Antioxidant activity of the juice and seed and skin extracts prepared with methanol, acetonitrile, and water of Viburnum opulus L. grown in Eastern Black Sea Region were studied with an on-line HPLC–ABTS method and off-line antioxidant methods, among which a linear positive correlation was observed. The fruit extracts were analysed with the HPLC–UV method optimised with 14 standard phenolics. Identification of the phenolic components in the juice was made using an HPLC–UV–ESI-MS method. Nineteen phenolic compounds in juice were identified by comparing the retention times and mass spectra with those of the standards and the phenolics reported in the literature. The major peaks in the juice belonged to coumaroyl-quinic acid, chlorogenic acid, procyanidin B2, and procyanidin trimer. Quite different antioxidant composition profiles were obtained from the extracts with the solvents of different polarities. The antioxidant activities of the seed extracts were higher than those of the skin extracts in general.

Several in vitro methods have been tested for their ability to predict drug penetration across the blood-brain barrier into the central nervous system. In this article, the performance of three stationary phases for immobilized artificial membrane (IAM) liquid chromatographic approaches were compared on a set of 49 compounds. IAM liquid chromatography measurements were performed with Dulbecco’s phosphate-buffered saline and methanol as organic modifier in the mobile phase. Transport across the blood-brain barrier (log BB) was predicted using computed descriptor data and the retention factor of all compounds. All data were correlated with experimental log BB values and the relative performance of the approaches was studied. The IAM.PC.DD2 column proved to be the best suited for prediction of log BB values, although all three columns performed very good.

This application note describes how the Agilent Bio-Monolith Protein A column was applied to the optimization of Chinese hamster ovary cell-culture conditions to produce a recombinant monoclonal antibody with desired structural characteristics, focusing on glycosylation. The workflow demonstrates the versatility and wide applicability of the column in biopharmaceutical and biosimilar development.

As preparative chromatography was the first "killer application" for supercritical fluid chromatography (SFC) and the use of CO2 as a mobile phase and mobile phase component. Developing a scalable, fast way for separation methods with enhanced fluid chromatography (EFLC) and SFC is an integral element of the progress. In studying the effect of sample solvents for EFLC/SFC, we selected polar modifier solvents such as water and methanol. Such polar solvents will produce possible splitting of early eluting peaks while working with EFLC /SFC in using similar injection volumes and mobile phase composition compared to HPLC. The separation of standard test mixtures of polyaromatic compounds were used to evaluate the effect on early eluting compounds. Naphthalene peak showed distortion and further splitting as the mobile phase composition gets a higher percent of CO2. For better chromatographic advantages while working in EFLC and SFC, a sample should be dissolved in the less polar solvent as much as possible consistent with the mobile phase composition. The use of pre-heated mobile phase is also another suggestion for handling polar mobile phase solvents at elevated temperature to minimize problems with early eluting peaks shape.

Two primary classes of perfluorinated acids are perfluorinated sulfonates: perfluorooctane sulfonate (PFOS) and perfluorinated carboxylic acids: perfluorooctanoate (PFOA, C8 acid). They are generally the most prominent perfluorinated contaminant in biological samples from around the world. In this study reversed phase and chiral columns were evaluated for the separation of isomers of branched perfluoroalkyl compounds using SFC-MS-TOF. On Cellulose tris (4methylbenzoate) stationary phase using MeOH/H2O (5 %)/ NH4HCO2 (10 mM)/CO2 mobile phase fast baseline separation of the isomers of Perfluoro-3-methylheptane sulfonate (P3FOS), Perfluoro-3-methylheptanoic acid (P3FOA), Perfluoro-4-methylheptane sulfonate (P4FOS), Perfluoro-4-methylheptanoic acid (P4FOA), Perfluoro-5-methylheptane sulfonate (P5FOS), and Perfluoro-5-methylheptanoic acid (P5FOA) in isocratic mode was developed with potential for related isomers.

