The two-phase amphiphilic preconcentration based on surfactants to enrich phenolic compounds from diluted plant extracts and rat urine by micellar electrokinetic chromatography.

A novel technology by two-phase amphiphilic preconcentration based on surfactants was established for enriching phenolic compounds by micellar electrokinetic chromatography (MEKC). The cationic surfactant cetyltrimethylammonium chloride (CTAC) was combined with the anionic analytes that existed in the sample solution before injection. The boundary was formed between CTAC and sodium dodecyl sulfate (SDS) in the background solution when the sample solution was injected into the capillary, where the analytes bound inside micelles were released due to the stronger electrostatic force between SDS and CTAC. This procedure accelerated the separation of analytes from CTAC and greatly improved the enrichment efficiency. The optimal conditions were obtained after a series of optimizations, and the sensitivity enrichment factors of the four analytes were in the range of 39-93 compared to typical injections in capillary zone electrophoresis. Good linearity for matrix-matched calibrations was established for all analytes with R2 values of 0.9993-0.9997. The limits of detection (S/N = 3) for kaempferol, quercetin, salvianolic acid C, and salvianolic acid B were 0.0166, 0.0292, 0.0215, and 0.0195 µg/ml, respectively. The intracapillary RSDs of the analytes ranged from 0.8% to 1.3% for migration time and from 0.4% to 1.8% for peak areas. The developed method was successfully applied to the determination of phenolic compounds, the main compounds of Salvia miltiorrhiza Bge., and had been validated for the determination of spiked recoveries in rat urine.

Soapwort (Saponaria officinalis L.) Extract vs. Synthetic Surfactants-Effect on Skin-Mimetic Models.

Our skin is continuously exposed to different amphiphilic substances capable of interaction with its lipids and proteins. We describe the effect of a saponin-rich soapwort extract and of four commonly employed synthetic surfactants: sodium lauryl sulfate (SLS), sodium laureth sulfate (SLES), ammonium lauryl sulfate (ALS), cocamidopropyl betaine (CAPB) on different human skin models. Two human skin cell lines were employed: normal keratinocytes (HaCaT) and human melanoma cells (A375). The liposomes consisting of a dipalmitoylphosphatidylcholine/cholesterol mixture in a molar ratio of 7:3, mimicking the cell membrane of keratinocytes and melanoma cells were employed as the second model. Using dynamic light scattering (DLS), the particle size distribution of liposomes was analyzed before and after contact with the tested (bio)surfactants. The results, supplemented by the protein solubilization tests (albumin denaturation test, zein test) and oil emulsification capacity (using olive oil and engine oil), showed that the soapwort extract affects the skin models to a clearly different extent than any of the tested synthetic surfactants. Its protein and lipid solubilizing potential are much smaller than for the three anionic surfactants (SLS, ALS, SLES). In terms of protein solubilization potential, the soapwort extract is comparable to CAPB, which, however, is much harsher to lipids.

Quantitative analysis of nine isoflavones in traditional Chinese medicines using mixed micellar liquid chromatography containing sodium dodecylsulfate/ß-cyclodextrin supramolecular amphiphiles.

Isoflavone is one of the phytoestrogens that have estrogenic effects, so it is usually served as an active ingredient for quality control of traditional Chinese medicines rich in isoflavones. Nine isoflavones commonly found in traditional Chinese medicines were separated in 30 min using mixed micellar liquid chromatography. The mobile phase consisted of 0.08 M sodium dodecylsulfate and 6.05 mM ß-cyclodextrin:methanol (87:13, v/v) at pH 3 and eluted isocratically at 1 mL/min through a C18 column. In this study, we systematically optimized the chromatographic conditions including the pH, the composition and concentration of surfactants, the type and ratio of organic solvents, and column temperature. The method was validated according to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use guidelines. There is no report using micellar liquid chromatography to detect isoflavones, and the optimized method has been successfully applied to quantify isoflavones in red clover and Radix Puerariae. This method is efficient, cheap, and convenient. Finally, we verified the existence of supramolecular amphiphilic vesicles in the mobile phase by transmission electron microscopy to explain the increased chromatographic efficiency.

An environmentally friendly sodium dodecyl sulfate-synergistic microwave-assisted extraction followed by ultra-high-performance liquid chromatography with photodiode array method for the simultaneous extraction and determination of iridoids, phenylpropanoids, and lignans from Eucommiae Cortex.

