Determination of α-tocopherol and ergocalciferol by thin-layer chromatography

A procedure was developed for determining α-tocopherol (vitamin E) and ergocalciferol (vitamin D₂) in oils. The procedure involved the alkaline hydrolysis of an oil according to GOST (State Standard) 30417 followed by the independent recovery of vitamins by extraction with organic solvents. The extracts were separated on Sorbfil plates with the stationary phase of silica gel with a particle size of 5–12 μm by eluting vitamin E with an octane-diethyl ether mixture (7: 1) and vitamin D₂ with a tetrachloromethane-diethyl ether mixture (4: 1). The zones of α-tocopherol and ergocalciferol were developed with conc. HNO₃ and conc. H₂SO₄, respectively. The plate scanning and image processing were performed with a Sorbfil Videodensitometer software. The calibration plots were linear in the range of 2.5–15 and 15–50 mg/mL for α-tocopherol and 0.08–0.13 mg/mL for ergocalciferol. The procedure was used to determine vitamins in bur marigold oily extract and grape seed oil.     

Capillary electrophoresis-mass spectrometry of Spirulina platensis proteins obtained by pressurized liquid extraction

In this work, the usefulness of CE-MS to monitor and optimize the pressurized liquid extraction (PLE) of proteins from Spirulina platensis microalga is demonstrated. Crude and purified PLE extracts from microalga were analyzed by CE-MS. It was observed that the use of purification protocols of phycobiliproteins (namely, ultrafiltration or precipitation-dialysis-freeze drying) resulted in better CE resolution and MS signals, demonstrating that sample matrix plays an important role in CE-MS of proteins in real samples. Ultrafiltration was found less laborious and much faster than precipitation-dialysis-freeze drying (1 vs. 48 h). Direct analysis of crude extracts was demonstrated to be also possible by CE-MS, providing less-quality information but enough to characterize PLE extracts in a much faster way. Therefore, the latter protocol was selected to monitor and optimize the extraction process of phycobiliproteins from S. platensis. To do that, different extraction conditions were tested, including time, temperature and pressure of extraction, nature of pressurized liquid, distribution of microalga inside the extraction cell, type of packing, etc. It is demonstrated that the combined use of PLE and CE-MS allows the attainment of extracts rich in phycobiliproteins in short extraction times (namely, yields of 20% can be obtained in less than 2 h under the optimum PLE process in an automatic way). To our knowledge, this work shows for the first time the usefulness of CE-MS for monitoring and optimizing a PLE process.     

Grape seed oil extraction: Interest of supercritical fluid extraction and gas-assisted mechanical extraction for enhancing polyphenol co-extraction in oil

The aim of this study is to compare three oil extraction methods and to evaluate their efficiency for producing an oil rich in polyphenols. The three extraction methods are screw pressing, extraction by supercritical CO₂ percolation and the combination of these two processes (Gas-Assisted Mechanical Expression: GAME). Screw pressing is the most efficient process for producing grape seed oil with a high yield, but supercritical CO₂ process permits an increase of polyphenol co-extraction with oil. The GAME process allows extraction of more polyphenols than screw pressing and constitutes an interesting process considering oil yield.     

Supercritical fluid extraction of grape seed oil and subsequent separation of free fatty acids by high-speed counter-current chromatography

Supercritical fluid extraction (SFE) of grape seed oil was performed to study the effect of various parameters such as pressure, temperature and the particle size of the sample on the yield and composition of oil using an analytical-scale SFE system. Then the extraction was scaled up by 125 times using a preparative SFE system under the optimized conditions of high pressure (30-40 MPa) and low temperature (35-40 degrees C) with medium particle size (20-40 mesh). The maximum yield of the oil can reach 6.2% with pure supercritical CO2 and 4.0% more oil can be obtained by adding 10% of ethanol as modifier. The unsaturated fatty acids (UFSs) make up about 70% in the oil on the basis of free fatty acids. The grape seed oil was then subjected to separation and purification for free fatty acids after saponification by high-speed counter-current chromatography coupled with evaporative light scattering detection (ELSD). The separation of 1.0 g of oil can yield about 430 mg pure linoleic acid at 99% purity. The fatty acids were analyzed by HPLC-ELSD.     

