Analytica Chimica Acta (v.556, #2)

Contents (iii-iv).

A brief review: HPLC methods to directly detect drug glucuronides in biological matrices (Part I) by Romina Kaushik; Barry Levine; William R. LaCourse (255-266).
Detection of a drug and its glucuronide metabolite(s) is of great importance in interpretive forensic and clinical toxicology. Until recently, glucuronides were determined by cleavage of the glucuronide with an enzyme (e.g., β-glucuronidase) to yield the parent compound, which was subsequently detected, or via derivatization to a more volatile or detectable analogue. Direct detection of the glucuronide conjugates overcomes the critical limitations of approaches that involve enzymatic cleavage procedures and/or derivatization. This review will focus on direct methods to determine glucuronides of various potentially abused drugs in human biological matrices. More specifically, this review will be restricted to methods that involve liquid chromatography (LC) coupled with various detectors. Papers are presented in chronological order for each compound class to emphasize the evolution of methodology and continued importance of the application.
Keywords: Forensic; Drugs; Glucuronides; Metabolite; Biomarker; Review;

A direct, versatile method for the determination of ethyl glucuronide (EtG), a biomarker of ethanol consumption, in urine has been developed using reversed-phase liquid chromatography with pulsed electrochemical detection (PED). EtG and methyl glucuronide (MetG), which serves as an internal standard, are readily separated using a mobile phase consisting of 1% acetic acid/acetonitrile (98/2, v/v). Post-column addition of NaOH allows for the detection of all glucuronides using PED at a gold working electrode. Upon optimization, EtG was found to have a limit of detection of 0.03 μg/mL (7 pmol; 50 μL injection volume) and repeatability at the limit of quantitation of 1.7%R.S.D. (relative standard deviation). Solid-phase extraction (SPE) using an aminopropyl phase was used to remove interferents in urine samples prior to their analysis. Compound recovery following SPE was approximately 50 ± 2%. The forensic utility of this method was further validated by the analysis of 29 post-mortem urine specimens, whose results agreed strongly with certified determinations.
Keywords: Forensic science; Biomarker; Ethanol; Glucuronide; Pulsed electrochemical detection;

Optimisation of a liquid chromatography–tandem mass spectrometric method for the determination of acrylamide in foods by Y. Govaert; A. Arisseto; J. Van Loco; E. Scheers; S. Fraselle; E. Weverbergh; J.M. Degroodt; L. Goeyens (275-280).
A robust and fitted routine method resulting from an analytical optimisation has been applied for the determination of acrylamide in several foods including mainly potato and cereal products. For the sample treatment, different materials were evaluated for filtration and purification of the extract. To increase the performances in terms of sensitivity, a preconcentration to small volume was introduced before liquid chromatography coupled to tandem mass spectrometry analysis on a μ-Bondapak C18 column using d3-acrylamide as internal standard. For identification, relative retention time and two diagnostic ions were monitored. A limit of detection of 10 μg kg−1, a limit of quantitation of 20 μg kg−1, mean recoveries ranging from 100 to 115%, coefficients of variation from 1.36 to 8.06% for repeatability and from 3.3 to 18.2% for reproducibility within the laboratory and a measurement uncertainty of 42% were obtained during an in-house validation procedure. Results of tests, validation data and Z-score obtained during participation to proficiency studies are presented.
Keywords: Acrylamide; Food safety; Liquid chromatography coupled to tandem mass spectrometry; Potato products; Cereal products;

Determination of the antidepressant mirtazapine and its two main metabolites in human plasma by liquid chromatography with fluorescence detection by Roberto Mandrioli; Laura Mercolini; Nadia Ghedini; Claudio Bartoletti; Salvatore Fanali; Maria Augusta Raggi (281-288).
A high-performance liquid chromatographic method for the determination in human plasma of the recent noradrenergic and specific serotonergic antidepressant (NaSSA) mirtazapine and its two main metabolites, N-desmethylmirtazapine and 8-hydroxymirtazapine, has been developed. Fluorescence detection was used, exciting at λ  = 290 nm and monitoring emission at λ  = 370 nm. Separation was obtained by using a reversed-phase column (C8, 250 mm × 4.6 mm I.D., 5 μm) and a mobile phase composed of 75% aqueous phosphate buffer containing triethylamine at pH 3.0 and 25% acetonitrile. Melatonin was used as the internal standard. A careful pre-treatment of plasma samples was developed, using solid-phase extraction with phenyl cartridges (100 mg, 1 mL). The calibration curves were linear over a working range of 5–150 ng mL−1 for mirtazapine and of 2.5–75.0 ng mL−1 for N-desmethylmirtazapine and 8-hydroxymirtazapine. The limit of quantitation (LOQ) was 2.5 ng mL−1 and the limit of detection (LOD) was 1.25 ng mL−1 for all analytes. The method was applied with success to plasma samples from depressed patients undergoing treatment with mirtazapine. Precision data, as well as accuracy results, were satisfactory and no interference from other drugs was found. Hence the method is suitable for therapeutic drug monitoring of mirtazapine and its metabolites in depressed patients’ plasma.
Keywords: Mirtazapine; Metabolites; High-performance liquid chromatography; Fluorescence detection; Human plasma; Solid-phase extraction;

