Analytica Chimica Acta (v.701, #2)

Microextraction by packed sorbent (MEPS): A tutorial by Mohamed Abdel-Rehim (119-128).
Display Omitted► Microextraction by packed sorbents (MEPS) is a new online sample preparation technique. ► MEPS is more greener than SPE and LLE and can be used for small sample volumes (10 μL). ► Extraction of drugs from whole blood samples is possible. ► This emerging technique has been used in a number of recent research works in pre-clinical, clinical and environmental analysis.This tutorial provides an overview on a new technique for sample preparation, microextraction by packed sorbent (MEPS). Not only the automation process by MEPS is the advantage but also the much smaller volumes of the samples, solvents and dead volumes in the system. Other significant advantages such as the speed and the simplicity of the sample preparation process are provided.In this tutorial the main concepts of MEPS will be elucidated. Different practical aspects in MEPS are addressed. The factors affecting MEPS performance will be discussed. The application of MEPS in clinical and pre-clinical studies for quantification of drugs and metabolites in blood, plasma and urine will be provided. A comparison between MEPS and other extraction techniques such as SPE, LLE, SPME and SBSE will be discussed.
Keywords: Sample preparation; MEPS; GC–MS; LC–MS; Drug analysis;

Evaluation of pulsed radiofrequency glow discharge time-of-flight mass spectrometry for precious metal determination in lead fire assay buttons by Sien Compernolle; Jorge Pisonero; Nerea Bordel; Dorine Wambeke; Ine De Raedt; Kristof Kimpe; Alfredo Sanz-Medel; Frank Vanhaecke (129-133).
Display Omitted► Use of pulsed rf glow discharge TOF mass spectrometry for an industrial application. ► Direct determination of the precious metals Ag, Au, Pd, Pt and Rh in solid lead buttons obtained by Pb fire assay. ► Spectral overlap was overcome by relying on different temporal profiles for analyte and interfering species. ► Limits of detection <0.1 μg g−1for all elements of interest.In this paper, an exploration of the capabilities and limitations of pulsed radiofrequency glow discharge time-of-flight mass spectrometry (GD-TOFMS) for the determination of the precious metals Ag, Au, Pd, Pt and Rh in lead buttons obtained by Pb fire assay is reported on. Since the matrix consists almost entirely of lead (>99%), the occurrence of doubly charged Pb (Pb2+) ions can hinder accurate determination of Rh. This problem was counteracted by relying on the time-resolved formation of different ion types over the pulse period of the glow discharge, which allows discrimination against the Pb2+ ions. The formation of ArCu+ ions as a result of the use of a copper anode was assessed to pose no threat to the accuracy of the results obtained for the set of samples analyzed as its contribution to the total signal at m/z  = 103 could be adequately corrected for. The method developed was evaluated in terms of accuracy and precision using a set of Pb button standards with analyte concentrations between 5 and 100 μg g−1. For the purpose of validation, a 10 μg g−1 standard was considered as a sample. Overall, an acceptable accuracy was obtained with a bias of <5% between the experimental results and the corresponding reference values, except for Au, for which a larger deviation occurred. Precision values (repeatability) of typically <5% relative standard deviation (RSD) (for N  = 3) were obtained and the limits of detection (LODs) vary from ∼0.020 μg g−1 for Ag to ∼0.080 μg g−1 for Pt.
Keywords: Precious metals; Lead fire assay; Pulsed radiofrequency glow discharge; Time-of-flight mass spectrometry;

Serum/plasma methylmercury determination by isotope dilution gas chromatography-inductively coupled plasma mass spectrometry by Douglas C. Baxter; Mikko Faarinen; Heléne Österlund; Ilia Rodushkin; Morten Christensen (134-138).
Display Omitted• We determine methylmercury in serum and plasma using isotope dilution calibration. • Separation by gas chromatography and detection by inductively coupled plasma mass spectrometry. • Data for 50 specimens provides first reference range for methylmercury in serum. • Serum samples shown to be stable for 11 months in refrigerator.A method for the determination of methylmercury in plasma and serum samples was developed. The method uses isotope dilution with 198Hg-labeled methylmercury, extraction into dichloromethane, back-extraction into water, aqueous-phase ethylation, purge and trap collection, thermal desorption, separation by gas chromatography, and mercury isotope specific detection by inductively coupled plasma mass spectrometry. By spiking 2 mL sample with 1.2 ng tracer, measurements in a concentration interval of (0.007–2.9) μg L−1 could be performed with uncertainty amplification factors <2. A limit of quantification of 0.03 μg L−1 was estimated at 10 times the standard deviation of concentrations measured in preparation blanks. Within- and between-run relative standard deviations were <10% at added concentration levels of 0.14 μg L−1, 0.35 μg L−1 and 2.8 μg L−1, with recoveries in the range 82–110%. Application of the method to 50 plasma/serum samples yielded a median (mean; range) concentration of methylmercury of 0.081 (0.091; <0.03–0.19) μg L−1. This is the first time methylmercury has been directly measured in this kind of specimen, and is therefore the first estimate of a reference range.
Keywords: Serum; Plasma; Methylmercury; Isotope dilution; Gas chromatography; Inductively coupled plasma mass spectrometry;

