts of Tagetes patula L.

The intact plant parts and genetically modified hairy root clone #TpA6 of Tagetes patula were extracted with supercritical fluid CO₂ extraction (SFE) and a conventional solvent extraction. SFE optimization included the variation of fluid CO₂ pressure, dynamic time, and the addition of methanol modifier co-solvent. The four characteristic thiophene metabolites, 5-(3-buten-1-ynyl)-2,2′-bithienyl (BBT), 2,2′:5′,2″-terthiophene (α-T), 5-(4-acetoxy-1-butynyl)-2,2′-bithienyl (BBTOAc), and 5-(3,4-diacetoxy-1-butynyl)-2,2′-bithienyl [BBT(OAc)₂], were analysed by GC–MS. The proposed SFE method allowed the selective extraction of thiophenes in 60 min dynamic time with supercritical CO₂ without modifier co-solvent, at 30 MPa and 40 °C. The SFE and the reference solvent extraction yielded similar results. The SFE of intact roots and flowers yielded 717 ± 31.3 and 480 ± 26.6 μg g⁻¹ α-T, respectively, while the leaves did not contain considerable amounts of thiophenes. Remarkable amounts of BBT, BBTOAc, and BBT(OAc)₂ were characteristic of the SFE of hairy root cultures.

     

Identification and quantification of the volatile constituents in Cnidium monnieri using supercritical fluid extraction followed by GC‐MS

The volatile components of Cnidium monnieri were obtained by supercritical fluid extraction (SFE) and analyzed by GC‐MS (identification and determination of metabolites). The compounds were identified according to their retention times and mass spectra. The effects of different parameters, such as extraction pressure, temperature, dynamic extraction time, flow rate of CO₂, on the SFE of C. monnieri extracts were investigated. A total of 14 compounds of SFE extracts were identified. Osthole (69.52%), bornyl acetate (10.03%), α‐pinene (4.71%), and imperatorin (2.42%) were the major compounds identified in C. monnieri SFE extracts. The quantitation of osthole and imperatorin were then accomplished. The linear calibration ranges were all 5–1000 μg/mL for osthole and imperatorin by GC‐MS analysis. The recovery of osthole and imperatorin were in the range 96.5–101.8%. The LODs for osthole and imperatorin were 1.0 and 0.6 μg/mL, respectively.     

Supercritical fluid extraction of atrazine and its metabolites from soil

Soxhlet (methanol) and SFE extraction with carbon dioxide in the presence of modifiers at different temperatures (100–200°C) for the extraction of atrazine and its main metabolites from a soil sample were compared. The most effective extraction conditions for both atrazine and its metabolites (i.e. deethylatrazine and deisopropylatrazine) were Co₂ modified with 20% molar methanol-trifluoroacetic acid (MeOH-TFA) (TFA 0.65M in MeOH) at 100°C, leading to an extraction efficiency comparable with that of Soxhlet extraction with MeOH for atrazine and ca. 20% higher for its main metabolites. The relative standard deviation (RSD) of SFE was lower than that obtained by Soxhlet extraction, probably because of less interference in the cGC-NPD determination. All the other modifiers evaluated (acetone, triethylamine, and methanol) were less effective than MeOH-TFA for the extraction of atrazine and its metabolites from a soil sample, even at high molar concentrations (20%) and use of higher extraction temperatures (200°C). These results indicate the importance of matrix effects and the need of the selection of an appropriate modifier in order to obtain quantitative extractions by SFE.     

Determination of fat content and fatty acid composition in meat and meat products after supercritical fluid extraction

Two different relatively simple, commercially available supercritical fluid extractors (SFE), Leco and Foss-Tecator, were tested for the determination of total fat content in meat and meat products. The fatty acid composition in meat and meat products was also determined after the Foss-Tecator extraction in an aliquot of the extract. Total fat was determined by weighing after the different extraction procedures and the fatty acid composition by gas chromatography after hydrolysis and methylation of the extract. The results for total fat content agreed well with results from a standard method of Schmid, Bondzynski, and Ratzlaff, which uses conventional solvent extraction. Fatty acid composition was compared with the Bligh and Dyer extraction, and showed good agreement. The average relative difference between SFE and Bligh and Dyer of all fatty acids in the sample was <3% for acids exceeding 0.5% of total fatty acid amount. The advantages of SFE over traditional methods are a much lower consumption of hazardous organic solvents and shorter extraction times. To obtain quantitative recoveries by SFE, ethanol was added to the extraction cells before extraction.     

