Clean-up and re-use of kieselguhr (Extrelut) for liquid–liquid extraction of urinary cortisol

Cortisol was isolated from human urine using kieselguhr (Extrelut)-filled columns. After use, Extrelut was cleaned-up once with distilled water and twice with ethanol. Before re-use, the cleaned-up kieselguhr was dried for 24 h by a warm air stream. The comparison of cortisol recovery from human urine and HPLC chromatograms of urinary extracts show that Extrelut can be repeatedly used for liquid–liquid extraction of urinary cortisol.     

In situ recovery of 2,3-butanediol from fermentation by liquid–liquid extraction

End-product conversion, low product concentration and large volumes of fermentation broth, the requirements for large bioreactors, in addition to the high cost involved in generating the steam required to distil fermentation products from the broth largely contributed to the decline in fermentative products. These considerations have motivated the study of organic extractants as a means to remove the product during fermentation and minimize downstream recovery. The aim of this study is to assess the practical applicability of liquid–liquid extraction in 2,3-butanediol fermentations. Eighteen organic solvents were screened to determine their biocompatibility, and bioavailability for their effects on Klebsiella pneumoniae growth. Candidate solvents at first were screened in shake flasks for toxicity to K. pneumoniae. Cell density and substrate consumption were used as measures of cell toxicity. The possibility of employing oleyl alcohol as an extraction solvent to enhance end product in 2,3-butanediol fermentation was evaluated. Fermentation was carried out at an initial glucose concentration of 80 g/l. Oleyl alcohol did not inhibit the growth of the fermentative organism. 2,3-Butanediol production increased from 17.9 g/l (in conventional fermentation) to 23.01 g/l (in extractive fermentation). Applying oleyl alcohol as the extraction solvent, about 68% of the total 2,3-butanediol produced was extracted. An erratum to this article can be found at     

Air–liquid and liquid–liquid interfaces influence the formation of the urothelial permeability barrier in vitro

Optimizing culture conditions is known to be crucial for the differentiation of urothelial cell cultures and the formation of the permeability barrier. However, so far, no data exist to confirm if air–liquid (AL) and liquid–liquid (LL) interfaces are physiologically relevant during urothelial differentiation and barrier formation. To reveal the influence of interfaces on the proliferation, differentiation, and barrier formation of the urothelial cells (UCs) in vitro, we cultured UCs under four different conditions, i.e., at the AL or LL interfaces with physiological calcium concentration and without serum or without physiological calcium concentration and with serum. For each of the four models, the urothelial integrity was tested by measuring the transepithelial resistance (TER), and the differentiation stage was examined by immunolabeling of differentiation-related markers and ultrastructural analysis. We found that the UCs at a LL interface, regardless of the presence or absence of calcium or serum, form the urothelium with more cell layers and achieve a higher TER than UCs at an AL interface. However, UCs grown at an AL interface with physiological concentration of calcium in medium form only one- to two-layered urothelium of UCs, which are larger and express more differentiation-related proteins uroplakins than UCs in other models. These results demonstrate that the interface itself can play a major, although so-far neglected, role in urothelial physiology, particularly in the formation of the urothelial permeability barrier in vitro and the regulatory mechanisms related with urothelial differentiation. In the study, the culturing of UCs in three successive steps is proposed.     

Atomistic simulations of liquid–liquid coexistence in confinement: comparison of thermodynamics and kinetics with bulk

We review a few simulation methods and results related to the structure and non-equilibrium dynamics in the coexistence region of immiscible symmetric binary fluids, in bulk as well as under confinement, with special emphasis on the latter. Monte Carlo methods to estimate interfacial tensions for flat and curved interfaces have been discussed. The latter, combined with a thermodynamic integration technique, provides contact angles for coexisting fluids attached to the wall. For such three-phase coexistence, results for the line tension are also presented. For the kinetics of phase separation, various mechanisms and corresponding theoretical expectations have been discussed. A comparative picture between the domain growth in bulk and confinement (including thin-film and semi-infinite geometry) has been presented from molecular dynamics simulations. Applications of finite-size scaling technique have been discussed in both equilibrium and non-equilibrium contexts.     