In the work, for the first time, two solvent-free sample preparation techniques of microwave distillation (MD) and solid-phase microextraction (SPME) were combined, and developed for determination of essential oil compounds in traditional Chinese medicine (TCM). Using the proposed method, isolation, extraction and concentration of TCM essential oil compounds can be carried out in a single step. To demonstrate its feasibility, MD-SPME was compared with a conventional technique, steam distillation (SD), for gas chromatography-mass spectrometry (GC-MS) analysis of essential oil compounds in a TCM, Artemisia Selengensis Turcz. Forty-nine compounds in the TCM were separated and identified by the present method, while only 26 compounds were detected by SD method. Relative standard deviation (R.S.D.) values less than 9% show that the present method has good precision. The SD method required long time (6 h) to isolate of the essential oil, and large amount of organic solvent for further extraction, while MD–SPME needed little time (only 3 min) to prepare sample, and no organic solvent. These results show that MD–SPME–GC-MS is a simple, rapid and solvent-free method for determination of TCM essential oil.
Keywords: Extraction methods; Traditional chinese medicine; Essential oil; Microwave distillation; Solid-phase microextraction; Gas chromatography-mass spectrometry;

Determination of low carboxyhemoglobin blood levels by gas chromatography by Jan Czogała; Władysław Wardas; Maciej Łukasz Goniewicz (295-300).
Carbon monoxide (CO) is one of the anthropogenic environmental pollutant. An essential source of this compound, especially in indoor air, is tobacco smoke, which itself contains high amount of the compound. CO from tobacco smoke states the risk not only for active smokers but also for passive ones. Carboxyhemoglobin HbCO blood level is a biomarker of exposure to CO. The aim of the study was to develop method of HbCO analysis by gas chromatography based on quantitative determination of released CO from analyzed HbCO. Before developing of the method the authors stated that the method should require only small amount of the blood for analysis and be enough sensitive to determined HbCO level in persons exposed to low concentrations of CO including passive smokers. A specific reactor for quantitative release of CO from blood sample was developed. The process of CO release was caused by internal mixing of the blood sample with releasing agent K3[Fe(CN)6] and stimulated by internal mixing of the blood sample with external rotating magnetic field. The method for preparing calibration solutions was developed. The method was calibrated for two ranges of HbCO concentrations (0–2.5% and 0–15%). The HbCO levels were determined in 0.15 ml blood samples taken from the finger tips of passive smokers exposed to environmental tobacco smoke with different intensities. The determined HbCO levels varied from 0.31% to 2.19% of total hemoglobin.
Keywords: Carboxyhemoglobin; Carbon monoxide; Gas chromatography; Blood;

Microchip device for rapid screening and fingerprint identification of phenolic pollutants by Joseph Wang; Weena Siangproh; Antonio Javier Blasco; Orawon Chailapakul; Alberto Escarpa (301-305).
A new microchip protocol has been developed for rapid measurements of the ‘total’ content of phenolic compounds, as well as for a detailed fingerprint identification of the ‘individual’ ones. The protocol involves the use of a microchip flow-injection analysis for fast screening and early detection of phenols and switching to the separation (fingerprint) mode once such compounds are detected. This is readily accomplished by exchanging the run buffers in the separation channel. While operating with an acidic run buffer (pH 5) offers high speed flow-injection measurements of the ‘total’ phenolic content, on chip switching to a basic buffer (pH 8) leads to ionization of the phenolic compounds and to their effective separation and detection. Under optimum conditions, assay rates of about 120 and 18 samples/h can be realized for the ‘total’ and ‘individual’ measurements, respectively. The effect of the buffer pH, switching (washing) time, applied voltages and other relevant variables, is described. The concept is illustrated in connection to amperometric detection and is attractive for a wide range of environmental-monitoring applications.
Keywords: Capillary electrophoresis; Flow-injection microchip; Phenolic; Microanalyzer; Amperometric detection; Run buffer;

Colloidal gold nanoparticles were conjugated with oligonucleotides to create biorecognition nanomodules. The efficiency of conjugation was determined by fluorescence using a FITC-labelled thiolated model probe and by enzyme-linked nanoparticle assay (ELINA) using a digoxigenin-labelled thiolated model probe. The thermal stability of the conjugation was determined by displacement and fluorescence measurement of the FITC probe. Functionality for hybridisation was determined by enzyme-linked oligonucleotide assay (ELONA). It was found that the equilibrium oligonucleotide surface coverage reached 37% of the total nanoparticle area. These results could be verified by ELINA. Under hybridisation conditions that allowed the detection of 4-point mutations on a target 19-mer sequence (1 h at 65 °C), it was found that the biofunctionalised nanomodules lost between 10 and 30% of the conjugated biorecognition molecules.
Keywords: Colloidal gold; Bioconjugation; Nanoparticle; ELINA; ELONA; Biorecognition nanomodules; Oligonucleotide;