HRGC/FID and HRGC/MSD Analysis of the Secondary Metabolites Obtained by Different Extraction Methods from Lepechinia schiedeana,and in Vitro Evaluation of Its Antioxidant Activity

Steam distillation (SD), simultaneous distillation-solvent extraction (SDE), microwave-assisted solvent extraction (MWE), and supercritical (CO₂) extraction (SFE) were used to isolate secondary metabolites from Lepechinia schiedeana. The various extracts were analyzed by capillary gas-chromatography, on poly (dimethylsiloxane) (DB-1) and poly(ethyleneglycol) (INNOWAX), 60 m columns, using FID or MSD (EI, 70 eV). Kováts indexes, mass spectra, or standard compounds were employed for compound identification. 43, 61, 67, and 79 compounds at concentrations above 0.01% were detected in the SD, SDE, MWE, and SFE extracts, respectively. Ledol, C₁₅H₂₆O, was the major constituent (20.04–36.87%) in all extracts. Oxygenated sesquiterpenes (24.36–43.14%), C₁₀H₁₆, monoterpenes (27.70–39.87%), and C₁₅H₂₄, sesquiterpenes (10.04–22.22%) were the main groups of compounds present in SD, SDE, MWE, and SFE extracts. Heavy hydrocarbons (C_n > 15), diterpenoids, and phytosterols were found only in MWE and SFE extracts. The antioxidant activity of Lepechinia schiedeana was measured by the HRGC quantification of the volatile carbonyl compounds, final products of lipoxidation, released in a model lipid system (sunflower oil) by the effect of the Fenton reagent. The concentration of volatile carbonyl compounds decreased by 65% when lipid oxidation was induced in the presence of macerated Lepechinia plant. The protection of polyunsaturated acids in sunflower oil was also studied by measuring their concentrations after heating of the oil (180°C, 2 h) with and without macerated Lepechinia plant.     

Supercritical CO2 Extraction of Triterpenoids from Chaga Sterile Conk of Inonotus obliquus

Triterpenoids are among the bioactive components of Chaga, the sterile conk of the medicinal fungus Inonotus obliquus. Supercritical fluid extraction of Chaga triterpenoids was carried out with supercritical CO₂, while a modified Folch method was used as a comparison. Three temperature-pressure combinations were tested varying between 314–324 K (40–50 °C) and 281–350 bars, using time- and volume-limited extractions. Six triterpenoids were identified with GC-MS and quantified with GC-FID: ergosterol, lanosterol, β-sitosterol, stigmastanol, betulin, and inotodiol. The Folch extraction resulted in recovery of trametenolic acid, which was not extracted by supercritical CO₂. Inotodiol was the major triterpenoid of all the extracts, with a yield of 87–101 mg/100 g and 139 mg/100 g, for SFEs and the Folch method, respectively. The contents of other major triterpenoids, lanosterol and ergosterol, varied in the ranges 59–63 mg/100 g and 17–18 mg/100 g by SFE, respectively. With the Folch method, the yields were 81 mg/100 g and 40 mg/100 g, respectively. The highest recovery of triterpenoids with SFE in relation to Folch was 56% and it was obtained at 324 K (50 °C) and 350 bar, regardless of extraction time or volume of CO₂. The recoveries of lanosterol and stigmastanol were unaffected by SFE conditions. Despite the lower yield, SFE showed several advantages including shorter extraction time and less impact on the environment. This work could be a starting point for further studies on green extraction methods of bioactive triterpenoids from Chaga.     

Composition and antioxidant activity of Thymus vulgaris volatiles: Comparison between supercritical fluid extraction and hydrodistillation

Supercritical fluid extraction (SFE) of the volatile oil from Thymus vulgaris L. aerial flowering parts was performed under different conditions of pressure, temperature, mean particle size and CO₂ flow rate and the correspondent yield and composition were compared with those of the essential oil isolated by hydrodistillation (HD). Both the oils were analyzed by GC and GC‐MS and 52 components were identified. The main volatile components obtained were p‐cymene (10.0–42.6% for SFE and 28.9–34.8% for HD), γ‐terpinene (0.8–6.9% for SFE and 5.1–7.0% for HD), linalool (2.3–5.3% for SFE and 2.8–3.1% for HD), thymol (19.5–40.8% for SFE and 35.4–41.6% for HD), and carvacrol (1.4–3.1% for SFE and 2.6–3.1% for HD). The main difference was found to be the relative percentage of thymoquinone (not found in the essential oil) and carvacryl methyl ether (1.0–1.2% for HD versus t?0.4 for SFE) which can explain the higher antioxidant activity, assessed by Rancimat test, of the SFE volatiles when compared with HD. Thymoquinone is considered a strong antioxidant compound.     

