Analytical and Bioanalytical Chemistry (v.383, #4)
3D characterization of natural colloids by FlFFF-MALLS-TEM by Mohammed Baalousha; Frank V. D. Kammer; Mikael Motelica-Heino; Philippe Le Coustumer (549-556).
Understanding the role of colloids in the environment needs a more complete characterization of their size and shape than the usually used hydrodynamic diameter. A novel methodology to determine three dimensional description of colloids has been successfully experimented in this work by coupling Flow-Field Flow Fractionation (FlFFF) to Multi Angle Laser Light Scattering (MALLS), and Transmission Electron Microscope (TEM). This methodology was evaluated for a soil colloidal sample. Results show that, the particles surface areas determined by FlFFF-MALLS-TEM, are 3–4 times greater than those calculated using the hydrodynamic diameter determined by FlFFF. Further, results indicated that, the surface area/volume values are 3–5 times greater than those calculated based on the hydrodynamic diameter. Therefore, a correction factor must be applied when the particle surface area is calculated from the hydrodynamic diameter determined by FlFFF.
Keywords: FlFFF; MALLS; TEM; Natural colloids; 3D characterization
Determination of carbamates and organophosphorus pesticides by SDME–GC in natural water by Carmen López-Blanco; Sonia Gómez-Álvarez; María Rey-Garrote; Beatriz Cancho-Grande; Jesús Simal-Gándara (557-561).
Water contamination due to the wide variety of pesticides used in agriculture practices is a global environmental pollution problem. Analytical methods with low quantification limits are necessary. The application of a new extraction technique, solvent drop microextraction (SDME), followed by gas chromatography with a nitrogen-phosphorus detector, was assessed for determining carbamates and organophosphorus pesticides in natural water. Experimental parameters which control the performance of SDME such as selection of microextraction solvent, optimization of organic drop volume, effects of sample stirring, salt addition, and, finally, sorption time profiles were studied. Once SDME was optimized, analytical parameters such as linearity (r 2>0.99), precision (<13%), and detection limits (0.2 to 5 μg/L), plus matrix effects were evaluated (no matrix effects were found). SDME is a dynamic technique able to extract pesticides from water in 14 min; the use of organic solvents and water samples for SDME is negligible compared to other extraction techniques.
Keywords: Carbamates; Organophosphorus pesticides; Natural water analysis; Solvent drop microextraction
Development of an on-line UV decomposition system for direct coupling of liquid chromatography to atomic-fluorescence spectrometry for selenium speciation analysis by Stephane Simon; Aurelie Barats; Florence Pannier; Martine Potin-Gautier (562-569).
Speciation analysis of four selenium species (selenite, selenate, selenocystine, and selenomethionine) has been performed by on-line coupling of liquid chromatography (LC), UV decomposition, hydride generation (HG), and atomic-fluorescence spectrometry (AFS). Because only selenite (SeIV) can generate hydrides, on-line conversion of organic and inorganic selenium species is discussed. Preliminary study showed that the use of only UV light was not sufficient to reduce selenate, because no absorption is observed for this compound at the main wavelength of the low-pressure mercury lamp (253.7 nm). Thus, new conditions based on addition of a reducing reagent (I−) were developed. Mechanisms of action are proposed to explain selenium species conversions. Because of their compatibility with on-line treatment, phosphate buffers were used for chromatographic separation on an anion exchange column (Hamilton PRP-X100). Detection limits (19–60 pg Se) and repeatability of the technique were close to those obtained by conventional quadrupole ICPMS. Applications to real samples such as water and oysters are presented and emphasize the robustness of the system.
Keywords: Selenium; Speciation; Liquid chromatography; Hydride generation; Atomic-fluorescence spectrometry
A comparative study of extraction apparatus in HPLC analysis of ochratoxin A in muscle by E. Moreno Guillamont; C. M. Lino; M. L. Baeta; A. S. Pena; M. I. N. Silveira; J. Mañes Vinuesa (570-575).