A bifurcated optical fiber chemical sensor for continuous monitoring of bisphenol A (BPA) has been proposed based on the fluorescence quenching (λ ex/λ em  = 286/390 nm) of pyrene/dimethyl β-cyclodextrin (HDM-β-CD) supramolecular complex immobilized in a plasticized poly(vinyl chloride) (PVC) membrane, in which pyrene served as a sensitive fluorescence indicator probe. The decrease of the fluorescence intensity of pyrene/HDM-β-CD complex upon the addition of BPA was attributed to the displacement of pyrene by BPA, which has been utilized as the basis of the fabrication of a BPA-sensitive fluorescence sensor. The response mechanism of the sensor was discussed in detail. The sensor exhibited a dynamic detection range from 7.90 × 10−8 to 1.66 × 10−5  mol L−1 with a detection limit of 7.00 × 10−8  mol L−1, and showed excellent reproducibility, reversibility, selectivity, and lifetime. The proposed sensor was successfully used for the determination of BPA in water samples and landfill leachate.
Keywords: Chemical sensor; Dimethyl β-cyclodextrin; Pyrene; Bisphenol A; Competitive complexation;

A sensitive biosensor has been developed for the neurotoxin β-N-oxalyl-α,β-diaminopropionic acid (β-ODAP) contained in the seeds of grass pea (Lathyrus sativus) and for l-glutamate based on glutamate oxidase (GlOx) and a Prussian blue (PB) modified glassy carbon (GC) electrode. The configuration of the system is so as to detect the hydrogen peroxide released during the enzymatic cycle at a low applied potential, −50 mV versus Ag|AgCl, in the flow injection mode. For this purpose GlOx was coupled to PB electrodeposited onto a glassy carbon electrode and stabilised by treatment with tetrabutylammonium toluene-4-sulfonate (TTS) during one of the steps in the electrodeposition. GlOx was cross-linked with glutaraldehyde (GA), bovine serum albumin (BSA) and Tween-20 on the surface of the PB modified GC electrodes. Addition of 0.01% and 0.001% polyethyleneimine (PEI) to the immobilisation mixture resulted in an enhancement of the response signal with about 35% and 62% for glutamate and β-ODAP, respectively, when using 0.01% PEI and with 164% and 200% for glutamate and β-ODAP, respectively, when using 0.001% PEI. The linear response range for β-ODAP was extended from 0.05–0.5 mM to 0.01–1 mM, when 0.001% PEI was used. However, a higher concentration of PEI, 0.1%, caused a decrease in the sensitivity of the biosensor.
Keywords: Glutamate oxidase; β-ODAP; Polyethyleneimine; Enzyme modified electrodes; Prussian blue;

Microsensors based on carbon fiber microelectrodes coated with enzyme-entrapping redox hydrogels facilitate the in vivo detection of substances of interest within the central nervous system, including hydrogen peroxide, glucose, choline and glutamate. The hydrogel, formed by cross-linking a redox polymer, entraps the enzymes and mediates electron transfer between the enzymes and the electrode. It is important that the enzymes are entrapped in their enzymatically active state. Should entrapment cause enzyme denaturation, the sensitivity and the selectivity of the sensor may be compromised. Synthesis of the redox polymer according to published procedures may yield a product that precipitates when added to aqueous enzyme solutions. Casting hydrogels from solutions that contain the precipitate produces microsensors with low sensitivity and selectivity, suggesting that the precipitation disrupts the structure of the enzymes. Herein, we show that a surfactant, sodium dodecyl sulfate (SDS), can prevent the precipitation and improve the sensitivity and selectivity of the sensors.
Keywords: Microsensor; Enzyme-modified electrode; Redox polymer; Hydrogel; Horseradish peroxidase; Ascorbate oxidase;

An amperometric immunosensor for the specific and simple detection of 3,4-methylenedioxyamphetamine (MDA) and its analogues, 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyethylamphetamine (MDEA) in saliva and urine was developed. A direct competitive assay in which free analyte and horseradish peroxidase labelled species were simultaneously added to an immobilised polyclonal antibody was employed. Both MDA and MDMA could be labelled with the enzyme and the use of an MDMA–HRP tracer greatly enhanced the sensitivity of the assay. Amperometric detection was performed at +100 mV versus Ag/AgCl, using tetramethylbenzidine (TMB)/H2O2 as substrate. The antibody, raised specifically against the methylenedioxy moiety of an MDA–BSA immunogen allowed highly specific detection of these analogues with negligible cross-reactivity towards any other amphetamine related compounds. Total assay time was 45 min and the standard curve using MDA could be evaluated within the range 0.61–400 ng ml−1 with corresponding limit of detection (LOD) of 0.36 and 0.042 ng ml−1 for saliva and urine, respectively. The cross-reactivity pattern of the analytes was determined and showed the order of sensitivity increased with increased alkyl chain length (MDA < MDMA < MDEA). The overall performance of the sensor, working range, precision and sensitivity demonstrate its usefulness for rapid and direct measurement of methylenedioxy analogues of ecstasy in saliva and urine. The sensor has better specificity than any previous method for ecstasy, with greater sensitivity than ELISA methods, is less expensive/assay with an “easier to use” format than previous methods. The detection works in saliva or urine, eliminating requirement of blood sampling, with improved precision.
Keywords: Ecstasy; Immunosensor; Screen-printed electrode; Saliva; Urine;

