Separation and characterization of antioxidants from Spirulina platensis microalga combining pressurized liquid extraction, TLC, and HPLC-DAD

A new procedure has been developed to separate and characterize antioxidant compounds from Spirulina platensis microalga based on the combination of pressurized liquid extraction (PLE) and different chromatographic procedures, such as TLC, at preparative scale, and HPLC with a diode array detector (DAD). Different solvents were tested for PLE extraction of antioxidants from S. platensis microalga. An optimized PLE process using ethanol (generally recognized as safe, GRAS) as extraction solvent has been obtained that provides natural extracts with high yields and good antioxidant properties. TLC analysis of this ethanolic extract obtained at 115 degrees C for 15 min was carried out and the silica layer was stained with a DPPH (diphenyl-pycril-hydrazyl) radical solution to determine the antioxidant activity of different chromatographic bands. Next, these colored bands were collected for their subsequent analysis by HPLC-DAD, revealing that the compounds with the most important antioxidant activity present in Spirulina extracts were carotenoids, as well as phenolic compounds and degradation products of chlorophylls.     

Pressurized liquid extracts from Spirulina platensis microalga. Determination of their antioxidant activity and preliminary analysis by micellar electrokinetic chromatography

In this work, different extracts from the microalga Spirulina platensis are obtained using pressurized liquid extraction (PLE) and four different solvents (hexane, light petroleum, ethanol and water). Different extraction temperatures (115 and 170 degrees C) were tested using extraction times ranging from 9 to 15 min. The antioxidant activity of the different extracts is determined by means of an in vitro assay using a free radical method. Moreover, a new and fast method is developed using micellar electrokinetic chromatography with diode array detection (MEKC-DAD) to provide a preliminary analysis on the composition of the extracts. This combined application (i.e., in vitro assays plus MEKC-DAD) allowed the fast characterization of the extracts based on their antioxidant activity and the UV-vis spectra of the different compounds found in the extracts. To our knowledge, this work shows for the first time the great possibilities of the combined use of PLE-in vitro assay-MEKC-DAD to investigate natural sources of antioxidants.     

Quantitation of chiral amino acids from microalgae by MEKC and LIF detection

In this work, chiral and nonchiral MEKC methods have been combined with LIF detection (MEKC-LIF) to identify and quantify a group of D- and L-amino acids (D/L-aa) in different microalgae samples. The combination of the nonchiral and chiral-MEKC-LIF methods made the identification of the microalgae amino acids easier, previously derivatized with FITC, providing a double proof on the correct detection of these analytes. Three microalgae species, Spirulina platensis, Dunaliella salina, and Tetraselmis suecica, were compared in terms of their content in D-Arg, L-Arg, D-Lys, L-Lys, D-Ala, L-Ala, D-Glu, L-Glu, D-Asp, and L-Asp. Also, a comparison between two Spirulina platensis samples dried under different conditions (i.e., hot air or lyophilized) was carried out in order to investigate the effect of the thermal processing on the amino acid content. Moreover, two procedures for the extraction of amino acids from microalgae (i.e., a classical procedure and pressurized liquid extraction (PLE)) together with different conditions for amino acid derivatization were studied in order to increase the sensitivity of the whole analytical method. By using the selected chiral-MEKC-LIF conditions (100 mM sodium tetraborate, 30 mM SDS, and 20 mM beta-CD at pH 9.7) the main microalgae D/L-aa are separated in less than 25 min with efficiencies up to 840 000 plates/m and good sensitivity (i.e., 330 ng of D-Arg per gram of microalga could be detected by this procedure for an S/N of 3). Several D-aa were detected in all the microalgae, observing interesting differences in their D/L-aa profiles, what corroborates the usefulness of the chiral-MEKC-LIF approach to characterize different microalgae species as well as different microalgae drying processes. Moreover, the use of PLE can selectively extract different free amino acids from microalgae.     

