Journal of Chromatography B (v.770, #1-2)

Preface (1).

The effect of flow-rate on quantitatively determined binding parameters for several biocomplementary pairs in affinity mode high-performance monolithic disk affinity chromatography (HPMDAC) has been investigated using frontal analysis approach. Affinity interactions were evaluated from linearized adsorption isotherms and dynamic dissociation constants of the complexes K diss. and the theoretical adsorption capacities Q max were calculated. HPMDAC isolation of a typical protein trypsin from both buffered solution and artificial mixture as well as biospecific extraction of antibodies against bovine serum albumin and recombinant protein G from such complex mixtures as blood serum and cellular lysate were examined. Immobilized counterparts soybean trypsin inhibitor, bovine serum albumin, and human immunoglobulin G were used in chromatographic experiments. The maximum adsorption capacities obtained at different flow-rates were compared with those determined at static conditions. The dependence of quantitative parameters on the surface density of immobilized ligands has also been explored. Finally, a series of experiments was carried out to evaluate the dependence of dynamic affinity binding on temperature for two complementary pairs.

Supercritical fluid chromatography coupled to a hybrid mass spectrometer (Q-Tof2) equipped with electrospray ion source has been used to separate and characterise a wide range of pharmaceutical racemates. We have chosen diverse molecular structures to demonstrate the potential of such experimental arrangement for high throughput analyses. The use of three different chiral stationary phases and different pressure/temperature working conditions provided clear indications on how such a high throughput method can be developed. The use of mass spectrometry was found to be essential for an unambiguous assignment of the eluting components particularly in the case of complex mixtures. The direct coupling of both systems without the need for a special interface resulted in similar peak shapes and peak widths in the UV and total ion current (TIC) chromatograms.

Separation of phospholipid classes in sea urchin, Paracentrotus lividus by high-performance liquid chromatography by A. Rodrı́guez-Bernaldo de Quiros; J. López-Hernández; J. Simal-Lozano (71-75).
A simple high-performance liquid chromatography (HPLC) method for determination of major phospholipid classes in sea urchin Paracentrotus lividus is described. The separation was performed on a Tracer Extrasil SI 5 μm 25×0.4 cm column and an isocratic mobile phase of acetonitrile–methanol 85%–phosphoric acid (50:50:1.8, v/v). The HPLC method utilizes UV detection at 205 nm. Five phospholipids were identified and quantified: phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC) and sphingomyelin (SM). Fresh and canned samples were analyzed. Student’s t-test showed no significant difference (P≤0.05) between the mean phospholipid contents of raw and canned sea urchin.
Keywords: Phospholipids;

Solid-phase extraction procedure to remove organic acids from honey by Silvia Suárez-Luque; Inés Mato; José F. Huidobro; Jesús Simal-Lozano (77-82).
A solid-phase extraction procedure was applied to remove organic acids from honey. Malic, maleic, citric, succinic and fumaric acids were isolated with an anion-exchange cartridge. The different parameters which affected the extraction procedure were studied and optimised to establish the optimal conditions for maximum recovery of organic acids and minimum extraction of interferences. The optimised procedure used a cartridge which was activated with 10 ml of 0.1 M sodium hydroxide solution (percolation rate 3 ml/min). A 10 ml volume of honey solution was passed at a flow-rate of 0.5 ml/min. The cartridge was washed with 10 ml of water (3 ml/min) and organic acids were eluted with 4 ml of 0.1 M sulfuric acid (0.5 ml/min). This solution was injected directly into the chromatograph. When this procedure was carried out on standard solutions of organic acids, recoveries between 99.2 and 103.4% were found. If this procedure was applied to honey samples these recoveries were also satisfactory and ranged from 62.9 to 99.4%.
Keywords: Organic acids;