Immunoassay of P-glycoprotein on single cell by capillary electrophoresis with laser induced fluorescence detection by Hua Xiao; Xin Li; Hanfa Zou; Ling Yang; Yanqin Yang; Yulin Wang; Hailin Wang; X. Chris Le (340-346).
The cellular mechanism based on P-glycoprotein (PGP) for its drug pump function has become very important in multidrug resistance (MDR) research. A method has been established to characterize PGP on single K562 cell by coupling capillary electrophoresis with laser induced fluorescence detection. A permeable intact cell after the immunoassay binding with fluorescence labeling antibody was injected into the capillary and directly separated without lysis. It was found that once 5–10 optional cells were detected in batch, the PGP amount on this cell line could be outlined and calculated clearly. The PGP amount on K562 MDR cell line is 3.88 times higher than that on K562 sensitive cell line. These two cell lines with immunoassay binding were also analyzed by injection of multi-cells in order to improve the throughput. A resistance factor so called multidrug resistance multiple (MRM) was introduced to evaluate the MDR difference between cell lines. The MRM values of the cell line K562 measured by single cell analysis are well correlated with those by flow cytometry, which also prove the validity of our method in single cell analysis for the possibility of cancer diagnosis, pharmacokinetics and drug screening in future.
Keywords: Capillary electrophoresis; Laser induced fluorescence detection; Mulitidrug resistance; P-glycoprotein; Resistance factor;

Immunochemical determination of four organophosphorus insecticide residues in olive oil using a rapid extraction process by Marta Garcés-García; Eva M. Brun; Rosa Puchades; Ángel Maquieira (347-354).
Sensitive, simple and rapid ELISA methods have been developed for the determination of four organophosphorus pesticides in extra virgin olive oil. The analytical procedure involves simultaneous extraction of the analytes from oil matrix with methanol and a freezing clean-up step (−80 °C), followed by immunoassay determination using standards in matrix. The methodology is specific for diazinon, fenthion, malathion and chlorpyrifos showing little or no cross-reactivity against other organophosphorus compounds. Limits of detection for the pesticides in olive oil are from 46 ng ml−1 for diazinon to 10 ng ml−1 for fenthion, all of them under the established MRLs for olives. The excellent recoveries (between 94 and 122%) obtained by the complete analytical protocol confirm the potential of this approach for detecting these compounds in olive oil, being useful as screening and complementary method in pesticide regulatory and food safety programs. The proposed methodology also correlates well with the reference chromatographic (GC–MS) methods.
Keywords: Immunoassay; Insecticide; Organophosphorus; Diazinon; Fenthion; Malathion; Chlorpyrifos; Olive oil; Food analysis;

Ozone tropospheric degradation of organic compound is very important in environmental chemistry. The lifetime of organic chemicals in the atmosphere can be calculated from the knowledge of the rate constant of their reaction with free radicals such as OH and NO3 or O3. In the present work, the rate constant for the tropospheric degradation of 137 organic compounds by reaction with ozone, the least widely and successfully modeled degradation process, are predicted by quantitative structure activity relationships modeling based on a variety of theoretical descriptors, which screened and selected by genetic algorithm variable subset selection procedure. These descriptors which can be used as inputs for generated artificial neural networks are; HOMO–LUMO gap, number of double bonds, number of single bonds, maximum net charge on C atom, minimum (>0.1) bond order of C atom and Minimum e–e repulsion of H atom. After generation, optimization and training of artificial neural network, network was used for the prediction of log KO3 for the validation set. The root mean square error for the neural network calculated log KO3 for training, prediction and validation set are 0.357, 0.460 and 0.481, respectively, which are smaller than those obtained by multiple linear regressions model (1.217, 0.870 and 0.968, respectively). Results obtained reveal the reliability and good predictivity of neural network model for the prediction of ozone tropospheric degradations rate constant of organic compounds.
Keywords: Artificial neural network; Multiple linear regressions; Quantitative structure–activity relationship; Ozone degradation; Genetic algorithm;

Monitoring alcoholic fermentation by joint use of soft and hard modelling methods by Marcelo Blanco; Antonio C. Peinado; Jordi Mas (364-373).
The strengths of hard and soft modelling were exploited by using both types of methods in combination to monitor alcoholic fermentations under Saccharomyces cerevisiae yeasts. Experimental work was performed in two steps. In the first, fermentation processes were conducted under identical conditions except for the initial glucose concentration in order to tests various previously reported empirical hard modelling methods. The product inhibition model of Hinshelwood was found to provide the best results in terms of goodness of fit and consistency in parameter values between runs under these conditions. In the second step, fermentation processes conducted at variable temperature and pH were monitored in-line by using an immersion NIR probe. The results were processed by using multivariate curve resolution–alternating least-squares (MCR–ALS) methodology in combination with the hard modelling information obtained in the first step as spectral equality constraints. Notwithstanding the complexity of the fermentation matrix introduced by variability in the species involved in yeast metabolism, the extracted profiles exhibited highly significant correlation with the reference values provided by a validated reference method for the determination of glucose, ethanol and biomass. The results testify to the efficiency of the joint use of soft and empirical hard modelling for studying evolving biological systems and opens up new avenues for application to other bioprocesses.
Keywords: NIR spectroscopy; Hard modelling; Soft modelling; ALS; PLS; In-line monitoring;