.Display Omitted• Aim of the work: to discriminate anthocyanins from black rice or grapevine in wine. • Two series of adulterated samples were analysed by FT-NIR and 1H NMR spectroscopies. • Feature selection-classification on NIR spectra reached a 70% prediction efficiency. • Feature selection-classification on NMR spectra reached a 95% prediction efficiency. • 2D correlation analysis recognised the zones bringing the same chemical information.In the Italian oenological industry, the regular practice used to naturally increase the colour of red wines consists in blending them with a wine very rich in anthocyanins, namely Rossissimo. In the Asian market, on the other hand, anthocyanins extracted by black rice are frequently used as correctors for wine colour. This practice does not produce negative effects on health; however, in many countries, it is considered as a food adulteration.The present study is therefore aimed to discriminate wines containing anthocyanins originated from black rice and grapevine by using reliable spectroscopic techniques requiring minimum sample preparation. Two series of samples have been prepared from five original wines, that were added with different amounts of Rossissimo or of black rice anthocyanins solution, until the desired Colour Index was reached. The samples have been analysed by FT-NIR and 1H NMR spectroscopies and the resulting spectra matrices were subjected to multivariate classification. Initially, PLS-DA was used as classification method, then also variable selection/classification methods were applied, i.e. iPLS-DA and WILMA-D. The classification with variable selection of NIR spectra permitted to classify the test set samples with an efficiency of about 70%. Probably these not excellent performances are due to the matrix effect, together with the lack of sensitivity of NIR with respect to minor compounds. On the contrary, very satisfactory results were obtained on NMR spectra in the aromatic region between 6.5 and 9.5 ppm. The classification method based on wavelet-based variables selection, permitted to reach an efficiency in validation greater than 95%.Finally, 2D correlation analysis was applied to FT-NIR and 1H NMR matrices, in order to recognise the spectral zones bringing the same chemical information.
Keywords: Wine adulteration; Anthocyanins; Fourier Transform-Near InfraRed; 1H NMR; Multivariate classification; Feature selection;

Increased sensitivity of anodic stripping voltammetry at the hanging mercury drop electrode by ultracathodic deposition by José A. Rodrigues; Carlos M. Rodrigues; Paulo J. Almeida; Inês M. Valente; Luís M. Gonçalves; Richard G. Compton; Aquiles A. Barros (152-156).
.Display Omitted► At very cathodic accumulation potentials (overpotential deposition) the voltammetric signals of Zn2+, Cd2+, Pb2+ and Cu2+ increase. ► 5 to 10-fold signal increase is obtained. ► This effect is likely due to mercury drop oscillation at such cathodic potentials. ► This effect is also likely due to added local convection at the mercury drop surface caused by the evolution of hydrogen bubbles.An improved approach to the anodic stripping voltammetric (ASV) determination of heavy metals, using the hanging mercury drop electrode (HMDE), is reported. It was discovered that using very cathodic accumulation potentials, at which the solvent reduction occurs (overpotential deposition), the voltammetric signals of zinc(II), cadmium(II), lead(II) and copper(II) increase. When compared with the classical methodology a 5 to 10-fold signal increase is obtained. This effect is likely due to both mercury drop oscillation at such cathodic potentials and added local convection at the mercury drop surface caused by the evolution of hydrogen bubbles.
Keywords: Anodic stripping voltammetry (ASV); Cadmium (Cd); Copper (Cu); Hanging mercury drop electrode (HMDE); Lead (Pb); Overpotential; Zinc (Zn);