Comparison of the effect of <Emphasis Type="Italic">Pleurotus citrinopileatus</Emphasis> extract and vitamin E on the stabilization properties of camellia oil

We selected Camellia tenuifolia (Hayata) seed oil to compare the effects of mushroom extract and vitamin E on its stabilization properties. Camellia tenuifolia was selected for its higher oil content, but its proportions of unsaturated fatty acids and natural antioxidants as well as its oxidation stability are lower than those of Camellia oleifera oil. Our aim was to improve the oxidation stability, thermal stability, and photodegradation of C. tenuifolia seed oil and then compare the advantages of mixing traditional antioxidant (vitamin E) and mushroom natural antioxidant components (mushroom extract) in the oil. The focus was on the analysis of the effects of Pleurotus citrinopileatus (Singer) extract and vitamin E on the stabilization properties of C. tenuifolia seed oil, which involved some degradation research, such as evaluating the thermal, oxidation, and antioxidant effects as well as the irradiative (pulsed light) stability of the original oil and oil mixed with additives for comparing the differences by differential scanning calorimetry tests and isothermal microcalorimeter (TAM Air) analyses. We determined the effects of stabilization additives vitamin E and various PC extract doses by using pulsed light irradiation (0, 30, and 60 pulses) and found that the 3 mass% PC extract had the best antiphotodegradation characteristics, and the 0.1 mass% vitamin E indicated the outstanding oxidation stability for among all of the additives in this study. Overall, we obtained the following suitable conditions to stabilize camellia oil: addition of vitamin E, addition of 3 mass% PC extract, and a nitrogen atmosphere.     

Pressurized liquid extraction of mate tea leaves

The objective of this work is to investigate the influence of process parameters on the pressurized liquid extraction (PLE) of Ilex paraguariensis leaves. A factorial 2⁶⁻² experimental design was employed using responses as the extraction yield and the chromatographic profile of the extracts. The extraction time, polarity of solvent, amount of sample, numbers of PLE cycles, flushing volume and extraction temperature were selected as independent variables (factors). Results obtained indicated that the solvent polarity was the most significant variable in the study, while the amount of sample and extraction temperature also showed significant effect. The other variables did not present significant influence in the yield of extraction. GC/MS analysis of the extract enabled the identification of saturated hydrocarbons, fatty acids, fatty acid methyl esters, phytosterols and theobromine in the extracts. Quantitative analysis of four compounds presented in the extracts (caffeine, phytol, vitamin E and squalene) was performed by the GC/MS in the SIM mode.     

Selective pressurized liquid extraction of polychlorinated dibenzo-p-dioxins, dibenzofurans and dioxin-like polychlorinated biphenyls from food and feed samples

Selective pressurized liquid extraction (PLE) of polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (dl-PCBs) from various food and feed samples was performed with a selective PLE method previously developed for bulk PCBs. The method utilizes sulfuric acid impregnated silica inside the extraction cell to oxidize coextracted fat. Extractions were performed at 100 degrees C with n-heptane for 5 min in two cycles. Data obtained by selective PLE combined with gas chromatography/high-resolution mass spectrometry (GC-HRMS) were compared to concentrations derived from reference laboratories applying conventional sample preparation and GC-HRMS. Experiments performed on spiked vegetable oil, naturally contaminated crude fish oil and oil containing compound feed samples showed good results for these relatively simple matrices. The accuracy was generally +/-20% as compared to spiked levels or to values obtained by the reference laboratories. The precision, measured as the relative standard deviation (RSD) for 2,3,7,8-tetrachlorodibenzo-p-dioxin toxic equivalency values (TEQs), was below 10% in all cases. The method was also tested on naturally contaminated herring tissue, chicken tissue, pork tissue and sepiolitic clay, which all caused some trouble. It was observed that sufficient amounts of sodium sulfate should be used for dehydration of tissue samples and additionally, the cells should not be packed too dense in order to avoid suppressed extraction efficiency. Once this was attended to, satisfactory data could be obtained, except for sepiolithic clay. This study demonstrates that selective PLE can be applied with success to a number of food and feed matrices in analysis of PCDD/Fs and dl-PCBs. Since the fat removal step is on-line, the selective PLE method will reduce time and solvent consumption for sample preparation as compared to traditional clean-up.     

Pressurized-Liquid Extraction as an Efficient Method for Valorization of Thymus serpyllum Herbal Dust towards Sustainable Production of Antioxidants

The aim of this study was to valorize Thymus serpyllum herbal dust, a particular fraction distinguished as an industrial waste from filter-tea production. Pressurized liquid extraction (PLE) was used with the aim of overcoming certain obstacles of conventional extraction techniques in terms of shortening extraction time, reducing solvent consumption and energy costs, using “green” solvents and obtaining high yield and quality products. In order to optimize PLE of T. serpyllum herbal dust, the preliminary screening of the independent variables in order to define the most influential parameters and their domain was done first. After the screening, the optimization study using the face-centered central composite experimental design (CCD) with response surface methodology (RSM) was implemented. Additionally, taking into account the high awareness of the positive influence of antioxidants on the human health and associating it with high content of polyphenolic compounds in various members of Lamiaceae family, PLE has proven to be a great approach for antioxidants recovery from T. serpyllum herbal dust.     