Effect of temperature,collection mode,and modifier on the supercritical CO2 extraction of dicofol residues from fish samples

Toxicity evaluation of Dicofol to Astyanax bimaculatus schubarti, a characteristic fish species living in tropical rivers and lakes was carried out through LC₅₀ – 96 Hours. These experiments were performed under laboratory controlled conditions with atmospheric air flow and dilution water at 25°C in the static mode, supercritical fluid extraction (SFE) with pure CO₂ and CO₂ modified with hexane and methanol were used at 50, 70, 80, and 100°C and 300 atm. Several collection modes were studied to extract Dicofol from fish samples. The extraction efficiencies were directly comparedd with those obtained after 8 h of Soxhlet extraction using the same clean-up with Florisil and analysis by HRGC/ECD and HRGC/MS as a confirmatory analytical technique. The SFE recoveries at temperatures lower than 80°C were typically lower than soxhlet recoveries; however a temperature increase enhanced the efficiency of SFE. The results showed that under certain conditions, supercritical fluid gave higher extractio power (extracted 11 % more pesticide), shorter extraction time, and lower solvent consumption than Soxhlet, thus affording an excellent alternative to the conventional method for extracting Dicofol from fish sample.     

Extraction of Peppermint Essential Oils and Lipophilic Compounds: Assessment of Process Kinetics and Environmental Impacts with Multiple Techniques

Consumers are becoming more mindful of their well-being. Increasing awareness of the many beneficial properties of peppermint essential oil (EO) has significantly increased product sales in recent years. Hydrodistillation (HD), a proven conventional method, and a possible alternative in the form of microwave-assisted hydrodistillation (MWHD) have been used to isolate peppermint EO. Standard Soxhlet and alternatively supercritical fluid (SFE), microwave-assisted, and ultrasound-assisted extraction separated the lipid extracts. The distillations employed various power settings, and the EO yield varied from 0.15 to 0.80%. The estimated environmental impact in terms of electricity consumption and CO₂ emissions suggested that MWHD is an energy efficient way to reduce CO₂ emissions. Different extraction methods and solvent properties affected the lipid extract yield, which ranged from 2.55 to 5.36%. According to the corresponding values of statistical parameters, empiric mathematical models were successfully applied to model the kinetics of MWHD and SFE processes.     

Modeling of supercritical fluid extraction of flavonoids from Calycopteris floribunda leaves

The aim of this study was to obtain flavonoids extracts from Calycopteris floribunda leaves using supercritical fluid extraction (SFE) with CO₂ and a co-solvent. Pachypodol, a potential anticancer drug lead compound separated from the extracts, was examined. Classical organic solvent extraction (CE) with ethanol was performed to evaluate the high pressure method. HPLC analysis was introduced to interpret the differences between SFE and CE extracts in terms of antioxidant activity and the concentration of pachypodol. SFE kinetics and mathematical modeling of the overall extraction curves (OEC) were investigated. Evaluation of the models against experimental data showed that the Sovová model performs the best. The supercritical fluid extraction process was optimized using a central composite design (CCD), where temperature and pressure were adjusted. The optimal conditions of SFE were: pressure of 30 MPa and temperature of 35°C.     

