Simultaneous measurement of fluoroquinolones in eggs by a combination of supercritical fluid extraction and high pressure liquid chromatography

Simultaneous detection of the fluoroquinolone antibiotics ciprofloxacin, enrofloxacin, ofloxacin, and norfloxacin in eggs by a combination of supercritical fluid extraction (SFE) and high pressure liquid chromatography (HPLC) was studied. Lipid matrices that have been considered to result in poor extraction and isolation of fluoroquinolones in eggs were removed first by SFE with supercritical CO(2) alone, and then the fluoroquinolones were extracted by SFE with supercritical CO(2) containing 20% (v/v) methanol for HPLC analysis. A time-course study of the extraction of lipid matrices of eggs suggested that the SFE method successfully removed the matrices within 20 min. When the fluoroquinolones added to control eggs were extracted by SFE, the extraction efficiency was similar to that by the solvent extraction method, giving the recovery percentages from 83 to 96% in a 40 min-extraction time. The fluoroquinolones extracted from eggs by SFE were analyzed simultaneously by HPLC equipped with a fluorescence detector with detection sensitivity at about 10 ppb for the detection limit. The standard calibration profiles of fluoroquinolones showed linear responses to HPLC, showing more than 0.995 for the mean r(2) value. This is the first report of the simultaneous measurement of fluoroquinolones in eggs by a combination of SFE and HPLC. Using the SFE method allowed us to avoid extensive sample preparation such as solvent extraction and chromatographic cleanup that are basically required in extraction of fluoroquinolones.     

Antileishmanial activity of fucosterol recovered from Lessonia vadosa Searles (Lessoniaceae) by SFE,PSE and CPC

Fucosterol, a triterpene derivative encountered in several alga species, provides a wide range of biological activities, such as protection against metabolic syndrome, or against UV-induced skin damage. We describe here the comparison of extraction by supercritical fluid (SFE) and pressurized solvent (PSE) of the brown alga Lessonia vadosa mainly abundant in the coastal water of Patagonia, followed by the isolation of fucosterol, using centrifugal partition chromatography (CPC), in association with HPLC–UV quantification. After collection, the seaweed was dried, ground and extracted either by PSE or by SFE under various conditions. The yield and the content in fucosterol of each extract were determined by HPLC–UV. Optimization of a biphasic solvent system and K_D calculation led to the isolation of pure fucosterol with high recovery rate. Extraction by SFE using CO₂ at 180 bar and 50 °C with 20 to 30 % of cellulose as modifier and CPC purification by cyclohexane/acetone/methanol/water 10/1/10/1 with lower layer as mobile phase led to the best results in terms of yield, purity, time and solvent consumption. Natural and semisynthetic steroid derivatives have been previously shown to be potential drug candidates against parasitic diseases including leishmaniasis. In this context fucosterol was evaluated and demonstrated noticeable antileishmanial activity (IC₅₀ < 10 μM) against intracellular amastigotes with limited or no cytotoxicity in host cell macrophages. These results make this compound a valuable starting scaffold for pharmacomodulation. Adaptation of the procedure by slight modifications of the extraction and/or isolation conditions could permit the exploitation of other alga species as raw material. Since SFE and CPC are available for pilot and batch production, this work may serve as a model for further scale-up and industrial development.     

Supercritical fluid extraction of selected pharmaceuticals from water and serum

Selected drugs from benzodiazepine, anabolic agent and non-steroidal anti-inflammatory drug (NSAID) therapeutic classes were extracted from water and serum using a supercritical CO₂ mobile phase. The samples were extracted at a pump pressure of 329 MPa, an extraction chamber temperature of 45°C, and a restrictor temperature of 60°C. The static extraction time for all samples was 2.5 min and the dynamic extraction time ranged from 5 to 20 min. The analytes were collected in appropriate solvent traps and assayed by modified literature HPLC procedures. Analyte recoveries were calculated based on peak height measurements of extracted vs. unextracted analyte. The recovery of the benzodiazepines ranged from 80 to 98% in water and from 75 to 94% in serum. Anabolic drug recoveries from water and serum ranged from 67 to 100% and 70 to 100%, respectively. The NSAIDs were recovered from water in the 76 to 97% range and in the 76 to 100% range from serum. Accuracy, precision and endogenous peak interference, if any, were determined for blank and spiked serum extractions and compared with classical sample preparation techniques of liquid-liquid and solid-phase extraction reported in the literature. For the benzodiazepines, accuracy and precision for supercritical fluid extraction (SFE) ranged from 1.95 to 3.31 and 0.57 to 1.25%, respectively (n=3). The SFE accuracy and precision data for the anabolic agents ranged from 4.03 to 7.84 and 0.66 to 2.78%, respectively (n=3). The accuracy and precision data reported for the SFE of the NSAIDs ranged from 2.79 to 3.79 and 0.33 to 1.27%, respectively (n=3). The precision of the SFE method from serum was shown to be comparable to the precision obtained with other classical preparation techniques.     