Modern extraction techniques, supercritical fluid extraction (SFE) and solid-phase microextraction (SPME) were used for isolation of four corticosteroids from biological matrices. SFE was applied for extraction from solid matrices — hydromatrix and pig muscle. The effects of various extraction conditions were studied. Good recoveries of corticosteroids from hydromatrix were obtained under moderate extraction conditions and without modification of carbon dioxide. On the contrary, the best recoveries from spiked pig muscle were obtained with modified carbon dioxide. SPME was used for extraction from liquid samples — water and urine. The eventuality of the use of this fast solvent-free technique in steroid analysis is demonstrated. Several extraction conditions were optimized. Extracted steroids were analyzed by HPLC–UV and a special SPME–HPLC interface was used for combination with SPME.
Keywords: Steroids;

Effect of column and software on gas chromatographic determination of fatty acids by M. Vecka; E. Tvrzická; B. Staňková; A. Žák (91-99).
Four capillary columns (A: CP-WAX 52 CB 25 m×0.25 mm; B: CP WAX 52 CB 30 m×0.25 mm; C: CP-WAX 58 CB 25 m×0.25 mm, Chrompack; D: OMEGAWAX™ 320 30 m×0.32 mm, Supelco) and two integration software (Mosaic v.5.10, Chrompack and CSW v.1.7, Data Apex®) were compared for analysis of fatty acids. Column A was mounted stepwise in two different instruments. Fatty acids of blood plasma phosphatidylcholine and standard mixture of saturated fatty acids were analysed as methyl esters under identical chromatographic conditions. Both integrating software did not differ significantly in most results; differences were observed only for minor components: 16:1n9 (0.10±0.020 vs. 0.17±0.005 M%, P<0.0001, column A1; 0.09±0.011 vs. 0.16±0.007 M%, P<0.0001, column A2; 0.09±0.010 vs. 0.17±0.003 M%, P<0.0001, column C; 0.09±0.008 vs. 0.19±0.003 M%, P<0.0001, column D), 20:0 (0.10±0.001 vs. 0.06±0.005 M%, P<0.05, column C) and 20:2n6 (0.43±0.030 vs. 0.91±0.016 M%, P<0.0001, column A2). Increased values for 16:1n9 and 20:2n6 integrated by MOSAIC are caused by cointegration of two poorly resolved peaks: fatty acid and impurity from sample matrix. Lower values for 20:0 are caused by incomplete integration of minor peak. Differences between columns were observed mostly for minor fatty acids. The results indicate that CSW is more suitable software for integration of complicated chromatograms. Linear calibration dependences measured with standard mixture of saturated fatty acids (carbon number 10–24) were observed in wide range of concentrations (three orders). Slope close to unity and minimal value of intercept confirmed theoretical relations when analyses are run under optimal conditions. Use of one column is advisable in small intervention or experimental metabolic studies.
Keywords: Fatty acids;

Affinity chromatography was used to study an interaction of aspartic proteinases with immobilized aromatic amino acids and their derivatives. The following ligands were used: l-tyrosine, 3-iodo-l-tyrosine, 3,5-diiodo-l-tyrosine, l-phenylalanine, p-iodo-l-phenylalanine and N-acetyl-l-phenylalanine. With the exception of the last one, ligands were coupled directly to divinyl sulfone activated Sepharose 4B. For the preparation of immobilized N-acetyl-l-phenylalanine, divinyl sulfone activated Sepharose 4-B with linked ethylene diamine was used. Porcine pepsin was used for the evaluation of the capacity of the prepared affinity carriers. The capacity of the immobilized amino acid derivatives significantly increased in comparison with the non-derivatized amino acids. The prepared immobilized ligands were further used for the separation of human pepsinogens.
Keywords: Aromatic amino acids; Aspartic proteinases; Enzymes;

Determination of dissociation constant of phosphinate group in phosphinic pseudopeptides by capillary zone electrophoresis by Dušan Koval; Václav Kašička; Jiřı́ Jiráček; Michaela Collinsová; Timothy A. Garrow (145-154).
Capillary zone electrophoresis (CZE) was used for determination of dissociation constant of phosphinate group in phosphinic pseudopeptides, i.e. peptides where one peptide bond is substituted by phosphinic acid moiety –PO2 –CH2–. The dissociation constants were determined for a set of newly synthesized pseudopeptides derived from a structure N–Ac–Val–Alaψ(PO2 –CH2)Leu–His–NH2 by nonlinear regression of experimentally measured pH dependence of their effective electrophoretic mobilities. CZE experiments were carried out in Tris–phosphate background electrolytes in the pH range 1.4–3.2. The pseudopeptides were synthesized as a mixture of four diastereomers, the separation of which was achieved in most cases. Moreover, differences of the effective mobilities of the pseudopeptide diastereomers enabled simultaneous determination of the dissociation constant of their phosphinate group without necessity of previous isolation of individual isomers.
Keywords: Phosphinic pseudopeptides; Phosphinate;

