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Analytical and Bioanalytical Chemistry (v.376, #7)

Becoming an integral part of your field (or networking for more success) by John Fetzer (pp. 943-944).

Proteomics by Helmut E. Meyer (pp. 945-945).

An improved method for tracking and reducing the void volume in nano HPLC–MS with micro trapping columns by Goran Mitulović; Marek Smoluch; Jean-Pierre Chervet; Ines Steinmacher; Andreas Kungl; Karl Mechtler (pp. 946-951).

Tandem mass spectrometry coupled to HPLC is the state of the art technique in proteomic research. Here we describe a highly sensitive nano liquid chromatography system (nano HPLC) for analysis of protein digests. Using preconcentration in a column-switching set-up, we were able to inject large sample volumes (≤250 µL) without significant loss of sensitivity. The major problem with this type of preconcentration is usually the occurrence of void volumes. In order to diagnose void volumes a simple and easy test was developed by which the UV trace and the pressure profile in the separation column were monitored. Part by part replacement of connection tubing restored a void volume-free system. A major pre-requisite for handling samples in the femtomol range was found to be the use of protein/peptide-saturated columns tryptic digests of cytochrome C were injected directly onto the reversed-phase nano separation column (75 µm inner diameter) and the separation results were compared with chromatograms obtained from separations using column switching. By using column switching we were able to inject large sample volumes in a short time period without losing resolution.

Keywords: Nano-HPLC; Sensitivity; Trapping column; Proteomics; Void volumes

A novel MALDI LIFT-TOF/TOF mass spectrometer for proteomics by Detlev Suckau; Anja Resemann; Martin Schuerenberg; Peter Hufnagel; Jochen Franzen; Armin Holle (pp. 952-965).

A new matrix-assisted laser-desorption/ionization time-of-flight/time-of-flight mass spectrometer with the novel "LIFT" technique (MALDI LIFT-TOF/TOF MS) is described. This instrument provides high sensitivity (attomole range) for peptide mass fingerprints (PMF). It is also possible to analyze fragment ions generated by any one of three different modes of dissociation: laser-induced dissociation (LID) and high-energy collision-induced dissociation (CID) as real MS/MS techniques and in-source decay in the reflector mode of the mass analyzer (reISD) as a pseudo-MS/MS technique. Fully automated operation including spot picking from 2D gels, in-gel digestion, sample preparation on MALDI plates with hydrophilic/hydrophobic spot profiles and spectrum acquisition/processing lead to an identification rate of 66% after the PMF was obtained. The workflow control software subsequently triggered automated acquisition of multiple MS/MS spectra. This information, combined with the PMF increased the identification rate to 77%, thus providing data that allowed protein modifications and sequence errors in the protein sequence database to be detected. The quality of the MS/MS data allowed for automated de novo sequencing and protein identification based on homology searching.

Keywords: TOF/TOF; MS/MS; Tryptophan oxidation; de novo Sequencing; Automation; MALDI

Identification of phosphorylation and acetylation sites in αA-crystallin of the eye lens (mus musculus) after two-dimensional gel electrophoresis by Heike Schaefer; Katrin Marcus; Albert Sickmann; Marion Herrmann; Joachim Klose; Helmut E. Meyer (pp. 966-972).

Posttranslational modifications are of great interest because of their relevance in biological systems as proteins are commonly activated or deactivated by phosphorylation, glycation and acetylation [1, 2]. During eye lens aging the number of the αA-crystallin isoproteins increases. This could be observed by the use of 2D-PAGE (two-dimensional gel electrophoresis). The number of αA-crystallin spots in the gel increased during eye lens aging. For further analysis the spots of 2D-PAGE were cut out and the identification of the proteins was done using nanoLC-ESI-MS/MS (liquid chromatography electrospray ionization tandem mass spectrometry). The created MS/MS-data were analyzed using the Sequest algorithm. Searches with different parameters were done to preferably get the complete sequence coverage and to identify posttranslational modifications of the αA-crystallin. The acetylated N-terminus of this protein could be detected. Furthermore, phosphorylation of serine 122 and 148 was identified in two different spots.

Keywords: αA-crystallin; Posttranslational modifications; NanoLC-MS/MS; Sequest

Differential analysis of phosphorylated proteins in resting and thrombin-stimulated human platelets by Katrin Marcus; Jan Moebius; Helmut E. Meyer (pp. 973-993).