. A hybrid method coupling electrochemical “adsorption–desorption” and colorimetric analyses may be used for the in situ determination of heavy metal ions in turbid water samples.Display Omitted• A novel hybrid analytical method was applied to detect Cd2+, Pb2+ and Cu2+ in turbid, polluted water. • Combing electrochemical “adsorption–desorption” with colorimetric methods. • Portable screen-printed electrodes (SPEs) used as working devices. • Practicality was confirmed by detecting real samples and results agreed with ICP-AES.A novel and facile hybrid analytical method coupling electrochemical “adsorption–desorption” and colorimetric analyses was developed to detect heavy metal ions in turbid water samples. The target metal ions were deposited onto an electrode inserted into the original sample, which was referred to as the “adsorption” process. After changing the medium, the concentrated target metal ions were dissolved in a new, clean buffer (blank buffer), which was referred to as the “desorption” process. The concentrations of the target metal ions were measured by colorimetric analyses after the addition of specific indicator amounts. We demonstrated the applicability of this method by detecting Cd2+, Pb2+ and Cu2+ with co-depositing Bi3+ on portable screen-printed electrodes (SPEs). A good correlation (correlation coefficient of R  = 0.997) was observed between concentrations ranging from 1 to 200 μM and absorbance values. After the multiple “desorption” process, the even better detection limits as low as 10, 10 and 100 nM were achieved for Cd2+, Pb2+ and Cu2+, respectively. The practicality of this hybrid method was confirmed by the detection of Cd2+, Pb2+ and Cu2+ in wastewater samples, and these results were in agreement with inductively coupled plasma atomic emission spectroscopy (ICP-AES). Overall, this hybrid method provides a simple, selective and effective technique for environmental pollutant analyses.
Keywords: Heavy metal ions; Hybrid methods; Adsorption–desorption; Colorimetric analysis; Screen-printed electrode;

Display Omitted• We had explored a novel NDA-FM-TOX method for measurement of toxicity. • This method did not require both of air supply and deaerated equipment for rapid measuring of real polluted wastewater. • It has been developed successfully for the detection of various toxic substances.In this paper, a mediated method by using ferricyanide under non-deaerated condition for biotoxicity measurement was proposed. Ultramicroelectrode array (UMEA) was employed for effectively amplify the electrochemical signal from the total limiting currents to distinguish a little change in toxicity. Five species of microorganisms including two bacilli (Escherichia coli and Enterobacter cloacae), two pseudomonas (Pseudomonas fluorescens and Pseucomonas putida) and one fungus (Trichosporon cutaneum) were employed. 3,5-dichlorophenol (DCP) was taken as the reference toxicant. The IC50 values we obtained were similar with the values obtained using in the deaerated method. E. coli was used as model test microorganism. The final concentration of ferricyanide is 45 mM, E. coli OD600 8 and 1 h incubation were taken in optimum conditions in this study. Four heavy metal ions (Cr6+, Hg2+, Cd2+ and Bi3+) were examined under the optimum conditions. Comparison with the results reported previously has confirmed that this method provided a simple and rapid alternative to toxicity screening of chemicals, especially advantageous for in situ monitoring of water system.
Keywords: Biotoxicity; Non-deaerated; Ultramicroelectrode; Electrochemical; Metal ion;

Vitamin C derivatives as new coreactants for tris(2,2′-bipyridine)ruthenium(II) electrochemiluminescence by Yali Yuan; Haijuan Li; Shuang Han; Lianzhe Hu; Saima Parveen; Guobao Xu (169-173).
Display Omitted► Ru(bpy)3 2+ electrochemiluminescence of vitamin C derivatives have been investigated. ► Ascorbyl phosphate and ascorbyl palmitate show intense electrochemiluminescence. ► Ascorbyl 2-phosphate was detected with high sensitivity. ► This study provides a new way to detect vitamin C derivatives.Vitamin C derivatives (VCDs) have been widely used as the alternative and stable sources of vitamin C, and accordingly exhibit many new applications, such as anti-tumor and central nervous system drug delivery. In this study, their Ru(bpy)3 2+ electrochemiluminescence (ECL) properties have been investigated for the first time using well-known ascorbyl phosphate and ascorbyl palmitate as representative VCDs. Ascorbyl phosphate and ascorbyl palmitate are VCDs with different substituted positions. Both of them increase Ru(bpy)3 2+ ECL, indicating that other VCDs may also enhance Ru(bpy)3 2+ ECL signal. The calibration plot for ascorbyl phosphate is linear from 3 × 10−6 to 1.0 × 10−3  M with a detection limit of 1.4 × 10−6  M at a signal-to-noise ratio of 3. The relative standard deviation is 3.6% for six replicate measurements of 0.01 mM ascorbyl 2-phosphate solution. The proposed method is about one order of magnitude more sensitive than electrochemical and UV–vis methods for the determination of ascorbyl phosphate, and is used successfully for the determination of ascorbyl phosphate in whitening and moisturising body wash.
Keywords: Tris(2,2′-bipyridine)ruthenium; Electrochemiluminescence; Vitamin C derivatives; Ascorbyl palmitate; Ascorbyl phosphate;