Two different porphyrin derivatives (H2TPP(m-OPh)4 and Rh(III)TPP(m-OPh)4) were investigated with respect to their capability to help resolution of five model aromatic peptides in capillary electrophoresis/open tubular capillary electrochromatography. Though the main separation mechanism was preferentially based on the ionic properties of the separated analytes, involvement of particularly H2TPP(m-OPh)4–peptide interactions at alkaline pH (8.0) was clearly demonstrated. In combination with Tris–phosphate buffer, a speed up of the separation was observed at pH 2.25 (particularly if Rh(III)TPP(m-OPh)4 was used as capillary coating); in spite of the speed up of the separation the selectivity of the system was sufficient and resulted in a complete separation of the five model peptides. It can be expected that Rh(III)TPP(m-OPh)4 capillary coating in combination with Tris–phosphate buffer can be generally used for a considerable speeding up of lengthy separations of peptides in acidic media with some decrease in the separation power of the system.
Keywords: Peptides; Aromatic amino acids; Porphyrins;

Capillary electrochromatographic study of the interactions of porphyrin derivatives with amino acids and oligopeptides by Jana Charvátová; Václav Kašička; Vladimı́r Král; Zdeněk Deyl (165-175).
Open-tubular capillary electrochromatography (OT-CEC) was used to study the interactions of synthetic (metallo)porphyrin derivatives (immobilized by physical adsorption to the fused-silica capillary wall) with three aromatic amino acids (phenylalanine, tyrosine, tryptophan), three aliphatic amino acids (β-alanine, proline, valine) and two oligopeptides (diglycine, triglycine). The effective mobilities of amino acids and peptides measured in OT-CEC mode in the acid and alkaline background electrolytes (BGEs) were compared with those obtained by capillary zone electrophoresis (CZE) in the bare fused-silica capillary in the same BGEs. In this way the influence of the peripheral substituents and the character of the central metal atom in porphyrin derivatives on the interactions with amino acids and peptides in the acid and alkaline media was investigated. Three types of noncovalent interactions, axial ligation to the central metal atom, π–π stacking and electrostatic repulsion seem to take part in the interactions of analyzed amino acids and peptides with porphyrin derivatives, resulting in a better separation of these analytes by OT-CEC than by CZE.
Keywords: Porphyrins; Amino acids; Peptides;

Enzymes immobilized on magnetic carriers: efficient and selective system for protein modification by Z. Bı́lková; M. Slováková; D. Horák; J. Lenfeld; J. Churáček (177-181).
In order to obtain an economical, efficient and selective system for glycoprotein modification we prepared reactors with immobilized neuraminidase or (and) galactose oxidase. High storage and operational stability of the enzyme reactors was obtained by their immobilization through the carbohydrate parts of the enzyme molecules to hydrazide-modified supports. Magnetic and non-magnetic forms of bead cellulose and poly(HEMA-co-EDMA) microspheres were used for immobilization. These reactors can be used almost universally for the activation of ligands and for labelling of substances having a carbohydrate moiety.
Keywords: Neuraminidase; Galactose oxidase; Enzymes; Glycoproteins;

