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ABSTRACT: Hyphenation of micro-liquid and ultra-performance liquid chromatography with a hybrid (QqTOF) tandem mass spectrometry was studied from the viewpoint of their applicability for monitoring of anthocyanin dyes in various cultivars and different vintages of red wine. After appropriate optimization both techniques proved to be suitable for this task. UHPLC system provided lower LOD and LOQ values as well as higher productivity and precision of retention parameters and peak areas with respect to micro-LC method. On the other hand, micro-LC method offers significant savings of mobile phase, less contamination of ion source of mass spectrometer and makes the nebulization process in electrospray much easier. In combination with principal component analysis (PCA), the method allows to elucidate long-term relations in a complete set of certified red wine cultivars grown in South Moravia (certified in Czech Republic until year 2005). Among the studied varieties Rubinet exhibit a specific anthocyanin profile. Especially interesting is a high content of 3-coumaroylglucoside-5-glucosides of malvidin and peonidin. Those dyes can be used as markers of artificial color enhancement. Principal component analysis applied to data processed with regard to enzymatic activity markedly facilitate classification among varieties. This approach allows resolution of teinturiers, variety Blaufränkish and partial classification of other varieties.
Journal of chromatography. A 07/2011; 1218(42):7581-91. · 4.19 Impact Factor
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ABSTRACT: A novel long drop time mercury electrode has been constructed from common fused-silica capillary (50 μm I.D., 360 μm E.D.). Proposed device provides reproducible mercury drops with typical lifetime 40-120 s. The electrode was used for a set of electrocapillary measurements aimed at determination of critical micelle concentration of anionic surfactants by a convection controlled drop-time technique. A critical micelle concentration of sodium dodecyl sulfate 5.6 ± 0.4 mmol L(-1) and 4.3 ± 0.4 mmol L(-1) were obtained in 1 mmol L(-1) and 5 mmol L(-1) phosphate buffer (pH 7.0), respectively. The values were comparable to those obtained from conductometric measurements under the same conditions (7.0 ± 0.1 mmol L(-1) and 5.2 ± 0.1 mmol L(-1), respectively) and the difference was explained in accordance with theory of hemi-micelle formation.
Talanta 03/2011; 84(1):187-91. · 3.79 Impact Factor
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ABSTRACT: An analytical protocol for the isolation and quantification of indole-3-acetic acid (IAA) and its amino acid conjugates was developed. IAA is an important phytohormone and formation of its conjugates plays a crucial role in regulating IAA levels in plants. The developed protocol combines a highly specific immunoaffinity extraction with a sensitive and selective LC-MS/MS analysis. By using internal standards for each of the studied compounds, IAA and seven amino acid conjugates were analyzed in quantities of fresh plant material as low as 30 mg. In seeds of Helleborus niger, physiological levels of these compounds were found to range from 7.5 nmol g(-1) fresh weight (IAA) to 0.44 pmol g(-1) fresh weight (conjugate with Ala). To our knowledge, the identification of IAA conjugates with Gly, Phe and Val from higher plants is reported here for the first time.
Talanta 12/2009; 80(2):651-5. · 3.79 Impact Factor
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ABSTRACT: N,N-dialkylaminoethane-2-thiols belong to the group of precursors and degradation products of chemical warfare agents (CWAs). These compounds were analyzed by means of electrospray ionization-multiple stage mass spectrometry (ion trap) and proposed fragments were confirmed by accurate mass measurement using a QqTOF system. The fragmentation pathways of studied compounds and the products of oxidation (formation of -S-S- linkage) were described. Some minor interesting processes, such as rearrangement of SH group, were observed and proved. A new microLC/MS method, based on ion-pairing chromatography, was developed. Trifluoroacetic acid was employed as an ion-pairing agent to increase the low retention of compounds of interest in the reverse-phase system. The technique was compared with the UPLC/MS method, allowing fast analysis of all the studied thiols as well as an explorative control of originated disulfides.