A novel silver-coated solid-phase microextraction metal fiber based on electroless plating technique by Juanjuan Feng; Min Sun; Jubai Li; Xia Liu; Shengxiang Jiang (174-180).
A novel silver coated SPME fiber with high extraction efficiency and excellent stability was prepared by a simple electroless plating process. Coupled to GC analysis, the fiber showed wide linear ranges for PAHs and PAEs because of its large surface area and the special physicochemical characteristics of the silver coating. Compared with commercially available fibers and other novel SPME fibers reported lately, the silver-coated fiber kept higher extraction efficiency and lower LODs towards the same analytes.Display Omitted► Silver metal is used as a novel SPME coating. ► A simple electroless plating method based on the silver mirror reaction is applied in the preparation of the proposed fiber. ► The silver-coated SPME fiber exhibits high extraction efficiency and excellent stability and durability to harsh conditions.A novel silver-coated solid-phase microextraction fiber was prepared based on electroless plating technique. Good extraction performance of the fiber for model compounds including phthalate esters (dibutyl phthalate, dioctyl phthalate, dicyclohexyl phthalate and diallyl phthalate) and polycyclic aromatic hydrocarbons (naphthalene, fluorene, phenanthrene, fluoranthene) in aqueous solution was obtained. Under the optimized conditions (extraction temperature, extraction time, ionic strength and desorption temperature), the proposed SPME-GC method showed wide linear ranges with correlation coefficients (R 2) ranging from 0.9745 to 0.9984. The limits of detection were at the range of 0.02 to 0.1 μg L−1. Single fiber repeatability and fiber-to-fiber reproducibility as well as stability to acid, alkali and high temperature were studied and the results were all satisfactory. The method was applied successfully to the aqueous extracts of disposable paper cup and instant noodle barrel. Several kinds of analytes were detected and quantified.
Keywords: Silver; Electroless plating; Solid-phase microextraction; Gas chromatography; Phthalate esters; Polycyclic aromatic hydrocarbons;

Display Omitted► The opium determination is of a great deal of importance from toxicological and pharmaceutical standpoints. ► Thebaine, an opium alkaloid, has been suggested as a good urinary marker of poppy capsule use. ► Electromembrane microextraction method was coupled with high performance liquid chromatography and ultraviolet detection. ► The method was developed for determination of thebaine in water, urine, poppy capsule, street heroine, and codeine tablet. ► Detection limits and intra-day precision (n  = 3) less than 15 μg L−1 and 8.9%, were obtained respectively.Opium determination is of great importance from toxicological and pharmaceutical standpoints. In present work, electromembrane extraction (EME) coupled with high-performance liquid chromatography (HPLC) and ultraviolet (UV) detection was developed for determination of thebaine as a natural alkaloid, in different matrices containing water, urine, poppy capsule, street heroine, and codeine tablet. Thebaine migrated from 3 mL of sample solutions, through a thin layer of 2-nitrophenyl octyl ether (NPOE) immobilized in the pores of a porous hollow fiber, and into a 15 μL acidic aqueous acceptor solution present inside the lumen of the fiber. The variables of interest, such as chemical composition of the organic liquid membrane, stirring speed, extraction time and voltage, pH of donor and acceptor phases and salt effect in the EME process were optimized. Under optimal conditions, thebaine was effectively extracted from different matrices with recoveries in the range of 45–55%, which corresponded to preconcentration factors in the range of 90–110. Good linearity was achieved for calibration curves with a coefficient of estimation higher than 0.997. Detection limits and intra-day precision (n  = 3) were less than 15 μg L−1 and 8.9%, respectively.
Keywords: Electromembrane extraction; Thebaine; Urine; Opium; High-performance liquid chromatography;