Oxycodone (OCOD) and its metabolites, including oxymorphone (OMOR), noroxycodone (NOCOD) and noroxymorphone (NOMOR), are opioids that carry an OH group at position 14. Using capillary electrophoresis (CE) with a binary phosphate buffer containing 60% ethylene glycol (pH 7.9), the migration order of OCOD and OMOR with respect to their N-demethylated analogs was found to be reversed compared to that observed for codeine, dihydrocodeine, morphine and dihydromorphine, compounds that do not have an OH group at position 14. OCOD and structurally related compounds can also be distinguished from these opioids by their absorbance spectra at low wavelengths and via a characteristic neutral H2O loss at the MS2 level. Using the binary phosphate buffer, CE with UV detection is shown to be capable of monitoring OCOD, NOCOD, OMOR (after hydrolysis only) and NOMOR (after hydrolysis and in patient urine only) in alkaline liquid–liquid extracts of urines that were collected after ingestion of 10 mg OCOD hydrochloride and in a patient urine collected at steady state (80 mg OCOD hydrochloride daily). Using an aqueous pH 9 ammonium acetate buffer, these results were confirmed by CE–MS3. Based on CE–MS, MS2 and MS3 data, the absorbance spectra measured across the CE peaks and the relative position within the electropherogram, two peaks monitored in the UV absorbance electropherograms could be assigned to the two keto-reduced metabolites 6oxycodol (6OCOL) and nor6oxycodol, for which no standards were available. Comparison of data obtained with urines pretreated with two different enzyme products (β-glucuronidase and β-glucuronidase/arylsulfatase) suggest that OCOD, NOCOD and 6OCOL are mainly glucuronidated, whereas OMOR mainly forms other conjugates. Furthermore, in a first attempt to directly measure conjugates of the compounds of interest, solid-phase extracts were analyzed by CE–MS4, which revealed the presence of the acyl glucuronides of 6OCOL and OMOR and an unidentified OMOR conjugate. The quantitation of free OCOD and NOCOD by CE–MS using deuterated internal standards is also discussed briefly.
Keywords: Oxycodone;

Retention parameters of 45 barbituric acid derivatives were determined on an amide embedded RP silica column using non-buffered water–dioxan eluent systems. Linear correlations were calculated between the logarithm of the capacity factor and the dioxan concentration in the eluent. Six different retention parameters of each barbituric acid derivative were correlated with different conventional and quantum chemical structural descriptors using quantitative structure–retention relationship (QSRR). The different parameters were: intercept (log k 0) and slope (b) values of the linear, the combined retention parameter (log k 0/b), asymmetry factor (AF 5) and theoretical plate values (N USP and N JP, according to the United States and Japanese Pharmacopoeia calculations). Stepwise regression analysis (SRA) and principal component analysis (PCA) followed by two-dimensional nonlinear mapping were used to determine the retention behavior of barbituric acid derivatives. SRA and PCA led to similar results. The results indicated that the retention of barbituric acid derivatives are mainly governed by the polaric and steric parameters of the substituents.
Keywords: Barbituric acid;

Two analytical separation techniques are being investigated for their potential in determining a wide range of endocrine disrupting chemicals (EDCs) in the environment. Capillary electrophoresis (CE) in the micellar mode in conjunction with a cyclodextrin (CD) modifier is shown to have potential for determination of alkylphenol breakdown products. Gas chromatography with mass spectrometric (GC–MS) detection is being utilised for validation of the CE method development and in addition as a separation technique to optimise preconcentration using solid-phase extraction. GC has demonstrated potential for the separation of 26 priority chemicals suspected as being endocrine disrupting compounds. The challenge of the method development process lies in the fact that these compounds are of differing polarities, size and charge and therefore are difficult to separate in a single run. Capillary electrophoresis in the CD–MEKC (micellar electrokinetic chromatography) mode is showing potential in this regard. Limits of determination are in the low mg/l range for CE and GC, however, using preconcentration it is possible to improve detection sensitivity with >80% recovery for some analytes and up to 100% recovery for most target species.

