Abstract Capillary liquid chromatography (CLC) in reversed separation mode was applied to the quantification and impurity profile determination of eight newly synthesized pyridoquinolines. The CLC separation system consisted of Nucleosil C18 stationary phase and methanol containing 1% ( v/v ) of triethylamine (TEA) as the mobile phase. The optimized separation system enabled the separation of impurities from the main component and their quantification in a reasonable analysis time. The presence of TEA in the mobile phase greatly improved the peak shape and decreased the analysis time of the studied derivatives on the C18 stationary phase. Calibration curves of pyridoquinolines were plotted in a concentration range from 1.0×10 –6 to 1.0×10 –3 mol/L at two parallel detector wavelengths of 254 and 265 nm and subsequently used for quantification of pyridoquinoline derivatives in human urine. No pretreatment of the real biological sample was needed. Detection and quantification limits were calculated for all the derivatives and the detection limits of most of the pyridoquinolines were found to lie in the μmol/L concentration range. The proposed CLC separation system has proved to be a suitable method for quantification of test derivatives in samples containing human urine matrix.
The retention behavior of several series of free α‐ and ω‐amino acids and positional isomers of amino pentanoic acid in the hydrophilic interaction chromatography mode (HILIC) was studied. The study was carried out on three stationary phases followed by post‐column derivatization with fluorescence detection in order to describe the retention mechanism of the tested amino acids. The effect of chromatographic conditions including acetonitrile content in the mobile phase, mobile phase pH (ranging from 3.5 to 6.5) and concentration of buffer in the mobile phase was investigated. The effect of the number of carbon atoms ( n C ) in aliphatic chains of the individual homologue of α‐ and ω‐amino acids and the logarithm of the partition coefficient (log D ) on retention was also a part of the presented study. A good correlation ( r > 0.98) between the log k and log D values of amino acids or n C , respectively, was observed. The described linear relationships were subsequently applied to predict the retention behavior of individual members of the homologous series of amino acids and to optimize the mobile phase composition in HILIC. The obtained results confirmed that the retention mechanism of α‐amino acids, ω‐amino acids and positional isomers of amino acids was based on the log D values and the number of carbon atoms in the aliphatic chains of amino acids. The elution order of ω‐amino acids and positional isomers of amino pentanoic acid was strongly dependent on the mobile phase pH in the investigated range whereas the retention factors of all α‐amino acids remained essentially unchanged on all tested stationary phases.
Although the proteome of each organism is unambiguously coded in its genome, the proteome shows the real biology in action in each particular organism. New powerful tools are being developed for biochemists and biologists to analyze complex biological samples for studying the complete protein supplement of the genome, i. e., the proteome. There are several methods available for proteome analysis including 2-DE and several forms of MS. In recent years, technologies such as microfluidics and array-based systems have appeared in the field of analysis, identification, and quantification of proteins. These novel approaches might help in solving current technical challenges in proteomics. This paper presents a practical application of the first commercially available microfluidic nano-ESI device coupled with nano-LC (i. e., HPLC-chip) for the analysis of samples of some biological protein mixtures.
Whenever a mobile phase contains more than one component, additional signals commonly called system peaks can appear. The origin of these signals is explained through loss of equilibrium in the separation column caused by injection of analyte dissolved in a different solvent than the mobile phase. The system peaks are then generated by a relaxation process started by the non-equilibrium state. An overview of the theory and applications of the system peaks in separation methods, mainly in liquid chromatography, is presented in this paper. Only a brief theoretical discussion of the system peak origin is given as the theoretical aspects of system peak formation have already been published in many papers. The main focus of this review is to summarize applications, in which system peaks were used to measure physical or physicochemical data. Signals of system peaks are often misinterpreted but they offer valuable information about thermodynamics and kinetics of the separation process that takes place in chromatographic column.