Development of a combined technique using a rapid one-step immunochromatographic assay and indirect competitive ELISA for the rapid detection of baicalin by Madan Kumar Paudel; Waraporn Putalun; Boonchoo Sritularak; Osamu Morinaga; Yukihiro Shoyama; Hiroyuki Tanaka; Satoshi Morimoto (189-193).
Display Omitted• We developed a combined immunoassay using an immunochromatographic assay (ICA) and indirect competitive ELISA (icELISA) using anti-baicalin monoclonal antibody (anti-BA MAb). • The ICA enables a short assay time (15 min), no dependence on any instrumental systems. • In addition, the icELISA is suitable for quantitative analysis of BA and baicalein with reliability and accuracy. • The developed immunochemical system can provide practical and efficient applications toward monitoring the quality of Scutellariae Radix and ensure proper use of Kampo medicines containing S. Radix.A colloidal gold conjugated anti-baicalin monoclonal antibody (anti-BA MAb) was prepared and used in an immunochromatographic assay (ICA) for BA in Scutellariae Radix and Kampo medicines. This competitive ICA uses an anti-BA MAb which shows a high specificity for BA and baicalein. Its advantages include a short assay time (15 min), no dependence on any instrumental systems, and it can detect BA in plant materials and Kampo medicines. The limit of detection for the ICA was found to be around 0.6 μg mL−1of baicalin. Moreover, the usefulness of the combination of indirect competitive ELISA and the ICA using anti-BA MAb as a quality control method was confirmed for analysis of BA in Scutellariae Radix and Kampo medicines with a sufficient sensitivity (200 ng mL−1 to 2 μg mL−1), obtainable in an easy and timely manner.
Keywords: Baicalin; Scutellaria Radix; Immunochromatographic strip; Anti-baicalin monoclonal antibody; Enzyme-linked immunosorbent assay;

Display Omitted• A long-wavelength fluoroimmunoassay for the determination of soy protein is reported. • The tracer is constituted by antibodies bound to nile blue-doped silica nanoparticles. • The number of assay steps is lower than that required for reported ELISAs. • The use of shallow well plates allows the reduction of immuno-reagent volumes. • Front-surface fluorescence is used as the detection system.A long-wavelength fluoroimmunoassay for the determination of soy protein is reported for the first time using a conjugate composed of anti-soy protein antibodies bound to nile blue-doped silica nanoparticles (NPs). These NPs have been synthesized by a reverse-micelle microemulsion method and functionalized by using 3-(aminopropyl)triethoxysilane (APS) and 3-(trihydroxysilyl)propyl methylphosphonate (THPMP) to avoid NP aggregation. The tracer has been obtained by linking the functionalized NPs with anti-soy protein antibodies previously oxidised with sodium periodate. The immunoassay has been developed in 96-well microplates using a heterogeneous competitive format with antibody capture. Soy proteins are immobilised onto the wells and bovine serum albumin is added to block the surface, thus minimising non-specific binding. After washing, the microplates can be stored ready to use. At the analysis time, soy protein standards or sample and tracer are added and incubated and, after the corresponding washing and drying steps, the fluorescence is measured onto the solid surface at λ ex 620 and λ em 680 nm. The method features a dynamic range of 0.1–10 mg L−1 and a detection limit of 0.05 mg L−1. The precision of the method has been assayed at 0.5 and 5 mg L−1 protein concentrations, obtaining the values of relative standard deviation of 9.6% and 6.1%, respectively. This new immunoassay has been applied to the analysis of food containing soy protein and the results obtained have been compared to those provided by a commercial ELISA kit with no statistically differing results. Also, a recovery study has been performed, providing percentages in the range of 81.5–111.0%.
Keywords: Soy protein; Food samples; Long-wavelength fluorimetry; Heterogeneous immunoassay; Nile blue-doped silica nanoparticles;