A simple, optimized method for the determination of sulphide in whole blood by GC–MS as a marker of bowel fermentation processes by Radomı́r Hyšpler; Alena Tichá; Monika Indrová; Zdeněk Zadák; Lidmila Hyšplerová; Jiřı́ Gasparič; Jaroslav Churáček (255-259).
Hydrogen sulphide is produced in human large intestine by anaerobic fermentation and may play a pathogenic role. An analytical method for determination of sulphide in whole blood using an extractive alkylation technique was optimised and validated for this purpose. The sample was mixed with organic phase containing pentafluorobenzyl bromide as an alkylating agent. The benzalkonium chloride was used as a phase-transfer catalyst. The quantitative determination was performed using GC–MS technique in selected ion monitoring mode. The blood levels of sulphide of healthy controls were measured (35–80 μM/l). The method is versatile, reproducible (RSD=2.7%) and suitable for research of anaerobic fermentation in vivo.
Keywords: Sulphide;

Fractionation of phosphorus and trace elements species in soybean flour and common white bean seeds by size exclusion chromatography–inductively coupled plasma mass spectrometry by Richard Koplı́k; Hana Pavelková; Jana Cincibuchová; Oto Mestek; František Kvasnička; Miloslav Suchánek (261-273).
Soluble species of phosphorus, sulfur, selenium and eight metals (Mn, Fe, Co, Ni, Cu, Zn, Mo and Cd) in soybean flour and common white bean seeds were investigated by size exclusion chromatography (SEC) and inductively coupled plasma mass spectrometry (ICP-MS). Samples were extracted by 0.02 mol l−1 Tris–HCl buffer solution (pH 7.5). Fractionation of sample extracts by preparative scale SEC was accomplished using a Fractogel EMD BioSEC column (600×16 mm) and 0.02 mol l−1 Tris–HCl buffer solution (pH 7.5) as mobile phase (flow rate: 2 ml min−1). A 2-ml sample was injected. Contents of elements in chromatographic fractions were determined by AAS, ICP-AES and ICP-MS. The elution profiles of P, Fe, Co, Ni, Cu, Zn and Mo in both samples were similar. Main species of Co, Ni, Cu, Zn and Mo were found in the low molecular weight region (2–5 kDa), whereas Fe is predominantly bound to high molecular weight compounds (180 kDa). The dominant phosphorus fraction was detected in the medium molecular weight region (10–30 kDa) and the other fraction in the low molecular weight region. Isotachophoretic analysis of chromatographic fractions revealed that the main phosphorus compound in the medium molecular weight region is phytic acid. SEC on Superdex 75 and Superdex Peptide columns (300×10 mm) was performed in on-line hyphenation with ICP-MS. The same mobile phase was used with a flow rate of 0.5 ml min−1; volume of injected sample was 200 μl. Element specific chromatograms were obtained by continuous nebulization of effluent into ICP-mass spectrometer measuring intensities of 47(PO)+ and 48(SO)+ oxide ions and 55Mn, 57Fe, 59Co, 62Ni, 65Cu, 66Zn, 82Se, 95Mo and 114Cd nuclides. Chromatographic profiles of elements are generally analogous to those obtained with a Fractogel column, but better chromatographic resolution of separated species was achieved so that slight differences between samples were revealed. Estimated molecular weights of major phosphorus species in soybean flour and common white bean seed extracts are 6 and 3.6 kDa, respectively, whereas those of minor phosphorus species in both samples are 0.7 kDa. Traces of phosphorus were also detected in the high molecular weight region (130 kDa). Chromatograms of P, Ni, Cu, Zn and Mo compounds in both extracts are similar but not identical. Molecular weights of major Cu and Zn species are ∼1 and 0.4 kDa for soybean flour and white bean seeds, respectively. In cases of Mn, Fe, Co and Se, the element profiles of soybean flour and white bean seed extracts are significantly different.
Keywords: Phosphorus; Trace elements;

Identification of xanthans isolated from sugarcane juices obtained from scalded plants infected by Xanthomonas albilineans by Blanca Fontaniella; C.W. Rodrı́guez; Dolores Piñón; C. Vicente; Marı́a-Estrella Legaz (275-281).
The exudate gum produced by Xanthomonas albilineans, a specific sugarcane pathogen, has been isolated from juices of diseased sugarcane stalks, hydrolyzed with hydrochloric acid, and the hydrolysate analyzed by capillary electrophoresis. Sucrose, cellobiose, mannose, glucose, glucose-1-P and glucuronic acid were identified as the major components of the polysaccharide isolated from diseased stalks. Juices from healthy stalks contained maltose instead of cellobiose. The chemical nature of this polysaccharide is discussed.
Keywords: Xanthans;