Geographical classification of crude oils by Kohonen self-organizing maps by Ana M. Fonseca; José L. Biscaya; João Aires-de-Sousa; Ana M. Lobo (374-382).
In the analysis of an environmental disaster caused by spillage of crude oil, limitation of the possible sources to a few geographical origins can help in the identification of the polluting vessel from a group of potential candidates. In this paper we show that Kohonen self-organizing maps (or Kohonen neural networks) can classify samples of crude oils on the basis of gas chromatography–mass spectrometry (GC–MS) descriptors, in terms of geographical origin, with a high degree of accuracy. Two data sets were investigated – one from Instituto Hidrográfico (Lisbon, Portugal) with 188 samples from 20 geographical origins, and another from EUROCRUDE® with 374 samples. After training the Kohonen self-organizing maps with a training set, predictions were obtained for an independent test set. Correct predictions were obtained for 70% and 60% of the test sets for the two studies, respectively. Ensembles of networks were highly interesting for the calculation of a prediction score, which can be used as a measure of the reliability of the prediction. For the samples with high prediction scores, the percentage of correct predictions jumped to 94–96%. The ability of the maps to identify a given origin is very much dependent on the availability of samples from that class in the training set. Equally good predictions were obtained for a small test set of weathered samples. This investigation adds value to the GC–MS descriptors already in use for practical analytical work, suggesting new ways to ferret out useful knowledge from them.
Keywords: Crude oils; Self-organizing maps; Neural network; GC–MS; Geographical classification; Spills;

The implementation of maximum likelihood parallel factor analysis (MLPARAFAC) in conjunction with the direct exponential curve resolution algorithm (DECRA) is described. DECRA takes advantage of the intrinsic exponential structure of some bilinear data sets to produce trilinear data by a simple shifting scheme, but this manipulation generates an error structure that is not optimally handled by traditional three-way chemometrics methods such as TLD and PARAFAC. In this work, the effects of these violations are studied using simulated and experimental data used in conjunction with the well-established TLD and PARAFAC. The results obtained by both methods are compared with the results obtained by MLPARAFAC, which is a method designed to optimally accomodate a variety of measurement error structures. The impact on the estimates of different parameters linked to the data sets and the DECRA method is investigated using simulated data. The results indicate that PARAFAC produces estimates of much poorer quality than TLD and MLPARAFAC. Also, it was found that the quality TLD estimates was comparable or only marginally poorer than the MLPARAFAC estimates. A number of commonly used algorithms were also compared to MLPARAFAC using two sets of published experimental data from kinetic studies. The MLPARAFAC estimates of rate constants were more precise than the other methods examined.
Keywords: Direct exponential curve resolution algorithm; DECRA; MLPARAFAC; PARAFAC; Weighted PARAFAC; Maximum likelihood; Three-way data; Trilinear data; Measurement errors; Error covariance; Parallel factor analysis;

Heptakis (2,6-di-O-methyl)-β-cyclodextrin (DM-β-CD) blending with hydroxy-terminated silicone oil (OH-TSO) coated solid-phase microextraction (SPME) fiber (DM-β-CD/OH-TSO) was first prepared with sol–gel technology and applied to headspace SPME for analysis of ephedrine (EP) and methamphetamine (MA) in human urine by gas chromatography (GC). By exploiting the advantages of the unique cavity-shaped cyclic molecular structure of CD and the superiorities of sol–gel coating technique, the novel fiber showed desirable extraction ability and operational stability. Influence of relevant experimental parameters (extraction time, extraction temperature, basicity, ionic strength, etc.) was systematically investigated. In the optimal conditions the proposed headspace SPME–GC method provided good linearity over four orders of magnitude with limit of detection (LOD) of ng/ml (0.33 ng/ml for EP, 0.60 ng/ml for MA). The recoveries of EP and MA in urine were 98.0% and 98.2%. And the relative standard deviations (R.S.D., n  = 6) for EP and MA were 3.9% and 5.0%, respectively.
Keywords: Solid-phase microextraction; β-Cyclodextrin; Ephedrine; Amphetamine;

A novel method for monitoring fibre performance and death, based on a Shewhart control plot, for use in headspace analysis by solid phase microextraction-gas chromatography (SPME-GC) is presented. The method is also demonstrated to be effective in standardising fibre response in extended time–course experiments, where several fibres may be required to complete a lengthy study. Extraction conditions that gave good total recovery and diversity of compounds, from olive oil headspace were found to be: 1 g oil sample in 10 mL vessel; exposure of DVB-CAR-PDMS, 50/30 μm fibre for 30 min at 40 °C; and desorption for 3 min at 250 °C. Calibration curves were constructed for 25 compounds commonly reported in olive oil headspace; with coefficients of determination (R 2) ranging from 0.959 to 0.994 and limits of detection (LOD) from 0.01 to 0.59 μg/g. The method was applied to monitor the change in concentration of select C6 volatile compounds, which have implications in sensory quality –E-2-hexenal, hexanal, E-2-hexen-1-ol, hexanol – over a period of 12 months storage.
Keywords: Headspace SPME-GC; Olive oil; DVB-CAR-PDMS fibre; Fibre death; Shewhart control chart; Time–course monitoring;