Biological Mass Spectrometry 10/2009; 44(11):1604-12. · 3.41 Impact Factor
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ABSTRACT: The development of a rapid, simple and accurate analytical method aimed at the detection and quantification of bovine milk in either ovine or caprine milk samples by means of CE-MS analyses of whey proteins with high-ionic strength and presence of acidic running buffer is described. The high-ionic strength buffer was used in order to minimize the problems with the adsorption of the proteins onto the fused-silica capillary wall. The acidic running electrolyte, pH 1.9, was used to support the production of positive ions in electrospray. Highly linear dependences of the ratio of the sum of non-bovine beta-lactoglobulins (ovine or caprine) to the total beta-lactoglobulins in milk mixture (bovine plus ovine or bovine plus caprine) vs. the volume percentage of added bovine milk in ovine (or caprine) milk were obtained. This technique allowed the fast and reliable evaluation of milk adulteration. The amount of bovine milk added into the "non-bovine" ones can be well within the concentration range of 5-95%.
Electrophoresis 06/2008; 29(10):2088-93. · 3.30 Impact Factor
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ABSTRACT: Selected precursors and degradation products of chemical warfare agents namely N,N-dialkylaminoethane-2-ols, N,N-dialkylaminoethyl-2-chlorides and some of related N-quaternary salts were studied by means of electrospray ionization-multiple tandem mass spectrometry (ESI-MS(n)). Proposed structures were confirmed with accurate mass measurement. General fragmentation patterns of these compounds are discussed in detail and suggested processes are confirmed using deuterated standards. The typical processes are elimination of alkene, hydrogen chloride, or water, respectively. Besides, elimination of ethene from propyl chain under specific conditions was observed and unambiguously confirmed using exact mass measurement and labelled standard. The potential of mass spectrometry to distinguish the positional isomers occurring among the studied compounds is reviewed in detail using two different MS instruments (i.e. ion trap and hybrid quadrupole-time of flight (Q-TOF) analyzer). A new microcolumn liquid chromatography (microLC)/MS(n) method was designed for the cases where the resolution based solely on differences in fragmentation is not sufficient. Low retention of the derivatives on reversed phase (RP) was overcome by using addition of less typical ion pairing agent (1 mM/l, 3,5-dinitrobenzoic acid) to the mobile phase (mixture water : acetonitrile).
Journal of Mass Spectrometry 01/2008; 42(12):1550-61. · 3.27 Impact Factor
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ABSTRACT: Chemical warfare agents and their degradation products represent a broad group of compounds with different chemical properties (polarity, volatility, thermostability, etc.). These chemicals often have to be detected and determined in complex matrices and therefore highly efficient separation techniques hyphenated to selective and sensitive detectors play an indispensable role. This review offers an overview of selected papers devoted to the title subject. It cannot be considered as a comprehensive literature compilation but should allow the reader to obtain an insight into the application of separation techniques in the important area of human protection and control of chemical weapons.
Journal of Separation Science 08/2006; 29(11):1531-8. · 2.73 Impact Factor
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ABSTRACT: Chemical warfare agents and their degradation products represent a broad group of compounds with different chemical properties (polarity, volatility, thermostability, etc.). These chemicals often have to be detected and determined in complex matrices and therefore highly efficient separation techniques hyphenated to selective and sensitive detectors play an indispensable role. This review offers an overview of selected papers devoted to the title subject. It cannot be considered as a comprehensive literature compilation but should allow the reader to obtain an insight into the application of separation techniques in the important area of human protection and control of chemical weapons.
Journal of Separation Science 06/2006; 29(11):1531 - 1538. · 2.73 Impact Factor
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ABSTRACT: Hyphenation of capillary electrophoresis with electrospray ionization mass spectrometry was utilized for the monitoring of anthocyanins in wine and wine musts. CE/MS was performed in two electrolytes: 1) an acidic one (chloroacetate-ammonium, pH 2) and 2) a basic one with high selectivity towards derivatives containing vicinal hydroxy groups (borate-ammonium, pH 9). The setup of MS was optimized and the fragmentation of common anthocyanins was studied in detail. Attention was also focused on the fragmentation of anthocyanidin skeleton. The anthocyanidins substituted with hydroxy groups fragment via a cascade of neutral losses of water and carbon monoxide. Fragmentation of anthocyanidins containing a methoxy group on their B-ring starts with the cleavage of methane and/or methyl radical. The optimized method was utilized for the monitoring of changes in anthocyanin profile in red wines as well as the process of release of anthocyanins to wine must.