Detection of β-blockers in urine by solid-phase extraction-supercritical fluid extraction and gas chromatography-mass spectrometry

The most convenient way to perform supercritical fluid extraction (SPE) of liquid sample matrices is to combine it with solid-phase extraction (SPE). β-Blockers from urine were collected on an Empore disc, which was then placed into an extraction cell for derivatization and SPE. SPE recovery was best at pH 10. Effects of temperature, pressure and volume of pyridine on the acetylation and SFE processes were studied. Without acetylation the β-blockers were not significantly soluble in CO₂. SFE temperatures of 70°C and 150°C together with 200 μl of acetic anhydride and 400 μl pyridine gave the best results. With the SPE-SFE-GC-MS method developed here, β-blockers like oxprenolol, metoprolol and propranolol could easily be detected in urine samples, and the limit of detection (LOD) for these compounds was found to be 20 ng/ml, 30 ng/ml and 40 ng/ml, respectively.     

Extraction of astaxanthin from Haematococcus pluvialis by supercritical carbon dioxide fluid with ethanol modifier

Conventional solvent extraction methods cannot attain high‐quality antioxidant extracts from microalgae and also require solvent recovery and posttreatment. In this study, we utilized environmental friendly supercritical carbon dioxide fluid extraction (SFE‐CO₂) techniques to obtain pigment (i.e. astaxanthin) from Haematococcus pluvialis. The effects of key operating parameters on the extraction efficiency of astaxanthin were investigated, giving an optimal condition of H. pluvialis weight, 6.5 g; CO₂‐flow rate, 6.0 NL/min; extraction time, 20 min; extraction pressure, 4500 psi; volume of ethanol modifier added, 9.23 mL/g; extraction temperature, 50°C; modifier composition, 99.5%. Under these optimum conditions, the astaxanthin yield was 73.9% (10.92 mg/g dry H. pluvialis powder) after eight cycle of extraction cycles. The saponification index (C_S/C₀, representing the ratio of astaxanthin concentration after and before the saponification procedures) of the extract could be increased from 1 to 12.78 by saponification with 3.5 M NaOH.     

Extraction and isolation of avermectins and milbemycins from liver samples using unmodified supercritical CO2 with in-line trapping on basic alumina

A multi-residue supercritical fluid extraction (SFE) method has been developed for the extraction and isolation of eprinomectin, moxidectin, abamectin, doramectin and ivermectin residues from animal liver. Liver samples are mixed with hydromatrix and packed into a vessel containing 2 g of basic alumina. The samples are extracted at 100°C using unmodified supercritical carbon dioxide (SF-CO₂) at a pressure of 300 bar and flow-rate of 5.0 l/min. The analytes are adsorbed in-line on the basic alumina trap, which is later eluted with 4 ml of methanol–ethyl acetate (70:30, v/v). After evaporating to dryness, sample extracts are derivatised using methylimidazole, trifluoroacetic anhydride and acetic acid at 65°C for 30 min. Derivatised sample extracts are analysed by high-performance liquid chromatography (HPLC) with fluorescence detection. The method was validated using bovine liver fortified at levels of 4 and 20 μg/kg with the drugs. The mean recovery ranged between 76 and 97%. The intra- and inter-assay variations showed RSD values <10 and <16%, respectively. The procedure was also applied to ovine and porcine liver, giving similar results. The limit of quantitation of the method is 2 μg/kg.     

Quantification of seed oil from species with varying oil content using supercritical fluid extraction