A simple and green sodium dodecyl sulfate-synergistic microwave-assisted extraction method was developed to extract and determine the iridoids, phenylpropanoids, and lignans in Eucommiae Cortex followed by ultra-high-performance liquid chromatography with photodiode array detection. The biodegradable solution (sodium dodecyl sulfate) was used as a promising alternative to organic solvents. The response surface methodology provided the optimum extraction conditions (2 mg/mL sodium dodecyl sulfate, 1100 W microwave power, and 6 min extraction time). The recoveries of three types of components ranged from 95.0 to 105% (RSDs < 5%). The intra- and inter-day precision and accuracy were less than 3.40% and within the range of 97.1-105%, respectively. Compared with other extraction methods, this newly established method was more efficient and environmental friendly. The results demonstrated that sodium dodecyl sulfate-synergistic microwave-assisted extraction followed by ultra-high-performance liquid chromatography with photodiode array method was applicable for the simultaneous extraction and determination of these three types of compounds for quality evaluation of Eucommiae Cortex.

Mimicry of a biophysical pathway leads to diverse pollen-like surface patterns.

A ubiquitous structural feature in biological systems is texture in extracellular matrix that gains functions when hardened, for example, cell walls, insect scales, and diatom tests. Here, we develop patterned liquid crystal elastomer (LCE) particles by recapitulating the biophysical patterning mechanism that forms pollen grain surfaces. In pollen grains, a phase separation of extracellular material into a pattern of condensed and fluid-like phases induces undulations in the underlying elastic cell membrane to form patterns on the cell surface. In this work, LCE particles with variable surface patterns were created through a phase separation of liquid crystal oligomers (LCOs) droplet coupled to homeotropic anchoring at the droplet interface, analogously to the pollen grain wall formation. Specifically, nematically ordered polydisperse LCOs and isotropic organic solvent (dichloromethane) phase-separate at the surface of oil-in-water droplets, while, different LCO chain lengths segregate to different surface curvatures simultaneously. This phase separation, which creates a distortion in the director field, is in competition with homeotropic anchoring induced by sodium dodecyl sulfate (SDS). By tuning the polymer chemistry of the system, we are able to influence this separation process and tune the types of surface patterns in these pollen-like microparticles. Our study reveals that the energetically favorable biological mechanism can be leveraged to offer simple yet versatile approaches to synthesize microparticles for mechanosensing, tissue engineering, drug delivery, energy storage, and displays.

QbD Based Approach to Enhance the In-Vivo Bioavailability of Ethinyl Estradiol in Sprague-Dawley Rats.

Lyophilized nanosuspension of poorly soluble Ethinyl estradiol (EE) was fabricated to enhance its solubility and bioavailability using a quality-by-design (QbD) approach. With the help of the Ishikawa diagram, prospective risk factors were identified and screened by Placket-Burman design to investigate the effects of formulation and process variables on dependent variables. The number of cycles (X4), the concentration of soya lecithin (X5) and the concentration of tween 80 (X7) were identified as significant factors (P<0.05), which were further optimized using Central Composite Design. The mean particle size, zeta potential, drug content and entrapment efficiency of optimized lyophilized EE nanosuspension (EENPs) was 220±0.37 nm, -19.3±6.73 mV, 92.23±0.45%, 99.52±0.52%, respectively. Significantly, EENPs enhances Cmax and AUC0-t by 1.5, 1.7 folds and relative bioavailability by 2-fold with its distribution being at higher concentrations in the liver, spleen, and stomach. Thus, QbD based approach for the development of nanosuspension could be an absolute, optimistic approach to identify the critical process parameters and critical quality attributes.

Sonodynamic therapy in atherosclerosis by curcumin nanosuspensions: Preparation design, efficacy evaluation, and mechanisms analysis.