Characterization by high-performance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry of the lipid fraction of Spirulina platensis pressurized ethanol extract

Microalgae have been suggested as a potential source for new functional ingredients, making possible the development of new functional foods from natural origin. Among the natural ingredients, polyunsaturated fatty acids (PUFAs) have generally been identified as an interesting group of compounds with biological activity, mainly related to their anti-inflammatory properties. In this regard, the use of environmentally friendly extraction procedures (e.g. pressurized liquid extraction, PLE) to obtain such natural ingredients is also becoming necessary. In this work, an exhaustive characterization of the lipid fraction of a pressurized ethanolic extract of the microalga Spirulina platensis is carried out. To achieve this objective high-performance liquid chromatography (HPLC) coupled to quadrupole time-of-flight mass spectrometry (QTOF-MS) is employed. The use of the QTOF analyzer allows the selection and isolation of precursor ions as well as providing the high efficiency, sensitivity and mass accuracy required. By means of this powerful hyphenated technique, it was possible to identify several polar lipids in an extract of S. platensis (some of them, to our knowledge, described for the first time in this work), including four free fatty acids, four monogalactosyl monoacylglycerols, three phosphatidylglycerols and two sulfoquinovosyl diacylglycerols.     

Characterization of proteins from Spirulina platensis microalga using capillary electrophoresis-ion trap-mass spectrometry and capillary electrophoresis-time of flight-mass spectrometry

In this work, a new capillary electrophoresis-mass spectrometry (CE-MS) procedure is developed to analyze proteins in Spirulina platensis microalgae. It is demonstrated that a fine optimization of several separation parameters is essential in order to achieve suitable CE-MS analysis of these proteins in natural extracts from microalgae. Namely, optimization of the composition of the separation buffer, electrospray conditions, and washing routine between runs are required in order to obtain reliable and reproducible CE-MS analyses of the main proteins found in this microalga (namely, allophycocyanin-alpha chain, allophycocyanin-beta, c-phycocyanin-alpha, and c-phycocyanin-beta). The relative molecular mass of these biopolymers is determined using two different MS instruments coupled to CE, i.e., CE-ion trap-MS and CE-time of flight-MS (CE-TOF-MS). A comparison between the results obtained with both instruments is carried out. The high resolution of the TOF-MS enables the distinction of small modifications in proteins and, thus, a more accurate mass determination. Interestingly, molecular mass values obtained by both CE-MS procedures agree very well while these experimental values are only in partial agreement with those theoretically expected (i.e., genetically derived masses). Some protein modifications due to amino acids exchange induced by nucleotide codon mutations are proposed to explain this difference.     

Pressurized liquid extraction of medicinal plants

The suitability of pressurized liquid extraction (PLE) in medicinal plant analysis was investigated. PLE extracts from a selection of representative herbs were compared with extracts obtained according to Pharmacopoeia monographs with respect to yield of relevant plant constituents, extraction time and solvent consumption. In all cases a significant economy in time and solvents was realized, while extraction yields of the analytes were equivalent or higher.     

Pressurized liquid extraction of vitamin E from Brazilian grape seed oil

The goal of this paper is to optimize the pressurized liquid extraction (PLE) of vitamin E from grape seed oil from residues of the wine industry. For this purpose an experimental planning to optimize the extraction of Brazilian grape seed oil by means of PLE with hexane as solvent was applied and the results are compared with conventional methods (Soxhlet and mechanical press extraction). Vitamin E was separated and analyzed using HPLC with UV detection. This study demonstrates the ability of the PLE in extracting grape seed oil rich in vitamin E.     

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.     

Appraisal of “classic” and “novel” extraction procedure efficiencies for the isolation of polycyclic aromatic hydrocarbons and their derivatives from biotic matrices

In this study the extraction efficiency of pressurized liquid extraction (PLE), employing different extraction solvent mixtures under different extraction conditions, was compared with extraction efficiencies of commonly used procedures, Soxhlet extraction and extraction enhanced by sonication. Spruce needles and fish tissue were selected as test samples. Purification of obtained extracts was carried out by gel permeation chromatography (GPC) employing gel Bio-Beads S-X3. Identification and quantitation of target PAHs was performed by high-performance liquid chromatography with fluorescence detection (HPLC–FLD).

Within optimisation of PLE conditions, temperature of extraction, type of solvent, duration and number of static cycles as well as the influence of sample pre-treatment (drying, homogenisation, etc.) were tested. Comparison of the extraction efficiency of PLE with the efficiencies of the other techniques was done under the optimised conditions, i.e. sample slurry obtained by desiccation with anhydrous sodium sulphate, extracted at 100 °C in 1 cycle lasting 5 min. Hexane:acetone (1:1, v/v) was chosen as the most suitable extraction solvent for isolation of analytes from test samples.