Blood platelets are important components of haemostasis. After their activation they cause healing of wounds by forming plugs and initiate repair processes. One important event in regulating this activation is the phosphorylation/dephosphorylation of multiple proteins on various tyrosine, serine and threonine residues. To understand the exact molecular mechanisms in platelet activation it is essential to identify proteins involved in the signalling pathways and to localise and characterise their phosphorylation sites. After treatment with ³²P and separation by 2D-PAGE using different pI ranges, phosphorylated platelet proteins were detected by autoradiography. Phosphotyrosine-containing proteins were assigned by immunoblotting with an anti-phosphotyrosine antibody. Another approach for the identification of phosphorylated proteins was immunoprecipitation of tyrosine-phosphorylated proteins using an anti-phosphotyrosine antibody. Protein spots/bands of interest were excised from the gel, digested with trypsin and analysed by MALDI-TOF-MS and nano-LC-ESI-MS/MS, respectively. Several phosphorylated proteins could be identified and the localisation of some in vivo phosphorylation sites was possible.

Keywords: Mass spectrometry; Protein phosphorylation; Proteomics; Two-dimensional polyacrylamide gel electrophoresis; Platelets; Protein phosphorylation; 2D-PAGE; Immunoprecipitation; Nano-LC-ESI-MS/MS

Acetylation of the HIV-1 Tat protein: an in vitro study by Wilma Dormeyer; Alexander Dorr; Melanie Ott; Martina Schnölzer (pp. 994-1005).

In the last few years, the understanding of lysine acetylation as a regulatory post-translational modification of proteins in cell signalling cascades has increased. It is now known that not only histones but also non-histone factors can serve as substrates of different acetyltransferase enzymes. Acetylated lysine residues in non-histone factors are often identified using radioactive labelling experiments and immunochemical analysis of synthetic peptides. In this study of the human immunodeficiency virus 1 (HIV-1) Tat protein, we demonstrate the benefits of matrix-assisted laser desorption/ionisation mass spectrometry, proteolytic digestion and Edman sequencing for the mapping of acetylation sites. We confirmed that the HIV-1 Tat protein is acetylated in vitro by the acetyltransferase p300 at a specific lysine residue at position 50 in its RNA binding region. Furthermore, we showed that the Tat cysteine-rich region is acetylated at multiple cysteine residues in the absence of enzyme. Since this non-enzymatic cysteine acetylation occurs independently from the surrounding peptide sequence, we consider the presence of cysteine residues in acetylated peptides an important factor for the interpretation of in vitro acetylation assays in general.

Keywords: Acetylation; MALDI-TOF mass spectrometry; Edman sequencing; HIV-1 Tat; Histone acetyltransferase p300

Epitope-targeted proteome analysis: towards a large-scale automated protein–protein-interaction mapping utilizing synthetic peptide arrays by Krzysztof Bialek; Andrzej Swistowski; Ronald Frank (pp. 1006-1013).

We describe the development of a process for the genome-wide mapping of interactions between protein domains and peptide ligands entirely based on high-throughput biochip technologies. A phage library displaying protein domains from a randomly fragmented and cloned cDNA library will be "panned" on an array of synthetic peptide ligands. After multiplexed affinity enrichment, peptide-specific phage populations will be automatically eluted, propagated, labelled and identified by hybridisation to a DNA microarray. Peptide arrays are synthesized in situ by SPOT synthesis on a planar substrate. By utilizing a commercially available library of human brain cDNA plus a set of distinct model domains cloned into T7-phage, we could show that a single panning round on an array of known peptide ligands for these model domains synthesized on a cellulose membrane can yield an enrichment of better than a factor of 1,000. This is sufficient to detect peptide-specific enrichment of Cy3(post-panning) against Cy5(pre-panning)-labelled phage DNA inserts on a cDNA microarray. Thus, the proof-of-principle of our approach could be successfully demonstrated and first interaction data are being collected.

Keywords: Peptide arrays; DNA microarrays; Protein domains; Epitope; Phage display; Automation; Proteomics; Functional genomics

Interpretation of mass spectrometry data for high-throughput proteomics by Daniel C. Chamrad; Gerhard Koerting; Johan Gobom; Herbert Thiele; Joachim Klose; Helmut E. Meyer; Martin Blueggel (pp. 1014-1022).

