Journal of Chromatography B (v.800, #1-2)

Foreword by V.A. Davankov (1).

One century of liquid chromatography by Heinz Engelhardt (3-6).
Keywords: Liquid chromatography, history;

A review is presented on the CE analysis of colloidal/nano particles. Topics discussed include the CE separation of polymeric, inorganic, microbial (i.e. viruses, bacteria, fungi, and whole cells), and sub-cellular particles (i.e. mitochondria and nuclei). Several of the encountered difficulties in analysis are presented as well as the methods employed to overcome them.
Keywords: Reviews; Colloids; Nano-particles; Microbes;

Magnetic hydrophilic methacrylate-based polymer microspheres designed for polymerase chain reactions applications by Alena Španová; Daniel Horák; Eva Soudková; Bohuslav Rittich (27-32).
Magnetic hydrophilic non-porous P(HEMA-co-EDMA), P(HEMA-co-GMA) and PGMA microspheres were prepared by dispersion (co)polymerization of 2-hydroxyethyl methacrylate (HEMA) and ethylene dimethacrylate (EDMA) or glycidyl methacrylate (GMA) in the presence of several kinds of magnetite. It was found that some components used in the preparation of magnetic carriers interfered with polymerase chain reaction (PCR). Influence of non-magnetic and magnetic microspheres, including magnetite nanoparticles and various components used in their synthesis, on the PCR course was thus investigated. DNA isolated from bacterial cells of Bifidobacterium longum was used in PCR evaluation of non-interfering magnetic microspheres. The method enabled verification of the incorporation of magnetite nanoparticles in the particular methacrylate-based polymer microspheres and evaluation of suitability of their application in PCR. Preferably, electrostatically stabilized colloidal magnetite (ferrofluid) should be used in the design of new magnetic methacrylate-based microspheres by dispersion polymerization.
Keywords: Polymerase chain reaction inhibition; Magnetic microspheres; Methacrylates;

Micro-thermal focusing field-flow fractionation by Josef Janča; Irina A. Ananieva; Anastasija Yu. Menshikova; Tatiana G. Evseeva (33-40).
Focusing mechanism was effectively exploited to separate large (micrometer-size) particles by using new micro-thermal field-flow fractionation (micro-TFFF). It has been shown that the retention order of micrometer-size particles at high field strength can be explained by the mechanism of steric exclusion only at lowest flow rates of the carrier liquid. A simplistic, purely mechanical model of steric exclusion is not accurate to describe the retention at higher flow rates where the focusing phenomenon appears. Despite the fact that the thickness of the channel for micro-FFF cannot be reduced without taking into account a possible deterioration of the separation due to the contribution of “steric exclusion” mechanism, this paper demonstrates, in agreement with our previous results, that if the operational conditions were conveniently chosen, namely a low flow rate, a reasonable fit of the experimental retention data with the theory of steric exclusion mechanism in FFF was found and the separation of micron-size particles can be accomplished. However, high selectivity and resolution and high-speed separation were achieved if the focusing effect has clearly dominated the FFF mechanism. As a result, it seems that the micro-TFFF is the most universal technique which can be applied for the separation of the synthetic and natural macromolecules within an extended range of molar masses up to ultra-high molar masses and for the particles of various chemical nature and origin in a nano-size range as well as for large (micrometers) particles. Until nowadays, only sedimentation and flow field-flow fractionation techniques in so called “steric” modes were applied for the separations of large size particles. This application of micro-TFFF in focusing mode for the separation of large size particles is the first one described in the literature.
Keywords: Micro-thermal field-flow fractionation;

In the present study neurons from the olfactory system of the fish crucian carp, Carassius carassius L. were used as components in an in-line neurophysiologic detector (NPD) to measure physiological activities following the separation of substances by high-performance liquid chromatography (HPLC). The skin of crucian carp, C. carassius L. contains pheromones that induce an alarm reaction in conspecifics. Extra-cellular recordings were made from neurons situated in the posterior part of the medial region of the olfactory bulb known to mediate this alarm reaction. The nervous activity of these specific neurons in the olfactory bulb of crucian carp was used as an in-line neurophysiologic detector. HPLC was performed with an HP 1100 model equipped with a diode array detector (DAD) and ChemStation software. An adsorbosphere nucleotide–nucleoside 7 μm column was used to separate the substances in the skin extract using artificial pound water (APW) as the mobile phase. UV spectral detection was performed at 214, 254 and 345 nm, and scans (190–400 nm) were collected continuously. This system enabled the selection of peaks in the chromatogram with fish alarm pheromone activity. The neurons in parts of the olfactory system from different aquatic organisms and vertebrates can be used for the detection of species-specific stimuli such as sexual and alarm signals, food odours, and other physiologically significant substances. NPDs clearly offer new and promising options for in-line HPLC as highly selective and sensitive detectors in biological, medical and pharmaceutical research.
Keywords: Neurophysiologic detector; Alarm pheromones;

Bionanocompositional chitosan-silica sorbent for liquid chromatography by S.Sh Rashidova; D.Sh Shakarova; O.N Ruzimuradov; D.T Satubaldieva; S.V Zalyalieva; O.A Shpigun; V.P Varlamov; B.D Kabulov (49-53).
Preparation of hybrid nanocompositional chitosan/silica sorbent was carried out. It was shown that formation of gel in sol–gel process of hydrolytic polycondensation of tetraethoxysilane (TEOS) with including of chitosan consists of two stages. Suppression of crystallization of chitosan in obtained two-phase system and changes in IR spectra are evidenced of interactions between molecules of chitosan and silanol groups of silica network. The resulting hybrid chitosan/silica sorbent was tested by high-performance liquid chromatography (HPLC).
Keywords: Sorbents; Silica; Chitosan;