Ochratoxin A (OTA) is a secondary fungal metabolite produced by several moulds, mainly by Aspergillus ochraceus and by Penicillium verrucosum, that occurs in meat products. The aim of this work was to optimize an efficient extraction procedure for the determination of OTA in muscle tissue in order to assess its occurrence in meat samples. Three different apparatus, a Waring blender, a switching apparatus, and an ultrasonic processor, were evaluated to verify the efficiency of extraction. The analytical methods proposed involve the extraction with chloroform-orthophosphoric acid, cleanup through an immunoaffinity column, high-performance liquid chromatography/fluorescence detection for separation and identification of OTA, and confirmation with liquid chromatography/FD after methylation of OTA in muscle tissue. The limit of quantification of the proposed method was 0.04 μg kg−1. Recoveries of OTA, using switching apparatus, ranged from 90.3 to 103.2% for chicken muscle spiked at 2.4 and 0.48 μg kg−1, respectively, with a within-day relative standard deviation of 17 and 15.3%. The proposed method was applied to 38 chicken, swine, and turkey muscle samples and the presence of OTA was confirmed in five samples. Finally, the estimated daily intake of OTA in this study was between 23 pg kg−1 body weight per day for swine samples and 18 pg kg−1 body weight per day for turkey samples.
Keywords: Ochratoxin A; Muscle; Waring blender; Switching apparatus; Ultrasonic processor
Application of computer imaging, stripping voltammetry and mass spectrometry to study the effect of lead (Pb-EDTA) on the growth and viability of early somatic embryos of Norway spruce (Picea abies /L./ Karst.) by Jiří Petřek; Jan Víteček; Helena Vlašínová; René Kizek; Karl J. Kramer; Vojtěch Adam; Bořivoj Klejdus; Ladislav Havel (576-586).
Image analysis (IA) was used to determine the areas and circumferences of clusters of early somatic embryos (ESEs) of the Norway spruce (Picea abies /L./Karst.). Results obtained from IA were compared with the fresh weights of the ESE clusters and their esterase activities. The areas of the ESE clusters correlated well with both the increases in fresh weight (R 2=0.99) of the ESEs and their esterase activities (R 2=0.99). In addition, we studied the viability of the ESEs, which was determined by (a) double staining with fluorescein diacetate and propidium iodide (the resulting fluorescence was quantified by IA) and (b) determining esterase activity using a spectrofluorimetric detector. The results obtained with IA and esterase assay were comparable (the deviation between the tangents of the bisectors was 6.4%). IA was also used to study the effect of Pb–EDTA chelate (50, 250 and 500 μM) on the viability of the ESEs and on the growth of clusters. The presence of Pb–EDTA markedly slowed the growth of ESEs clusters (by more than 65% with 250 μM of Pb–EDTA after 288 h of cultivation) and decreased the viability of ESEs (by more than 30% with 500 μM of Pb–EDTA after 288 h of cultivation). The lead concentration in the ESEs was determined by differential pulse anodic stripping voltammetry and increased with the external lead concentration and the time of treatment from 100 to 600 pg Pb/100 mg of fresh weight of ESEs. Glutathione is a diagnostic marker of the influence of Pb–EDTA on ESEs and its content was determined by high–performance liquid chromatography coupled with mass spectrometry. The glutathione content changed linearly with treatment time and the applied external lead concentration. The highest glutathione content was obtained at 250 μM of Pb–EDTA after 192 h of cultivation.
Keywords: Early somatic embryo of Spruce; Computer image analysis; Lead; Glutathione; Differential pulse anodic stripping voltammetry
Modelling of proton and metal exchange in the alginate biopolymer by Concetta De Stefano; Antonio Gianguzza; Daniela Piazzese; Silvio Sammartano (587-596).