Recent developments in proteomics have revealed a bottleneck in bioinformatics: high-quality interpretation of acquired MS data. The ability to generate thousands of MS spectra per day, and the demand for this, makes manual methods inadequate for analysis and underlines the need to transfer the advanced capabilities of an expert human user into sophisticated MS interpretation algorithms. The identification rate in current high-throughput proteomics studies is not only a matter of instrumentation. We present software for high-throughput PMF identification, which enables robust and confident protein identification at higher rates. This has been achieved by automated calibration, peak rejection, and use of a meta search approach which employs various PMF search engines. The automatic calibration consists of a dynamic, spectral information-dependent algorithm, which combines various known calibration methods and iteratively establishes an optimised calibration. The peak rejection algorithm filters signals that are unrelated to the analysed protein by use of automatically generated and dataset-dependent exclusion lists. In the "meta search" several known PMF search engines are triggered and their results are merged by use of a meta score. The significance of the meta score was assessed by simulation of PMF identification with 10,000 artificial spectra resembling a data situation close to the measured dataset. By means of this simulation the meta score is linked to expectation values as a statistical measure. The presented software is part of the proteome database ProteinScape which links the information derived from MS data to other relevant proteomics data. We demonstrate the performance of the presented system with MS data from 1891 PMF spectra. As a result of automatic calibration and peak rejection the identification rate increased from 6% to 44%.

Keywords: Bioinformatics; Database; High throughput; Mass spectrometry; Protein identification; Proteomics

Characterization of the sorption of uranium(VI) on different complexing resins by Maria Pesavento; Raffaela Biesuz; Giancarla Alberti; Michela Sturini (pp. 1023-1029).

The sorption of uranium(VI) on two cationic resins containing different complexing groups, the iminodiacetic resin Chelex 100 and the weak carboxylic resin Amberlite CG-50, was investigated. The Gibbs–Donnan model was used to describe and to predict the sorption through the determination of the intrinsic complexation constants. These quantities, even though non-thermodynamic, characterize the sorption as being independent of experimental conditions.The sorption mechanism of the metal on the complexing resins was also studied by adding a competitive soluble ligand that shifts the sorption curves to higher pH values. The ligand competes with the resin for the complexation with the metal ion. Uranium is also strongly sorbed on Chelex 100 at very acid pH, through formation of two complexes in the resin phase: ML with logβ _110i=−1.16, in more acidic solution, and ML₂with logβ _120i=−5.72. Only the presence of the competitive ligand in solution makes the determination of the second complex possible. Also on Amberlite CG-50 the sorption is strong and involves the formation of the complex ML₂, in more acidic solution, with logβ _120i=−3.16. In the presence of the ligand EDTA, the complex ML₂(OH)₂was characterized with logβ _12–2i=−5.15. In all the experiments the hydrolysis reaction in the aqueous phase was quantitatively considered.

Keywords: Sorption of uranium(VI) on chelating resins; Gibbs–Donnan model; Competition of ligand for sorption; Chelex 100; Amberlite CG-50

Speciation of phytate ion in aqueous solution. Alkali metal complex formation in different ionic media by Concetta De Stefano; Demetrio Milea; Alberto Pettignano; Silvio Sammartano (pp. 1030-1040).