Field gas chromatography–mass spectrometry for fast analysis by Alexei L. Makas; Mikhail L. Troshkov (55-61).
The objective of this presentation is to demonstrate the original device and procedure for fast gas chromatography–mass spectrometry (GC–MS) analysis of gaseous and liquid samples and to discuss its features and capabilities. The concept was developed in order to expand the range of compounds suitable for GC separation and to reduce the time of analysis. Field GC–MS, consisting of original “concentrator–thermodesorber” (CTD) unit, multiple module GC system and compact magnetic mass spectrometer with powerful two-stage vacuum system and multicollector ion detector, is represented. The whole weight of the device is 90 kg. Power consumption is 250 W. The device and analytical procedures allow high speed screening of toxic substances in air and extracts within 100 s per sample. The examples of applications are described, including fast screening of tributyl phosphate (TBP) in air at low ppt level at the rate 1 sample/min.
Keywords: Field analysis; Real-time analysis; Portable instrumentation;

Miniaturized mass-selective detector with atmospheric pressure chemical ionization by A.L Makas; M.L Troshkov; A.S Kudryavtsev; V.M Lunin (63-67).
The miniaturized mass-spectrometric detector with atmospheric pressure chemical ionization (APCI) is described. The analyzer employed in this instrument is the monopole with rod 54 mm in length and 2 mm in radius, which retains its efficiency up to 0.13 Pa (≈10−3  Torr). Together with the ion source, channeltron ion detector, radio frequency power supply and preamplifier, it is packed into a case with dimension of 185  mm×100  mm×70  mm. Mass spectrometer with the whole vacuum system weighs about 20 kg. In spite of low power of the vacuum system, the limit of detection at ppt level is achieved. The “strong” fragmentation mode is suggested for high-specific detection of phosphor-containing substances. The detector conforms to multicapillary column attachment.
Keywords: Miniature instrumentation; Collisional-induced dissociation;

Interaction properties of the novel HPLC silica gel–poly(ethylene glycol) (PEG) bonded phase were evaluated applying polymeric test substances, viz. polystyrenes, poly(methyl methacrylate)s, poly(ethylene oxide)s and poly(2-vinyl pyridine)s, and eluents of different polarities. Silanols on the silica gel surface are well shielded by the PEG phase, and silanophilic adsorption of macromolecules is suppressed in comparison with most silica C18 bonded phases. The adsorption of solutes on the OH groups of the PEG phase seems to be low as well. The partition of macromolecules in favor of the PEG phase is inferior to that observed in case of the silica C18 phases. The volume of the PEG bonded phase is small and it is supposed that the PEG chains assume flat conformation on the silica gel surface.
Keywords: Retentivity; Macromolecular probes; Poly(ethylene glycol);

Description of a gas chromatograph designed for express analysis of explosives (2,4-dinitrotoluene, 2,4,6-trinitrotoluene, pentaerythritol tetranitrate), chemical warfare agents (mustard gas, lewisite, sarin) and drugs (heroin, cocaine hydrochloride, crack) is given. The devices comprises a multicapillary chromatographic column and an ion mobility increment spectrometer (MCC–IMIS). The main analytical characteristics of an IMIS (estimated detection limit (DL), linear dynamic range (LDR), speed of response) and a chromatographic column (separation power, degree of separation, a number of possible peaks at a chromatogram section, divided by analysis time) are determined. The maximum value of DL equal to 5 pg/ml was registered for cis-α-LW, and the lowest one of 0.001 pg/ml was for cocaine. The maximum value of LDR equal to 1000 was registered for sarin and the lowest one of 150 was for the ions of lewisite. Speed of response of one compound detection with the IMIS was 0.7 s.
Keywords: Ion mobility increment spectrometry; Explosives; Chemical warfare agents; Heroin; Cocaine; Crack;

Plastic substrates based separation channels in electromigration techniques by Jana Charvátová; Zdeněk Deyl; Miroslav Klevar; Ivan Mikšı́k; Adam Eckhardt (83-89).
Three types of plastic materials (polyester, polyurethane and polymethylmethacrylate) were tested as materials for manufacturing separation columns (polyester and polyurethane capillaries were used) or separation channels (polymethylmethacrylate) in the chip format. A set of 11 fluorescein isothiocyanate amino acid derivatives was used as the test mixture. Using α-cyclodextrin additive to the background electrolyte in the case of the chip separation was also tested. The main problem with all plastic separation media was the selectivity of the separation. The best results, practically identical with bare fused silica capillary, were obtained with the polymethylmethacrylate chip, provided that α-cyclodextrin in a concentration 40 mmol/l was added to the background electrolyte. An important observation was that in SDS containing background electrolyte all the plastic materials used exhibited a distinct electroosmotic flow, which was ascribe to the sorption of the negatively charged constituents of the background electrolyte to the capillary wall. Regarding the order in which the individual components of the test mixture were brought to the detector only a single change was observed. Histidine migrated in the polystyrene and polymethylmethacrylate separation channels more slowly than in the bare silica or polyurethane based capillaries.
Keywords: Plastic separation channels; Fluorescein isothiocyanate amino acid derivatives;

The separation of dansyl (DNS-AAs) and carbobenzoxy (CBZ-AAs) amino acids using micellar electrokinetic chromatography employing polyelectrolyte-surfactant complexes (PSC) formed in the reaction between polyacrylic acid (PAA) and dodecyltrimethylammonium bromide (DTAB) as pseudo-stationary phases was described. The PSCs were stabilized by hydrophobic interactions of alkyl chains of the surfactant ions and converted to an intramolecular micellar-like phase. The running buffer was a 50 mM solution of sodium phosphate (pH 6.0) containing 4.6–20.2 mM PSC, in which a part of carboxyl groups of PAA was blocked by aliphatic amines. For the systems with 7.9 mM of PAA/DTAB complex (ϕ=0.30, ϕ-composition of water-soluble polyelectrolyte complex) as a pseudo-stationary phase, the peaks of six dansyl amino acids (DNS-AAs) were baseline resolved. The separation in this case is based on a complex distribution mechanism of the dansyl derivatives between the free buffer and the intramolecular micellar-like phase of the water-soluble PSC. On the other hand, the additives of PAA/DTAB complex (ϕ=0.30) to the running buffer does not essentially affect on the electrophoretic behaviour of the CBZ-AAs, the variant MEKC is not realized. The influence of the concentration of the complex of PAA/DTAB on the electrophoretic behaviour of analytes was investigated. Relative retentions and relative selectivities were used for describing electrophoretic behaviour of the amino acid derivatives.
Keywords: Pseudo-stationary phases; Micellar electrokinetic chromatography; Polyelectrolyte complexes; Polyacrylic acid; Amino acids;