Cyclic dipeptide-based small molecules modulate zinc-mediated liquid–liquid phase separation of tau

Liquid–liquid phase separation (LLPS) is a complex physicochemical phenomenon mediated by multivalent transient weak interactions among macromolecules like polymers, proteins, and nucleic acids. It has implications in cellular physiology and disease conditions like cancer and neurodegenerative disorders. Many proteins associated with neurodegenerative disorders like RNA binding protein FUS (FUsed in Sarcoma), alpha-synuclein (α-Syn), TAR DNA binding protein 43 (TDP-43), and tau are shown to undergo LLPS. Recently, the tau protein responsible for Alzheimer's disease (AD) and other tauopathies is shown to phase separate into condensates in vitro and in vivo. The diverse noncovalent interactions among the biomolecules dictate the complex LLPS phenomenon. There are limited chemical tools to modulate protein LLPS which has therapeutic potential for neurodegenerative disorders. We have rationally designed cyclic dipeptide (CDP)-based small-molecule modulators (SMMs) by integrating multiple chemical groups that offer diverse chemical interactions to modulate tau LLPS. Among them, compound 1c effectively inhibits and dissolves Zn-mediated tau LLPS condensates. The SMM also inhibits tau condensate-to-fibril transition (tau aggregation through LLPS). This approach of designing SMMs of LLPS establishes a novel platform that has potential implication for the development of therapeutics for neurodegenerative disorders.     

Rare events out of equilibrium: mobility through the liquid–liquid interface

Transition state theory provides a well established means to compute the rate at which rare events occur; however, this is strictly an equilibrium approach. Here we consider a nonequilibrium problem of this nature in the form of transport through a liquid–liquid interface. When two immiscible liquids are coexisting in equilibrium, there will be a certain amount of mixing between the two phases, resulting in a finite linear mobility across the liquid–liquid interface. We derive an exact relationship between the mobility and the local diffusion in the direction perpendicular to the interface. We compute the mobility using both nonequilibrium molecular dynamics and a variety of linear response type approaches, with accurate agreement being obtained for the best of these. Our analysis makes it clear how the local diffusion is influenced by the inhomogeneities of the interface, even when at a distance from it. This nonlocal character to the mobility has not been appreciated before and results in a strong variation in the local diffusion, which is formally coupled to the variation in the potential of mean force. The nonlocal aspect of the diffusion requires the velocity autocorrelation function to be integrated out to far longer times than is the case for homogeneous liquids, and requires special care with regard to the choice of numerical approach.     

Biophysics of endocytic vesicle formation: A focus on liquid–liquid phase separation

Endocytosis is a fine-tuned mechanism of cellular communication through which cells internalize molecules on the plasma membrane, such as receptors and their bound ligands. Through receptor clustering in endocytic pits, recruitment of active receptors to different endocytic routes and their trafficking towards different fates, endocytosis modulates cell signaling and ultimately leads to a variety of biological responses. Many studies have focused their attention on specialized endocytic mechanisms depending on the nature of the internalizing cargo and cellular context, distinct sets of coat proteins, endocytic adaptors and membrane lipids. Here, we review recent advances in our understanding of the principles underlying endocytic vesicle formation, integrating both biochemical and biophysical factors, with a particular focus on intrinsically disordered regions (IDRs) creating weakly interconnected protein networks assembled through liquid–liquid phase separation (LLPS) and driving membrane bending especially in clathrin-mediated endocytosis (CME). We finally discuss how these properties impinge on receptor fate and signaling.     

Equilibrium studies on enantioselective liquid–liquid extraction of homophenylalanine enantiomers with metal-BINAP complexes

Enantioselective liquid–liquid extraction of homophenylalanine (Hph) enantiomers was investigated with metal-BINAP complexes as enantioselective extractants. The metal complexes were synthesized by the complexation of (s)-2,2′-Bis(diphenylphosphino)-1,1′-binaphthalene (BINAP) with different central ions, among which, copper(I) complex allowed the separation of the Hph enantiomers with the highest operational selectivity. Efficiency of the extraction depends, often strongly, on a number of process variables, including types of organic solvents, pH of the aqueous phase, concentration of host and substrate, and temperature. In order to better understand the extraction process, equilibrium of the system were modeled by a homogeneous reaction model and an interfacial reaction model, respectively. Important parameters required by the modeling, such as complexation equilibrium constant and physical distribution coefficients were determined experimentally. When coupled with the parameters, extraction performance can be predicted by the models. Comparison between the experimental values and the model predictions indicates that the homogeneous reaction model can predict more accurately. By modeling and experiment, an optimal extraction condition concerning pH of 8 and host concentration of 2 mmol/L was obtained with high enantioselective (α) of 1.837 and performance factor (pf) of 0.086.     