Liquid chromatographic analysis of supercritical carbon dioxide extracts of Schizandra chinensis by Milena Bártlová; Lubomı́r Opletal; Vladimı́r Chobot; Helena Sovová (283-289).
Six major lignans (schizandrin, gomisin A, deoxyschizandrin, γ-schizandrin, gomisin N, wuweizisu C) in the caulomas and leaves of Schizandra chinensis (Turcz.) Baill., and cinnamic acid in the leaves of the plant, were quantitatively analysed by high-performance liquid chromatography in reversed-phase mode with UV detection. Resolution of the determined lignans was evaluated for two multistep gradients applied. Samples for HPLC analysis were prepared by extraction with supercritical carbon dioxide at pressures of 20–27 MPa and temperatures of 40–60 °C. Kinetics of the extraction of individual components was measured and simulated with a model.
Keywords: Carbon dioxide;

High-performance liquid chromatographic analysis and separation of N-feruloylserotonin isomers by Milan Pavlı́k; Věra Laudová; Karel Grüner; Karel Vokáč; Juraj Harmatha (291-295).
The N-feruloylserotonin containing fraction was isolated from seeds of Leuzea carthamoides (Willd.) DC by solvent extraction followed by column chromatography on silica gel or on Sephadex LH-20. Nuclear magnetic resonance spectroscopic analysis of the isolated fraction showed the presence of four structurally related compounds. These compounds were identified as four isomers of N-feruloylserotonin: N-(Z)-feruloylserotonin, N-(Z)-isoferuloylserotonin, N-(E)-feruloylserotonin and N-(E)-isoferuloylserotonin. They were analyzed by HPLC on Separon SGX C18, Separon SGX and Separon SGX phenyl, using various mobile phases. Separon SGX phenyl phase was found the most efficient for a rapid analysis and for the final separation of the N-feruloylserotonin isomers.
Keywords: N-Feruloylserotonin; N-Isoferuloylserotonin;

Influence of storage conditions on the stability of monomeric anthocyanins studied by reversed-phase high-performance liquid chromatography by Helena Morais; Cristina Ramos; Esther Forgács; Tibor Cserháti; José Oliviera (297-301).
The effect of light, storage time and temperature on the decomposition rate of monomeric anthocyanin pigments extracted from skins of grape (Vitis vinifera var. Red globe) was determined by reversed-phase high-performance liquid chromatography (RP-HPLC). The impact of various storage conditions on the pigment stability was assessed by stepwise regression analysis. RP-HPLC separated well the five anthocyanins identified and proved the presence of other unidentified pigments at lower concentrations. Stepwise regression analysis confirmed that the overall decomposition rate of monomeric anthocyanins, peonidin-3-glucoside and malvidin-3-glucoside significantly depended on the time and temperature of storage, the effect of storage time being the most important. The presence or absence of light exerted a negligible impact on the decomposition rate.
Keywords: Anthocyanins;

Capillary electromigration methods, zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC), have been used for evaluation of the efficiency of different extraction agents applied to the extraction of pollen allergens and organic pollutants from dust samples collected during different periods (before, during and after pollen seasons) and in different locations in air-filtration devices (car-traffic tunnel in Prague and a metro station in Paris). Water and acetic acid extracts were analyzed by CZE using acetic acid as background electrolyte (BGE). Water and alkaline water–SDS–buffer extracts were analyzed by MEKC in Tris–phosphate BGE with anionic detergent sodium dodecylsulfate (SDS) micellar pseudophase. More material was extracted and more components were found in the water–buffer extracts than in the water extracts, and better resolution of the components was achieved by MEKC than by CZE. Significant differences have been found in the analyses of dust extracts of different origin. More material and more components have been found in the extracts of the dust collected in the pollen-rich period (March, April) than in the pollen-free period (December, January).
Keywords: Pollen allergens; Organic pollutants;