The growing interest in high throughput assays is the result of the increasing numbers and complexity of samples being produced by modern combinatorial synthetic procedures. The low viscosities and high diffusivities of enhanced fluid mixtures allow highly efficient separations to be achieved with analysis time gain as compared to High-performance liquid chromatography (HPLC). In this study, possibilities and limitations of HPLC mobile phases ethanol/water, acetonitrile/water, and methanol/water at higher proportion aqueous content and acetone/ acetonitrile as non-aqueous mobile phases were evaluated to compare liquid chromatography with green enhanced fluidity liquid chromatography (EFLC) separations by adding different concentrations of carbon dioxide as ternary mobile phase. The techniques were evaluated via van Deemter plots on reversed phase columns. EFLC allows reduce analysis time reduction and to obtain improved column efficiencies by effectively increasing the permeability of the system and by ensuing faster diffusion kinetics and further better selectivity. Similarly the impact on retention and separation in reversed phase using C18 and Naphtylethyl (Pi NAP) stationary phases were explored. A mixture of 16 priority PAH pollutants were used to investigate these effects. Next to interesting changes in selectivity improvements in analysis time and shifting Van Deemter curves could be measured in this way demonstrating the potential of this new green variant of HPLC.

The most common chromatographic methods for the analysis of oligonucleotides have been developed using ion-exchange liquid chromatography (IEXLC) and ion paring reverse phase liquid chromatography (IP-RPLC) with UV detection. Both techniques have demonstrated enough peak capacity to separate completely oligonucleotides up to 20 mer [1]. The raise in the usage of oligonucleotides in the therapeutic field in the last years, requires to couple a more sensitive and specific detector such as the mass spectrometer (MS) for the analysis of these molecules, their metabolites and impurities in complex matrixes. IP-RPLC using volatile buffers has been the only suitable method for coupling a MS detector; conversely, the high concentration of the ion paring reagent lowers the MS sensitivity. Hydrophilic interaction liquid chromatography (HILIC) has demonstrated to be a MS friendly chromatographic technique. HILIC methods are not popular for the analysis of oligonucleotides due to the poor peak capacities that have been obtained. Some methods have used polymer based zwitterionic stationary HILIC phases [2], obtaining with this greater peak capacities; however, this kind of chromatographic columns show les reproducibility between batches and are more expensive than silica columns, which are more suitable to implement in routine analysis. A comprehensive study of HILIC using silica as stationary phase for the separation of oligonucleotides has been done. The separation of adenosine and thymidine oligonucleotides (up to 30 mer) by HILIC has been evaluated using fractional factorial designs. The separation has been performed in a 50 mm x 4.6 mm, Ascentis® HILIC column packed with silica of particle size 3.5 μm with an average pore diameter of 100 °A. A linear gradient elution using water and acetonitrile has been performed. The detection has been done at 260 nm using a single wave length detector. The studied separation parameters were: the column temperature; pH, ionic strength, initial water composition and gradient steepness of the mobile phase. The critical parameters where identified using screening designs, further a surface response has been build for the optimization of peak capacity. The main governing factors in oligonucleotides ( 20mer). The effect of the column length, the particle size and the average pore diameter were also evaluated in order to achieve higher peak capacities for oligonucleotides (>20 mer). The HILIC shows a promising perspective for comprehensive (LC x LC) separation of complex oligonucleotides samples.

We recently described Green Enhanced Fluidity Hydrophilic Interaction Liquid Chromatography [1] for the analysis for highly polar and ionisable solutes. In this technique, high concentrations of carbon dioxide are added to mobile phases typically used in Hydrophilic Interaction Liquid Chromatography (HILIC). Most noteworthy observation was that better results could be obtained with CO2-ethanol–ammonium formate buffer mixtures compared to acetonitrile–ammonium formate buffer mixtures. This green mode of HILIC has been further evaluated on both porous and superficially porous silica particles. Fundamental studies (H-u and kinetic plots) have been carried to define the performance of both column formats and their applicability is illustrated with pharmaceutical samples. Moreover, the border lines between Enhanced Fluidity Chromatography (EFC) and Suband Supercritical Fluid Chromatography have been studied. [1] Alberto dos Santos Pereira, Ana Jimenez Giron, Engdawork Admasu 3 , Pat Sandra, J. Sep. Science 33 (2010) 834.