Journal of Separation Science 09/2005; 28(12):1291-9. · 2.73 Impact Factor
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ABSTRACT: The octanol-water partition coefficients of seven anthocyanins (in neutral form, pH = 7.0) were measured by means of MEKC. A new iterative method for the estimation of migration time of electroosmotic flow (EOF) and micelles in MEKC is presented. This calculation is based on Gauss-Newton linearization of the dependence between migration indices and migration times of a set of suitable standards and the application of an advanced statistical evaluation procedure. The values of partition coefficients obtained with the aid of an iterative process are compared with values obtained on use of EOF and micelle markers.
Journal of Separation Science 09/2005; 28(12):1285-90. · 2.73 Impact Factor
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ABSTRACT: This paper deals with the use of solid-phase extraction (SPE) for the extraction and purification of 3-indolylacetic acid (IAA), the main and most important representative of the plant growth regulators auxins. The procedure is based on the use of C18-SPE columns in two steps. In the first one, raw extract in methanol:water (4:1) is applied on the column in order to remove neutral ballast. In the second step the eluate is diluted with water to a final methanol concentration of 20% (to decrease the elution strength of the solvent) and acidified with formic acid to a final concentration of 1% (to convert the IAA from the anionic to the neutral form). Neutral IAA is then retained on the second SPE column, eluted by (acidified) methanol, and subjected to final analysis. Scintillation counting of tritium labeled IAA standard was used for the investigation and optimization of the SPE procedure. Gas chromatography with mass spectrometric detection was suggested as a convenient method for routine determination of IAA after its methylation with diazomethane. Overall recovery of the procedure was estimated as 89–94% and a physiological level of IAA equal to 0.48 nmol/g (84 ng/g) fresh weight was found using an optimized SPE-GC-MS method.
Journal of Separation Science 07/2005; 28(12):1370 - 1374. · 2.73 Impact Factor
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ABSTRACT: Capillary zone electrophoresis with UV detection was used for determination of dissociation constants of alkaloids sanguinarine and chelerythrine. Despite the limited solubility of the uncharged forms of the alkaloids resulting in insufficient analytical signal at higher pH the reliable dissociation constants were obtained when acidified samples containing low amount of the alkaloid were injected into the capillary. The precipitation of the alkaloid in the capillary induced by injecting sample of low pH into the background electrolyte of higher pH does not affect the mobility of the alkaloid if its concentration injected exceeds the solubility only to a small extent. Dissociation constants (pK(R+)) of sanguinarine and chelerythrine calculated to 8.3 +/- 0.1 and 9.2 +/- 0.1, respectively, are relevant to Good buffers of ionic strength of 30 mM.
Journal of Chromatography 07/2004; 1040(1):141-5. · 4.53 Impact Factor
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ABSTRACT: Derivatization of orotic acid (OA) into various forms (trimethylsilylderivate, alkyl ester and per-methylated derivate) and their evaluation by GC/MS is described. The tested approach includes ion-exchange SPE clean-up, evaporation and chemical reaction with different types of derivatization agents (N,O-bis-(trimethylsilyl)trifluoroacetamide with trimethylchlorosilane, butanol with acetylchloride and ethereal solution of diazomethane). Derivate originated in the reaction with diazomethane was used for determination of urinary orotic acid by GC/MS. Detection limit of 0.28 micromol l(-1) was reached using the ion 82 m/z in single ion monitoring (SIM) mode. Linearity of the method was tested within the range of 3.4-2503.4 micromol l(-1) covering physiological and pathological levels of orotic acid in urine sample. Recoveries were within the range 93.7-110.6%. Application of the method on the patient with defect of ornithine transcarbamylase (OTC) was demonstrated as well.
Journal of Chromatography B 02/2004; 799(2):303-9. · 2.89 Impact Factor
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ABSTRACT: Using mercaptoethanol and (L)-cysteine as representatives of mercapto compounds and capillary zone electrophoresis as experimental technique, it was evidenced that sanguinarine and chelerythrine do not react with the mercapto group of organic compounds at pH 7.4. Their interaction is fast and reversible complexation based on non-bonding intermolecular interaction in which enter uncharged forms of sanguinarine or chelerythrine. A negatively charged group, either bound to the mercapto ligand or supplied from solution, participates in the complexation. Simple 1:1 interaction scheme reported in literature holds therefore only for mercapto compounds bearing anionic group. Stoichiometric binding constants corrected for the abundance of the uncharged alkaloid form at pH 7.4 are of the order of magnitude of 10(4) l/mol and depend on both cations and anions of the background electrolyte. Interaction of sanguinarine and chelerythrine with human or bovine serum albumins does not qualitatively differ from their interaction with simple mercapto compounds. Stoichiometric binding constants for the binding of sanguinarine with human and bovine serum albumins in sodium phosphate buffer pH 7.4, corrected for the abundance of the interacting uncharged form, are 332,000+/-38,400 and 141,000+/-14,400 l/mol, respectively. The former agrees well with the value K=385,000 (or log K=5.59) from static photometric experiments. Constants for the complexation of uncharged chelerythrine with human and bovine serum albumins are 2,970,000+/-360,000 and 1,380,000+/-22,600 l/mol, respectively.