Previous studies have shown that curcumin (Cur) induced by ultrasound has protective effects on atherosclerosis even if low bioavailability of the Cur. The enhancement of bioavailability of the Cur further improved the curative effect of sonodynamic therapy (SDT) on atherosclerosis through nanotechnology. Nanosuspensions as a good drug delivery system had obvious advantages in increasing the solubility and improving the effectiveness of insoluble drugs. The aim of this study was to develop curcumin nanosuspensions (Cur-ns) which used polyvinylpyrrolidone (PVPK30) and sodium dodecyl sulfate (SDS) as stabilizers to improve poor water solubility and bioavailability of the Cur. And then the therapeutic effects of Cur-ns-SDT on atherosclerotic plaques and its possible mechanisms would be investigated and elucidated. Cur-ns with a small particle size has been successfully prepared and the data have confirmed that Cur-ns could be more easily engulfed into RAW264.7 cells than free Cur and accumulated more under the stimulation of the ultrasound. Reactive oxygen species (ROS) inside RAW264.7 cells after SDT led to the decrease of mitochondrial membrane potential (MMP) and the higher expression of cleaved caspase-9/3. The results of in vivo experiments showed that Cur-ns-SDT reduced the level of total cholesterol (TC) and low density lipoprotein (LDL) and promoted the transformation from M1 to M2 macrophages, relieved atherosclerosis syndrome. Therefore, Cur-ns-SDT was a potential treatment of anti-atherosclerosis by enhancing macrophages apoptosis through mitochondrial pathway and inhibiting the progression of plaques by interfering with macrophages polarization.

Cocrystal Solubility Advantage Diagrams as a Means to Control Dissolution, Supersaturation, and Precipitation.

Cocrystals are often more soluble than needed and pose unnecessary risks for precipitation of less soluble forms of the drug during processing and dissolution. Such conversions lead to erratic cocrystal behavior and nullify the cocrystal solubility advantage over parent drug (SA = Scocrystal/Sdrug). This work demonstrates a quantitative method for additive selection to control cocrystal disproportionation based on cocrystal solubility advantage (SA) diagrams. The tunability of cocrystal SA is dependent on the selective drug-solubilizing power of surfactants (SPdrug = (ST/Saq)drug). This cocrystal property is used to generate SA-SP diagrams that facilitate surfactant selection and provide a framework for evaluating how SA influences drug concentration-time profiles associated with cocrystal dissolution, drug supersaturation, and precipitation (DSP). Experimental results with indomethacin-saccharin cocrystal and surfactants (sodium lauryl sulfate, Brij, and Myrj) demonstrate the log-linear relationship characteristic of SA-SP diagrams and the dependence of σmax and dissolution area under the curve (AUC) on SA with characteristic maxima at a threshold supersaturation where drug nucleation occurs. This approach is expected to streamline cocrystal formulation as it facilitates additive selection by considering the interplay between thermodynamic (SA) and kinetic (DSP) processes.

Novel sodium alkyl-1,3-disulfates, anionic biosurfactants produced from microbial polyesters.

A sodium alkyl disulfate mixture (SADM) synthesised from microbially produced 3-hydroxy fatty acids methyl esters (HFAMEs), showed 13-fold surface tension decrease when compared with the reference surfactant sodium dodecyl sulfate (SDS). Polyhydroxyalkanoates, accumulated by bacteria intracellularly when supplied with a mixture of fatty acids derived from hydrolysed rapeseed oil, were isolated, depolymerised and methylated to produce HFAMEs in very high yield (90%). A sequential chemical reduction and sulfation of the HFAMEs produced the sodium alkyl disulfates in high yields (>65%). SADM performs also 1.3-times better than dodecyl (1,3) disulfate, in surface tension tests. SADM shows also the formation of a specific critical micelle concentration (CMC) at a concentration 21-fold lower than SDS. The wettability of the SADM mixture is similar to SDS but the foaming volume of SADM is 1.5-fold higher. The foam is also more stable with its volume decreasing 3 times slower over time compared to SDS at their respective CMC values. Established sulfation technologies in chemical manufacturing could use the 3-hydroxy fatty acids methyl esters moiety (3-HFAME) given its origin from rapeseed oil and the extra OH residue on 3-position in the molecule, which affords the opportunity to produce disulfate surfactants with a proven superior performance to monosulphated surfactants. Thus, not only addressing environmental issues by avoiding threats of deforestation and monocultivation associated with palm oil use but also achieve a higher performance with lower use of surfactants.

Influences of alkali on the quality and protein polymerization of buckwheat Chinese steamed bread.