Simultaneous determination of the herbicides isoproturon, dichlorprop-p, and bifenox in soils using RP-HPLC

Summary A simple and fast analytical method for the simultaneous determination of the herbicides isoproturon (N-(4-isopropylphenyl)-N'N'-dimethyl-urea), dichlorprop-p ((+)-2-(2,4-dichlorophenoxy)-propionic acid), bifenox (methyl-5-(2,4-dichlorophenoxy)-2-nitro-benzoate), and its main metabolite bifenox acid (5-(2,4-dichloro-phenoxy)-2-nitro-benzoic acid) in soils is described. The herbicides differ substantially in their physical and chemical properties. The soil extraction with a mixture of CH₂Cl₂, C₂H₅OH, and acetone, followed by an isocratic RP-HPLC determination yields high, reproducible recoveries for all compounds. The method can be used for different soil moisture and pH conditions. The detection limits range from 0.01 to 0.03 mg active compound/kg dry soil. An additional clean-up using size exclusion techniques can improve the detection limits to 0.003 to 0.007 mg active compound/kg dry soil. The method is suitable for routine residue analyses and applicable to other herbicides as well.     

Optimization of supercritical fluid extraction of saikosaponins from Bupleurum falcatum with orthogonal array design

Supercritical fluid extraction (SFE) was used to extract saikosaponins a, c and d from the root of Bupleurum falcatum. An orthogonal array design L₉(3)⁴ was employed as a chemometric method for the optimization of the SFE conditions. The effects of four factors including pressure (30–40 MPa), temperature (40–50°C), ethanol concentration (60–100%) and time (2.5–3.5 h) on the yields of saikosaponins were investigated by a preparative SFE system in the SFE mode. Under the optimized conditions, namely 35 MPa of pressure, 45°C of temperature, 80% of ethanol concentration and 3.0 h of time, the yields of saikosaponin c, saikosaponin a, saikosaponin d, total saikosaponins and SFE extract were 0.16, 0.12, 0.96, 1.24 and 16.48 mg/g, respectively. Determinations of the saikosaponins were performed by HPLC.     

Fat contents of cereal foods: comparison of classical with recently developed extraction techniques

Eight cereal products ranging from a fat-free ready-to-eat (RTE) cereal (frosted flakes) to a full-fat high-ratio cake mix were subjected to analyte separation by anhydrous diethyl ether extraction (EE), acid hydrolysis/mixed ether extraction (AH), solid-phase extraction (SPE), and supercritical fluid extraction (SFE) with CO2 and with CO2 modified with 15% ethanol (SFEmod). Gravimetric analyses of extracts indicated higher (P < or = 0.05) crude fat values by AH than by EE, SFE, or SFEmod. Extractions followed by fatty acid methyl ester analysis also produced higher (P < or = 0.05) triglyceride means by AH than by other methods used for heat-treated RTE cereals and for ready-to-cook (RTC) cream of wheat. Therefore, for labeling purposes, AH seems most appropriate because in most cases it achieves higher triglyceride values than extraction with solvent(s) alone. SFE and SPE, on the other hand, yield fat values similar to those obtained by EE and offer the advantages of reduced solvent volume, short extraction times, and ease of laboratory automation.     

HRGC/FID/NPD and HRGGC/MSD study of Colombian ylang-ylang (Cananga odorata) oils obtained by different extraction techniques

Steam distillation (SD), simultaneous distillation-solvent extraction (SDE), and supercritical (CO₂) extraction (SFE) were used to isolate volatile secondary metabolites from fresh, totally mature flowers of Colombian ylang-ylang (Cananga odorata). The various extracts were analyzed by capillary chromatography (DB-1, DBWAX, 60 m columns) using FID, NPD or MSD (EI, 70 eV). Kováts indexes, mass spectra, or standard substances were employed for compound identification. 51, 70, and 73 compounds at concentrations above 100 ppb were detected in the SD, SDE, and SFE extracts, respectively. The main constituents of these extracts were linalool (20.7, 28.0, and 16.5%), germacrene-D (10.1, 3.1, and 20.3%) benzyl benzoate (14.1, 2.9, and 3.9%), benzyl acetate (9.6, 17.0, and 6.2%), caryophyllene (3.1, 2.9, and 3.9%), and p-methylanisole (6.8, 6.1, and 2.7%). 85% of the composition of SDE extracts was represented by oxygenated compounds. Heavy hydrocarbons (C_n >20) and fatty acids were found only in the SFE extracts, which also had a higher content of nitrogenated compounds (phenylacetonitrile, 4-methylbenzaldoxime, indole, 2-phenyl-nitroethane, and methyl anthranilate) and sesquiterpenes (43% vs 19.5% in SD and 8.1% in SDE) and 1.5 – 2 times lower concentration of monoterpenes and light oxygenated compounds than the SD (49.7%) and SDE (64.5%) extracts.