The acid–base properties of phytic acid [myo-inositol 1,2,3,4,5,6-hexakis(dihydrogen phosphate)] (H₁₂Phy; Phy¹²⁻=phytate anion) were studied in aqueous solution by potentiometric measurements ([H⁺]-glass electrode) in lithium and potassium chloride aqueous media at different ionic strengths (0<I mol L⁻¹≤3) and at t=25 °C. The protonation of phytate proved strongly dependent on both ionic medium and ionic strength. The protonation constants obtained in alkali metal chlorides are considerably lower than the corresponding ones obtained in a previous paper in tetraethylammonium iodide (Et₄NI; e.g., at I=0.5 mol L⁻¹, logK ₃ H=11.7, 8.0, 9.1, and 9.1 in Et₄NI, LiCl, NaCl and KCl, respectively; the protonation constants in Et₄NI and NaCl were already reported), owing to the strong interactions occurring between the phytate and alkaline cations present in the background salt. We explained this in terms of complex formation between phytate and alkali metal ions. Experimental evidence allows us to consider the formation of 13 mixed proton–metal–ligand complexes, M_jH_iPhy^{(12−i−j)−}, (M⁺=Li⁺, Na⁺, K⁺), with j≤7 and i≤6, in the range 2.5≤pH≤10 (some measurements, at low ionic strength, were extended to pH=11). In particular, all the species formed are negatively charged: i+j−12=−5, −6. Very high formation percentages of M⁺–phytate species are observed in all the pH ranges investigated. The stability of alkali metal complexes follows the trend Li⁺≥Na⁺K⁺. Some measurements were also performed at constant ionic strength (I=0.5 mol L⁻¹), using different mixtures of Et₄NI and alkali metal chlorides, in order to confirm the formation of hypothesized and calculated metal–proton–ligand complex species and to obtain conditional protonation constants in these multi-component ionic media.

Keywords: Phytate; Protonation constants; Alkali metal complexes; Dependence on ionic medium; Dependence on ionic strength; Speciation; Predictive relationships

Determination of protonation constants of some fluorinated polyamines by means of ¹³C NMR data processed by the new computer program HypNMR2000. Protonation sequence in polyamines by Chiara Frassineti; Lucia Alderighi; Peter Gans; Antonio Sabatini; Alberto Vacca; Stefano Ghelli (pp. 1041-1052).

The pK _a values of 6-fluoro-4,8-diazadodecane-1,12-diamine (6-fluorospermine) (1), 6,6-difluoro-4,8-diazadodecane-1,12-diamine (6,6-difluorospermine) (2), 6-fluoro-4-azaoctane-1,8-diamine (6-fluorospermidine) (3) and 6,6-difluoro-4-azaoctane-1,8-diamine (6,6-difluorospermidine) (4) in D₂O solution have been determined at 40 °C from ¹³C NMR chemical shifts data using the new computer program HypNMR2000. The enthalpies of protonation of compounds 1–4 and the parent amines spermine (5) and spermidine (6) have been determined from microcalorimetric titration data. The values of ΔH° were used to derive basicity constants relative to 25 °C. The NMR data have been analysed by two different methods to obtain information on the protonation sequence in the polyamines 1–5. The protonation sequence for spermine is related to its biological activity.

Keywords: ¹³C NMR; Fluorinated compounds; Polyamines; Protonation constants; Protonation enthalpies; Protonation sequence

Splitless on-line coupling of capillary high-performance liquid chromatography, capillary electrochromatography and pressurized capillary electrochromatography with nuclear magnetic resonance spectroscopy by Erdmann Rapp; Andreas Jakob; Alexandre Bezerra Schefer; Ernst Bayer; Klaus Albert (pp. 1053-1061).

A mixture of unsaturated fatty acid methyl esters was separated with a new splitless capillary set-up. With the employed apparatus configuration different capillary separation techniques such as capillary high-performance liquid chromatography (cHPLC), capillary electrochromatography (CEC) and pressurized capillary electrochromatography (pCEC) could be applied. The detection and identification of the sample compounds were accomplished by hyphenating these capillary separation techniques with nuclear magnetic resonance (NMR) spectroscopy using a novel configuration of the detection capillary set-up. Using modified electrokinetically driven separation techniques, the electric field was applied solely across the separation column. With this improved interface for capillary liquid chromatography-NMR on-line coupling, the stereochemical assignment of the cis and trans configuration of unsaturated fatty acids could be easily accomplished. Finally, the results of cHPLC-NMR, CEC-NMR and pCEC-NMR coupling experiments were compared.

Keywords: Hyphenated techniques; NMR; Capillary high-performance liquid chromatography; Capillary electrochromatography; Pressurized capillary electrochromatography

A microfluidic biosensor based on nucleic acid sequence recognition by Sylvia Kwakye; Antje Baeumner (pp. 1062-1068).