Comparison of mentioned isolation techniques with respect to the amount of co-extracts, procedure blank levels and time and solvent volume demands was also done.     

Evaluation of pressurized liquid extraction and pressurized hot water extraction for tanshinone I and IIA in Salvia miltiorrhiza using LC and LC-ESI-MS

Pressurized liquid extraction (PLE) and pressurized hot water extraction (PHWE) using a laboratory-made system are applied for the extraction of thermally labile components such as tanshinone I and IIA in Salvia miltiorrhiza. PLE and PHWE are carried out dynamically at a flow of 1 mL/min, temperature between 95-140 degrees C, applied pressure of 10-20 bars, and extraction times of 20 and 40 min, respectively. Effects of ethanol added into the water used in PHWE are explored. PLE is found to give comparable or higher extraction efficiencies compared with PHWE with reference to Soxhlet extraction for tanshinone I and IIA in Salvia miltiorrhiza. The tanshinone I and IIA present in the various medicinal plant extracts are determined by liquid chromatography and liquid chromatography-mass spectrometry.     

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.     

The Use of Carrier Ampholyte-Free Isoelectric Focusing for Proteomic Analysis

Carrier ampholyte-free isoelectric focusing was applied for pre-concentration, purification and micropreparation of phycobiliproteins (C-phycocyanin, allophycocyanin, B-phycoerythrin) extracted from cyanobacteria Anabeana doliolum and from red microalga Porphyridium cruentum. The extraction of phycobiliproteins was carried out in deionized water. The sonication in the ultrasonic bath and liquid nitrogen freeze grind was used for extraction of proteins of interest. Pre-concentrated and pre-separated proteins were collected and analyzed via MALDI-TOF-TOF mass spectrometer after their proteolytic digestion via trypsin. Based on tandem mass spectrometric analysis, the C-phycocyanin, allophycocyanin and B-phycoerythrin were identified unambiguously.

     

The Use of Carrier Ampholyte-Free Isoelectric Focusing for Proteomic Analysis

Carrier ampholyte-free isoelectric focusing was applied for pre-concentration, purification and micropreparation of phycobiliproteins (C-phycocyanin, allophycocyanin, B-phycoerythrin) extracted from cyanobacteria Anabeana doliolum and from red microalga Porphyridium cruentum. The extraction of phycobiliproteins was carried out in deionized water. The sonication in the ultrasonic bath and liquid nitrogen freeze grind was used for extraction of proteins of interest. Pre-concentrated and pre-separated proteins were collected and analyzed via MALDI-TOF-TOF mass spectrometer after their proteolytic digestion via trypsin. Based on tandem mass spectrometric analysis, the C-phycocyanin, allophycocyanin and B-phycoerythrin were identified unambiguously.     

Optimization of pressurized liquid extraction of Piper gaudichaudianum Kunth leaves

Piperaceae family is original from tropical regions and it shows more than 700 species around the world. Piper gaudichaudianum Kunth is the specie more abundant in Brazil, occurring from Northeast to South Brazil. In this paper, it was investigated the influence of some experimental parameters on the pressurized liquid extraction (PLE) of P. gaudichaudianum Kunth leaves, using petroleum ether as extractor solvent. The optimization of the main variables involved in the PLE process (extraction temperature and time) has been done by response surface methodology (RSM) using, as responses, the extraction yield and the chromatographic profile (GC/MS) of the extracts. The optimized procedure employed 3 g of ground leaves, 10 min of extraction and one cycle of extraction at 85 degrees C. The major compounds present in the petroleum ether extracts were: palmitic acid, stearic acid and nerolidol. The results presented in this work show the possibility of using a fast and easy process to recover compounds from P. gaudichaudianum Kunth.     

Characterization via liquid chromatography coupled to diode array detector and tandem mass spectrometry of supercritical fluid antioxidant extracts of Spirulina platensis microalga

Spirulina platensis microalga has been extracted on a pilot scale plant using supercritical fluid extraction (SFE) under various extraction conditions. The extraction yield and the antioxidant activity of the extracts were evaluated in order to select those extracts with both the highest antioxidant capacity and a good extraction yield. These extracts were characterized using LC coupled to diode array detection (DAD) and LC coupled to mass spectrometry (MS) with two different interfaces, atmospheric pressure chemical ionization (APCI) and electrospray (ESI) which allowed us to perform tandem MS by using an ion trap analyzer. The best extraction conditions were as follows: CO2 with 10% of modifier (ethanol) as extraction solvent, 55 degrees C (extraction temperature) and 220 bar (extraction pressure). Fractionation was achieved by cascade depressurization providing two extracts with different activity and chemical composition. Several compounds have been identified in the extracts, corresponding to different carotenoids previously identified in Spirulina platensis microalga along with chlorophyll a and some degradation products. Also, the structure of some phenolic compounds could be tentatively identified. The antioxidant activity of the extracts could be attributed to some of the above mentioned compounds.     