A novel and simple method has been developed for the simultaneous determination of beta-lactam antibiotics (BLAs) (penicillin G, amoxicillin, ampicillin, penicillin V, oxacillin, cloxacillin, dicloxacillin and nafcillin) in wastewater. The method is based on solid-phase extraction (SPE) and high performance liquid chromatography with UV-diode array detection (UV-DAD). Two SPE cartridges have been compared for sample clean up and preconcentration: a reversed-phase silica-based cartridge (Bond Elut C18, Varian Inc.) and a strong polymeric mixed mode anion exchanger (Oasis MAX, Waters). The penicillins have been separated using a LUNA™ C18 (2) (150 mm × 4.6 mm, 5 μm) HPLC column and gradient elution with mobile phases consisting of aqueous trifluoroacetic acid and acetonitrile. The analytical wavelength was set at 220 nm. Under optimised conditions it was possible to preconcentrate up to 1000 mL of Milli-Q water in the Oasis MAX cartridges with recoveries in the range 82–97% (R.S.D. 2–9%) for all the antibiotic tested, except amoxicillin (52%, R.S.D. 8%), and limits of detection in the range of 8–24 ng L−1. The matrix components in industrial and urban wastewater samples reduce the preconcentration efficiency in both sorbents, especially for the Bond Elut C18. The use of the Oasis MAX allowed detection limits between 2.9–25.6, 2.5–12.4 and 2.2–12.7 μg L−1, when processing 250 mL of industrial, influent and effluent sewage treatment plant (STP) samples. Recoveries ranged between 46–91, 28–91 and 39–114% (industrial, influent and effluent STP, respectively) for samples spiked with all the antibiotics at 25 and 75 μg L−1 (n  = 3 for each level).
Keywords: Penicillins; Multi-residue analysis; Solid phase extraction (SPE); HPLC-UV-DAD; Oasis MAX cartridge; Wastewater;

Novel preconcetration method involving porous polytetrafluoroethylene (PTFE) filter tube impregnated bis(2-ethylhexyl) hydrogen phosphate (HDEHP) as a sorbent was studied to establish it as a practical preconcentration method for ultra trace analysis with ICP-MS. A 1 ng portion of In(III) in 1000 ml of matrix free solution or 700 ml of synthetic seawater was quantitatively complexated with HDEHP adsorbed onto porous PTFE filter tube by passing the solution through the micro pore of the filter tube. Preconcentrated In(III) was then quantitatively recovered provided that the elution, which consists of a cyclical filtering 0.1 ml of 8 mol dm−3 hydrochloric acid through the filter tube for 1 min; therefore, up to 10000- and 7000-fold of enrichment was attained for matrix free solution and synthetic seawater, respectively. To introduce a 0.1 ml of the eluted solution to ICP-MS, flow injection method with air segmented discrete sample introduction (ASDI) was also studied by using manually operated simple valve system. By using ASDI, good linearity of calibration curve (r  = 0.99997) was observed from 0.01 to 5.0 ng ml−1 of In(III). Good reproducibility was also shown in measurements of 0.1 ml of 5 ng ml−1 of In(III) (R.S.D. = 1.9%, n  = 5). The average recovery and R.S.D. of the results for the five duplicates determination of 0.1 ng of In(III) spiked to 200 ml of synthetic seawater were 99 and 2.4%, respectively. The method was applied to the determination of In(III) in coastal seawater sampled at north east of Hachijyo Island, Japan; using 200 ml of sample, 2000-fold preconcentration of In(III) was performed within 30 min for five samples. The analytical detection limit and the blank were 9.8 and 21 pg l−1, respectively. The average concentration was determined to be 96 pg l−1, and R.S.D. of the results was 3.7% (n  = 3).
Keywords: Preconcentration; Liquid–solid extraction; Bis(2-ethylhexyl) hydrogen phosphate; HDEHP; PTFE; Filter; In(III); ICP-MS;

Separation and determination of some metal ions on new chelating resins containing N, N donor sets by Sadhan Pramanik; Sanjoy Dhara; Shuvendu S. Bhattacharyya; Pabitra Chattopadhyay (430-437).
Two new stable chelating resins have been synthesized incorporating the imidazolylazobenzene and 1,4-bis(imidazolylazo)benzene as functional group into Merrifield polymer through C―N covalent bond and characterized by elemental analyses, IR and thermal study. A comparison of sorption capacity of newly formed resins towards the cations Ag(I), Cu(II), Zn(II), Cd(II), Hg(II) and Pb(II) as a function of pH has been studied. Kinetic studies show the time for the completeness of metal ion saturation with the resin phase. Cd(II) in trace quantities has been successfully separated and determined in different biological samples and Zn(II) in medicinal samples. It is also found that Cd(II) can be removed from water at usual pH of natural water. Both the resins can be employed for water purification as the resins reveal sorption ability towards toxic metal ions and exhibit no affinity to alkali or alkaline earth metal ions.
Keywords: Sorption behavior; Chelating resin; Imidazolylazobenzene; Metal ions;