Gas chromatography–mass spectrometry (GC–MS) with positive ion chemical ionization (PICI) using isobutane as reagent gas was applied for analysis of isobutoxycarbonyl/isobutyl derivatives of 13 fatty, 6 dicarboxylic and 13 amino acids in a single run. For all investigated compounds (except several amino acids) the quasimolecular ions [MH]+ were registered. Asparagine underwent fragmentation via decarboxylation followed by elimination of OC4H9 ([M−117]+), whereas serine and tyrosine produced the cluster ions [M+C4H9OCO]+. Estimated detection limits were 6–250 pg in the total ion current (TIC) mode and 3–10 times lower using the selected-ion monitoring (SIM) mode.
Keywords: Derivatization; Fatty acids; Dicarboxylic acids; Amino acids; Isobutyl chloroformate;

Affinity chromatography of porcine protease and its zymogen was carried out on immobilized components of specific substrate used for the pepsin determination. For the immobilization of N-acetyl-l-phenylalanine and iodinated derivative of l-tyrosine, divinyl sulfone activated Sepharose was used. Ligands with blocked amino group and free carboxyl one were linked to Sepharose via ethylene diamine spacer using carbodiimide reaction. Conditions of affinity chromatography of porcine pepsin and pepsinogen on the prepared carriers were optimized: the effect of pH, ionic strength and a nature of the buffers used on adsorption of the enzyme and zymogen to an affinity carrier, as well as their elution was studied. The following parameters were taken into consideration: capacity of the prepared affinity matrices, reproducibility of experiments and the enzyme stability. Pepsin was adsorbed to both immobilized ligands at pH 3.5–4.0; for the elution of the enzyme it was necessary to increase ionic strength (up to 0.5 M). For the adsorption of pepsinogen pH 5.2 was found to be optimum, for its desorption, an increase of ionic strength was used.
Keywords: Porcine pepsin; Porcine pepsinogen;

An efficient optimization method was suggested to separate biologically active peptides by RP-HPLC. In this work, the binary mobile phase of water and acetonitrile was used with the buffer of trifluoroacetic acid (TFA). The elution profiles were calculated by the plate theory based on the linear and quadratic equations of retention factor, ln  k=A+BF, ln  k=A+BF+CF2 , and F was the vol.% of acetonitrile. We modified the plate theory to calculate elution profile in both isocratic and gradient mode. From the final calculated results, the first mobile phase composition was water in 0.1% TFA/acetonitrile in 0.1% TFA, 81/19 vol.%, then after 7–8 min, the second composition of mobile phase was linearly changed to 79/21 vol.%, and finally after 8 min, it was kept at the isocratic mode. In the experimental conditions, the agreement between the experimental data and the calculated values was relatively good.
Keywords: Mobile phase composition; Plate theory; Peptides;

Four subunits of Schizosaccharomyces pombe RNA polymerases I–III shared by all three enzymes (Rpb5, Rpb8, Rpb10 and Rpc10 [Rpb12]) have been overexpressed in Escherichia coli expression vectors pQE or pET as hexahistidine fusions. The recombinant proteins have been purified to near homogeneity using metal–chelate affinity chromatography and gel filtration. Homogeneity and identity of the purified protein preparations was demonstrated by denaturing polyacrylamide gel electrophoresis and TOF-MALDI mass spectrometry. The proteins were obtained in large amounts, and their preparations are currently in use for monoclonal antibody production and physico-chemical studies of these individual components of eukaryotic transcription enzymes.
Keywords: Expression; Purification; Cloning; Nuclear RNA polymerases;

3-Amino derivative of β-cyclodextrin: thermodynamics of copper(II) complexes and exploitation of its enantioselectivity in the separation of amino acid racemates by ligand exchange capillary electrophoresis by Vincenzo Cucinotta; Alessandro Giuffrida; Diego La Mendola; Giuseppe Maccarrone; Antonino Puglisi; Enrico Rizzarelli; Graziella Vecchio (127-133).
The systems that the 3-amino derivative of β-cyclodextrin (CD3NH2) forms with the proton, the copper(II) ion and each of the enantiomers of certain amino acids (alanine, phenylalanine, tyrosine and tryptophan) were investigated. The enantioselectivity shown by the potentiometric measurements carried out on the phenylalanine ternary systems was exploited in capillary electrophoresis by ligand exchange capillary electrophoresis (LECE) to obtain the separation of phenylalanine racemate. The tyrosine racemate was also separated by LECE. The comparison between thermodynamic and capillary electrophoresis (CE) results is discussed, in order to get a better insight into the separation mechanism.
Keywords: Enantiomer separation; Copper(II) complexes; β-Cyclodextrins; Amino acids;