Evaluation of surfactant assisted pressurized liquid extraction for the determination of glycyrrhizin and ephedrine in medicinal plants

Surfactant assisted pressurized liquid extraction (PLE) with a laboratory made system was applied for the extraction of glycyrrhizin in Radix glycyrrhizae/liquorice and ephedrine in Ephedra sinica. The proposed system set-up for this current work was simpler as no heating and back pressure regulator was required. Extraction with surfactant assisted PLE was carried out dynamically at a flow of 1.5 mL min⁻¹, at room temperature, under an applied pressure of 10-20 bar with an extraction time of 45-50 min. The extraction efficiencies of the proposed method using surfactants such as sodium dodecyl sulfate (SDS) and Triton X-100 were compared with sonication using organic solvent for different batches of medicinal plants materials. For the determination of glycyrrhizin in R. glycyrrhizae, the extraction efficiencies of surfactant assisted PLE with SDS and Triton X-100 was observed to be comparable with sonication. The method precision was found to vary from 1.6 to 2.6% (R.S.D., n = 6) on different days. For ephedrine in E. sinica, surfactant assisted PLE with SDS was found to give higher extraction efficiencies compared to Triton X-100. The overall method precision for surfactant assisted PLE with SDS for ephedrine in E. sinica was found to vary from 1.5 to 4.1% (R.S.D., n = 6) on different days. The marker compounds present in the various medicinal plant extracts were determined by gradient elution HPLC. Our data showed the possibility of PLE at room temperature and the advantages of eliminating the use of organic solvents in the extraction process.     

Comparison of soxhlet, ultrasound-assisted and pressurized liquid extraction of terpenes, fatty acids and Vitamin E from Piper gaudichaudianum Kunth

This paper describes a comparative study of extraction methods of terpenes (terpenic alcohols and phytosterols), fatty acids and Vitamin E from leaves of Piper gaudichaudianum Kunth. The analysis of extracts was done by gas chromatography with mass spectrometric detection. The identification and quantification was made by co-injections of the extract with certified standards. The use of pressurized liquid extraction (PLE; Dionex trade name: ASE, for accelerated solvent extraction) decrease significantly the total time of extraction, the amount of solvent and the manipulation of sample and solvents in comparison with soxhlet (SE) and ultrasound-assisted (USE). In addition, PLE was more effective for the extractions of terpenes (terpenic alcohols and phytosterols), fatty acids and Vitamin E.     

Pressurized liquid extraction as a sample preparation method for the analysis of isoflavones in pulses

In this work, we describe a rapid and simple analytical method that exploits pressurized liquid extraction (PLE) and liquid chromatography with diode array detection for the determination of isoflavones in samples of Spanish pulses. Confirmation of the analytes present was performed using ion-trap mass spectrometry. To optimize the PLE extraction, variables such as the dispersing agent, type of solvent and sample amount, and the experimental parameters, such as temperature and the number of extraction cycles, were studied. Separation was carried out using a reverse-phase C18 with polar endcapping as the stationary phase and acetonitrile/water with 0.2?% of formic acid, under a gradient regime, as the mobile phase. Optimal extraction of formononetin and biochanin-A from chickpeas with PLE was achieved using Hydromatrix as a dispersant agent, methanol/water (50:50), a temperature of 90?°C, and three cycles. The same optimal conditions-except methanol/water (75:25)-for solvent extraction were obtained for the extraction of daidzin, genistin, and formononetin from lentils. Recoveries ranged from 97 to 110?%, and standard deviations lower than 20?% were obtained. The contents obtained for daidzin in lentils using the proposed method were not significantly different from those obtained using another official method of analysis.     

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.     

Chemical characterization and phase behaviour of grape seed oil in compressed carbon dioxide and ethanol as co-solvent

The aim of this work is to report phase equilibrium experimental results for the systems grape oil/carbon dioxide and (grape oil/carbon dioxide + ethanol). The oil was obtained by supercritical extraction from the grape seed residue from wine production. The static synthetic method using a variable-volume view cell was employed for obtaining the experimental bubble and dew (cloud) points transition data over the temperature range of (313.15 to 343.15) K and pressures up to 20.6 MPa. The experiments were carried out using (ethanol + CO₂) overall mass fractions ranging from 0.50 to 0.99, keeping a fixed ethanol to carbon dioxide molar ratio at 1:3. Results indicate the existence of complex phase behaviour for all temperatures investigated with the occurrence of vapour–liquid, liquid–liquid and vapour–liquid–liquid phase transitions observed.     