The development of a generic semi-disposable microfluidic biosensor for the highly sensitive detection of pathogens via their nucleic acid sequences is presented in this paper. Disposable microchannels with defined areas for capture and detection of target pathogen RNA sequence were created in polydimethylsiloxane (PDMS) and mounted onto a reusable polymethylmethacrylate (PMMA) stand. Two different DNA probes complementary to unique sequences on the target pathogen RNA serve as the biorecognition elements. For signal generation and amplification, one probe is coupled to dye encapsulated liposomes while the second probe is coupled to superparamagnetic beads for target immobilization. The probes hybridize to target RNA and the liposome–target-bead complex is subsequently captured on a magnet. The amount of liposomes captured correlates directly to the concentration of target sequence and is quantified using a fluorescence microscope. Dengue fever virus serotype 3 sequences and probes were used as a model analyte system to test the sensor. Probe binding and target capture conditions were optimized for sensitivity resulting in a detection limit of as little as 10 amol μL⁻¹ (10 pmol L⁻¹) . Future biosensors will be designed to incorporate a mixer and substitute the fluorescence detection with an electrochemical detection technique to provide a truly portable microbiosensor system.

Keywords: Biosensor; Microfluidics; Rapid detection; Probe hybridization; Microfabrication; Liposomes

High-performance liquid chromatography-fluorescent assay of catechol-O-methyltransferase activity in rat brain by Mayumi Masuda; Makoto Tsunoda; Kazuhiro Imai (pp. 1069-1073).

A method to measure catechol-O-methyltransferase (COMT) activity using high performance liquid chromatography—fluorescence detection with norepinephrine (NE) as a natural substrate was optimized for both soluble (S-) and membrane-bound (MB-) COMT activities in rat brain areas, cerebral cortex, cerebellum, hippocampus, brain stem, hypophysis, and hypothalamus. The highest S-COMT activity in Sprague-Dawley rat brain was found in hippocampus. MB-COMT activities in all brain areas were about 3–8 times lower than S-COMT activities. However, considering Vmax/Km values, specificity constants for NE to S- and MB-COMT contributes mainly to the metabolism of NE in cerebral cortex and cerebellum.

Keywords: Norepinephrine; Catechol-O-methyltransferase; Brain; Hypertension

Fluorescence-labelled antigen-binding fragments (Fab) from monoclonal antibody 5F12 detect human erythropoietin in immunoaffinity capillary electrophoresis by Claus Bornemann; Tilman Burggraef; Günter Heimbüchel; Franz-Georg Hanisch; Sandra Winkels (pp. 1074-1080).

A faster and more convenient method is required for the detection of recombinant erythropoietin (Epo) in human body fluids. In the present study we wanted to elucidate the principal suitability of immunoaffinity capillary electrophoresis (CE) in this respect. CE offers itself as a high-speed, high-throughput technique provided a suitable affinity reagent is available. We chose monoclonal antibody 5F12 from Amgen which binds to a conformation-independent epitope in the N-terminal region of the human Epo protein. For CE with laser-induced fluorescence detection it was necessary to produce fluorescently labelled antibody with one single antigen binding site. Monomeric antigen-binding fragments (Fab) were obtained by site-selective cleavage of the pure antibody and labelled with the fluorescent dye, Alexa Fluor 488. The mixture of labelled isomers was partially resolved by ion exchange HPLC and isoelectric focusing. The fluorescent Fab could be used to detect erythropoietin by immunoaffinity capillary isoelectric focusing and zone capillary electrophoresis via its antigen complex.

Keywords: Human erythropoietin; Immunoaffinity capillary electrophoresis; Fluorescent antibody; Isoelectric focusing ; Doping control

Effect of extraction vessel geometry and flow homogeneity on recoveries of polycyclic aromatic hydrocarbons in pressurised hot water extraction by Terhi Andersson; Tina Pihtsalmi; Kari Hartonen; Tuulia Hyötyläinen; Marja-Liisa Riekkola (pp. 1081-1088).

Extraction vessels of different length, internal diameter and volume were tested to evaluate the effect of vessel geometry on the recovery of polycyclic aromatic hydrocarbons (PAHs) from certified sediment by pressurised hot water extraction (PHWE). Pressurised hot water extractions were performed at 300 °C with both liquid water (pressure 250 kg cm⁻²) and steam (pressure 50 kg cm⁻²). In addition, the effects on the recoveries of sediment packing and water flow direction were examined in two vessels. The geometry of the vessel, the packing of the sediment and the flow direction of the water had only minor effect on the recoveries.

