Analytical and Bioanalytical Chemistry (v.394, #4)

is Professor of Analytical Chemistry at Bologna University. His main research interests include the development of ultrasensitive bioanalytical methods employing bioluminescence, chemiluminescence, and electrochemiluminescence detection, for example immunoassays, gene hybridization assays, imaging techniques for localization of proteins and nucleic acids in cells and tissues, genetically engineered whole-cell biosensors, and whole-animal imaging. Proteomic studies for biomarker discovery based on liquid chromatography and mass spectrometry are also performed in his laboratory

Solid-phase microextraction with micellar desorption and HPLC-fluorescence detection for the analysis of fluoroquinolones residues in water samples by Sarah Montesdeoca Esponda; Ma Esther Torres Padrón; Zoraida Sosa Ferrera; José Juan Santana Rodríguez (927-935).
A sensitive and useful method based on solid-phase microextraction with micellar desorption (SPME-MD) coupled to HPLC with fluorescence detection was developed for the determination of five fluoroquinolones (levofloxacin, norfloxacin, ciprofloxacin, enrofloxacin, and sarafloxacin) in environmental water matrices. The SPME extraction efficiency was optimized with regard to time, temperature, pH, and ionic strength using a CW-TPR fiber. A detailed study about the optimum conditions for micellar desorption (surfactant type, concentration, and desorption time) were made. Among different surfactants studied, Polyoxyethylene 10 lauryl ether showed the best responses to extract fluoroquinolones using SPME-MD. Relative standard deviations of the developed method were below 9%. Limits of detection and quantification were between 0.01–0.2 and 0.03–0.6 ng mL−1, respectively. The recoveries achieved for all five compounds were in the 81–116% range. The proposed method was compared using conventional desorbing agent as methanol. Finally, the SPME-MD methodology was applied to the determination of these target analytes in several environmental liquid samples, including seawater, groundwater, and wastewater samples with excellent results.
Keywords: Solid-phase microextraction; Surfactants; Fluoroquinolones; High-performance liquid chromatography; Fluorescence; Water analysis

The beneficial effects of 1-butyl-3-methylimidazolium tetrafluoroborate (BMIm-BF4) ionic liquid (IL) as mobile phase additive, desorption solvent, and memory effect suppressor in solid-phase microextraction (SPME)–high-performance liquid chromatography with fluorescence detection for the determination of six heterocyclic aromatic amines have been evaluated for the first time. Several chromatographic parameters have been evaluated in the presence or absence of IL or using triethylamine as the most common mobile phase additive, with a Nova-Pak® C18 stationary phase. This IL was found to be clearly superior to triethylamine for efficiency as well as peak shape enhancement and sensitivity increase. SPME was chosen because it is faster than conventional extraction techniques and allowed us to minimize the use of organic solvents. However, memory effect may become a problem when a high-sensitivity detector is used. The appropriate conditions for the desorption step and to eliminate the memory effect involving BMIm-BF4 were established and optimized. The method was applied for the determination of these compounds in commercial meat extracts.
Keywords: Ionic liquids memory effect; Solid phase microextraction coupled with high-performance liquid chromatography; Fluorescence detection; Heterocyclic aromatic amines

Study of the acute cardiovascular effects of several antihypertensive agents with the measurement of plasma catecholamines in mice by Makoto Tsunoda; Masanori Yamagishi; Kazuhiro Imai; Teruyuki Yanagisawa (947-952).
A highly sensitive determination method was established for catecholamines (norepinephrine (NE), epinephrine, and dopamine) with high-performance liquid chromatography-peroxyoxalate chemiluminescence reaction detection. In this study, the method was applied to mouse plasma, and it was determined that only 10 μl of mouse plasma was necessary for the selective and reproducible determination of catecholamines. Studies were then conducted in acute cardiovascular effects of sodium nitroprusside, nicardipine, captopril (angiotensin-converting enzyme (ACE) inhibitor), candesartan, and olmesartan (type 1 angiotensin receptor antagonists (AT1 antagonists)) by this method. Sodium nitroprusside and nicardipine elevated plasma NE concentrations significantly, whereas the ACE inhibitor and the AT1 antagonists did not change plasma NE concentrations in anesthetized mice. These results suggested that angiotensin II-induced augmentation may be mainly carried through the central baroreflex pathway.
Keywords: HPLC; Chemiluminescence; Sympathetic nervous system; Angiotensin II

Hybrid gravitational field-flow fractionation using immunofunctionalized walls for integrated bioanalytical devices by Barbara Roda; Sonia Casolari; Pierluigi Reschiglian; Mara Mirasoli; Patrizia Simoni; Aldo Roda (953-961).
In this work, the biospecific recognition antigen–antibody reaction was implemented in gravitational field-flow fractionation (GrFFF), a flow-assisted separation technique for micron-sized particles, in order to realize a hybrid immunomodulated GrFFF system in which two different principles are combined to achieve highly versatile fractionation. Micron-sized polystyrene beads coated with horseradish peroxidase (HRP) were used as a model sample, and anti-HRP antibodies were immobilized on the accumulation wall of the GrFFF channel. Ultrasensitive chemiluminescence imaging was employed to visualize the beads during elution and to optimize experimental conditions. The same principle was then applied to real biological samples composed by Yersinia enterocolitica and Escherichia coli cells. Results show the possibility to modify the elution of selected sample components and even to retain them into the channel. The hybrid immunomodulated GrFFF system is a step towards the development of a module that could be integrated in a lab-on-a-chip-based point-of-care testing device which includes sample pre-analytical cleanup and analysis.
Keywords: Gravitational field-flow fractionation; Chemiluminescence; Imaging analysis; Cell sorting; Antibody; Point-of-care testing