First direct fluorescence polarization assay for the detection and quantification of spirolides in mussel samples by Paz Otero; Amparo Alfonso; Carmen Alfonso; Rómulo Aráoz; Jordi Molgó; Mercedes R. Vieytes; Luis M. Botana (200-208).
Display Omitted► A direct assay based in the binding of nAChR to spirolide toxins by FP is described. ► A direct relationship between FP and 13-desMeC in the range of 10-500 nM is obtained. ► FP is dependent on the 13, 19-didesMeC in a higher concentration range than 13-desMeC. ► FP assay is a sensitive method to detect and quantify 13-desMeC in mussel samples.In 2009, we achieve the first inhibition FP assay to detect imine cyclic toxins. In the present paper we propose a new FP assay for direct quantify spirolides. This new method has resulted in significant improvement of sensitivity, rapidity and accessibility. In the method design, nicotinic acetylcholine receptor from Torpedo marmorata membranes labelled with a derivative of fluorescein was used. Spirolides, 13-desmethyl spirolide C (13-desMeC) and 13,19-didesmethyl spirolide C (13,19-didesMeC) were extracted and purified from cultures of the Alexandrium ostenfeldii dinoflagellate. Data showed the decrease of FP when toxin concentration was increased. Thus, a relationship between the FP units and the spirolides amount present in a sample was obtained. This direct assay is a reproducible, simple and very sensitive method with a detection limit about 25 nM for 13-desMeC and 150 nM for 13,19-didesMeC. The procedure was used to measure spirolides in mussel samples using an extraction and clean up protocol suitable for the FP assay. Results obtained show that this method is able to quantify 13-desMeC in the range of 50–350 μg kg−1 meat. Other liposoluble toxins did not interfere with the assay, proving a specific method. Moreover, the matrix do not affect in the range of toxin concentrations that involving risk of spirolides intoxication.
Keywords: 13-desmethyl spirolide C; 13,19-didesmethyl spirolide C; Nicotinic acetylcholine receptor; Fluorescence polarization; Liquid chromatography–mass spectrometry;

Pretreatment-free immunochromatographic assay for the detection of streptomycin and its application to the control of milk and dairy products by N.A. Byzova; E.A. Zvereva; A.V. Zherdev; S.A. Eremin; P.G. Sveshnikov; B.B. Dzantiev (209-217).
Display Omitted► A rapid pretreatment-free immunochromatographic assay was developed for streptomycin control. ► A change in the hapten:protein ratio was applied to vary the cut-off level of the assay. ► The applicability of the assay for whole, drinking and sour clotted milk was confirmed.A rapid pretreatment-free immunochromatographic assay was developed for the control of the streptomycin (STR) content in milk and dairy products. The assay is based on the competition between an immobilized STR–protein conjugate and STR in a sample to be tested for the binding to monoclonal anti-STR antibodies conjugated to colloidal gold during the flow of the sample along a membrane strip with immobilized reactants. It is possible to improve the cut-off level of positive and negative samples distinguished by a change in the molar STR to protein ratio in the immobilized conjugate. The cut-off level (500 ng mL−1) thus achieved corresponds to the stated MRL of STR in milk and dairy products. For STR concentrations in the range of 16–250 ng mL−1 its content can be quantitatively measured based on the degree of binding of a colloidal gold label in the test strip zone with the immobilized STR–protein conjugate. The duration of the assay is 10 min. The selected sizes of membrane pores and colloidal gold particles allow the assay to be carried out at room temperature without additional reactants and pretreatment. The applicability of the assay for milk, whole milk, sour clotted milk, and kefir with different fat content (from 0.5% to 6%) was confirmed. The results of quantitative immunochromatographic assay show good correlation with traditional ELISA (r was equal to 0.935 and 0.940 for the series tested).
Keywords: Streptomycin; Immunochromatographic assay; Strip-test; Colloidal gold; Milk; Dairy products;

Prussian Blue acts as a mediator in a reagentless cytokinin biosensor by Marta Kowalska; Faming Tian; Mária Šmehilová; Petr Galuszka; Ivo Frébort; Richard Napier; Nicholas Dale (218-223).
Display Omitted• An electrochemical biosensor for detection of the plant hormone cytokinin. • Constitutive expression system for large-scale protein production. • CKX enzyme entrapment in sol–gel film on the surface of a PrB-modified electrode. • Prussian Blue as an electron mediator between the enzyme and the electrode. • The biosensor was sensitive to micromolar concentrations of several cytokinins.An electrochemical biosensor for detection of the plant hormone cytokinin is introduced. Cytokinin homeostasis in tissues of many lower and higher plants is controlled largely by the activity of cytokinin dehydrogenase (CKX, EC 1.5.99.12) that catalyzes an irreversible cleavage of N 6-side chain of cytokinins. Expression of Arabidopsis thaliana CKX2 from Pichia pastoris was used to prepare purified AtCKX2 as the basis of the cytokinin biosensor. Prussian Blue (PrB) was electrodeposited on Pt microelectrodes prior to deposition of the enzyme in a sol–gel matrix. The biosensor gave amperometric responses to several cytokinins. These responses depended on the presence of both the enzyme and the Prussian Blue. Thus Prussian Blue must act as an electron mediator between the FAD centre in CKX2 and the Pt surface.
Keywords: Phytohormone; Electrochemistry; Oxidase; Dehydrogenase; Quantitation; Electrode;