Introduction: The quantity and composition of seed oil affects seed viability and storability and hence the value of a species as a resource for nutrition and plant conservation. Supercritical fluid extraction with carbon dioxide (SFE‐CO₂) offers a rapid, environmentally friendly alternative to traditional solvent extraction. Objective: To develop a method using SFE‐CO₂ to quantify the seed oil content in a broad range of species with high to low oil contents. Methodology: Seed oil was extracted using SFE‐CO₂ from four crop species representing high, medium and low oil content: Helianthus annuus, Asteraceae, with ca. 55% oil; Brassica napus, Brassicaceae, with ca. 50% oil; Glycine max, Fabaceae, with ca. 20% oil; and Pisum sativum, Fabaceae, with ca. 2% oil. Extraction pressures of 5000, 6000 and 7500 psi and temperatures of 40, 60 and 80°C were examined and a second step using 15% ethanol as a modifier included. Oil yields were compared with that achieved from Smalley Butt extraction. The optimised SFE‐CO₂ method was validated on six species from taxonomically distant families and with varying oil contents: Swietenia humilis (Meliaceae), Stenocereus thurberi (Cactaceae), Sinapis alba (Brassicaceae), Robinia pseudoacacia (Fabaceae), Poa pratensis (Poaceae) and Trachycarpus fortunei (Arecaceae). Results: The two‐step extraction at 6000 psi and 80°C produced oil yields equivalent to or higher than Smalley Butt extraction for all species, including challenging species from the Brassicaceae family. Conclusion: SFE‐CO₂ enables the rapid analysis of seed oils across a broad range of seed oil contents. Copyright © 2008 John Wiley & Sons, Ltd.     

Supercritical fluid extraction and high performance liquid chromatographic determination of benzopyrans and phloroglucinol derivative in Hypericum polyanthemum

The aerial parts of Hypericum polyanthemum Klotzsch ex Reichardt (Guttiferae) were successively extracted with supercritical carbon dioxide (SC CO₂) under pressures of 90, 120, 150 and 200 bar at different temperatures (40, 50 and 60 °C), and compared with the n-hexane extract obtained by ultrasound-assisted extraction. The samples obtained were examined regarding extraction yield and HPLC quantification of the main secondary metabolites, the benzopyrans HP1 (6-isobutyryl-5,7-dimethoxy-2,2-dimethylbenzopyran), HP2 (7-hydroxy-6-isobutyryl-5-methoxy-2,2-dimethyl-benzopyran) and HP3 (5-hydroxy-6-isobutyryl-7-methoxy-2,2-dimethyl) and the phloroglucinol derivative, uliginosin B. SFE presented higher selectivity than the n-hexane maceration, and the best condition to extract the target metabolites has been determined to be at 50 °C and for the high molecular-weight compound, uliginosin B, higher pressures were required.     

超临界流体萃取——高效液相色谱法测定百合中秋水仙碱

分别用超临界二氧化碳流体和有机溶剂萃取百合中的秋水仙碱,然后用高效液相色谱法直接测定萃取物中秋水仙碱的含量,从而测得百合中秋水仙碱的含量。超临界流体萃取的条件是：用乙醇作提携剂,萃取压力为18MPa,萃取温度为40℃,高效液相色谱测定条件为：ODS柱,甲醇：磷酸二氢钾溶液作流动相,检测波长为220nm,此法快速,简便,准确,可应用于秋水仙碱原料,制剂及其它植物中秋水仙碱含量的测定。     

Extraction of Oils from Wheat Germ with Supercritical Carbon Dioxide

The separation of oil from wheat germ by extracting with supercritical carbon dioxide (C0₂) is described. The solubility of wheat germ oil in supercritical C0₂ at 200 atm and 40°C was about 0.35 weight%. The effect of pressure on the extraction process with liquid or supercritical C0₂ was of great significance. On the other hand, the effect of temperature on the extraction process was small. Oil extracted with supercritical C0₂ was lighter in color and contained less phosphorus than that extracted with hexane. The contents of α- and β-tocopherol in the oil extracted with supercritical CO₂. were comparable to those in the hexane-extracted oil     

Extraction of squalene from yeast by supercritical carbon dioxide

Squalene produced under anaerobic conditions, by a strain of Torulaspora delbrueckii was extracted from the biomass using supercritical carbon dioxide. Minimum use of solvent, lower time of isolation and a higher selectivity of extraction merit use of supercritical fluid extraction (SFE) technique over solvent extraction of squalene, as optimized and reported previously. A maximum squalene yield of 11.12 g g^–1 (dry weight) of yeast cells was obtained at a temperature of 60 °C and pressure of 250–255 bar at a constant flow rate of 0.2l min^–1 of carbon dioxide. Lyophilization prior to SFE increased the squalene yield to 430.52 g g^–1 dry weight of yeast cells, an amount that is far greater than that obtained by (2:1) chloroform–methanol solvent extraction.     