Determination of amino acids in fodders and raw materials using capillary zone electrophoresis by N.V Komarova; J.S Kamentsev; A.P Solomonova; R.M Anufrieva (135-143).
Two schemes were offered for analysis of amino acid contents in fodders and raw materials for mixed fodders by capillary zone electrophoresis (CZE). The first variant provides express analysis of four technologically important amino acids (lysine, methionine, threonine, cystine) in borate buffer on characteristic absorption of aminogroup (190 nm), with limits of quantitation being on average 0.2%. The second scheme includes pre-capillary derivatization of amino acids using phenylisothiocyanate (PITC) and separation of phenylthiocarbamyl (PTC)-derivatives obtained by CZE with a detection on 254 nm, which allows to widen a list of detectable components up to 19 (without tryptophan) and significantly improve detection limits down to 0.01%. Acid hydrolysis was used for a sample preparation. The results of analysis of fodders were compared using such methods, as CZE, ion exchange chromatography (amino acid analyzer) and reversed-phase (RP)-HPLC (with gradient technique of elution).
Keywords: Derivatization; Fodder; Amino acids;

Application of liquid chromatography–electrospray ionization mass spectrometry for study of steroid-converting enzymes by Ivan Mikšı́k; Kateřina Mikulı́ková; Jiřı́ Pácha; Marek Kučka; Zdeněk Deyl (145-153).
A high-performance liquid chromatography–atmospheric pressure ionization–electrospray ionization mass spectrometry (HPLC–API–ESI–MS) method was developed for the analysis of steroids in a study of steroid-converting enzymes. Separations ware done on a Zorbax Eclipse XDB-C18 column (eluted with a linear methanol–water–acetic acid gradient) and identification of the steroids involved was done by API–ESI–MS using positive ion mode and extracted ion analysis. The applicability of the present method for studying steroid metabolism was proven in assaying two steroid-converting enzymes (20β-hydroxysteroid dehydrogenase and 11β-hydroxysteroid dehydrogenase) in various biological samples (rat and chicken intestine, chicken oviduct).
Keywords: Steroids; Enzymes, steroid-converting;

Peptide mapping by capillary electrophoresis with Pluronic F127 by Ivan Mikšı́k; Jana Charvátová; Adam Eckhardt; Zdeněk Deyl (155-160).
Separation of peptides and proteins by capillary zone electrophoresis suffers from the interaction of these solutes with the capillary wall which results in the formation of broad peaks and low resolution. To minimize the protein/peptide–capillary wall interaction we tried to use Pluronic F127, a triblock copolymer of the general formula (polyethylene oxide) x (polypropylene oxide) y (polyethylene oxide) z when x=106, y=70 and z=106 which can be considered a surfactant capable of self-association both into isotropic and anisotropic gels. The analytes studied were enzymatic digests (obtained by trypsin or pepsin treatment) of insoluble matrix proteins from avian eggshell. The best separations were obtained by a system exploiting 10% Pluronic F127 in 20 mmol/l phosphate buffer, pH 2.5. Electrophoretic peptide profiles obtained were very complex owing to the complicated nature of the samples (the exact composition of the proteinous insoluble part of the eggshell is still unknown). The separation in phosphate buffer only offered complex maps of incompletely resolved peaks. The use of Pluronic F127 distinctly improved the separation with a considerably better resolution regarding both the number of peaks obtained and the quality of the separation.
Keywords: Peptide mapping; Pluronic F127;

Capillary electrophoretic separation of proteins and peptides by ion-pairing with heptanesulfonic acid by I. Mikšı́k; J. Charvátová; A. Eckhardt; T. Cserháti; E. Forgács; Z. Deyl (161-167).
Heptanesulfonic acid as ion-pairing agent was used for the separation of mixtures of low and high molecular mass peptides/proteins by capillary electrophoresis. The separation conditions used were: capillary 37 cm (30 cm to the detector) × 75 μm i.d., voltage 10 kV, phosphate buffer 50 mmol/l, ion-pairing agent heptanesulfonic acid at three different concentrations, namely, 0, 20 or 100 mmol/l, pH 2.5. The separation reflected the ion-pairing equilibria between the ion-pairing agent and the peptide/protein analytes. The influence of ion-pairing on sample mobility (running time) was more pronounced in case of the higher-molecular peptides as compared to the low molecular ones. This difference offers the possibility to separate low and high molecular peptides/proteins that under the absence of the ion-pairing agent would co-migrate. The principle of this approach was demonstrated on a randomly selected set of peptides/proteins; the practical applicability was demonstrated on a set of CNBr peptides arising from a naturally occurring mixture of collagen types I and III.
Keywords: Ion-pairing reagents; Peptides; Proteins; Heptanesulfonic acid;

Cleavage of double stranded plasmid DNA by lanthanide complexes by Bohuslav Rittich; Alena Španová; Martin Falk; Milan J Beneš; Martin Hrubý (169-173).
The use of free lanthanide ions and their complexes for plasmid DNA pBR322 and chromosomal DNA cleavage was studied. Plasmid pBR322 DNA was treated by lanthanide chlorides (Eu3+, La3+, Nd3+, Pr3+, Gd3+) in HEPES buffer (pH 7.0, 7.5 and 8.0) at 24, 37, 50, 63, and 76 °C. The formation of linear and nicked plasmid forms was investigated depending on the reaction conditions. Heterogeneous lanthanide complexes of ethylenediamine tetraacetic acid (EDTA) immobilized on insoluble methacrylate support and iminodiacetic acid (IDA) immobilized on styrene support were used as catalysts plasmid for DNA pBR322 cleavage, too. The temperature of reaction mixture had substantial influence on cleavage rate. The precipitation of DNA occurred during the measurement of interactions between chromosomal DNA and La3+ ions.
Keywords: Cleavage; Lanthanide complexes; Plasmid DNA pBR322;

Investigation of chromatographic conditions for the separation of cefuroxime axetil and its geometric isomer by Ljiljana Zivanovic; Ivana Ivanovic; Sote Vladimirov; Mira Zecevic (175-179).
The optimal chromatographic conditions for the separation of the syn- and anti-geometric isomers of cefuroxime axetil applying RP-HPLC and micellar liquid chromatography (MLC) methods were investigated. The possibility to separate diastereoisomers of syn- and anti-cefuroxime axetil was observed. Investigations were performed using three columns, two classical silicas and one with hybrid particle technology. Three aqueous-organic and one micellar mobile phases were used. The best results were achieved using micellar mobile phase. Optimization study was performed using different micellar mobile phases. MLC method is sensitive and applicable in purity and stability testing.
Keywords: Optimization; Cefuroxime axetil; Anti-cefuroxime axetil;