Bixin extraction from defatted annatto seeds

Bixin is the major carotenoid in the seed of the Annatto plant (Bixa orellana L.). The aim of this study was to obtain extracts containing bixin from seeds that had been partially defatted by supercritical fluid extraction. Pressurized liquid extraction (PLE) and low-pressure solvent extraction (LPSE) methods were used, and the effects of the solvent, temperature, pressure, solvent mass to feed mass (S/F) ratio and ultrasonication were evaluated for the global yield (X₀(%)) and the bixin yield (BY(%)). Extraction conditions producing high yields of bixin were established for both the PLE and LPSE methods. Analysis of variance was used to examine the influence of the individual extraction variables in LPSE and PLE. For LPSE; significant effects were found for solvent, temperature, and the interactions of temperature with solvent and temperature with S/F. Solvent was the only variable that significantly affected X₀(%) and BY(%), for PLE. While ultrasonication did not significantly affect X₀(%) or BY(%), scanning electron microscopy analysis revealed structural changes in the vegetal matrix following this treatment.     

New possibilities for the valorization of olive oil by-products

In this contribution, the capabilities of pressurized liquid extraction (PLE) using food-grade solvents, such as water and ethanol, to obtain antioxidant extracts rich on polyphenolic compounds from olive leaves are studied. Different extraction conditions were tested, and the PLE obtained extracts were characterized in vitro according to their antioxidant capacity (using the DPPH radical scavenging and the TEAC assays) and total phenols amounts. The most active extracts were obtained with hot pressurized water at 200 °C (EC(50) 18.6 μg/mL) and liquid ethanol at 150 °C (EC(50) 27.4 μg/mL), attaining at these conditions high extraction yields, around 40 and 30%, respectively. The particular phenolic composition of the obtained extracts was characterized by LC-ESI-MS. Using this method, 25 different phenolic compounds could be tentatively identified, including phenolic acids, secoiridoids, hydroxycinnamic acid derivatives, flavonols and flavones. Among them, hydroxytyrosol, oleuropein and luteolin-glucoside were the main phenolic antioxidants and were quantified on the extracts together with other minor constituents, by means of a UPLC-MS/MS method. Results showed that using water as extracting agent, the amount of phenolic compounds increased with the extraction temperature, being hydroxytyrosol the main phenolic component on the water PLE olive leaves extracts, reaching up to 8.542 mg/g dried extract. On the other hand, oleuropein was the main component on the extracts obtained with ethanol (6.156-2.819 mg/g extract). Results described in this work demonstrate the good possibilities of using PLE as a useful technique for the valorization of by-products from the olive oil industry, such as olive leaves.     

Chemical Composition,Antioxidant, Antimicrobial and Cytotoxic/Cytoprotective Activity of Non-Polar Extracts of Grape (Vitis labrusca cv. Bordeaux) and Blackberry (Rubus fruticosus) Seeds

The aim of this study was to compare the influence of the extraction method, chemical composition, antimicrobial effects, antioxidant activity, and cytotoxicity on human cells of the non-polar extracts of grape (Vitis labrusca) and blackberry (Rubus fruticosus) seeds. The Soxhlet (Sox), Bligh–Dyer (BD), and ultrasound (US) methods were used for extractions. For blackberry non-polar seed extract, extraction via the BD method showed the highest mean values of total phenolic content (TPC), expressed in milligrams of gallic acid equivalent per 100 mL of non-polar seed extracts (102.37 mg GAE/100 mL), and higher antioxidant activity in relation to the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, expressed in milligrams of gallic acid equivalent per 100 mL of non-polar seed extracts (11.50 mg AAE/100 mL), if compared with the Sox and US extractions. Similar results were obtained for the non-polar grape seed extracts, where BD extraction obtained the highest values for TPC (28.61 mg GAE/100 mL) and DPPH (35.36 mg AAE/100 mL). The type of extraction method had an impact on the composition of fatty acids. Only the non-polar blackberry and grape seed extracts obtained via the Sox method showed some in vitro inhibitory effect against Escherichia coli (IAL 2064) and Staphylococcus aureus (ATCC 13565). Regardless of the extraction method used, the non-polar blackberry and grape seed extracts did not decrease the cell viability (IC₅₀ >1000 µg/mL) of cancer and normal cell lines, thus indicating the relative safety of the extracts. All the seed extracts decreased the generation of reactive oxygen species in the cell lines. Blackberry and grape seed lipid fractions can be utilized as antioxidants, and the extraction methods used cause significant changes in relation to their bioactivity and chemical composition.     