Keywords: Polycyclic aromatic hydrocarbons; Pressurised hot water extraction; Extraction vessel geometry; Water flow direction; Sediment packing

Analysis of alcohol ethoxylates and alkylamine ethoxylates in agricultural soils using pressurised liquid extraction and liquid chromatography–mass spectrometry by Kristine A. Krogh; Betty Bügel Mogensen; Bent Halling-Sørensen; Amparo Cortés; Karl V. Vejrup; Damià Barceló (pp. 1089-1097).

Nonionic surfactants e.g. alcohol ethoxylates (AEOs) and alkylamine ethoxylates (ANEOs) are commonly utilised as adjuvants in pesticide formulations to enhance their effectiveness. In this study, analytical methods for AEO and ANEO determination in soil samples using pressurised liquid extraction (PLE) were developed and used in connection with LC–MS. The recovery of the method, which was highly dependent on the soil properties, varied in the range 47–106% for AEO and 27–109% for ANEO. Detection limits (LOD) were 7–13 µg kg⁻¹ for AEO and 24–43 µg kg⁻¹ for ANEO. The developed method has been applied to determine AEOs and ANEOs in surface soil samples from fields sprayed with glyphosate herbicides. Tallowalkylamine ethoxylates (an ANEO) were detected in the soil before and after pesticide application, with increasing concentrations after treatment. The highest concentration in the soil samples was observed for the ANEO homologues with the longest ethoxy chains; in the clay soil the concentration decreased with the length of the ethoxy chain. ANEOs added to pesticide formulations as a technical mixture will, as demonstrated in this study, behave as individual homologues, which is reflected in their behaviour in the environment.

Keywords: Alcohol ethoxylates; Alkylamine ethoxylates; Pressurised liquid extraction; Adjuvants; Environmental analysis; Surfactants; Pesticides; Soil

Screen-printed multienzyme arrays for use in amperometric batch and flow systems by S. Sapelnikova; E. Dock; R. Solná; P. Skládal; T. Ruzgas; J. Emnéus (pp. 1098-1103).

Screen-printing technology for electrode fabrication enables construction of amperometric devices suitable for combination of several enzyme electrodes. To develop a biosensor array for characterisation of wastewaters, tyrosinase and horseradish peroxidase (HRP) or cholinesterase-modified electrodes were combined on the same array. The behaviour of the tyrosinase-modified electrode in the presence of hydrogen peroxide (required co-substrate for the HRP-modified electrode) and acetylthiocholine chloride (required co-substrate for cholinesterase) was studied. Performance of bi-enzyme biosensor arrays in the batch mode and in the flow-injection system are discussed.

Keywords: Flow-injection analysis; Amperometric batch and flow system; Screen-printed multienzyme array; Enzyme electrode; Tyrosinase and horseradish peroxidase-modified electrode; Tyrosinase and cholinesterase-modified electrode; Wastewater characterisation

Simple optical fibre biosensor based on immobilised enzyme for monitoring of trace heavy metal ions by Bambang Kuswandi (pp. 1104-1110).

A simple optical fibre biosensor based on immobilised enzyme for monitoring of trace heavy metal ions has been developed. The biosensor recognition system was designed based on the inhibition of urease activity, where the urease is immobilised on ultrabind membrane. The studies of inhibition by the heavy metal ions Hg(II), Ag(I), Cu(II), Ni(II), Zn(II), Co(II) and Pb(II) were performed using a fibre-optic biosensor configuration, where the pH change resulting from the bio-catalytic hydrolysis of urea was monitored at the wavelength 615 nm spectroscopically, using commercial pH indicator strip before and after the exposure to the heavy metal ions. The immobilised urease was regenerated by l-cysteine. The linear response range between 1×10^-9–1×10^-5 M and the limit of detection 1×10^-9M (0.2 μg/L) for Hg(II) ions was achieved by employing the flow method. The optimisation of experimental parameters, including flow method, is also discussed.

Keywords: Immobilised urease; pH indicator; Heavy metal ions; Optical fibre biosensor

Sensitive and simple determination of the vasodilator agent dipyridamole in pharmaceutical preparations by phosphorimetry by A. Salinas-Castillo; A. Segura Carretero; A. Fernández-Gutiérrez (pp. 1111-1114).

