Displacement chromatography (DC) has been widely used to separate metabolites with similar chemical characteristics. DC works with highly overloaded sample sizes, which are normal for samples not subjected to clean-up. DC successfully handles samples which contain high concentrations of salts and/or proteins, and results in consecutive steps of displaced compounds rather than Gaussian curves.Planar displacement chromatography (DTLC) is suitable for seeking new, i.e. hitherto unknown, metabolites in excreted body fluids. Transfer of the radiolabeled methyl group from l
-monomethylysine can easily be proven using spacer-displacement planar chromatography.
Among forced-flow layer chromatographic techniques, the centrifugally-driven variety was first developed, and named centrifugal layer chromatography, in 1947 by Hopf. In the 1980s Nyiredy further developed the technique and renamed it rotation planar chromatography. This paper summarizes the classifications and applicability of versions of this technique.
Authors:Huba Kalász, József Lengyel, Tibor Szarvas, György Morovján, and Imre Klebovich
Thin-layer chromatography has been used to monitor formaldehyde production during in-vivo metabolism of (−)-deprenyl. Formaldehyde was reacted with dimedone to yield its dimedone adduct, formaldemedone. Determination of
C-labeled formaldemedone was performed by planar displacement chromatography. The separated bands were detected either by digital autoradiography or on X-ray film after contact autoradiography. Two-dimensional separations have improved the evaluation.
Authors:Huba Kalász, Rafael Doležal, Kornélia Tekes, Kálmán Magyar, Tamás Csermely, and Sándor Hosztafi
Lipophilicity is an important physicochemical characteristic of compounds having potential use in the treatment of humans and animals. Morphine and its semisynthetic derivatives have effects on mu (μ), kappa (κ), and delta (δ) opioid receptors, and their physiological, pharmacological, and therapeutic effects mainly depend on their distribution in the body. The progress in separation techniques has made it possible to use modern methods to determine lipophilic/hydrophilic character of small amounts of organic compounds in just a few minutes. In addition to the classical determination of lipophilicity (shaken-flask method), other experimental methods (such as reversed-phase thin-layer chromatography, distribution-pH profile, etc.) and calculations based on the molecular composition are widely used. The lipophilic character of several morphine derivatives was determined using reversed-phase thin-layer chromatography and also high-performance liquid chromatography (HPLC) method. The results are graphically compared. Lipophilicity (log P) calculation using Pallas 3.8 program was successful only for certain morphine derivatives. Parameters derived from the experimental results of reversed-phase thin-layer chromatography and those of a special HPLC column were well comparable.
Authors:Huba Kalász, Attila Hunyadi, Kornélia Tekes, Rafael Dolesal, and Gellért Karvaly
Blood-brain penetration of 20-hydroxyecdysone 2,3;20,22-diacetonide (20DA) has been scouted using chromatographic methods. In vivo experiments were performed by treating male Wistar rats intraperitoneally (i.p.) with a dose of 50 mg kg−1 20DA. Control experiment was done by using 20-hydroxyecdysone (20E). Definite brain penetration of 20DA was found by using high-performance liquid chromatography (HPLC), while 20E does not show this type of distribution.
Authors:Tamás Csermely, Georg Petroianu, Kamil Kuca, Józsel Fűrész, Ferenc Darvas, Zsolt Gulyás, Rudolf Laufer, and Huba Kalász
Quaternary pyridinium aldoximes have been analyzed by thin-layer chromatography. Their separation was adequate when silica plates were used with a mobile phase with a high water content. As a consequence of their limited migration, reversed-phase TLC was not appropriate for determination of the lipophilicity of quaternary pyridinium aldoximes. Displacement TLC of some quaternary pyridinium aldoximes is, nevertheless, possible using silica as stationary phase with water-acetone-hydrochloric acid mobile phases. Normal-phase TLC with different concentrations of organic modifier gave a series of
values for the pyridinium aldoximes. Approximation of the different plots of
against organic modifier concentration to straight lines afforded
values and the slopes of the lines. The
values and the slopes both serve as indicators of the hydrophilic character of the compounds.
Authors:Krisztina Boór, Zsolt Rónai, Mária Sasvári-Székely, Péter Gaszner, and Huba Kalász
The polymorphic human DNA sequence in the promoter region of the dopamine D4 receptor gene has been investigated by means of non-invasive DNA sampling, amplification by means of the polymerase chain reaction, and subsequent separation of the reaction products by horizontal slab-gel electrophoresis. The single nucleotide polymorphism at the −521
position upstream of the initiation codon of the dopamine D4 receptor gene has two variations in the human population, a −521 C form and a −521 T form. Genotyping of −521 C/T single nucleotide polymorphism was performed by two independent methods — use of bidirectional allele-specific amplification, as described recently, and by restriction fragment site polymorphism using
I enzyme. Only results giving the same genotypes by both methods were accepted. The genotyping methods were used to compare allele and genotype frequencies in 47 drug-user males and 118 controls. Our preliminary results showed a tendency for a higher frequency of the −521 C allele among drug users, but further investigations are needed to determine whether the results are statistically significant.
Authors:Gellért Balázs Karvaly, Kornélia Tekes, Zoltán Szimrók, József FŰrÉsz, Kamil KuČa, and Huba Kalász
Mono- and bis-pyridinium quaternary aldoximes (K-oximes) have long been employed as cholinesterase reactivator components of antidotes against lethal cholinesterase-inhibiting organophosphorous chemicals. Their positive charge poses difficulties in their chromatographic analysis, resulting in the publication of different approaches for each K-oxime. A multiplexed method is presented for the rapid quantitation of 10 K-oximes in blood with its utility demonstrated in vivo. Liquid chromatography with absorbance detection was employed. Reversed-phase separation was achieved on a highly nonpolar stationary phase. Method validation was based on the respective guideline of the European Medicines Agency. Times to peak concentrations and 120-min areas under the time–concentration curves were determined in rats following intraperitoneal administration. Adequate retention and separation of K-oximes with acceptable peak shapes in short isocratic runs was achieved by adjusting ionic strength, organic content and the concentration of the ion-pairing agent of the mobile phase. Chromatographic properties were governed by optimizing the concentration of dissolved ions. Accurate adjustment of the organic content was indispensable for avoiding peak drifting and splitting. Dose-adjusted exposure to K-347 and K-868 was exceptionally low, while exposure to K-48 was the highest. The method is suitable for screening systemic exposure to various K-oximes and can be extended.