Anticircular high performance thin-layer chromatography

The principal analytical details for the third of three possible modes in high performance thin-layer chromatography are given, namely the anticircular mode. Separation is achieved by allowing the mobile phase to enter the plate layer on a precise outer circle line, from where it flows towards the centre with nearly constant speed. This technique is theoretically and practically the fastest of all three possible in HPTLC. It permits maximum sample capacity with a minimum of time, layer and mobile phase consumption. It is therefore the most economical HPTLC technique. A new carrier-free mobile phase transfer principle is used. The conditions for qualitative and quantitative analysis are good: repeatability, reproducibility and accuracy of routine TLC analyses are superior to those achieved by the classical trough technique. The specially narrow spot-path in anticircular HPTLC facilitates automated quantitation. Compared with the linear and circular modes, the anticircular mode shows better separation and significantly increased sensitivity at higher Rf-values. The drawback, however, is that the separation power (expressed by the separation number) is lower compared with the other two modes.     

Comparison of optical in-situ scanning of conventional and high-performance thin-layer chromatograms

Conventional TLC, linear and circular HPTLC are compared by using In-situ reflectance measurements. Chromatographic resolution, time requirement and reproducibility of quantitative scanning of the different TLC methods are investigated by means of three separation problems: benzodiazepines, corticosteroids, polycyclic aromatic hydrocarbons. In all three application examples, which were chosen without prejudice, the circular developing mode with subsequent quantitative scanning showed certain advantages over the corresponding linear HPTLC techniques. These were in the case of

• Benzodiazepines: Better reproducibility of quantitative assessment.
• Corticosteroids: Better resolution and slightly better quantitative reproducibility.
• Polycyclic aromatic hydrocarbons: Chromatography considerably faster.

     

Determination of the 7-amino-metabolites of the 7-nitro-benzodiazepines in human plasma by thin-layer chromatography

A sensitive thin-layer chromatographic method is described for the determination of the 7–amino-metabolites of flunitrazepam and clonazepam. The same procedure is applicable to other 7-nitro-benzodiazepines such as nitrazepam. A purified plasma extract of the 7-amino-metabolites is separated by thin-layer chromatography. The primary aromatic amines are diazotized and coupled with N-(1-naphthyl) ethylene-diamine on the thin-layer plate. The quantities of the 7-amino-metabolites are directly evaluated by colorimetric densitometry. The lower limit of detection is in order of 0.5–10 ng cm^?3 of plasma. The relative standard deviation of the whole procedure is less than ±15% in the range of 0.5–10 ng cm^?3 for a single determination. The unchanged drugs can be reduced on the thin-layer plate and detected by the same method. Since this procedure is rather difficult to perform, it is advantageous to determine the 7-nitro-benzodiazepines in plasma by gas chromatography. The thin-layer chromatographic method was used to measure the 7-amino-metabolites in plasma of patients on either a flunitrazepam or a clonazepam oral dosing regimen.     

Parameters influencing theoretical determination of RM-values of naphthalene derivatives in thin-layer chromatography by using binary mobile phase

The purpose of the studies being carried out is to find some regularities of a general character which determine the relationship between the structures of the chromatographed substances and the composition of the mobile phase, i.e., between the log of the partition coefficient of the sample (A_z), the K₁-values which characterize the adsorption equilibrium of component “1” in a given chromatographic system, the structures of the mobile phase components and the structures of the substances being chromatographed. This paper deals with the relationship between the A_z and K₁ parameters and the structure of naphthalene and a number of its derivatives. It has been shown that there is a close relationship between the kind, size, frequency and site of the substituent on the one hand and the A_z and K₁ values on the other. In order to eliminate the possibility of unwanted side effects, the investigations were carried exclusively using non-active binary mobile phases of the type N + N or N + /B/. From these investigations, a comparison between the parameter A_z and the surface of adsorbed molecules (A_s) was also possible.     

High resolution gas chromatography/mass spectrometric characterization of urinary metabolites of N,N-diethyl-m-toluamide (DEET) in man

Studies of the pharmacology and toxicology of the popular insect repellant, N,N-diethyl-m-toluamide (DEET), have largely been done in animal models using radioactive tracing without the structural elucidation of its metabolites. This paper describes a high resolution gas chromatography/mass spectrometry (GC/MS) technique and reports the results of the preliminary characterization of the metabolites of DEET in the urine of a 30-year-old man who had been exposed to DEET contained in a commercial product. The metabolites were extracted and separated with an OV-101 glass capillary column, 30 m × 0.3 mm, and mass spectrometric elucidations were carried out with both Electron Impact (EI) and Chemical Ionization-Methane (MCI) modes. Oxidation of the benzylic moiety and hydroxylation of the sidechain of DEET molecules appeared to be the predominate routes of metabolism in man. The artifacts were also proposed.     