Extraction of indole alkaloids fromCatharanthus roseus by using supercritical carbon dioxide

Summary Indole alkaloids, particularly vindoline and catharanthine, were extracted from the leaves ofCatharanthus roseus by supercritical extraction with CO₂. The contents of vindoline and catharanthine in the extracts were determined by HPLC and identified by LC/MS. About 52 %(w/w) of the initial vindoline content, 1.5 mg vindoline/g dry wt leaves, was recovered after extracting this material for 10 h with the CO₂ flow rate of 400 ml/min at 40°C and 150 bar. Vindoline concentration in the extract was 67 %(w/w).     

Isolation of off-flavors and odors from tuna fish oil using supercritical carbon dioxide

Off-flavors and unfavorable odors in tuna fish oil were successfully removed and identified using supercritical carbon dioxide extraction, while retaining variable compounds, polyunsaturated fatty acids such as EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid). Samples of oil were extracted in a 100 mL semi-batch stainless steel vessel under conditions which ranged from 8 to 20 MPa and 20 to 60°C with solvent (CO₂) flows from 10 g/min. GC-MS was used to identify the main volatile components contributing to the off-flavors and odors which included 2-methyl-1-propanol, 2,4-hexadienal, cyclopropane, and octadiene. Analyses of oil extracted at 40°C, 20 MPa showed a 99.8% reduction in dimethyl disulfide. Other significant off-flavors identified were 2-methyl-butene, 3-hydroxy butanal and ethylbenzene.     

Comparison of three different extraction methods and HPLC determination of the anthraquinones aloe‐emodine,emodine, rheine,chrysophanol and physcione in the bark of Rhamnus alpinus L. (Rhamnaceae)

Introduction – Rhamnus alpinus L. (Rhamnaceae), a traditional plants in the flora of the Abruzzo region, is known to contain active anthraquinone secondary metabolites. However, the content of anthraquinones varies among R. alpinus samples depending on collection season and site. Thus, using simple, reliable and accurate analytical methods for the determination of anthraquinones in R. alpinus extracts allows comparative study of different methods of extraction. Objective – After a partial validation of an HPLC method for the simultaneous determination of five anthraquinones, aloe‐emodine, rheine, emodine, chrysophanol and physcione, in the bark of R. alpinus, we compared three different methods of extraction. Methodology – Anthraquinones were extracted from the bark of R. alpinus using different techniques (methanol maceration, ultrasonic and supercritical CO₂ extraction). Separation and quantification of anthraquinones were accomplished using a reversed‐phase C₁₈ column with the mobile phase of H₂O–methanol (40 : 60, v/v, 1% formic acid) at a wavelength of 254 nm. The qualitative analyses were also achieved at wavelength of 435 nm. Results – All calibration curves were linear over the concentration range tested (10–200 mM) with the determination coefficients ≥0.991. The detection limits (S/N = 3) were 5 mM for each analytes. All five anthraquinones were found in the samples tested at concentrations reported in experimental data. Conclusion – The described HPLC method and optimised extraction procedure are simple, accurate and selective for separation and quantification of anthraquinones in the bark of R. alpinus and allow evaluation of the best extraction procedure between the tested assays. Copyright © 2009 John Wiley & Sons, Ltd.     

Supercritical extraction and supercritical antisolvent fractionation of natural products from plant material: comparative results on Persea indica

Supercritical fluids (SCFs) are alternative solvents in the field of Green Chemistry that are being developed as advanced separation techniques due to their combined properties such as the penetrability of a gas and the solvent power of a liquid. These characteristics are used in the supercritical fluid extraction (SFE), where compounds of interest can be extracted after mixing a SCF as a solvent with a matrix in a pressurized vessel. Supercritical antisolvent fractionation (SAF) uses the SCF as antisolvent, allowing for the precipitation of insoluble compounds in the SCF-organic solution mixture. An updated overview of SFE and SAF of natural products is presented in this article. Additionally we compare the results of SFE, SAF and organic soxhlet extraction (OSE) of Persea indica, a Macaronesian paleoendemism with strong insecticidal components. The composition of the extracts was analyzed by HPLC-MS, showing in the SFE extracts as major components ryanodol, cinnzeylanol and alkyl-γ-lactones, with their abundance varying with the extraction conditions. On the other hand, the SAF allowed for the fractionation of a liquid solution to give a ryanodol enriched extract (31 % more ryanodol than the initial ethanolic solution). The antifeedant and postingestive effects of these extracts on Spodoptera littoralis showed that the SAF extracts were the most active followed by SFE and OSE and their different effects can be partially explained by their composition.     