Acid–base behaviour of a commercial sodium alginate extracted from brown seaweed (Macrocystis pyrifera) has been investigated at different ionic strengths (0.1≤I/mol l−1≤1.0) and in different supporting electrolytes (Et4NI, NaCl, KCl, LiCl, NaCl+MgCl2), with the aim of examining the influence of ionic medium on the proton-binding capacity and of quantifying the strength of interaction with light metal ions in the perspective of speciation studies in natural aqueous systems. Potentiometric ([H+]-glass electrode) and titration calorimetric data were expressed as a function of the dissociation degree (α) using different models (Henderson–Hasselbalch modified, Högfeldt three parameters and linear equations). The dependence on ionic strength of the protonation constants was taken into account by a modified specific interaction theory model. Differences among different media were explained in terms of the interaction between polyanion and metal cations of the supporting electrolytes. Quantitative information on the proton-binding capacity, together with the stabilities of different species formed, is reported. Protonation thermodynamic parameters, at α=0.5, are log K H=3.686±0.005, ΔG 0=−21.04±0.03 kJ mol−1, ΔH 0=4.8±0.2 kJ mol−1 and TΔS 0=35.7±0.3 kJ mol−1, at infinite dilution. Protonation enthalpies indicate that the main contribution to proton binding arises from the entropy term. A strict correlation between ΔG and TΔS was found, TΔS=−9.5–1.73 ΔG. Results are reported in light of building up a chemical complexation model of general validity to explain the binding ability of naturally occurring polycarboxylate polymers and biopolymers. Speciation profiles of alginate in the presence of sodium and magnesium ions, naturally occurring cations in natural waters, are also reported.
Keywords: Alginic acid; Proton- and metal-binding capacity; Thermodynamic parameters; Ionic strength dependence; Models for medium dependence and ion association
Acetylcholinesterase sensor based on screen-printed carbon electrode modified with prussian blue by E. Suprun; G. Evtugyn; H. Budnikov; F. Ricci; D. Moscone; G. Palleschi (597-604).
Acetylcholinesterase (ChE) sensor based on Prussian blue (PB) modified electrode was developed and tested for the detection of organophosphorus and carbamic pesticides. The signal of the sensor was generated in PB mediated oxidation of thiocholine recorded at+200 mv in DC mode. ChE from electric eel was immobilized by cross-linking with glutaraldehyde in the presence of bovine serum albumin (BSA) on the surface of screen-printed carbon electrode covered with PB and Nafion. The content of the surface layer (specific enzyme activity, Nafion and BSA amounts) was optimized to establish high and reliable response toward the substrate and ChE inhibitors. The ChE/PB sensor makes it possible to detect Aldicarb, Paraoxon and Parathion-Methyl with limits of detection 30, 10 and 5 ppb, respectively (incubation 10 min). The feasibility of practical application of the ChE/PB sensor developed for the monitoring of degradation of the pesticides in wine fermentation was shown. To diminish matrix interferences, the electrolysis of the grape juice with Al anode and evaporation of ethanol were suggested, however the procedures decrease the sensitivity of pesticide detection and stability of the sample tested.
Keywords: Cholinesterase sensor; Biosensor; Pesticide detection; Prussian Blue; Wine contamination
Automatic initialisation of buffer composition estimation for on-line analysis of unknown buffer solutions by Usama E. Zaher; Peter A. Vanrolleghem (605-618).
An automatic initialisation procedure for extracting useful information about buffer composition from a titration experiment is presented in this paper. The initialisation procedure identifies which buffering components are present in the sample from a relatively long list of buffers expected in the system monitored. The procedure determines approximate pK a values of the buffers and evaluates their maximum and minimum concentrations. This information is then used to start an optimisation procedure to fit the model of the buffer components to the titration data and to accurately determine buffer concentrations and pK a values. The procedure has been integrated as a software layer around the buffer capacity optimum model builder (BOMB) that fits a buffer-capacity model to a measured buffer-capacity curve to estimate model properties (pK a values and concentrations). The reliability and robustness of the resulting buffer capacity software (BCS) were tested using a titrimetric analyser simulator (TAS). The BCS was then validated off-line and on-line.
Keywords: Anaerobic digestion; Buffer capacity; Software sensor; Titration; Wastewater treatment
Preconcentration and separation of Cr(III) and Cr(VI) using sawdust as a sorbent by Saima Q. Memon; M. I. Bhanger; M. Y. Khuhawar (619-624).