Journal of Chromatography B 12/2003; 797(1-2):357-66. · 2.89 Impact Factor
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ABSTRACT: A novel Headspace Solid Phase Microextraction (HS-SPME) protocol is proposed for the analysis of floral scent. Volatile compounds emitted from the flower are collected on a Carboxen/PDMS fiber for 1 hour, transferred to the GC, and analyzed by GC/MS. The method completely eliminates the use of organic solvents, does not require special instrumentation, and may readily be performed in the field without access to mains electricity and other energy supplies. The method is robust, sensitive, and reduces the sampling stress on the investigated plant. Since enzymatic reactions in living flowers may cause changes in the composition of emitted fragrance, dried rosemary (Rosmarinus officinalis L.) was used as a stable standard for the method development and optimization. In addition, grape wine was also suggested as homogeneous, bio-compatible, and relatively stable standard of pronounced and typical scent for the same purpose. The optimized method was used for the comparative investigation of the fragrances emitted by two different species – Lathyrus vernus (L.) and Orchis pallens (L.). Several monoterpenes (C10 compounds) were found as the main fragrance components of lathyrus, while sesquiterpenes (C15 compounds) were typical for the orchid.
Journal of Separation Science 06/2003; 26(8):715 - 721. · 2.73 Impact Factor
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ABSTRACT: HPLC and CE methods were employed to determine the quaternary benzo[c]phenanthridine alkaloid sanguinarine in biological matrices (rat hepatocytes, human gingival fibroblasts, feed, porcine faeces, body fluids and tissues). HPLC was carried out on a C18 column using gradient elution and ion pairing techniques with 1-heptanesulfonic acid as ion pairing agent under acidic conditions. The detection limit for fluorimetric detection at λex = 327 nm and λem = 577 nm was 3 nM sanguinarine. CE analyses were performed in 50 mM phosphate-Na buffer pH 2.5, with 150 mM SDS used for pre-concentration by the sweeping effect. This experimental configuration allows injecting the total capillary length with sanguinarine sample. The detection limit for UV detection at 285 nm was 12 nM. Both methods are suitable for analysing submicromolar quantities of sanguinarine in biological materials. The HPLC method is more sensitive than CE because it uses fluorescence detection.
Journal of Separation Science 06/2003; 26(8):679 - 685. · 2.73 Impact Factor
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ABSTRACT: Capillary electrophoresis with indirect UV detection was applied to the analysis of a mixture of 3-quinuclidinol and its four quaternary derivatives (N-methyl, N-ethyl, N-propyl, and N-isopropyl derivative). 10 mM imidazole acetate buffer, pH = 4.0 offers suitable detection sensitivity (LOD = 1 μmol L–1) and permits separation of the mixture except for the pair 3-quinuclidinol–N-methyl derivative. The separation of all analytes was achieved on addition of 15 mmol L–1 β-cyclodextrin or 25% (w/w) polyethylene glycol 2000 to the background electrolyte. The optimized method was employed for the analysis of pond water spiked with these analytes. Actual ionic mobilities of the studied compounds were measured using mobility standards (potassium, sodium, tetramethyl- and tetrabutylammonium). The migration index was derived as another identification parameter based on migration data and the precision of the obtained values is discussed in brief.