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.     

Sensitive determination of herbicides in food samples by nonaqueous CE using pressurized liquid extraction

We have developed a method involving extraction with mixtures of solvents under pressure (pressurized liquid extraction (PLE)) for the determination of triazine herbicides in a series of samples from the food industry. The organic extracts obtained were subjected to a clean-up step with SPE, using Oasis MCX sorbents, after which they were analyzed by NACE. Potato was chosen as a representative matrix of horticultural products since it has a high water content. Spiked potato samples were used to optimize extraction conditions. In order to compare the results obtained with NACE, different studies were also conducted using HPLC. The detection limits in NACE were similar to those found with HPLC and were of the order of 10-15 microg/kg, depending on the analyte. Satisfactory results were obtained on applying the method proposed for the potato matrix (PLE with separation by electrophoresis) to other food matrices such as other tubercles, fruits, vegetables and cereals.     

Analysis of pesticides in fruits by pressurized liquid extraction and liquid chromatography-ion trap-triple stage mass spectrometry

A multi-residue method using pressurized liquid extraction (PLE) and liquid chromatography-quadrupole ion trap-triple stage mass spectrometry (LC-IT-MS(3)) has been developed for determining trace levels of pesticides in fruits. The selected pesticides can be distinguished in: benzimidazoles and azoles, organophosphorus, carbamates, neonicotinoids, and acaricides. PLE has been optimized to extract these pesticide residues from oranges and peaches by studying the effect of experimental variables on PLE efficiency. Samples were extracted at high temperature and pressure (75 degrees C and 1500psi) using ethyl acetate as extraction solvent and acidic alumina as drying agent. The recoveries obtained by PLE ranged from 58% to 97% and the relative standard deviation (RSDs) from 5% to 19%. The limits of quantification (LOQs) of the compounds were from 0.025 to 0.25mgkg(-1), which are well-below the maximum residue limits (MRLs) established by the European Union (EU) and the Spanish legislations.     

In-line solid-phase extraction preconcentration in capillary electrophoresis-tandem mass spectrometry for the multiresidue detection of quinolones in meat by pressurized liquid extraction

We have developed and validated a CE-MS/MS method using an in-line SPE device (analyte concentrator, AC) to determine eight quinolones of veterinary use whose maximum residue levels in animal edible tissues are established by the EU Council Regulation 2377/90, i.e., danofloxacin, sarafloxacin, ciprofloxacin, marbofloxacin, enrofloxacin, difloxacin, oxolinic acid, and flumequine. Different parameters affecting the AC performance, such as its design (in this case frit-free), the kind of sorbent (Oasis MCX), sample pH, volume, and composition of the elution plug and injection time were studied. The method was validated using standard solutions obtaining LODs between 17 and 59 ng/L. Finally, a pressurized liquid extraction (PLE) method was developed to determine these antibiotics in chicken muscle samples. The whole analytical method was validated in terms of linearity (r2 >or= 0.992), recoveries (63-112%), repeatability and intermediate precision (RSD 相似文献   

Comparison of pressurized liquid extraction and microwave assisted extraction for the determination of organochlorine pesticides in vegetables

A method to determine organochlorine pesticides in horticultural samples (lettuce, tomato, spinach, potato, turnip leaf and green bean) using pressurized liquid extraction (PLE) is described and compared with microwave assisted extraction (MAE). Significant parameters affecting PLE procedure such as temperature, static extraction time and extraction solvent were optimised and discussed. Clean-up of extracts was performed by solid phase extraction (SPE) using a carbon cartridge as adsorbent. Pesticides were determined by gas chromatography and electron capture detection (GC-ECD). Analytical recoveries obtained were ca. 100% and the relative standard deviations were lower than 15% for most of the studied pesticides with the proposed methods in each analysed matrix.