Several in vitro methods have been investigated for mimicking drug penetration across the blood-brain barrier (BBB) into the central nervous system (CNS). Both micellar liquid chromatography (MLC) and immobilized artificial membrane (IAM) liquid chromatography were tested in this contribution in order to construct models for BBB transfer prediction. MLC measurements were performed on a C18-column with sodium dodecyl sulfate (SDS), polyoxyethylene (23) lauryl ether (Brij35) or sodium deoxycholate (SDC) as surfactants in the micellar mobile phase. IAM liquid chromatography measurements were performed with a Dulbecco’s phosphate-buffered saline (DPBS) and a certain percentage of methanol as organic modifier in the mobile phase. This study aimed to obtain a high correlation between in vivo and predicted log BB values (= concentration of the drug molecule in the brain to concentration in the blood).

Enhanced fluidity liquid chromatography (EFLC) is a variant of HPLC which allows reduce analysis time reduction and to obtain improved column efficiencies by effectively increasing the permeability of the system and by the ensuing faster diffusion kinetics. The phenomenon can very effectively be obtained by mixing the HPLC mobile phase, prior to injection with a stream of supercritical (green) CO2, on a conventional HPLC system. However, as much uncertainty remained about the applicability range CO2 based EFLC in reversed phase LC, allegedly hindered by miscibility limitations, this study was aimed at investigating these effects for various HPLC mobile phases, when applying methanol, ethanol, acetonitrile and acetone as RPLC modifiers together with the addition of various amount of supercritical CO2 to establish de-mixing limits and therefore workable EFLC conditions. The work was performed under isocratic conditions on conventional 250 x 4.6 mm, 5 μm C18 column. Representative RPLC test mixtures were analysed and UV detection was thereby employed in order be able to investigate in relevant aspects such as peak broadening, detector noise levels, retention factor shifts, selectivity changes etc. It appeared that in the aqueous reversed phase mode up to 1540 % supercritical CO2 could be added, while the miscibility further much increasing under nonaqueous RPLC conditions. This technique offers promising prospects in further developments of greener and more efficient HPLC approaches.

One of the most critical parameters in GC-MS application to metabolimic studies is the sample preparation. Often a combination of oximation and silylation is used and this method is applicable to acids (e.g. fatty acids), sterols, amines, amino acids, sugars, etc. Automation of the derivatisation process enhances repeatability and the time between sample preparation and analysis can be kept short and constant. For analysis, retention time locked (RTL) gas chromatography/mass spectrometry was used. For data analysis, AMDIS deconvolution followed by 2 multivariate statistical approaches were used and compared. Arabidopsis thaliana, a popular model organism in plant biology and genetics, was used for testing. The data analysis software allowed the detection of several features that were up- or downregulated in the different species.

The issue of controlling genotoxic impurities in novel active pharmaceutical ingredients (APIs) is a significant challenge. Much of the current regulatory concern, has been focused on the formation and control of genotoxic sulfonate esters. This is linked with the withdrawal of Viracept (Nefinavir mesilate) from European markets in mid-2007, over concerns about elevated levels of ethyl methanesulfonate (EMS). This issue has resulted in calls from European regulators to assess risk mitigation strategies for all marketed products employing a sulfonic acid counter-ion to ensure that the sulfonate esters that could be potentially formed are controlled to threshold of toxicological concern (TTC)-based limits. This has even led to calls to avoid sulfonic acids as salt counter-ions. However, sulfonic acid salts possess a range of properties that are useful to both synthetic and formulation chemists. Whilst sulfonate salts are not a universal panacea to some of the problems of salt formation they do offer significant advantages as alternatives to other salt forming moieties under certain circumstances. This review thus sets out to define some of the advantages provided through utilization of sulfonic acids, explaining the importance of their retention as part of a thorough salt selection process.