The effects of alkali (NaHCO3 or Na2CO3) on the quality and protein polymerization of buckwheat Chinese steamed bread (CSB) were investigated. The alkali addition increased the specific volume of CSB, for NaHCO3 from 1.84 ml/g (control) to 2.66 ml/g. Image analysis showed that alkali increased the pore area fractions. The addition of Na2CO3 exhibited a greater pore count and lower pore average size. The texture properties were improved with the increase of crumb hardness and resilience. The extractability of protein in sodium dodecyl sulfate containing medium (SDSEP) decreased. Furthermore, SDS-PAGE showed an obvious decrease in the intensity of protein bands with lower molecular weight, while intensity of higher molecular protein bands increased. This demonstrated that alkali addition promoted protein aggregation in buckwheat CSB. In addition, the content of degydroalanine and lanthionine increased and free SH decreased, which indicated alkali addition prompted protein to form dehydroalanine-derived and disulfide cross-linkings.

Lecithin soybean phospholipid nano-transfersomes as potential carriers for transdermal delivery of the human growth hormone.

Pharmaceutical molecules such as peptides and proteins are usually injected into the body. Numerous efforts have been made to find new noninvasive ways to administer these peptides. In this study, highly flexible vesicles (transfersomes [TFs]) were designed as a new modern transdermal drug delivery system for systemic drug administration through the skin, which had also been evaluated in vitro. In this study, two growth hormone-loaded TF formulations were prepared, using soybean lecithin and two different surfactants; F1 _sodium deoxycholate and F 2 _sodium lauryl sulfate. Thereafter, the amount of skin penetration by the two formulas was assessed using the Franz diffusion cell system. TF formulations were evaluated for size, zeta potential and in vitro skin penetration across the rat skin. Results indicated that vesicle formulations were stable for 4 weeks and their mean sizes were 241.33 ± 17 and 171 ± 12.12 nm in the F 1 and F 2 formulation, respectively. After application to rat skin, transport of the human growth hormone (hGH) released from the TF formulations was found to be higher than that of the hGH alone. Maximum amounts of transdermal hormone delivery were estimated to be 489.54 ± 8.301 and 248.46 ± 4.019 ng·cm-2 , for F 1 and F 2 , respectively. The results demonstrate the capability of the TF-containing growth hormone in transdermal delivery and superiority of the F 1 to F 2 TFs.

Effect of synthetic surfactants and soapwort (Saponaria officinalis L.) extract on skin-mimetic model lipid monolayers.

The effect of a saponin-rich extract from rhizomes of Soapwort (Saponaria officinalis L) and four synthetic surfactants: sodium lauryl sulphate (SLS), sodium laureth sulphate (SLES), ammonium lauryl sulphate (ALS) and cocamidopropyl betaine (CAPB) on two model lipid monolayers is analyzed using surface pressure, surface dilatational rheology and fluorescence microscopy. The following monolayers were employed: dipalmitoylphosphatidylcholine/cholesterol mixture in a molar ratio of 7:3 (DPPC/CHOL) and Ceramide [AP]/stearic acid/cholesterol in a molar ratio of 14:14:10 (CER/SA/CHOL). They mimicked a general bilayer structure and an intercellular lipid mixture, respectively. Both lipid mixtures on Milli-Q water were first compressed to the initial surface pressure, Π0 = 30 mN/m and then the subphase was exchanged with the respective (bio)surfactant solution at 1% (w/w). All four synthetic surfactants behaved in a similar way: they increased surface pressure to about 40 mN/m and reduced the storage modulus of surface dilational surface rheology, E', to the values close to zero. The corresponding fluorescence microscopy pictures confirmed that the lipids mimicking the stratum corneum components were almost completely removed by the synthetic surfactants under the present experimental conditions. The components of the Soapwort extract (SAP) increased surface pressure to significantly higher values than the synthetic surfactants, but even more spectacular increase was observed for the storage modulus of the SAP-penetrated lipid monolayers (up to E'= 715 mN/m).

Sodium dodecyl sulfate sensitized switchable solvent liquid-phase microextraction for the preconcentration of protoberberine alkaloids in Rhizoma coptidis.