Journal of Separation Science 05/2003; 26(8):709 - 714. · 2.73 Impact Factor
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ABSTRACT: A method based on the coupling of capillary electrophoresis with mass spectrometry (CE/MS) was developed for the monitoring of 3-quinuclidinol and its four N-alkyl derivatives (methyl, ethyl, propyl and isopropyl derivatives). A fragmentation study (collision-induced dissociation of ions in an ion trap) and optimization of the ion optics set-up for CE/MS experiments using direct infusion of a methanolic solution of the standards into the mass spectrometer were carried out in advance. Molecular ions of all quaternary compounds and the quasi-molecular ion [M + H]+ of free 3-quinuclidinol prevail in the mass spectra. In the MS/MS of propyl and isopropyl derivatives, the elimination of the alkyl chain dominates, leading to the ion at m/z 128. The fragmentation of the other compounds is more complex. Previous CE separation of the mixture of isobaric propyl and isopropyl derivatives is necessary for their unambiguous identification. A 10 mM ammonium acetate buffer (pH 4.0) is the optimum running electrolyte, allowing the CE separation of methyl, ethyl, propyl and isopropyl derivatives. A 0.5% (v/v) solution of acetic acid in methanol provides sufficient detection sensitivity when used as the sheath liquid. Limits of detection of 0.1 ppm for 3-quinuclidinol and 0.05 ppm for quaternary derivatives were achieved under the optimum conditions. The optimized method was applied to the determination of 3-quinuclidinol and related quaternary derivatives spiked into a sample of pond water. The experimental set-up for CE/MS/MS was investigated, which strongly increases the identification capability of the technique.
Journal of Mass Spectrometry 01/2003; 37(12):1213-8. · 3.27 Impact Factor
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ABSTRACT: A method based on the coupling of capillary electrophoresis with mass spectrometry (CE/MS) was developed for the monitoring of 3-quinuclidinol and its four N-alkyl derivatives (methyl, ethyl, propyl and isopropyl derivatives). A fragmentation study (collision-induced dissociation of ions in an ion trap) and optimization of the ion optics set-up for CE/MS experiments using direct infusion of a methanolic solution of the standards into the mass spectrometer were carried out in advance. Molecular ions of all quaternary compounds and the quasi-molecular ion [M + H]+ of free 3-quinuclidinol prevail in the mass spectra. In the MS/MS of propyl and isopropyl derivatives, the elimination of the alkyl chain dominates, leading to the ion at m/z 128. The fragmentation of the other compounds is more complex. Previous CE separation of the mixture of isobaric propyl and isopropyl derivatives is necessary for their unambiguous identification. A 10 mM ammonium acetate buffer (pH 4.0) is the optimum running electrolyte, allowing the CE separation of methyl, ethyl, propyl and isopropyl derivatives. A 0.5% (v/v) solution of acetic acid in methanol provides sufficient detection sensitivity when used as the sheath liquid. Limits of detection of 0.1 ppm for 3-quinuclidinol and 0.05 ppm for quaternary derivatives were achieved under the optimum conditions. The optimized method was applied to the determination of 3-quinuclidinol and related quaternary derivatives spiked into a sample of pond water. The experimental set-up for CE/MS/MS was investigated, which strongly increases the identification capability of the technique. Copyright © 2002 John Wiley & Sons, Ltd.
Biological Mass Spectrometry 11/2002; 37(12):1213 - 1218. · 3.41 Impact Factor
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ABSTRACT: We developed a capillary electrophoresis method as a diagnostic tool for purine and pyrimidine metabolic disorders. Optimal conditions allowed the separation of the major diagnostic metabolites in urine samples within an analysis time of 10 min and with a separation efficiency of about 350,000 theoretical plates/m. The diagnostically important metabolites (adenine, adenosine, 2-deoxyadenosine, 2-deoxyguanosine, 2,8-dihydroxyadenine, guanosine, hypoxanthine, orotidine, orotic acid, and creatinine) were detectable at concentrations of 1.0-5.7 micromol/L. The method gives a linear calibration curve for tested purine and pyrimidine derivatives within the range of 5-500 micromol/L (r > 0.996) The coefficients of variation for the within- and between-day imprecisions were less than 3.2 and 5.8%, respectively. Characteristic abnormalities were detected in the electropherograms of urine samples from patients with purine and pyrimidine enzyme deficiencies. We provide electrophoretic and spectral characteristics of intermediates in purine and pyrimidine metabolism and possible artifacts from medication and their UV-absorbing compounds. Our method allows the detection of the majority of inborn errors of purine and pyrimidine metabolism.
Electrophoresis 03/2002; 23(4):565-71. · 3.30 Impact Factor