Static and dynamic superheated water extraction of essential oil components from Thymus vulgaris L.

Superheated water extraction (SWE) performed in both static and dynamic condition (S‐SWE and D‐SWE, respectively) was applied for the extraction of essential oil from Thymus vulgaris L. The influence of extraction pressure, temperature, time, and flow rate on the total yield of essential oil and the influence of extraction temperature on the extraction of some chosen components are discussed in the paper. The SWE extracts are related to PLE extracts with n‐hexane and essential oil obtained by steam distillation. The superheated water extraction in dynamic condition seems to be a feasible option for the extraction of essential oil components from T. vulgaris L.     

Application of PLE for the determination of essential oil components from Thymus vulgaris L

Essential plants, due to their long presence in human history, their status in culinary arts, their use in medicine and perfume manufacture, belong to frequently examined stock materials in scientific and industrial laboratories. Because of a large number of freshly cut, dried or frozen plant samples requiring the determination of essential oil amount and composition, a fast, safe, simple, efficient and highly automatic sample preparation method is needed.Five sample preparation methods (steam distillation, extraction in the Soxhlet apparatus, supercritical fluid extraction, solid phase microextraction and pressurized liquid extraction) used for the isolation of aroma-active components from Thymus vulgaris L. are compared in the paper. The methods are mainly discussed with regard to the recovery of components which typically exist in essential oil isolated by steam distillation.According to the obtained data, PLE is the most efficient sample preparation method in determining the essential oil from the thyme herb. Although co-extraction of non-volatile ingredients is the main drawback of this method, it is characterized by the highest yield of essential oil components and the shortest extraction time required. Moreover, the relative peak amounts of essential components revealed by PLE are comparable with those obtained by steam distillation, which is recognized as standard sample preparation method for the analysis of essential oils in aromatic plants.     

Determination of endocrine-disrupting compounds in cereals by pressurized liquid extraction and liquid chromatography-mass spectrometry. Study of background contamination

A sensitive method based on pressurized liquid extraction (PLE) and liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) has been developed for the determination in cereal samples of seven endocrine-disrupting compounds: bisphenol A (BPA), 4-tert-butylbenzoic acid (BBA), 4-nonylphenol (NP), 4-tert-butylphenol (t-BP), 2,4-dichlorophenol (DCP), 2,4,5-trichlorophenol (TCP) and pentachlorophenol (PCP). For the PLE procedure, methanol was selected as the extraction solvent. An experimental design approach was applied to optimize other PLE parameters. The recoveries achieved for the all seven compounds were in the 81-104% range, with relative standard deviations of 4-9%. An additional preconcentration step, based on solid-phase extraction (SPE), after the PLE step proved to be a successful way for obtaining a more sensitive method. The detection limits achieved in corn breakfast cereals were in the 0.003-0.013 microg g(-1) range, except for BPA, with a detection limit of 0.043 microg g(-1), for a sample size of 2.5 g. These values are similar to or even lower than currently legislated limits for pesticides in cereals and cereal-based foodstuffs. We also investigated possible contamination during the experimental process by the target compounds released from purified water, plastics, syringes, peristaltic pump tubes, glassware and other laboratory materials in contact with the samples along the analytical process.     

UPLC-MS/MS测定葡萄籽提取物中78种农药残留

建立了超高效液相色谱串联质谱法(UPLC-MS/MS)检测葡萄籽提取物中78种农药残留物的分析方法。样品用乙腈振荡提取,经乙二胺-N-丙基甲硅烷(PSA)和石墨化炭(GCB)固相萃取柱串联净化,乙腈-甲苯(V/V3:1)洗脱,洗脱液浓缩至约0.5 mL后,于45℃下氮气吹干,1.0 mL乙腈定容,UPLC-MS/MS测定,外标法定量。葡萄籽提取物中,78种农药在0.001～0.2μg/mL范围内线性关系均良好;所有农药的方法定量限(LOQ)均低于17.0μg/kg;在5,25,50μg/kg的添加水平下,葡萄籽提取物中78种农药的平均回收率处于72.6%～113.5%之间,相对标准偏差不大于11%。方法适用于葡萄籽提取物中78种农药残留的快速筛查测定。