Influence of the extraction mode on the yield of some furanocoumarins from Pastinaca sativa fruits by Monika Waksmundzka-Hajnos; Anna Petruczynik; Anna Dragan; Dorota Wianowska; Andrzej L Dawidowicz; Ireneusz Sowa (181-187).
Analysis of plant material is an important task in chemotaxonomical investigations, in search of plants with pharmacological activity or in standardisation of plant drugs. The choice of optimal conditions for the analysis of plant material and effect of extraction method on the yield of furanocoumarins from Pastinaca sativa fruits were examined. The following extraction methods were used in experiments: exhaustive extraction in Soxhlet apparatus, ultrasonification (USAE) at 25 and 60 °C, microwave-assisted solvent extraction in open and closed system (MASE) and accelerated solvent extraction (ASE). In most cases, the yield of furanocoumarins was highest by use of ASE method as well as by ultrasonification at 60 °C.
Keywords: Solid–liquid extraction; Microwave-assisted solvent extraction; Accelerated solvent extraction; Ultrasonification; Furanocoumarins;

Liquid chromatography method for determination of mefenamic acid in human serum by Mohammad-Reza Rouini; Ali Asadipour; Yalda Hoseinzadeh Ardakani; Fakhredin Aghdasi (189-192).
A simple, rapid and specific method for analysis of mefenamic acid (I) in serum by a sensitive high-performance liquid chromatography is described. Only 70 μl of serum and a little sample work-up is required. A simple procedure of extraction by dichloromethane followed by evaporation to dryness under gentle stream of nitrogen and dissolving the dried residue in mobile phase was used. The mefenamic acid peak was separated from endogenous peaks on a C8 column by a mobile phase of acetonitrile–water (50:50, v/v, pH 3). Mefenamic acid and internal standard (IS) (diclofenac) were eluted at 7.4 and 5.4 min, respectively. The limit of quantitation of mefenamic acid in serum was 25 ng/ml at 280 nm. The method was linear over the range of 25–2000 ng/ml with r 2 of 0.998. Mean recovery for mefenamic acid was 110%.
Keywords: Pharmacokinetics; Mefenamic acid;

A rapid reversed-phase type HPLC method for the simultaneous separation and analysis of d- and l-thyroxine (d- and l-T4) and triiodothyronine (T3) was developed using a quinine-derived chiral stationary phase and applied for a quantitative assay of the enantiomeric impurity of the drugs in pharmaceutical formulations of levothyroxine. The influence of operating parameters has been studied for the optimization of the separation and also in order to gain an insight into the retention mechanism. Validation of the method included linearity, precision and accuracy which revealed R.S.D. values of <3.3% for intra-assay precision and percent error ranging from −6 to +2.1% for various defined validation samples, proving satisfactory accuracy. Quantitation was performed over the range of 0.5–500 μg ml−1 with limits of detection and quantitation lower than 0.1 and 0.5 μg ml−1, respectively, for both analytes. Further, the determination of 0.1% impurity, of d-T4 as well as l- and d-T3 in levothyroxine sodium tablets proved to be feasible.
Keywords: Enantiomer separation; Chiral stationary phases; LC; Thyroxine; Triiodothyronine;

Capillary electrophoresis method for identification and simultaneous determination of procaine, dihydrostreptomycin and penicillin G, present in multiantibiotic veterinary preparations, was elaborated. The influence of pH (5.0–9.75) and concentration of disodium tetraborate decahydrate in running buffers (0.02–0.1 M) as well as temperatures (25–40 °C) on separation efficacy were analyzed. For quantitative analysis, 0.08 M borate buffer (pH 8.0) at 35 °C and 15 kV were chosen. Method was validated, selectivity, precision, linearity, LOD, LOQ, accuracy and specificity of capillary zone electrophoresis (CZE) were evaluated.
Keywords: Dihydrostreptomycin; Procaine; Penicillin G;

Nature of the main contaminant in the anti malaria drug primaquine diphosphate: a qualitative isomer analysis by Ilia Brondz; Dimitris Mantzilas; Uwe Klein; Dag Ekeberg; Erlend Hvattum; Marina N Lebedeva; Felix S Mikhailitsyn; Gasan D Souleimanov; Johan Røe (211-223).
The main contaminant of primaquine (CAS 90-34-6) has been tentatively identified, by using two liquid chromatography (LC) methods and liquid chromatography–mass spectrometry (LC–MS), as the positional isomer quinocide (CAS 525-61-1). The first LC system was equipped with a chiral Chirex (S)-VAL and (R)-NEA column and the second system was equipped with an Adsorbosphere Nucleotide-Nucleoside 7 μ column. Comparison of the main contaminant of primaquine with an authentic quinocide standard by using co-chromatography in both LC systems and LC–MS (mass fragmentation) supported the hypothesis. The toxicity of quinocide batch 17172, primaquine batch 16039, and the drug primaquine diphosphate batch 20107 used in pharmaceutical industry, and the effect of the substances on respiratory and electron transport chain were compared in the eucaryotic unicellular fresh water green alga Chlamydomonas reinhardtii as a model system. These studies suggest that minor amount of other related substances can contribute more to the toxicity of the drug primaquine diphosphate than the positional isomer quinocide.
Keywords: Primaquine; Quinocide;