Data processing pipelines for comprehensive profiling of proteomics samples by label-free LC-MS for biomarker discovery

Label-free quantitative LC-MS profiling of complex body fluids has become an important analytical tool for biomarker and biological knowledge discovery in the past decade. Accurate processing, statistical analysis and validation of acquired data diversified by the different types of mass spectrometers, mass spectrometer parameter settings and applied sample preparation steps are essential to answer complex life science research questions and understand the molecular mechanism of disease onset and developments. This review provides insight into the main modules of label-free data processing pipelines with statistical analysis and validation and discusses recent developments. Special emphasis is devoted to quality control methods, performance assessment of complete workflows and algorithms of individual modules. Finally, the review discusses the current state and trends in high throughput data processing and analysis solutions for users with little bioinformatics knowledge.     

Development of standard operation procedures for the manufacture of n-octadecyl bonded silicas as packing material in certified reference columns for reversed-phase liquid chromatography

The development of standard operation procedures for the manufacture of a n-octadecyl bonded spherical silica packing from partially condensed tetraethoxysilane as silica source is described. The synthesis comprises five intermediate products and six synthesis steps which were examined according to their reproducibility and robustness. The results led to the optimisation of the manufacturing process for a n-octadecyl bonded silica. Correlations were drawn between the dynamic viscosity of the poly(ethoxy)siloxane (PES), the synthesis parameters, the resulting pore structural properties and particle size distribution of the silicas. Validated procedures were developed to manufacture spherical porous ultra-pure silicas with a specific surface area of 350 m2 g(-1) +/- 5% R.S.D., a specific pore volume of 1.0 ml (-1) +/- 3.7% R.S.D., an average pore diameter of 12.0 nm +/- 0.5% R.S.D. and an average particle diameter of 5 microm. Results are presented on trial batches and the final master batch which were both used as packing materials in reversed-phase liquid chromatography (RP-LC) columns. The latter columns were certified and accepted as an HPLC column as reference material (BCR-722) by the European Commission, Institute for Reference Materials and Measurements (IRMM), Geel, Belgium.     

Environmental application of elemental speciation analysis based on liquid or gas chromatography hyphenated to inductively coupled plasma mass spectrometry—A review

In recent years the number of environmental applications of elemental speciation analysis using inductively coupled plasma mass spectrometry (ICP-MS) as detector has increased significantly. The analytical characteristics, such as extremely low detection limits (LOD) for almost all elements, the wide linear range, the possibility for multi-elemental analysis and the possibility to apply isotope dilution mass spectrometry (IDMS) make ICP-MS an attractive tool for elemental speciation analysis. Two methodological approaches, i.e. the combination of ICP-MS with high performance liquid chromatography (HPLC) and gas chromatography (GC), dominate the field. Besides the investigation of metals and metalloids and their species (e.g. Sn, Hg, As), representing “classic” elements in environmental science, more recently other elements (e.g. P, S, Br, I) amenable to ICP-MS determination were addressed. In addition, the introduction of isotope dilution analysis and the development of isotopically labeled species-specific standards have contributed to the success of ICP-MS in the field. The aim of this review is to summarize these developments and to highlight recent trends in the environmental application of ICP-MS coupled to GC and HPLC.     

Production of monoclonal antibodies against hop-derived (Humulus lupulus L.) prenylflavonoids and the development of immunoassays

Monoclonal antibodies against the hop-derived prenylated chalcone xanthohumol (X) and the prenylated flavonoids isoxanthohumol (IX) and 8-prenylnaringenin (8-PN) were developed. Carboxylic acid haptens of X, IX and 8-PN were synthesized by linking a spacer to their C4′-OH group followed by subsequent coupling to bovine serum albumin (BSA) to form conjugates that were employed as immunogens in BALB/c mice to raise antibodies. The monoclonal antibodies that were secreted from the established hybridoma cell lines proved, in cross-reactivity studies, to possess highly specific binding capacities in an optimized competitive indirect ELISA. The immunoassays make use of immunogen-coated microtiterplates and a peroxidase-labeled anti-mouse IgG₁ secondary antibody with ABTS as a chromogenic substrate. For X the IC₅₀ value derived from the standard curve was 62.91 ng mL⁻¹, and for both IX and 8-PN 37.15 ng mL⁻¹. The assay was validated for the quantitative analysis of X, IX and 8-PN in urine and serum. A simple sample pretreatment procedure using a diethyl ether extraction was optimized and the recoveries and matrix effects were assessed. The validity of the established assay was tested and mean inter- and intra-assay variations in urine were 2.32% and 1.91%, respectively for X, 6.24% and 2.39%, respectively for IX and 7.18% and 0.74%, respectively for 8-PN. In serum, the mean inter- and intra-assay variations were 8.90% and 1.37%, respectively for X, 6.13% and 1.57%, respectively for IX and 6.13% and 2.43%, respectively for 8-PN. Furthermore, the method demonstrated excellent accuracy and significant correlation with measurements by an established and validated HPLC-MS method.