A sodium dodecyl sulfate sensitized switchable solvent liquid-phase microextraction method was developed and applied to the preconcentration of active alkaloids in Rhizoma coptidis followed by high performance liquid chromatography determination. Before extraction, nonionic triethylamine was converted to its cationic form in the presence of carbon dioxide. Then, the ionic solvent carrying target analytes was once more reverted to its nonionic form by adding sodium hydroxide, as well as phase separation and analytes enrichment were realized simultaneously. Several parameters affecting the approach, such as concentration of sodium dodecyl sulfate, extraction solvent volume, sodium hydroxide concentration, sample phase pH, injection solvent type, and extraction time, were investigated and optimized. The possible microextraction mechanism of double micelle supramolecular inclusion was explored. Under the optimum conditions, the enrichment factors of four protoberberine alkaloids were from 101.8 to 152.0. The linear ranges (with r2  ≥ 0.990) were 0.032-4.23, 0.031-4.33, 0.0026-10.04, and 0.0013-4.13 µg/mL for epiberberine, coptisine, palmatine, and berberine, respectively. The detection limits were in the range of 0.16-0.32 ng/mL. Satisfactory accuracies (recoveries 98.8-104.6%) and precisions (RSDs 1.9-10.9%) were also obtained. The results showed that the approach is rapid, effective, eco-friendly, and easy-to-handle for the enrichment and detection of active alkaloids in Rhizoma coptidis.

Development of analytical methods for evaluating the quality of dissociated and associated amphiphilic poly(γ-glutamic acid) nanoparticles.

A quantitative method of analyzing nanoparticles (NPs) for drug delivery is urgently required by researchers and industry. Therefore, we developed new quantitative analytical methods for biodegradable and amphiphilic NPs consisting of polymeric γ-PGA-Phe [phenylalanine attached to poly(γ-glutamic acid)] molecules. These γ-PGA-Phe NPs were completely dissociated into separate γ-PGA-Phe molecules by adding sodium dodecyl sulfate (SDS). The dissociated NPs were chromatographically separated to analyze parameters such as the γ-PGA-Phe content in the NPs, the impurities present [using reverse-phase (RP) HPLC with an ultraviolet (UV) detector], and the absolute MW [using size-exclusion chromatography (SEC) with refractive index detection (RI) and multiangle light scattering (MALS) detection, i.e., SEC-RI/MALS]. The chromatographic patterns of the NPs were equivalent to those of the component polymer (γ-PGA-Phe), and excellent chromatographic separation for the quantitative evaluation of NPs was achieved. To the best of our knowledge, this is the first report of the quantitative evaluation of NPs in the field of NP-based delivery systems. Furthermore, these methods were applied to optimize and evaluate the NP manufacturing process. The results showed that impurities were effectively removed from the γ-PGA-Phe during the manufacturing process, so the purity of the final γ-PGA-Phe NPs was enhanced. In addition, the appearance, clarity of solution, particle size, zeta potential, particle matter, osmolarity, and pH of the product were evaluated to ensure that the NPs were of the required quality. Our approach should prove useful for product and process characterization and quality control in the manufacture of NPs. γ-PGA-Phe NPs are known to be a powerful vaccine adjuvant, so they are expected to undergo clinical development into a practical drug-delivery system. The analytical methods established in this paper should facilitate the reliable and practical quality testing of NP products, thus aiding the clinical development of γ-PGA-Phe-based drug-delivery systems. Moreover, since these analytical methods employ commonly used reagents and chromatographic systems, the methods are expected to be applicable to other NP-based drug-delivery products too. Graphical abstract NPs were completely dissociated into separate γ-PGA-Phe polymeric molecules, which yielded a similar chromatogram to that seen for the NPs.

Permeability of glibenclamide through a PAMPA membrane: The effect of co-amorphization.

Co-amorphous systems are an attractive alternative for amorphous solid polymer dispersions in the formulation of poorly soluble drugs. Several studies have revealed that co-amorphous formulations can enhance the dissolution properties of poorly-soluble drugs and stabilize them in the amorphous form. However, the interplay between the drug dissolution rate, drug supersaturation and different co-formers on membrane permeability of the drug for co-amorphous formulations remains unexplored. By using side-by-side chambers, separated by a PAMPA (parallel artificial membrane permeability assay) membrane, we were able to simultaneously test dissolution and passive membrane permeability of the co-amorphous combinations (1:1 molar ratio) of a poorly soluble drug glibenclamide (GBC) in combination with two amino acids, either serine (SER) or arginine (ARG). In addition, a known passive permeability enhancer sodium lauryl sulfate (SLS) was included in the co-amorphous mixtures at two concentration levels. The mixtures were also characterized with respect to their solid-state properties and physical stability. It was found that GBC mixtures with ARG and SLS had superior dissolution and physical stability properties which was attributable to the strong intermolecular interactions formed between GBC and ARG. These formulations also had optimal permeability properties due to their high concentration gradient promoting permeation and possible permeation enhancing effect of the co-formers ARG and SLS. Thus, simultaneous testing of dissolution and permeation through a PAMPA membrane may represent a simple and inexpensive tool for screening the most promising amorphous formulations in further studies.