A water-in-oil emulsion containing Kelex-100 for the speciation analysis of trace heavy metals in water by Hiroaki Matsumiya; Ryohei Ohkouchi; Masataka Hiraide (438-443).
A water-in-oil (w/o) emulsion containing Kelex-100 (7-dodecenyl-8-quinolinol) and Span-80 (sorbitan monooleate, non-ionic surfactant) was ultrasonically prepared from 1.0 mol l−1 hydrochloric acid and a (1 + 3) mixture of toluene and n-heptane. The resulting emulsion was gradually injected into water sample and dispersed as numerous tiny globules (0.01–0.1 mm in diameter). Dissolved inorganic species (free metal species) of heavy metals (e.g., Fe, Co, Cu, Cd, and Pb) were selectively transported through the oil layer into the internal aqueous phase of the emulsion, leaving other species, such as humic complexes and suspended particles (larger than 1 μm), in the sample solution. After collecting the dispersed emulsion globules, they were demulsified and the heavy metals in the segregated aqueous phase were determined by graphite-furnace atomic absorption spectrometry. The emulsion-based separation method allowed the selective collection of free metal species with a high concentration factor of 100, whereas the conventional solvent extraction did not offer such discrimination. This unique property of the emulsion method was successfully applied to the selective determination of free species of heavy metals in fresh water samples.
Keywords: Emulsion; Speciation analysis; Heavy metal; Humic substance; Fresh water;

The optimised BCR sequential extraction procedure and a 4 h 1 mol L−1 HCl partial extraction have been performed on the NIST 2711 reference material for a suite of 12 elements (Cd, Sb, Pb, Al, Cr, Mn, Fe, Co, Ni, Cu, Zn, As) using magnetic sector ICP-MS. A pseudo-total aqua regia digest of NIST 2711 has also been undertaken for quality assurance purposes, and comparison of the sum of the four BCR fractions, which included an aqua regia digest on the residue, with the pseudo-total aqua regia digest has been used to assess the accuracy of the BCR partitioning approach. As a result of this work, discrepancies between previous studies about BCR partitioning of elements in NIST 2711 have been discussed and an increase in confidence about the use of BCR partitioning scheme on seven elements (Cd, Pb, Al, Mn, Fe, Cu, Zn) in this standard material has been obtained. On the other hand, BCR partitioning for Sb, Cr, Co, Ni and As has been provided for the first time. Partial extraction results are also reported for the same 12 elements analysed by the optimised BCR procedure, with the partial extraction results exhibiting a strong correlation with the sum of the three labile steps of the BCR procedure.
Keywords: NIST 2711; Sequential extraction; BCR sequential extraction; Partial extraction; ICP-MS;

Cathodic electrochemiluminescence at double barrier Al/Al2O3/Al/Al2O3 tunnel emission electrodes by Markus Håkansson; Qinghong Jiang; Johanna Suomi; Kari Loikas; Mauri Nauma; Timo Ala-Kleme; Jouko Kankare; Pentti Juhala; Jarkko U. Eskola; Sakari Kulmala (450-454).
Double insulating barrier tunnel emission electrodes were fabricated by adding a new pure aluminum layer upon oxidized aluminum electrodes by vacuum evaporation and thermally oxidizing the new aluminum layer in air at room temperature. Resulting Al/Al2O3/Al/Al2O3 electrodes allow the use of various aluminum alloys in the electrode body necessary for hardness or shaping ability of the electrode while obtaining the luminescence properties of pure aluminum oxide. During electrical excitation of luminescent labels by cathodic hot electron injection into aqueous electrolyte solution, the background noise is mainly based on high-field-induced solid-state electroluminescence and F-center luminescence of the outer aluminum oxide film. The more defect states and/or impurity centers the outer oxide film contains, the higher is the background emission intensity. The present electrode fabrication method provides a considerable improvement in signal-to-noise ratio for time-resolved electrochemiluminescence (TR-ECL) measurements when the original native oxide film of the electrode body contains luminescence centers displaying long-lived luminescence. The excellent performance of the present electrodes is demonstrated by extremely low-level detection of Tb(III) chelates, luminol, Pt(II) coproporphyrin and Tb(III) labels in an immunometric immunoassay by time-resolved electrochemiluminescence.
Keywords: Electrochemiluminescence; Hot electrons; Time-resolved detection; Labels; Bioaffinity assays;

Ion mobility spectrometry (IMS) is a proven technology for detection of vapor phase chemical warfare agents. The technology is suitable for field portable instrumentation due to its small size, high sensitivity, speed of analysis, and low power consumption. However, it suffers from a limited dynamic range and potential difficulties in identifying compounds in complex matrices. The use of gas chromatography (GC) coupled to IMS can overcome the difficulty of chemical identification in mixtures by separating the sample into individual components before detection. Using this approach, IMS technology has previously been adapted to detect biological aerosols using an open tube pyrolyzer and a short GC column (Py-GC–IMS). The open sample introduction tube of a Py-GC–IMS instrument would be a convenient configuration to accept aerosol particulates, and while viewed as needed for aerosol trapping, is not optimal for liquid chemical analyses. To examine the usefulness of an existing Py-GC–IMS system for analysis of chemicals in water, an existing open-port sample interface was replaced with a septum-equipped closed tube injector to contain analyte vapors resulting from liquid injection. Tributylphosphate (TBP) was used as a surrogate chemical warfare agent, and aqueous injections into both closed and open tube assemblies were performed. Sample introduction into the closed tube inlet was also accomplished using solid phase microextraction (SPME) preconcentration. The limit of detection for TBP using an open tube, closed tube, and closed tube configuration with SPME sample introduction was 0.980, 0.196, and 0.0098 mg/L, respectively.
Keywords: Field analysis; Gas chromatography; Ion mobility spectrometry; Solid phase microextraction; Resistive heating; Chemical warfare agent;