A simple, inexpensive method based on solid-phase extraction (SPE) on sawdust from Cedrus deodera has been developed for speciation of Cr(III) and Cr(VI) in environmental water samples. Because different exchange capacities were observed for the two forms of chromium at different pH—Cr(III) was selectively retained at pH 3 to 4 whereas Cr(VI) was retained at pH 1—complete separation of the two forms of chromium is possible. Retained species were eluted with 2.5 mL 0.1 mol L−1 HCl and 0.1 mol L−1 NaOH. Detection limits of 0.05 and 0.04 μg mL−1 were achieved for Cr(III) and Cr(VI), respectively, with enrichment factors of 100 and 80. Recovery was quantitative using 250 mL sample volume for Cr(III) and 200 mL for Cr(VI). Different kinetic and thermodynamic properties that affect sorption of the chromium species on the sawdust were also determined. Metal ion concentration was measured as the Cr(VI)–diphenylcarbazide complex by UV–visible spectroscopy. The method was successfully applied for speciation of chromium in environmental and industrial water samples.
Keywords: Preconcentration; Separation; Chromium; Sawdust; Sorption thermodynamics
Multiple-injection affinity capillary electrophoresis to estimate binding constants of receptors to ligands by Dinora Chinchilla; Jose Zavaleta; Karla Martinez; Frank A. Gomez (625-631).
Multiple-injection affinity capillary electrophoresis (MIACE) is used to determine binding constants (K b) between receptors and ligands using as model systems vancomycin and teicoplanin from Streptomyces orientalis and Actinoplanes teichomyceticus, respectively, and their binding to D-Ala-D-Ala peptides and carbonic anhydrase B (CAB. EC 184.108.40.206) and the binding of the latter to arylsulfonamides. A sample plug containing a non-interacting standard is first injected followed by multiple plugs of sample containing the receptor and then a final injection of sample containing a second standard. Between each injection of sample, a small plug of buffer is injected which contains an increasing concentration of ligand to effect separation between the multiple injections of sample. Electrophoresis is then carried out in an increasing concentration of ligand in the running buffer. Continued electrophoresis results in a shift in the migration time of the receptor in the sample plugs upon binding to their respective ligand. Analysis of the change in the relative migration time ratio (RMTR) or electrophoretic mobility (μ) of the resultant receptor–ligand complex relative to the non-interacting standards, as a function of the concentration of ligand yields a value for K b. The MIACE technique is a modification in the ACE method that allows for the estimation of binding affinities between biological interactions on a timescale faster than that found for standard ACE. In addition sample volume requirements for the technique are reduced compared to traditional ACE assays. These findings demonstrate the advantage of using MIACE to estimate binding parameters between receptors and ligands.
Keywords: Multiple-injection affinity capillary; Electrophoresis; Vancomycin; Teicoplanin; Carbonic anhydrase B; Binding constants
Profiling of human stratum corneum ceramides by means of normal phase LC/APCI–MS by Hany Farwanah; Johannes Wohlrab; Reinhard H. H. Neubert; Klaus Raith (632-637).
The ceramides of the stratum corneum are critical to maintaining the epidermal barrier function of the skin. A number of skin diseases and disorders are known to be related to impairments of the ceramide pattern. Therefore, obtaining mass spectrometric profiles of the nine ceramide classes known to exist aids our understanding of the underlying molecular mechanisms, which should eventually lead to new diagnostic opportunities: for example, the mass spectrometric profiles of patients suffering from serious skin diseases such as atopic dermatitis and psoriasis can be compared to those of healthy controls. Previous work on mass spectrometric analysis of ceramides relied mostly on GC/MS after hydrolysis and derivatization. The introduction of ESI–MS and LC/ESI–MS has provided new options for directly analyzing intact ceramides. However, some of the ceramide classes are not accessible to ESI–MS. However, as shown in this work, these limitations of GC/MS and ESI-MS can be overcome using a new approach based on normal phase LC interfaced with APCI–MS. Separation and online detection of the stratum corneum ceramide classes became possible in one run. Ceramide species with C26 and/or C28 fatty acid chains were the most abundant ones in Cer [NP], Cer [NH], Cer [AP], and Cer [AH]. The main component of Cer [AS] was C16. The ω-esterified ceramide classes Cer [EOS], Cer [EOP] and Cer [EOH] contained mostly species with fatty acids >C30. This was also the case for Cer [NS], suggesting an analogy to the ω-esterified ceramides. In addition, evidence for a new ceramide class Cer [NdS] was found.