Chiral analysis of theanine and catechin in characterization of green tea by cyclodextrin-modified micellar electrokinetic chromatography and high performance liquid chromatography.

Monomeric catechins are important compounds in green tea accounting for potential bioactivity against a wide range of diseases. Besides catechins, l-Theanine (γ-glutamylethylamide), a characteristic amino acid in tea leaves, has become a further focus of the phytochemical research for the reported beneficial effects mainly on cognitive performance, emotional state and sleep quality. In the present study has been developed a CD-MEKC method based on sodium dodecyl sulfate (SDS) and Heptakis (2,6-di-O-methyl)-ß-cyclodextrin for the separation of six major green tea catechins and enantiomers of theanine. The latter, because of the poor detectability was derivatized prior analysis by o-phthaldialdehyde in the presence of N-acetyl-l-cysteine which, under mild conditions (neutral pH, in two minutes) allowed two diastereomers isoindole derivatives to be obtained. The derivatization reaction was directly carried out on tea infusion and derivatized samples were analysed by CD-MEKC involving 65 mM SDS and 28 mM cyclodextrin in acidic buffer (pH 2.5). The separation of six major green tea catechins including enantioresolution of (±)-Catechin and d/l-Theanine was obtained in about 5 min allowing d-Theanine to be quantified at least at 0.5% m/m level with respect to l-Theanine. Since (-)-Catechin and d-Theanine can be considered as non-native enantiomers (distomers), their presence in real samples provides an indication of tea leaves treatments (thermal treatment, fermentation, etc.) and could represent an opportunity for grading tea. The obtained results were confirmed by a RP-HPLC approach; even though the chromatography was developed in achiral conditions, the derivatization approach applied to theanine (diastereomers formation), allowed for d/l-Theanine chiral analysis.

Simultaneous separation and determination of praeruptorin A, B and C by micellar electrokinetic chromatography using sodium dodecyl sulphate and sodium cholate as mixed micelles.

INTRODUCTION: Praeruptorin A, B and C are major bioactive constituents in Peucedani Radix. They display anti-inflammatory effect, anti-hypertension effect, antiplatelet aggregation, potential anti-cancer activities and so on. They are worthy of investigation as potentially novel and versatile drugs. OBJECTIVE: To develop a method using micellar electrokinetic chromatography (MEKC) for the application in simultaneously separation and determination of praeruptorin A, B and C from Peucedani Radix and its medicinal preparations. METHODS: Method optimisation was carried out by investigating influences of significant factors on the separation. The method was subjected to validation. The determination of praeruptorin A, B and C in Peucedani Radix and its drug formulations was accomplished by the developed method. RESULTS: The optimal separation condition was 20 mM borate buffer containing 40 mM sodium cholate (SC), 22 mM sodium dodecyl sulphate (SDS) and 25% (v/v) acetonitrile (pH 10.00); 15 kV of voltage; 25°C of temperature; detection at 224 nm. Under this condition, three analytes were baseline separated within 16 min. A good linearity was obtained with correlation coefficients from 0.9988 to 0.9995. The limits of detection (LODs) and limits of quantitation (LOQs) ranged from 0.50 to 0.80 µg/mL and from 1.50 to 2.50 µg/mL, respectively. The recoveries ranged between 95.3% and 103.4%. CONCLUSION: The proposed method has been successfully applied to the simultaneous determination of praeruptorin A, B and C in Peucedani Radix and its pharmaceutical preparations. Additionally, it could be a potential alternative to the quality control of Peucedani Radix.

A robotic magnetic nanoparticle solid phase extraction system coupled to flow-batch analyzer and GFAAS for determination of trace cadmium in edible oils without external pretreatment.