In today’s agriculture, maize is considered to be one of the major feed, food and industrial crops. Cultivation of maize by inappropriate agricultural practices and on unsuitable sites is connected with specific risks of soil degradation, mainly due to water erosion of the soil. The aim of this study was to evaluate the yielding parameters, fodder quality and anti-erosion efficiency of different methods of conservation tillage for maize in two areas (Jevíčko—JEV and Skoupý—SKO) with different climate and soil conditions in the Czech Republic in the period 2016–2018, using multivariate exploratory techniques such as principal component analysis (PCA) and factor analysis (FA). Four variants of soil tillage methods were analysed: Conventional Tillage (CT), two slightly different Strip-Till techniques (ST) and Direct Sowing (DS). The analysed parameters were: dry mass of the plants, height of the plants, starch content (SC), organic matter digestibility (OMD) and content of neutral detergent fibre (NDF), soil loss by erosion and surface runoff. The multivariate exploratory techniques PCA and FA significantly differed in two categories of techniques in both locations. The first category consists of soil conservation techniques (SCT): ST (JEV/SKO) and DS (JEV). These techniques are characterised by lower yields of dry mass, lower height of plants, forage quality equal to CT, but a high level of protection of the soil against erosion. The second category consists of CT (JEV and SKO) and partially of DS (SKO). These treatments are characterised by high dry mass production, higher plants, high forage quality, but a feeble capacity of protection of the soil against erosion. The results of the study confirm the presumption of the positive influence of introduction and application of new agronomical practices in the areas of interest and other areas with similar natural conditions in the sense of sustainable management for agricultural management of agricultural land for the conditions of the Czech Republic and therefore of Central and Eastern Europe. PCA and FA were used as an effective method for comprehensive evaluation of the use of STC in agricultural practice.
In the last decade, pharmaceutical regulatory agencies are focused on monitoring and evaluation of trace-level genotoxic impurities (GTIs) in drug substances, which requires manufacturers to deliver innovative approaches for their analysis and control. GTIs in the low p.p.m. level rising from the process of drug production have to be positively identified and quantified. Therefore, sensitive and selective analytical methods are necessary for required quantification level of these GTIs. Unfortunately, general guidance on how to develop strategy of the analysis and control of GTIs is currently missing in the pharmaceutical industry. Therefore, practical example of the analytical control of 2-chloro-N-(2-chloroethyl)ethanamine GTI in the vortioxetine (VOR) manufacturing process was demonstrated in this work. QDa mass detection with electrospray ionization in selected-ion recording mode was utilized for quantitation of GTIs. The method of hydrophilic interaction liquid chromatography coupled with mass spectrometry detection (HILIC–MS) was validated as per International Conference on Harmonization guidelines and was able to quantitate GTIs at 75 p.p.m. with respect to VOR. The HILIC–MS method was achieved using a Primesep B column (150 × 4.6 mm, 5.0 µm; Sielc, USA) using mobile phase consisting of 10 mM ammonium formate buffer pH 3.0 and acetonitrile (5 : 95, v/v) at 0.8 mL/min flow rate. The QDa mass detector was operated in the positive ion mode. Quadrupole mass analyzer was employed in selected-ion monitoring mode using target ion at m/z 142 as [M+H]+.
An analytical reversed-phase high-performance liquid chromatography (HPLC) method for the detection and quantitative determination of two genotoxic impurities at ppm level present in the vortioxetine manufacturing process is described. Applying the concept of threshold of toxicological concern, a limit of 75 ppm each for both genotoxic impurities was calculated based on the maximum daily dose of active pharmaceutical ingredients. The novel reversed-phase HPLC method with photochemically induced fluorescence detection was developed on XSELECT Charged Surface Hybrid Phenyl-Hexyl column using the mobile phase consisted a mixture of 10 mM ammonium formate pH 3.0 and acetonitrile. The elution was performed using an isocratic composition of 48:52 (v/v) at a flow rate of 1.0 mL/min. The photochemically induced fluorescence detection is based on the use of UV irradiation at 254 nm through measuring the fluorescence intensity at 300 nm and an excitation wavelength of 272 nm to produce fluorescent derivatives of both genotoxic impurities. The online photochemical conversion and detection is easily accomplished for two expected genotoxic impurities and provides a sufficiently low limit detection and quantification for the target analysis.
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