Keywords: Stratum corneum; Ceramides; Normal phase; APCI; Mass spectrometry
Quantification of urinary conjugates of bisphenol A, 2,5-dichlorophenol, and 2-hydroxy-4-methoxybenzophenone in humans by online solid phase extraction–high performance liquid chromatography–tandem mass spectrometry by Xiaoyun Ye; Zsuzsanna Kuklenyik; Larry L. Needham; Antonia M. Calafat (638-644).
Urinary concentrations of phenols or their metabolites have been used as biomarkers to assess the prevalence of exposure to these compounds in the general population. Total urinary concentrations, which include both free and conjugated (glucuronide and sulfated) forms of the compounds, are usually reported. From a toxicologic standpoint, the relative concentrations of the free species compared with their conjugated analogs can be important because conjugation may reduce the potential biologic activity of the phenols. In this study, we determined the percentage of glucuronide and sulfate conjugates of three phenolic compounds, bisphenol A (BPA), 2,5-dichlorophenol (2,5-DCP), and 2-hydroxy-4-methoxybenzophenone (benzophenone-3, BP-3) in 30 urine samples collected between 2000 and 2004 from a demographically diverse group of anonymous adult volunteers. We used a sensitive on-line solid phase extraction–isotope dilution–high performance liquid chromatography–tandem mass spectrometry method. These three phenols were detected frequently in the urine samples tested. Only small percentages of the compounds (9.5% for BPA, and 3% for 2,5-DCP and BP-3) were excreted in their free form. The percentage of the sulfate conjugate was about twice that of the free compound. The glucuronide conjugate was the major metabolite, representing 69.5% (BPA), 89% (2,5-DCP), and 84.6% (BP-3) of the total amount excreted in urine. These results are in agreement with those reported before which suggested that BPA-glucuronide was an important BPA urinary metabolite in humans. To our knowledge, this is the first study describing the distribution of urinary conjugates of BP-3 and 2,5-DCP in humans.
Keywords: Glucuronide; Sulfate; Metabolism; Exposure; Biomonitoring
Determination of ropivacaine in human plasma using highly selective molecular imprint-based solid phase extraction and fast LC–MS analysis by Zoe Cobb; Lars I. Andersson (645-650).
A method for determining ropivacaine in human plasma using highly selective molecular imprint-based solid phase extraction and LC–MS analysis was developed. The imprinted extraction material was prepared using a structural analogue of ropivacaine as the template. The efficient sample cleanup achieved allowed single MS mode operation and analytical separation under isocratic conditions with a total separation time of less than two minutes. The absence of ion suppression was confirmed for both the m/z of ropivacaine and the m/z of the internal standard. The solid phase extraction protocol was optimised for elution of ropivacaine in a small volume of aqueous-rich solvent suitable for injection into a reversed phase LC–MS system. The final method measured trace levels of ropivacaine in human plasma with a limit of quantification of 2.5 nmol/L and interassay accuracy and precision of 101.7–104.4% and 2.1–7.2%, respectively.
Keywords: Molecular imprinting; Solid phase extraction; Mass spectrometry; Bioanalysis; Affinity separation; Ion suppression
Glutaraldehyde-modified electrode for nonlabeling voltammetric detection of p16 INK4A gene by Cunwang Ge; Wujian Miao; Meiju Ji; Ning Gu (651-659).
A nonlabeling electrochemical detection method for analyzing the polymerase-chain-reaction-amplified sequence-specific p16 INK4A gene, in which the basis for the covalent immobilization of deoxyribonucleic acid (DNA) probe is described, has been developed. The self-assembly process was based on the covalent coupling of glutaraldehyde (GA) as an arm molecule onto an amino-functional surface. The p16 INK4A gene was used as the model target for the methylation detection of early cancer diagnosis. An amino-modified DNA probe was successfully assembled on the GA-coupling surface through the formation of Schiff base under potential control. The hybridization of amino-modified DNA probes with the target was investigated by means of electrochemical measurements, including cyclic voltammetry and square wave voltammetry. Furthermore, the functions of GA coupling for sequence-specific detection were compared with those obtained based on mercaptopropionic acid. Hybridization experiments indicated that the covalent coupling of GA was suitable for the immobilization of DNA probe and was sensitive to the electrochemical detection of single-base mismatches of label-free DNA targets in hybridization. Moreover, reported probe-modified surfaces exhibited excellent stability, and the hybridization reactions were found to be completely reversible and highly specific for recognition in subsequent hybridization processes. The strategy provided the potential for taking full advantage of existing modified electrode technologies and was verified in microarray technology, which could be applied as a useful and powerful tool in electrochemical biosensor and microarray technology.