The applicability of heavy atom-induced room-temperature phosphorescence to pharmaceutical samples is demonstrated in this work. Thus a new, simple, rapid, and selective phosphorimetric method for dipyridamole determination is proposed. The phosphorescence signals are a consequence of intermolecular protection when analytes are exclusively in the presence of heavy atom salts and sodium sulfite as an oxygen scavenger to minimize RTP quenching. The determination was performed in 0.1 mol L⁻¹ thallium(I) nitrate and 8 mmol L⁻¹ sodium sulfite at a measurement temperature of 20 °C. The phosphorescence intensity was measured at 635 nm, with excitation at 305 nm. Phosphorescence was easily developed; a linear concentration range was obtained between 0 and 100 ng mL⁻¹ with a detection limit of 940 ng L⁻¹, an analytical sensitivity of 2.5 ng mL⁻¹, and a standard deviation of 2.7% at 60 ng mL⁻¹ concentration. The method has been successfully applied to the analysis of dipyridamole in a unique Spanish commercial formulation containing 100 ng mL⁻¹ per capsule. The recovery was 101.6% with 6.5% standard deviation of analytical measurement. The method using the standard addition methodology has been validated.

Keywords: Pharmaceutical analysis; Dipyridamole; Heavy atom-induced; Room temperature phosphorescence (RTP)

Modification of catalytic adsorptive stripping voltammetric method of hexavalent chromium determination in the presence of DTPA and nitrate by Małgorzata Grabarczyk; Mieczysław Korolczuk (pp. 1115-1118).

The voltammetric procedure for determination of traces of Cr(VI) [Anal. Chim. Acta (1992) 262:103] was modified by changing the temperature of the measurements. It was found that at the temperature of 40 °C the time of decrease of the Cr(III) signal was shortened from 30 to 5 min. As a result the total analysis time was drastically shortened. The modified procedure does not show any disadvantage as compared to the original method. The results of Cr(VI) determination by the modified procedure are less affected by Cr(III) as compared to the original method. The detection limit of the method was 2.5 × 10^-11 mol L^-1 (1.2 ng L^-1). The validation of the modified procedure was performed by comparison of the results of analyses of tap and river water samples with those obtained using original procedure.

Keywords: Chromium (VI); Determination; Voltammetry; Modification

Determination of Cr (in small quantities) by adsorptive stripping voltammetry: a comparative study of square wave versus differential pulse by A. Sánchez Misiego; R. M. García-Moncó Carra; P. Ambel Carracedo; M. E. Majado Torre (pp. 1119-1125).

The usefulness of an analytical method must be measured according to its practical application possibilities. A comparative study has been carried out here between the SW (working in an open atmosphere) and DP (working with de-aerated solutions) variants of catalytic-stripping adsorptive voltammetry applied to the determination of chromium traces in triethylenetetraminehexaacetic acid (TTHA) medium. In order to optimise the analytical signal, accumulation potential, nitrate ion concentration, pH, and TTHA concentration parameters were evaluated. Four linearity ranges were established within the interval 0.5–2000 nmol L⁻¹ chromium concentration in the cell, each with the recommended accumulation time. Quality parameters such as repeatability, linear regression, validity limits, precision, and sensitivity were evaluated. The SW variant is significantly advantageous when the chromium concentration in cell is less than 10 nmol L⁻¹ and even more if analysis time, cost, and being able to work in an open atmosphere are considered. The results are comparable to those obtained using GTAAS. Employing a CRM (tomato leaves), the accuracy is 1–4%. The proposed procedure, using tree leaves as samples, has been successfully tested for the possible monitoring of chromium contamination of the atmosphere.

Keywords: Chromium; Adsorptive stripping voltammetry; Square wave; Triethylenetetraminehexaacetic acid (TTHA) complex

Rapid and sensitive spectrophotometric determination of trace amounts of iron(III) using leuco Xylene cyanol FF by T. N. Kiran Kumar; H. D. Revanasiddappa (pp. 1126-1130).