Micro-determination of plasma diphenylhydantoin by gas—liquid chromatography

A selective, sensitive and precise gas—liquid chromatographic method for the determination of diphenylhydantoin in micro samples of blood plasma is described. After a double extraction with chloroform containing an analogue of diphenylhydantoin as an internal standard, the drug and standard are N,N-dimethylated in alkaline aqueous solution with methyl iodide followed by extraction into acetone. The methylated derivatives are separated gas chromatographically and measured using a flame-ionization detector. The lowest concentration of diphenylhydantoin in plasma which can be measured in a 100-μl sample is 1 μg/ml, which is well below the normal therapeutic concentration of 10–20 μg/ml in plasma. The methylated derivatives of diphenylhydantoin and the internal standard have been identified by their proton magnetic resonance spectra and mass spectra.     

Comparison of simple perturbation-theory estimates for the liquid–solid and the liquid–vapor interfacial free energies of Lennard-Jones systems

The most naive perturbation method to estimate interfacial free energies is based on the assumption that the interface between coexisting phases is infinitely sharp. Although this approximation does not yield particularly accurate estimates for the liquid–vapor surface tension, we find that it works surprisingly well for the interface between a dense liquid and a solid. As an illustration we estimate the liquid–solid interfacial free energy of a Lennard-Jones system with truncated and shifted interactions and compare the results with numerical data that have been reported in the literature. We find that the agreement between theory and simulation is excellent. In contrast, if we apply the same procedure to estimate the variation of the liquid–vapor surface tension, for different variants of the Lennard-Jones potential (truncated/shifted/force-shifted), we find that the agreement with the available simulation data is, at best, fair. The present method makes it possible to obtain quick and easy estimate of the effect on the surface free energy of different potential-truncation schemes used in computer simulations.     

Fast liquid chromatographic–mass spectrometric determination of pharmaceutical compounds

We present fast LC–MS–MS analyses of multicomponent mixtures containing flavones, sulfonamides, benzodiazepines and tricyclic amines. Using a short microbore HPLC column with small particle size, five to eight compounds were partially resolved within 15 to 30 s. TurboIonSpray and atmospheric pressure chemical ionization interfaces were well suited to tolerate the higher eluent flow-rates of 1.2 to 2 ml/min. The methods were applied to biological sample matrices after clean-up using solid-phase or liquid–liquid extraction. Good precision and accuracy (average 8.9 and 97.7%, respectively) were achieved for the determination of tricyclic amines in human plasma. Benzodiazepines were determined in human urine with average precision of 9% and average accuracy of 95% for intra- and inter-assay. Detection limits in the low ng/ml range were obtained. An example for 240 injections per hour of demonstrated the feasibility of rapid LC–MS–MS analysis.     

Profiling degradants of paclitaxel using liquid chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry substructural techniques

A rapid and systematic strategy based on liquid chromatography–mass spectrometry (LC–MS) profiling and liquid chromatography–tandem mass spectrometry (LC–MS–MS) substructural techniques was utilized to elucidate the degradation products of paclitaxel, the active ingredient in Taxol. This strategy integrates, in a single instrumental approach, analytical HPLC, UV detection, full-scan electrospray MS, and MS–MS to rapidly and accurately elucidate structures of impurities and degradants. In these studies, degradants induced by acid, base, peroxide, and light were profiled using LC–MS and LC–MS–MS methodologies resulting in an LC–MS degradant database which includes information on molecular structures, chromatographic behavior, molecular mass, and MS–MS substructural information. The stressing conditions which may cause drug degradation are utilized to validate the analytical monitoring methods and serve as predictive tools for future formulation and packaging studies. Degradation products formed upon exposure to basic conditions included baccatin III, paclitaxel sidechain methyl ester, 10-deacetylpaclitaxel, and 7-epipaclitaxel. Degradation products formed upon exposure to acidic conditions included 10-deacetylpaclitaxel and the oxetane ring opened product. Treatment with hydrogen peroxide produced only 10-deacetylpaclitaxel. Exposure to high intensity light produced a number of degradants. The most abundant photodegradant of paclitaxel corresponded to an isomer which contains a C3–C11 bridge. These methodologies are applicable at any stage of the drug product cycle from discovery through development. This library of paclitaxel degradants provides a foundation for future development work regarding product monitoring, as well as use as a diagnostic tool for new degradation products.     