Impurity fingerprints for the identification of counterfeit medicines—A feasibility study by Pierre-Yves Sacré; Eric Deconinck; Michal Daszykowski; Patricia Courselle; Roy Vancauwenberghe; Patrice Chiap; Jacques Crommen; Jacques O. De Beer (224-231).
Display Omitted► Impurity profiles treated like fingerprints. ► 73 illegal and 10 genuine Viagra®, 44 illegal and 5 genuine Cialis®. ► Detection of illegal tablets obtained by PLS-DA on the log transformed data. ► 100% correct classification rates (CCR) with k-NN algorithm for Viagra®-like samples. ► For Cialis®, 100% CCR for external validation, with k-NN algorithm.Most of the counterfeit medicines are manufactured in non good manufacturing practices (GMP) conditions by uncontrolled or street laboratories. Their chemical composition and purity of raw materials may, therefore, change in the course of time. The public health problem of counterfeit drugs is mostly due to this qualitative and quantitative variability in their formulation and impurity profiles.In this study, impurity profiles were treated like fingerprints representing the quality of the samples. A total of 73 samples of counterfeit and imitations of Viagra® and 44 samples of counterfeit and imitations of Cialis® were analysed on a HPLC-UV system. A clear distinction has been obtained between genuine and illegal tablets by the mean of a discriminant partial least squares analysis of the log transformed chromatograms. Following exploratory analysis of the data, two classification algorithms were applied and compared. In our study, the k-nearest neighbour classifier offered the best performance in terms of correct classification rate obtained with cross-validation and during external validation. For Viagra®, both cross-validation and external validation sets returned a 100% correct classification rate. For Cialis® 92.3% and 100% correct classification rates were obtained from cross-validation and external validation, respectively.
Keywords: Impurities; Fingerprints; Classification; Counterfeit; Phosphodiesterase type 5 inhibitors;

Display Omitted• For the first time reverse micelles with Triton X-100 coupled with ultrasound-assisted backextraction were used for extraction of β-sitosterol and cholesterol. • This is a direct method for extraction and quantification of cholesterol and β-sitosterol in vegetable oil samples. • The method is fast and simple as it does not require a previous saponification process.Ultrasonic back-extraction of Triton X-100 reverse micelles by a water/chloroform binary system and gas chromatography with flame ionization detection (GC-FID) was developed for extraction and determination of β-sitosterol and cholesterol in soybean and sunflower oil samples. After the homogenization of the oil samples with Triton X-100, an aliquot of 200 μL of methanol was added to the samples to form two phases. The clear Triton X-100 extract obtained by centrifugation was treated with a mixture of water (1000 μL) and chloroform (300 μL) for back-extraction of the analytes into the chloroform phase by ultrasonication. After centrifugation, the sedimented chloroform layer was withdrawn easily by a microsyringe and directly injected into the GC-FID system. The influence of several important parameters on the extraction efficiencies of the analytes was evaluated. Under optimized experimental conditions, the calibration graphs were linear in the range of 1.0–30.0 mg L−1 with coefficient of determination more than 0.994 for both analytes. The method detection limit values were in the range of 0.2–0.7 mg L−1. The lower limit of quantification values were in the range of 0.7–2.4 mg L−1. Intra-day relative standard deviations were in the range of 1.0–2.7%. This procedure was successfully applied with satisfactory results to the determination of β-sitosterol and cholesterol in spiked oil samples. The relative mean recoveries of oil samples ranged from 93.6% to 105.0%.
Keywords: β-Sitosterol; Cholesterol; Reverse micelle; Ultrasound-assisted; Back-extraction; Triton X-100;