A lab-made magnetic-mechanical robotic (MMR) system coupled to a flow-batch analyzer (FBA) for magnetic nanoparticles solid phase extraction (MSPE) is presented. As an illustrative application, an NMR-FBA couple was connected to a graphite furnace atomic absorption spectrometer (GFAAS) for quantification of trace cadmium in edible oils. Factors affecting MSPE, such as the amount of adsorbent, the type, concentration and volume of the eluent and elution time were studied. Under the optimized experimental conditions, the interferents studied did not reveal a significant change in the analytical response, indicating that proposed method is selective. The sampling rate, characteristic mass, working linear range, limits of detection (LOD), and sensitivity were 10h-1, 0.18pg, 0.05-1.0µgkg-1, 0.006µgL-1, and 0.4197, respectively. An enrichment factor of 9 was achieved using a 2.5mL oil sample. In order to evaluate the accuracy, a certified reference material was analyzed by the proposed and a reference method. The values obtained were compared with the one provided from the manufacturer and no statistically significant differences were observed among three values at a confidence level of 95% using paired t-test. In addition, the precision intra-day and inter day of the proposed method and the robustness were assessed and again no statistically significant differences were observed at a confidence level of 95%. The use of a microcolumn to immobilize the MNPs is not needed with the proposed MMR-FBA-GFAAS system, thus avoiding the well-known problem of non-uniform packing of the MNPs presented in previous flow-based automatic methods. Despite a high organic load of edible oils, the method developed is simple, robust and presents satisfactory analytical features when compared with others that have been reported in the literature, suggesting that it is a potentially useful alternative to determine trace analytes in viscous matrices without external pretreatment.

Analyzing bean extracts using time-dependent SDS trapping to quantify the kinetic stability of phaseolin proteins.

In common beans and lima bean, the storage protein phaseolin is difficult to degrade and SDS-resistant, a sign of kinetic stability. Kinetically stable proteins (KSPs) are characterized by having a high-energy barrier between the native and denatured states that results in very slow unfolding. Such proteins are resistant to proteolytic degradation and detergents, such as SDS. Here the method SDS-Trapping of Proteins (S-TraP) is applied directly on bean extracts to quantify the kinetic stability of phaseolin in lima bean and several common beans, including black bean, navy bean, and small red bean. The bean extracts were incubated in SDS at various temperatures (60-75 °C) for different time periods, followed by SDS-PAGE analysis at room temperature, and subsequent band quantification to determine the kinetics of phaseolin unfolding. Eyring plot analysis showed that the phaseolin from each bean has high kinetic stability, with an SDS-trapping (i.e. unfolding) half-life ranging from about 20-100 years at 24 °C and 2-7 years at 37 °C. The remarkably high kinetic stability of these phaseolin proteins is consistent with the low digestibility of common beans and lima bean, as well as their relatively high germination temperatures. From a practical perspective, this work exemplifies that S-TraP is a useful and cost-effective method for quantifying the kinetic stability of proteins in biological extracts or lysates. Depending on the protein to be studied and its abundance, S-TraP may be performed directly on the extract without need for protein purification.

Optimized solubilization of TRIzol-precipitated protein permits Western blotting analysis to maximize data available from brain tissue.

BACKGROUND: Techniques simultaneously assessing multiple levels of molecular processing are appealing because molecular signaling underlying complex neural phenomena occurs at complementary levels. The TRIzol method isolates RNA and DNA, but protein retrieval is difficult due to inefficient solubilization of precipitated protein pellets. NEW METHOD: We optimized a buffer for the efficient solubilization of protein from TRIzol-precipitated brain tissue for Western blotting analysis, which was also more effective at directly homogenizing brain tissue than RIPA buffer. RESULTS: Protein yield during solubilization, in addition to protein yield via direct homogenization, is increased by optimizing concentrations of chemicals in a standard lysis buffer. Effective incubation parameters for both total protein yield and the analysis of post-translational modifications is remarkably flexible. Importantly, different neural cell types and protein classes are represented in solubilized protein samples. Moreover, we used dissociated mouse brain tissue to isolate microglia from other cell types and successfully resolved cell type-specific proteins from these small and difficult to attain samples. COMPARISON WITH EXISTING METHOD(S): Solubilization buffers to date have been comprised primarily of SDS or urea; the data herein demonstrate that components common to lysis buffers can also enhance protein solubilization both after direct homogenization and after precipitation. CONCLUSIONS: This method is suitable for assessing gene and protein expression from a single brain sample, allowing for a more comprehensive evaluation of neural phenomena while minimizing the number of subjects.