超临界CO_2萃取冬虫夏草子座挥发性成分的GC-MS研究

报道冬虫夏草挥发性成分的组成,为其进一步的研究工作奠定基础。采用超临界CO2萃取法从冬虫夏草子座中提取挥发性成分,气相色谱-质谱联用技术对其化学成分进行分析。超临界流体萃取物共鉴定了39种组分,占总馏出组分的86.6%以上,占色谱总馏出峰面积的98.56%以上。已鉴定组分中,含量最高的为油酸,相对含量25.6%;其次为亚油酸,相对含量22.67%;再次为棕榈酸11.86%。超临界CO2萃取法能更真实、全面的反映药材中的化学成分,适合于珍稀中药材相关组分的测定。     

Intracellular metabolite profiling and the evaluation of metabolite extraction solvents for Clostridium carboxidivorans fermenting carbon monoxide

Clostridium carboxidivorans ferments CO, CO₂, and H₂ via the Wood-Ljungdahl pathway. CO, CO_2, and H₂ are unique substrates, unlike other carbon sources like glucose, so it is necessary to analyze intracellular metabolite profiles for gas fermentation by C. carboxidivorans for metabolic engineering. Moreover, it is necessary to optimize the metabolite extraction solvent specifically for C. carboxidivorans fermenting syngas. In comparison with glucose media, the gas media allowed significant abundance changes of 38 and 34 metabolites in the exponential and stationary phases, respectively. Especially, C. carboxidivorans cultivated in the gas media showed changes of fatty acid metabolism and higher levels of intracellular fatty acid synthesis possibly due to cofactor imbalance and slow metabolism. Meanwhile, the evaluation of extraction solvents revealed the mixture of water-isopropanol-methanol (2:2:5, v/v/v) to be the best extraction solvent, which showed a higher extraction capability and reproducibility than pure methanol, the conventional extraction solvent. This is the first metabolomic study to demonstrate the unique intracellular metabolite profiles of the gas fermentation compared to glucose fermentation, and to evaluate water-isopropanol-methanol as the optimal metabolite extraction solvent for C. carboxidivorans on gas fermentation.     

Use of supercritical fluid extraction in the analysis of pesticides in soil

The applicability of supercritical fluid extraction (SFE) in pesticide residue analysis in soil was investigated by analysing real soil samples from field experiments. Additionally, radiotracer batch experiments were performed to study the release of non-extractable residues. High repeatability, accuracy and high selectivity were the most important advantages of SFE in residue analysis. Extracts with low amounts of coextractants from the soil matrix were achieved, allowing extracts to be pooled and concentrated without further clean up steps. Thus, the limited volume of extraction thimbles of the SFE apparatus used could be compensated and insufficiently high limits of determination could be improved. Although the application of methanol-modified supercritical CO(2) was a time-saving extraction procedure which reduced solvent usage and solvent waste, SFE efficiency proved only competitive to conventional slurry and Soxhlet extraction. No exhaustive release of non-extractable residues was achieved in radiotracer batch experiments.     

Brewing of saké from rice and rice-koji defatted by supercritical carbon dioxide treatment

Saké was brewed using rice and rice-koji defatted by extraction with supercritical carbon dioxide (SCCO₂). Extraction with SC:CO₂ at 300 atm and 40°C decreased the total contents of lipids of 90%- and 80%-polished rice to levels comparable to those of 80%- and 70%-polished rice. Addition of 5 wt% ethanol to SCCO₂ further decreased the total lipids. SCCO₂ extraction also removed lipids from rice-koji without decreasing its enzymatic activities. SCCO₂ treatment of rice improved the quality of saké, but SCCO₂ treatment of rice-koji did not.     

Isolation of chlorophylls and carotenoids from freshwater algae using different extraction methods

Usually marine algae are an excellent source of pigments for different commercial sectors. Freshwater macroalgae can be exploited as a good source of biologically active compounds provided an appropriate extraction method is developed. The efficiency of four methods, like microwave‐assisted (MAE), ultrasound‐assisted extraction (UAE), supercritical fluid extraction (SFE) with ethanol as a co‐solvent, as well as conventional Soxhlet extraction were studied in the same conditions (time, solvent and temperature) for the recovery of chlorophylls and carotenoids from three freshwater green algae species: Cladophora glomerata, Cladophora rivularis and Ulva flexuosa. UV‐Vis spectrophotometry was used to determine chlorophyll a, chlorophyll b and total carotenoid content in obtained extracts. The results of this study showed that the advantages of novel extraction techniques (MAE and UAE) include higher yield and, in consequence, lower costs compared to traditional solvent extraction techniques. These methods were much more efficient in freshwater green algae pigment recovery than the classic Soxhlet extraction as well as SFE.