Keywords: DNA hybridization; p16 INK4A gene; Coupling of glutaraldehyde; Nonlabeling voltammetric detection
Real-time monitoring of L-glutamate release from mouse brain slices under ischemia with a glass capillary-based enzyme electrode by Naoto Nakamura; Kayoko Negishi; Ayumi Hirano; Masao Sugawara (660-667).
Real-time monitoring of L-glutamate release from various neuronal regions of mouse hippocampal slices under ischemia (a glucose-free hypoxia condition) is described. A glass capillary microelectrode with a tip size of ∼10 μm containing a very small volume (∼2 μL) of a solution of glutamate oxidase (GluOx) and ascorbate oxidase was used. First, the amperometric response behavior of the electrode at 0 V versus Ag/AgCl was characterized with a standard glutamate solution in terms of continuous measurements, effect of oxygen, viscosity of solution and concentration dependence. The electrode was applied to the real-time monitoring of L-glutamate released from different neuronal regions of acute hippocampal slices submerged in a hypoxia solution. The time-resolved amounts of L-glutamate released at various neuronal regions (CA1, CA3 and DG) of mouse hippocampal slices were quantified and compared with the reported L-glutamate fluxes using difference-image analysis during ischemia.
Keywords: Glass capillary electrode; Enzyme sensor; L-glutamate; Mouse hippocampal slices; Ischemia
An on-membrane quantitative analysis system for glycyrrhizin in licorice roots and traditional Chinese medicines by Osamu Morinaga; Ayako Fujino; Hiroyuki Tanaka; Yukihiro Shoyama (668-672).
An on-membrane quantitative analysis system has been developed for determining glycyrrhizin (GC) in licorice roots and traditional Chinese medicines. A GC standard and the extracts of licorice roots and traditional Chinese medicines were applied to a polyethersulfone (PES) membrane and were developed by acetonitrile/water/formic acid (45:55:2, by volume), then treated with a NaIO4 solution followed by bovine serum albumin (BSA), resulting in a GC-BSA conjugate on a PES membrane. Anti-GC monoclonal antibody was bound and then a second antibody labeled with peroxidase directed against the first antibody. Finally a substrate reacted with the enzyme and gave staining. The stained membrane was scanned and coloring spots were analyzed quantitatively using graphic analysis by NIH Image software, indicating at least 0.5 μg of GC was clearly detectable. GC can be analyzed quantitatively between 1.0 and 8.0 μg.
Keywords: Glycyrrhizin; Eastern blotting; NIH Image software; Quantitative analysis
An optical glucose biosensor based on glucose oxidase immobilized on a swim bladder membrane by Zaide Zhou; Lina Qiao; Peng Zhang; Dan Xiao; Martin M. F. Choi (673-679).
An optical glucose biosensor using a swim bladder membrane as an enzyme immobilization platform and an oxygen-sensitive membrane as an optical oxygen transducer has been developed. During the enzymatic reaction, glucose is oxidized by glucose oxidase with a concomitant consumption of dissolved oxygen resulting in an increase in the fluorescence intensity of the optical oxygen transducer. The fluorescence intensity is directly related to the glucose concentration. The effects of pH, temperature, buffer concentration, and selectivity have been studied in detail. The immobilized enzyme retained 80% of its initial activity after being kept for more than 10 months at 4°C. The glucose biosensor has been successfully applied to the determination of glucose content in human blood serum and urine samples.
Keywords: Glucose biosensor; Swim bladder membrane; Enzyme immobilization; Oxygen-sensitive membrane
Preparation of tea glycoprotein and its application as a calibration standard for the quantification and molecular weight determination of tea glycoprotein in different tea samples by high-performance gel-permeation chromatography by Shaoping Nie; Mingyong Xie; Yuanxing Wang (680-686).