An HPLC method for Vitamins A and E in rat plasma has been developed. The main goals of the method are the small amount of sample, 50 μl, and the direct extraction of analytes in one step with acetone, which is a solvent compatible with the reverse-phase mobile phases. Recoveries, as compared with classical and more tedious methods, were near 100%. The method employs a Supelco Discovery® C18 column and methanol/water (95:5, v/v) as mobile phase. After being developed, the method was validated following ICH guidelines, with UV, fluorescence and electrochemical detectors. It proved to be selective, lineal, accurate and precise. This method greatly simplifies sample treatment and that is a critical point when working with a large number of samples.
Keywords: Retinol; Tocopherols; Vitamins;

New terpenoids in cultivated and wild chamomile (in vivo and in vitro) by Éva Szőke; Emőke Máday; Ernő Tyihák; Inna N Kuzovkina; Éva Lemberkovics (231-238).
The effect of Chamomilla recutita (L.) Rauschert is made up by several groups of active substances, among which terpenoids in the inflorescences are of greatest importance. Among cultivated species, the Hungarian BK-2 contains more chamazulene in its essential oil than the German Degumil type, which is mainly cultivated for its (−)-α-bisabolol. Both components have important antiinflammatory activities. Among wild chamomile populations in Hungary, a population was found in the area of Szabadkigyós containing significant amounts—on average 48%—of (−)-α-bisabolol in its inflorescence oil. In vitro cultures were made from this population to obtain propagation material containing a high number of active substances. The intact roots contained no (−)-α-bisabolol but the sesquiterpene alcohol β-eudesmol as new compound was identified by our group. Sterile plantlets, cultured in vitro, were multiplied for phytochemical investigations. Pharmacologically important compounds of the essential oils were followed in great detail. The amount of in vitro cultured terpenoids and polyin compounds was compared with that of in vivo plants. These volatile compounds were identified by comparing their retention times with those of authentic standards, essential oils of known composition and peak enrichment. The confirmation of identity was done by comparison of their mass spectra with those reported in the literature and reference compounds. The percentage evaluation of each component was made by area normalisation. Gas chromatography (GC) and mass spectrometry (MS) showed that sterile chamomile cultures generated the most important terpenoid and polyin compounds characteristic of the parent plant. We identified germacrene-D, berkheyaradulene, 4-(2′, 4′, 4′-trimethyl-bicyclo[4.1.0]hept-2′-en-3′-yl)-3-buten-2-one, geranyl-isovalerate and cedrol as new components in these sterile cultures.
Keywords: Chamomilla recutita; Terpenoids;

Determination of γ-hydroxybutyric acid in biological fluids by using capillary electrophoresis with indirect detection by Federica Bortolotti; Giorgia De Paoli; Rossella Gottardo; Maristella Trattene; Franco Tagliaro (239-244).
γ-Hydroxybutyric acid (GHB) is a central nervous system (CNS) depressant and hypnotic which, in recent times, has shown an increasing abuse either as recreational drug (due to its euphoric effects and ability to reduce inhibitions) or as doping agent (enhancer of muscle growth). Analogues of GHB, namely γ-butyrolactone (GBL) and 1,4-butanediol (1,4-BD), share its biological activity and are rapidly converted in vivo into GHB. At present, GHB and analogues are placed in the Schedules of Controlled Substances. Numerous intoxications in GHB abusers have been reported with depressive effects, seizures, coma and possibly death. The purpose of the present work was the development of a rapid analytical method based on capillary zone electrophoresis for the direct determination of GHB in human urine and serum at potentially toxic concentrations. Analytical conditions were as follows. Capillary: length 40 cm (to detector), 75 μm i.d.; buffer: 5.0 mM Na2HPO4, 15 mM sodium barbital adjusted to pH 12 with 1.0 M NaOH; voltage: 25 kV at 23 °C; indirect UV detection at 214 nm; injection by application of 0.5 psi for 5 s. α-Hydroxyisobutyric acid was used as internal standard (IS). Sample pretreatment was limited to 1:8 dilution. Under these conditions, the sensitivity was ∼3.0 μg/ml (signal-to-noise ratio >3). Calibration curves prepared in water, urine and serum were linear over concentration ranges 25–500 μg/ml with R 2≥0.998. Analytical precision was fairly good with R.S.D.<0.60% (including intraday and day-to-day tests). Quantitative precision in both intraday and day-to-day experiments was also very satisfactory with R.S.D.≤4.0%. No interferences were found neither from the most common “drugs of abuse” nor from endogenous compounds. In conclusion, capillary electrophoresis can offer a rapid, precise and accurate method for GHB determination of biological fluids, which could be important for screening purposes in clinical and forensic toxicology.
Keywords: Direct injection; γ-Hydroxybutyric acid;

Selective reversed-phase liquid chromatography method for the kinetic investigation of 3-hydroxyflavone photostability by M.L Calabrò; S Tommasini; D Raneri; P Donato; P Ficarra; R Ficarra (245-251).
This paper reports a fast and accurate RP-HPLC chromatographic method for the simultaneous determination of 3-hydroxyflavone (3-OH F) and its photodegradation products. Solutions (5×10−5  M) in acetonitrile (ACN) of the molecule were subjected to forced degradation by exposure to artificial UV-A light source (black-light, λ max 354 nm) and the changes appearing in chromatograms were monitored at selected irradiation times. A multistep gradient was optimised to achieve complete elution of all photoproducts in the shortest analysis time. UV spectra recorded by the diode array detector system (285 and 340 nm) clearly showed the structural changes in the new species formed, with respect to the parent compound. The analytical method was subjected to a validation procedure in which linearity and range, as well as specificity, precision and accuracy were determined according to ICH guidelines. Quantitative evaluation of the photochemical process was performed on the basis of the calculated kinetic parameters: photodegradation rate constant k, half-life time t 0.5, time degradation of 10% of the drug t 0.1.
Keywords: Kinetics; Photostability; Validation; 3-Hydroxyflavone;

The reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed and validated for the simultaneous determination of imatinib mesylate and of the impurity product in Glivec® capsules (Novartis, Switzerland). Separations were performed on a X Terra™ 150  mm×4.6  mm, 5 μm particle size column at 25 °C. The mobile phase was a mixture of methanol–water–triethylamine (25:74:1, v/v/v) with flow rate of 1.0 ml min−1. pH value of water–triethylamine (TEA) was adjusted to 2.4 with orthophosphoric acid before adding of methanol. UV detection was performed at 267 nm. Acetaminophen was used as an internal standard. The method was validated statistically for its selectivity, linearity, precision, accuracy and robustness. Due to its speed and accuracy, the method may be used for quality control analyses.
Keywords: Imatinib mesylate;

Simultaneous effect of organic modifier and physicochemical parameters of barbiturates on their retention on a narrow-bore PGC column by Esther Forgács; Tibor Cserháti; Ivan Miksik; Adam Echardt; Zdenek Deyl (259-262).
The retention time of 22 barbituric acid derivatives was measured on a narrow-bore porous graphitized carbon (PGC) column using water–dioxane mixtures as mobile phases. The capacity factor (k), theoretical plate number (N), and asymmetry factor (AF) were calculated for each solute in each mobile phase. The relationships between chromatographic characteristics and physicochemical parameters of solutes were elucidated by stepwise regression analysis (SRA). SRA indicated that the binding of barbiturates to the PGC surface is of mixed character electrostatic and apolar interactive forces are equally involved. Sterical correspondence between the surface of the stationary phase and the solutes also exert a significant influence on the retention behavior.
Keywords: Barbiturates; Porous graphitized carbon;

Use of the affinity chromatography principle in creating new thromboresistant materials by Nadezhda A Samojlova; Maria A Krayukhina; Igor A Yamskov (263-269).
The principle of affinity chromatography was used for preparation of thromboresistant bilayer coatings. The outer biospecific layer containing ε-aminocaproic acid residues (from 2.2 up to 5.5 nmol/cm2) was synthesized using a copolymer of maleic anhydride with N-vinylpyrrolidone and l-lysine dihydrochloride or N-ε-tert-BOC-l-lysine. This surface can selectively adsorb plasminogen (fibrinolytic zymogen) from blood. The biospecific layer (from 2.0 up to 3.6 μg/cm2) was applied for covering chitosan (native or modified) or albumin interlayer. Such bilayer coatings (BCs) were stable and represented the insoluble polyelectrolyte complexes. BCs were proposed for bilayer modification of synthetic vascular grafts, polyethylene, and other materials contacting with blood. This technique allowed us to significantly reduce thrombogenic properties of polyethylene surfaces.
Keywords: Affinity chromatography principle; Bilayer coatings; Thromboresistant materials; Lysine; Plasminogen;

A method for the determination of isosorbide-5-mononitrate (5-ISMN) in human plasma by capillary gas chromatography with electron-capture detection was developed and applied to clinical samples. 9-Fluorenone was used as an internal standard, ethyl acetate was employed for liquid–liquid extraction. The advantage of the extraction procedure is the possibility of a direct injection of the plasma extract, without solvent removal/reconstitution of the sample. The precision and accuracy of the method were satisfactory in the concentration range 10–1600 ng/ml. The lower limit of quantification was 10 ng/ml.
Keywords: Isosorbide mononitrate;

A rapid and sensitive method for quantification of homocysteine total forms and glutathione levels in blood plasma via HPLC was developed. Dithiotreitol as a water soluble agent has been used as a reductant for both protein and nonprotein disulphides. Dithiotreitol reacts with the mixed disulphides under 60 °C treatment within 10 min. Reduced aminothiols and homocystein were easily derivated with 5,5′-dithiobis-(2-nitrobenzoic acid) and the resultant ultraviolet absorbance within 330 nm was detected by the HPLC method. The concentration of total plasma homocysteine was significantly higher in groups of patients: with the end stage of renal disease: 45.5±40.9 μmol/l (n=79), with cerebral vascular disorders 12.3±7.0 μmol/l (n=65), and with coronary atherosclerosis 15.4±10.9 μmol/l (n=15) than that in healthy subjects (6.2±1.74 μmol/l, n=20). Some major advantages of the method include: simultaneous measurement of both total homocysteine and total glutathione, no loss of oxidized form during processing of blood plasma for aminothiols measurement, use of protein-bound aminothiols solution as a calibrator.
Keywords: Homocysteine; Glutathione; Dithiothreitol;

A method is described for the determination of metabolites of mesocarb in human urine by combining gradient liquid chromatography and electrospray ionization (ESI)—ion trap mass spectrometry. Seven metabolites (two isomers of hydroxymesocarb, p-hydroxymesocarb, two isomers of dihydroxymesocarb and two isomers of trihydroxymesocarb) and parent drug were detected in human urine after the administration of a single oral dose 10 mg of mesocarb (Sydnocarb®, two tablets of 5 mg). Various extraction techniques (free fraction, enzyme hydrolyses and acid hydrolyses) and their comparison were carried out for investigation of the metabolism of mesocarb. After extraction procedure the residue was dissolved in methanol and injected into the column HPLC (Zorbax® SB-C18 (Narrow-Bore 2.1×150 mm i.d., 5 μm particles)) with mobile phase (0.2 ml/min) of methanol/0.2 mM ammonium acetate. Conformation of the results and identification of all metabolites are performed by LC-MS and LC-MS/MS. The major metabolites of mesocarb in urine of the human were p-hydroxylated derivative of the phenylcarbamoyl group of the parent drug (p-hydrohymesocarb) and dihydroxylated derivative of mesocarb (two isomers of dihydroxymesocarb). This analytical method for dihydrohymesocarb was very sensitive for discriminating the ingestion of mesocarb longer than the parent drug or other metabolites in human urine. The dihydroxymesocarb was detected in urine until 168–192 h after administration of the drug.
Keywords: Mesocarb; Hydroxymesocarb;