A new, simple, sensitive, and reliable method is presented for the rapid spectrophotometric determination of trace amounts of iron(III) using leuco Xylene cyanol FF. The method is based on the oxidation of leuco Xylene cyanol FF (LXCFF) to its blue form of xylene cyanol FF by iron(III) in sulfuric acid medium (pH 2.0–3.0), the absorbance of the formed dye is measured in an acetate buffer medium (pH2.8–4.4) at 615 nm. The method obeys Beer's law over a concentration range of 0.15–0.9 μg mL^-1 iron, having a molar absorptivity of 5.6×10⁴ L mol^-1 cm^-1 and a Sandell's sensitivity of 0.0001 μg cm^-2. The optimum reaction conditions and other analytical parameters have been evaluated. The developed method has been successfully applied to the determination of iron in water, soil, industrial effluent, plant material, pharmaceutical preparations, synthetic mixtures, and aluminum alloys.

Keywords: Spectrophotometry; Iron(III) determination; Leuco Xylene cyanol FF

Kinetic spectrophotometric methods for the determination of dothiepin hydrochloride in bulk and in drug formulation by Elham A. Taha (pp. 1131-1136).

Two simple and sensitive kinetic methods for the determination of dothiepin hydrochloride are described. The first method is based on kinetic investigation of the oxidation reaction of the drug with alkaline potassium permanganate at room temperature for a fixed time of 25 min. The absorbance of the colored manganate ions is measured at 610 nm. The second method is based on the reaction of dothiepin hydrochloride with 4-chloro-7-nitrobenzofurazan (NBD-Cl) in the presence of 0.1 mol L⁻¹ sodium bicarbonate. Spectrophotometric measurement was achieved by recording the absorbance at 470 nm for a fixed time of 60 min. All variables affecting the development of the color were investigated and the conditions were optimized. Plots of absorbance against concentration in both procedures were rectilinear over the ranges 4–24 and 50–250 μg mL⁻¹, with mean recoveries 99.33±0.42 and 99.88±0.53, respectively. The proposed methods were successfully applied for the determination of dothiepin hydrochloride in bulk powder and in capsule dosage form. The results obtained were found to agree statistically with those given by the non-aqueous B.P. method. Furthermore the methods were validated according to USP guidelines and also assessed by applying the standard addition technique. The determination of dothiepin hydrochloride by the fixed concentration method is feasible with the calibration equations obtained, but the fixed time method proves to be more applicable.

Keywords: Kinetic determination; Dothiepin hydrochloride; Potassium permanganate; 4-Chloro-7-nitrobenzofurazan (NBD-Cl).

Evaluation and applications of a gaseous mercuric chloride source by Xinbin Feng; Julia Y. Lu; Yingjie Hao; Cathy Banic; William H. Schroeder (pp. 1137-1140).

In this study, a diffusion-type device for generating gaseous mercuric chloride (HgCl₂) was systematically evaluated and applied to validate the annular denuder method for sampling gaseous HgCl₂ species in a synthetic gas stream. The results show that it takes at least 48 h for the system to reach a steady-state condition after the diffusion cell reaches the temperature set-point and the carrier gas is activated. The primary Hg species from the source was proven to be HgCl₂. In the temperature range from −5.00 to 11.80 °C, the Hg emission rates from the source vary from 1.8 to 14.2 pg min⁻¹. It is shown that, under the experimental conditions examined, KCl-coated annular quartz denuders designed for ambient reactive gaseous mercury (RGM) collection could quantitatively collect HgCl₂. It is also demonstrated that the impactors used to remove coarse airborne particulate matter could lead to a loss of up to one third of the HgCl₂ in the gas stream.

Keywords: Gaseous mercuric chloride; Diffusion source; Annular denuder; Mercury speciation; RGM; Atmospheric mercury measurements

A validated spectrofluorometric method for the determination of celecoxib in capsules by Patricia Damiani; Mariela Bearzotti; Miguel A. Cabezón (pp. 1141-1146).

A rapid, selective, sensitive and simple fluorescence method was developed for the direct determination of celecoxib in capsules. The capsules were emptied, pulverized and dissolved in either ethanol or acetonitrile, sonicated and filtered. Direct fluorescence emission was measured at 355±5 nm (exciting at 272 nm). The method was fully validated and the recoveries were excellent, even in presence of excipients.

Keywords: Spectrofluorimetry; Spectrofluorometric method; Celecoxib

2004 Bunsen-Kirchhoff Award for Analytical Chemistry (pp. 1147-1147).

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Analytical and Bioanalytical Chemistry (v.376, #7)

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Analytical and Bioanalytical Chemistry (v.376, #7)