Tea glycoprotein (TGC) was purified by Sephadex G-100 gel filtration, and its purity and molecular weight (MW) were determined by high performance gel permeation chromatography. The gel permeation chromatography (GPC) elution behavior of TGC on an Ultrahydrogel 500 (7.8×300 mm) column was studied using a mobile phase with various concentrations of NaCl. A dextran T system was found to be unsuitable as MW calibration standards in GPC because the MW of TGC was changed with the change of ionic concentration in the mobile phase. Thus, the purified TGC obtained in this study was standardized and used instead as the calibration standards for the determination and comparison of TGCs in tea samples collected from different geographic locations and species varieties. The GPC was run at 35°C on an Ultrahydrogel 500 column (7.8×300 mm) with a refractive index detector. Distilled water was used as the mobile phase at a flow rate of 0.6 ml·min−1. The calibration curve was linear over the TGC concentration range of 0.604–6.04 mg·ml−1, with a correlation coefficient of r=0.9997. The TGC mass recovery ranged from 80.4 to 93.2%, with a relative standard deviation of 5.42%. The assay method developed in this paper was found to be simple, reproducible, and reliable and, thus, ideally suitable for the quality control of TGC-derived products and raw materials.
Keywords: High performance gel permeation chromatography (HPGPC); Tea glycoprotein (TGC); Preparation; Determination
Microemulsion liquid chromatographic method for characterisation of fosinopril sodium and fosinoprilat separation with chemometrical support by Biljana Janči´; Mirjana Medenica; Darko Ivanović; Anđelija Malenović; Slavko Marković (687-694).
The properties of the eluent are the essential factors governing the efficiency in the high-performance liquid chromatography (HPLC) method. A novel approach in retention modelling in the liquid chromatographic separation of fosinopril sodium and its degradation product, fosinoprilat, applying a microemulsion as the mobile phase, was used. The modifications of the mobile phase included the changes to the type of the lipophilic phase, the type and concentration of co-surfactant and surfactant, as well as the pH of the mobile phase. In this study, a full factorial 23 design, as the optimal method for screening of the experiment, was applied for selecting factors which had an influence on separation. Optimisation was done by a central composite design. An appropriate resolution with reasonable retention times was obtained with a microemulsion containing 0.9% w/w of cyclohexane, 2.2% w/w of sodium dodecyl sulphate (SDS), 8.0% w/w of n-butanol and 88.9% of aqueous 25 mM disodium phosphate, the pH of which was adjusted to 2.8 with 85% orthophosphoric acid. Separations were performed on an X-Terra 50-mm×4.6-mm, 3.5-μm particle size column at 30°C. UV detection was performed at 220 nm and with a flow rate of 0.3 mL min−1. The established method was validated and applied for analysis of appropriate tablets. The proposed chromatographic procedure for the separation of fosinopril sodium and its degradation product is less expensive compared with the conventional reversed-phase HPLC method, as well as being simple and rapid. The optimised and validated method can be used for separation, identification and simultaneous determination of fosinopril sodium and fosinoprilat in bulk drug and in pharmaceutical dose forms.
Keywords: Microemulsion; Fosinopril sodium; Fosinoprilat; Chemometrics
A sensitive determination of dopamine in the presence of ascorbic acid using a nafion-coated clinoptilolite-modified carbon paste electrode by Senol Alpat; Sibel Kilinc Alpat; Azmi Telefoncu (695-700).
A selective dopamine determination using a nafion-coated clinoptilolite-modified carbon paste electrode in the presence of ascorbic acid was studied. Both cyclic voltammetry (CV) and differential pulse anodic stripping voltammetry (DPASV) were used for measurements of dopamine. To improve the selectivity of the clinoptilolite-modified carbon paste electrode in presence of a high concentration of ascorbic acid, the electrode surface was coated with nafion membrane. Experimental parameters affecting the determination of dopamine, including the clinoptilolite ratio, nafion membrane thickness, preconcentration time, preconcentration solution pH, stripping solution pH and interferences are discussed. The developed sensor has a wide linear range, a low detection limit, and good stability and reproducibility. The sensor offers a good alternative to existing analytical methods for dopamine, permits a relatively short analysis time, and is simple, selective and inexpensive.