A method for calculation of the retention time of a compound in a temperature-programmed mode of GC analyses is proposed. The method is based on a detailed consideration of thermodynamics of chromatographic process and account of the actual state of a capillary column. The suggested approach permits estimation of probability of a poisonous agent in tests by means of information stored in the database, eliminating a need for reference standard of such an agent.
Keywords: Retention parameters; Thermodynamic parameters; Sarin; Soman; Sulfur mustard;

Needle concentrator for gas chromatographic determination of BTEX in aqueous samples by Róbert Kubinec; Victor G Berezkin; Renáta Górová; Gabriela Addová; Helena Mračnová; Ladislav Soják (295-301).
A simple method of solventless extraction of volatile organic compounds (BTEX) from aqueous samples was developed and validated. A new arrangement of the full volume inside needle capillary adsorption trap (INCAT) device with Porapak Q as a sorbent material and wet alumina as a source of desorptive water vapour flow in a closed analytical system is presented. The analytical characteristics of developed device and of compared purge-and-trap (PTI) device for BTEX compounds are similar; the limits of detection as well as quantification are lower than 1 μg l−1.
Keywords: Needle concentrator; Benzene; Toluene; Ethylbenzene; Xylene;

The aim of the present work was to study the possibility of using the fatty acid content in the basidiospores as a taxonomic tool. Basidiospores of Armillaria borealis, Amanita muscaria, Agaricus sylvicola, Hypholoma capnoides, Cortinarius nemorensis and Russula delica were used. The content of fatty acids as well as other substances may vary to a certain degree depending on the part (pileus, stipe, lamella) or stage of development of the actual basidiocarp analysed. Moreover, substances from fungivorous invertebrates, parasitic fungi or bacteria may be found in the chemical analyses of the basidiocarps. Chemotaxonomic conclusions may, therefore, be burdened with serious uncertainties. On the other hand, the ripe basidiospores are terminated structures and belong to the most homogenous structures encountered from a basidiocarp. Their shape, size, colour and ornamentation are considerably homogenous within an actual species. Therefore, the basidiospores are often used as a reliable differentiating characteristic separating species as well as taxa of higher categories. From a practical point of view, ripe spores are easy to obtain in relatively large quantities with simple techniques, and they are not so prone to decay as the carpophore tissue. In the present study, gas chromatography (GC) and gas chromatography–mass spectrometry (GC–MS), after methanolysis of the fungus spores, were used to map essential fatty acids in basidiomycetes. Gas chromatography and gas chromatography–mass spectrometry revealed the presence of fatty acids of C12:0–C24:0 size in the basidiospores of these higher basidiomycetes. The major fatty acid in H. capnoides is C18:2, and the major fatty acid in the other species is C18:1. The basidiospores proved to be a good source of fatty acids for chemotaxonomic investigations of agarics.
Keywords: Basidiomycetes; Chemotaxonomy; Fatty acids;

The objective of this paper was to prove that matrix solid-phase dispersion (MSPD) coupled with high performance liquid chromatography (HPLC) and column switching could be used for the determination and quantification of carbendazim residue in plant samples. By comparing results obtained after optimization of the extraction conditions on an acidic silica gel column, it was determined that sorption and retention of carbendazim were achieved via specific interactions. The method of standard additions was used for quantitative analysis. Its performance was evaluated and validated: the detection limit (UV-Vis detection at λ=279 nm) was 0.02 μg/g, the relative standard deviations (R.S.D.) were between 2.7 and 4.1% and the recoveries were ranging from 84.3 to 90.7% at the 0.04, 0.08 and 0.1 μg/g fortification levels. The method was successfully tested on cereal samples, and the results obtained with the present off-line MSPD–HPLC procedure were found to compare well with those obtained with procedure involving LLE.
Keywords: Optimization; Matrix solid-phase dispersion; Carbendazim;

Reversed-phase liquid chromatographic–mass spectrometric determination of microcystin-LR in cyanobacteria blooms under alkaline conditions by Werawan Ruangyuttikarn; Ivan Miksik; Jeeraporn Pekkoh; Yuwadee Peerapornpisal; Zdenek Deyl (315-319).
Reversed-phase HPLC coupled to the atmospheric pressure ionization–electrospray ionization (API–ESI) MS was used for microcystin-LR detection and quantitation in samples of dried Microcystis aeruginosa cells. An alkaline linear gradient (20 mmol/l ammonium hydroxide–acetonitrile, pH 9.7) was used for elution of the toxic peptides. Limit of detection was 1 μg/ml (20 ng per injection) in the scan mode of MS and 0.1 μg/ml (2 ng per injection) in the case of selective ion monitoring.
Keywords: Cyanobacteria; Microcystin;

The method of simultaneous determination of F, Cl, NO2 , Br, NO3 , HPO4 2−, SO4 2− ions on trace level by IC using preliminary concentration and water removal from concentrating column was proposed. Detection limits were 10−9 to 10−8% for sample volume 10 ml depending on the anion.
Keywords: Fluoride; Chloride; Nitrite; Bromide; Nitrate; Phosphate; Sulphate;

Gas chromatography–mass spectrometry with headspace for the analysis of volatile organic compounds in waste water by V.I Safarova; S.V Sapelnikova; E.V Djazhenko; G.I Teplova; G.F Shajdulina; F.Kh Kudasheva (325-330).
Headspace analysis combined with high-resolution gas chromatography and detection by mass spectrometry was evaluated for the analysis of 53 volatile organic compounds (VOCs) in river waters, waste waters and treated water samples down to 0.1 μg l−1 concentration levels. The conditions optimised included sample thermostatting time and temperature, autosampler parameters and the nature of salt, added to the sample. The pollutions origin and their seasonal rippling have been done. It was shown that the content of VOCs in river water mainly correlates to the content of these compounds in waste waters, which shows the anthropogenic character of the pollutions.
Keywords: Volatile organic compounds;