Enzymatic in situ saccharification of cellulose in aqueous-ionic liquid media

The enzymatic saccharification of a model cellulosic substrate, Avicel PH-101, using an ionic liquid (IL), 1-ethyl-3-methylimidazolium diethylphosphate, was explored. After mixing the IL solution of cellulose with different volumes of 10 mM citrate buffer (pH 5.0), cellulase was directly added to the aqueous-IL mixture at 40°C. When the volume of IL to water was greater than 3:2, little cellulase activity was observed. However, decreasing the volume ratio markedly enhanced enzymatic activity: an IL to water ratio of 1:4 (v/v) resulted in over 70% of the starting amount of cellulose (10 mg/ml) being converted to glucose and cellobiose.     

Quantification of thyrotropin-releasing hormone by liquid chromatography–electrospray mass spectrometry

Thyrotropin-releasing hormone (TRH) is involved in a wide range of biological responses. It has a central role in the endocrine system and regulates several neurobiological activities. In the present study, a rapid, sensitive and selective liquid chromatography–mass spectrometry method for the identification and quantification of TRH has been developed. The methodology takes advantage of the specificity of the selected-ion monitoring acquisition mode with a limit of detection of 1 fmol. Furthermore, the MS/MS fragmentation pattern of TRH has been investigated to develop a selected reaction monitoring (SRM) method that allows the detection of a specific b2 product ion at m/z 249.1, corresponding to the N-terminus dipeptide pyroglutamic acid–histidine. The method has been tested on rat hypothalami to evaluate its suitability for the detection within very complex biological samples.     

Investigations on the biodegradation of alkylpolyglucosides by means of liquid chromatography–electrospray mass spectrometry

The biodegradation of alkylpolyglucosides (APGs) was studied under the conditions of the OECD Screening Test with activated sludge as an inoculum. An influence of alkyl and sugar chain length on the biodegradation rate and a central scission pathway of the biodegradation were investigated. The liquid chromatography-electrospray mass spectrometry technique was used for alkylpolyglucoside analysis and for identification and semiquantitative determination of metabolites. It was found that APGs with a longer alkyl chain were biodegraded faster than those with a shorter one. However, a longer sugar chain caused slower biodegradation of APGs. The central scission pathway of biodegradation was also confirmed.     

Reversed-phase high-performance liquid chromatography of peptides of porcine pepsin prepared by the use of various forms of immobilized α-chymotrypsin

Reversed-phase high-performance liquid chromatography (RP-HPLC) separation was used for the comparison of peptide maps of pepsin after its digestions by different forms of immobilized α-chymotrypsin. Porcine pepsin was hydrolysed with soluble α-chymotrypsin, with α-chymotrypsins glycosylated with lactose or galactose coupled to hydrazide derivative of cellulose, with α-chymotrypsin attached to poly(acrylamide-allyl glycoside) copolymer or to glycosylated hydroxyalkyl methacrylate copolymer Separon or to agarose gel Sepharose 4B. Efficiency of enzymatic protein cleavage with regard to peptide mapping of porcine pepsin has been examined by the use of α-chymotrypsins immobilized by different methods. Best results were achieved after hydrolysis with α-chymotrypsin immobilized on poly(acrylamide-allyl glycoside) copolymers. α-Chymotrypsin immobilized by this way has further three times higher relative specific activity in comparison with the soluble one. Modified α-chymotrypsin was not suitable for efficient pepsin cleavage.     

High-performance liquid chromatographic analysis of β-phenylethylamine for the estimation of in vivo protein synthesis

A rapid, sensitive, and automated reversed-phase liquid chromatographic method was developed for the analysis of phenylalanine as β-phenylethylamine, for the measurement of in vivo protein synthesis. β-Phenylethylamine was derivatized with o-phthaldialdehyde (OPA) to form a fluorescent derivative that was successfully measured in tissue cell fluids and hydrolysates as the decarboxylation product of phenylalanine. The system was extremely sensitive enabling the accurate determination of 0.5 pmol in biological samples. Analysis time was less than 11 min, so that 130 samples can be analysed per day. The method eliminates the need for time-consuming column extraction procedures. This method offers substantial advantages over existing methods for the isolation and determination of β-phenylethylamine.