Quantitative analysis of malic and citric acids in fruit juices using proton nuclear magnetic resonance spectroscopy by Gloria del Campo; Iñaki Berregi; Raúl Caracena; J. Ignacio Santos (462-468).
1H NMR spectroscopy was applied to the quantitative determination of malic and citric acids in apple, apricot, pear, kiwi, orange, strawberry and pineapple juices. Aspartic acid was studied as a potential interference. The effect of the sample pH on the chemical shifts of signals from malic, citric and aspartic acids was examined and a value of 1.0 was selected to carry out the determination. Integration of NMR signals at 2.89–2.95 and 3.00–3.04 ppm were used for calculating the concentration of malic and citric acids, respectively. At this pH the integrated signals were not overlapped. Sodium 3-(trimethylsilyl)tetradeuteropropionate (TSP) was used as an internal reference. The obtained results applying NMR procedures to analyze the juices from different fruits were compared to those obtained using enzymatic methods and both were in close agreement. The intra- and inter-day repeatability was tested for apple juice (7.86 g l−1 malic acid, 0.32 g l−1 citric acid) and apricot juice (5.06 g l−1 malic acid, 4.79 g l−1 citric acid) obtaining coefficients of variation lower than 3.4% for intra-day measures (n  = 10) and lower than 3.8% for inter-day measures (n  = 20).
Keywords: Quantitative NMR spectroscopy; 1H NMR; Malic acid; Citric acid; Fruit juices;

Electrostatic interactions of proteins, including bovine serum albumin (BSA), human serum albumin (HSA), γ-globulin (γ-IgG), α-chymotrypsin (Chy), lysozyme (Lys) and cellulase (Cel), with multiply negatively charged chromophores were investigated based on the measurements of the enhanced resonance light scattering (RLS) signals. Using triply negatively charged water blue (WB) as an example, the factors were discussed that affect the enhanced resonance light scattering signals of the interactions between proteins and the negatively charged chromophores. It was found that the enhanced RLS signals with the maximum light scattering peak at 346.0 nm in these interacting systems are strongly dependent on the isoelectric points of proteins and show adverse linear relationships with increasing ionic strength depending on the positive charges of the inorganic metal ions used to control the ionic strength of the medium, sufficiently disclosing that the electrostatic attraction performs an important role in the combination of proteins with WB. Linear responses were discovered between the enhanced RLS signals and the protein molecular weights (M w), displaying the dimensions of scattered particles formed by proteins and WB make a key contribution to the RLS enhancements. An empirical equation is proposed which possibly displays the factors affecting the enhanced RLS signals of the interactions between proteins with negatively charged chromophores.
Keywords: Proteins; Water blue; Resonance light scattering (RLS) technique; Electrostatic interaction;

Development of a sampling and flow injection analysis technique for iron determination in the sea ice environment by Delphine Lannuzel; Jeroen de Jong; Véronique Schoemann; Anne Trevena; Jean-Louis Tison; Lei Chou (476-483).
A trace metal clean method for sampling and analysis of iron is set up and applied to sea ice and its associated snow, brine, and underlying seawater sampled during the Antarctic expedition “ARISE in the East” (Antarctic Remote Ice Sensing Experiment, AA03-V1, September–October 2003, 64–65°S/112–119°E, RV Aurora Australis). For clean sampling, a non-contaminating electropolished stainless steel ice corer is designed in conjunction with a polyethylene lathe equipped with Ti chisels to remove possibly contaminated outer layers of ice cores. A portable peristaltic pump with clean tubing is used on the ice to sample the underlying seawater (interface ice–water = 0, 1 and 30 m) and sea ice brine from access holes. Considering the extreme range of salinities (1–100) and Fe concentrations (0.1–100 nM) previously observed in similar environments, it is of paramount importance to set up a simple and sensitive Fe analyser adapted to such gradients. We use a flow injection analysis (FIA) technique and successfully demonstrate its capability to measure Fe concentrations directly in the sample without an on-line preconcentration/matrix separation step. We test the sensitivity, accuracy, precision and long-term stability of the analytical procedure. Also we explore and remediate interferences from a suite of other trace elements, such as Ni, Cd, Cr, Mn, Cu, Zn and Co. Analysis of reference materials NASS-5 and CASS-3 gives a good agreement with the certified values. Repeated measurements over a period of 5 months of an “in-house” Antarctic seawater standard yields a concentration of 1.02 ± 0.07 nM (n  = 17, 1σ). The detection limit (3σ of the blank) is on average 0.12 nM. We report here results of the Fe distribution in sea ice that are in good agreement with previously published data. To our knowledge, this work provides the first complete profiles of total dissolvable and dissolved Fe in sea ice.
Keywords: Iron; FIA; Sea ice; Direct measurement; Chemiluminescence;

Author Index (484-487).