Keywords: Dopamine; Nafion; Clinoptilolite; Carbon paste electrode; DPASV
Rapid differentiation among bacteria that cause gastroenteritis by use of low-resolution Raman spectroscopy and PLS discriminant analysis by Cesar Mello; Diórginis Ribeiro; Fábio Novaes; Ronei J. Poppi (701-706).
Use of classical microbiological methods to differentiate bacteria that cause gastroenteritis is cumbersome but usually very efficient. The high cost of reagents and the time required for such identifications, approximately four days, could have serious consequences, however, mainly when the patients are children, the elderly, or adults with low resistance. The search for new methods enabling rapid and reagentless differentiation of these microorganisms is, therefore, extremely relevant. In this work the main microorganisms responsible for gastroenteritis, Escherichia coli, Salmonella choleraesuis, and Shigella flexneri, were studied. For each microorganism sixty different dispersions were prepared in physiological solution. The Raman spectra of these dispersions were recorded using a diode laser operating in the near infrared region. Partial least-squares (PLS) discriminant analysis was used to differentiate among the bacteria by use of their respective Raman spectra. This approach enabled correct classification of 100% of the bacteria evaluated and unknown samples from the clinical environment, in less time (∼10 h), by use of a low-cost, portable Raman spectrometer, which can be easily used in intensive care units and clinical environments.
Keywords: PLS discriminant analysis; Raman spectroscopy; Bacteria; Gastroenteritis
In situ analysis of mediaeval wall paintings: a challenge for mobile Raman spectroscopy by Peter Vandenabeele; Karel Lambert; Sofie Matthys; Walter Schudel; Anna Bergmans; Luc Moens (707-712).
Raman spectroscopy is an analytical technique, which is gaining attention as a molecular technique for the investigation of objects of art. Especially the non-destructive properties of the method make this application suitable for the in situ analysis of artefacts. However, although using mobile, fibre optics Raman instrumentation for this type of research seems to be straightforward, some practical obstacles may hamper the investigation. In this paper, pitfalls and solutions are described when applying a dedicated spectrometer to the analysis of mediaeval wall paintings. It is shown how some practical problems may be overcome, and the results of the analysis are presented. Although the mediaeval wall paintings from the chapel of the castle of Ponthoz are well-preserved, still some interesting degradation phenomena could be observed: the identification of a black degradation product, likely to be meta-cinnabar, a degradation product of the red pigment vermilion (HgS); the formation of gypsum (CaSO4·2H2O) as a weathering product of calcium carbonate (CaCO3); the observation of copper(II)hydroxychlorides.
Keywords: Raman spectroscopy; Pigment analysis; Mediaeval wall paintings; Mobile instrumentation; In situ analysis
Ancient biodeterioration: an FT–Raman spectroscopic study of mammoth and elephant ivory by Howell G. M. Edwards; Susana E. Jorge Villar; Nik F. Nik Hassan; Nlin Arya; Sonia O'Connor; Donna M. Charlton (713-720).
Raman spectra of mammoth ivory specimens have been recorded using near-infrared excitation, and comparisons made with modern Asian and African elephant ivories. Whereas the most ancient mammoth ivory (60–65 ky) showed no evidence for an organic collagen component, more recent samples of mammoth ivory indicated that some preservation had occurred, although with biodeterioration of the protein structure exhibited by the amide I and III bands in the 1200–1700 cm−1 region of the Raman spectrum. The consequent difficulties encountered when applying chemometrics methods to ancient ivory analysis (which are successful for modern specimens) are noted. In the most ancient mammoth ivory specimens, which are extensively fragmented, evidence of mineralization is seen, with the production of gypsum, calcite and limonite; Raman microscopic analysis of crystalline material inside the fissures of the mammoth ivory shows the presence of gypsum as well as cyanobacterial colonisation. The application of Raman spectroscopy to the nondestructive analysis of archaeological materials in order to gain information of relevance to their preservation or restoration is highlighted.
Keywords: Elephant; Ivory; Mammoth; Biodeterioration; Raman spectroscopy
Redox-iodometry: a new potentiometric method by Waldemar Gottardi; Jörg Pfleiderer (721-722).