A MIP-based flow-through fluoroimmunosensor as an alternative to immunosensors for the determination of digoxin in serum samples by Gema Paniagua González; Pilar Fernández Hernando; Jesús Senén Durand Alegría (963-970).
This work reports a comparative study of two automated flow-through fluorosensors for the determination of digoxin in serum samples: an immunosensor with an anti-digoxin polyclonal antibody as the reactive phase permanently immobilised on controlled-pore glass and a sensor with a selective reaction system based on a methacrylic molecularly imprinted polymer (MIP) synthesised by bulk polymerisation. The variables affecting the sensitivity and dynamic range of the sensors (e.g. the carrier and elution solutions, flow rates, pH and reagent concentrations) were optimized, and the binding characteristics of their reactive phases were compared in a competitive fluorescent assay. Digoxin was reproducibly determined by both sensors at the milligram per litre level (detection limit = 1.20 × 10−3 mg L−1 and RSD = 4–7% for the immunosensor; detection limit = 1.7 × 10−5 mg L−1 and RSD = 1–2% for the MIP sensor). No cross-reactivity with digoxin-related compounds was seen for either sensor at a digoxin/interferent ratio of 1:100. The lifetime of the immunosensor was about 50 immunoassays; its shelf life, when unused, is about 3 months. The lifetime of the MIP sensor was over 18 months. Both sensors were used to determine the digoxin concentration of human serum samples with satisfactory results.
Keywords: Digoxin; Permanent immobilisation; Molecular imprinting; Flow-through fluorosensor; Serum analysis

New electrochemiluminescent biosensors combining polyluminol and an enzymatic matrix by Audrey Sassolas; Loïc J. Blum; Béatrice D. Leca-Bouvier (971-980).
Performant reagentless electrochemiluminescent (ECL) (bio)sensors have been developed using polymeric luminol as the luminophore. The polyluminol film is obtained by cyclic voltammetry (CV) on a screen-printed electrode either in a commonly used H2SO4 medium or under more original near-neutral buffered conditions. ECL responses obtained after performing polymerization either at acidic pH or at pH 6 have been compared. It appears that polyluminol formed in near-neutral medium gives the best responses for hydrogen peroxide detection. Polymerization at pH 6 by cyclic voltammetry gives a linear range extending from 8 × 10−8 to 1.3 × 10−4 M H2O2 concentrations. Based on this performant sensor for hydrogen peroxide detection, an enzymatic biosensor has been developed by associating the polyluminol film with an H2O2-producing oxidase. Here, choline oxidase (ChOD) has been chosen as a model enzyme. To develop the biosensor, luminol has been polymerized at pH 6 by CV, and then an enzyme-entrapping matrix has been formed on the above modified working electrode. Different biological (chitosan, agarose, and alginate) and chemical (silica gels, photopolymers, or reticulated matrices) gels have been tested. Best performances have been obtained by associating a ChOD-immobilizing photopolymer with the polyluminol film. In this case, choline can be detected with a linear range extending from 8 × 10−8 to 1.3 × 10−4 M.
Keywords: Luminol; Electropolymerization; Electrochemiluminescence; Optical detection; Screen-printed electrodes; Oxidase

Multiplex chemiluminescence microscope imaging of P16INK4A and HPV DNA as biomarker of cervical neoplasia by M. Mirasoli; M. Guardigli; P. Simoni; S. Venturoli; S. Ambretti; M. Musiani; A. Roda (981-987).
Classification of cervical intraepithelial neoplasia (CIN) lesions in low-grade (CIN1) or high-grade (CIN2-3) ones is crucial for optimal patient management, but current histological diagnosis on bioptic samples is often hampered by inter-observer variability. To allow objective classification, we have exploited the peculiar characteristics of chemiluminescence detection, such as high sensitivity and easy quantification of the luminescence signal, to perform sequentially in the same tissue section both an immunohistochemical quantitative detection of p16INK4A (a protein marker of high-grade CIN lesions) and an in situ hybridization for human papillomavirus (generally accepted as a necessary but insufficient cause of cervical carcinoma). Different label enzymes (alkaline phosphatase and horseradish peroxidase) were employed in order to avoid any interference between the two assays, and quantitative chemiluminescence image analysis was used to obtain objective evaluation of sample positivity. The multiplexed method allowed detection of two complementary biomarkers and provided discrimination between different lesions (non-neoplastic, low-grade and high-grade CIN). This assay might thus represent an accurate and objective diagnostic test providing important information for counseling, selection of therapy and follow up after surgical treatment.
Keywords: Chemiluminescence; Imaging; Immunohistochemistry; In situ hybridization; Cervical intraepithelial neoplasia; Human papillomavirus

Kinetic determination of the GTPase activity of Ras proteins by means of a luminescent terbium complex by Christian Spangler; Corinna M. Spangler; Michael Spoerner; Michael Schäferling (989-996).
Guanine nucleotide binding proteins, such as Ras proteins, play a pivotal role in maintaining the regular life cycle of cells. The involvement of Ras mutants in the progress of cancer has attracted many efforts to find detection methods for Ras activity. In this study we present a luminescent microwell plate assay for monitoring GTPase activity of Ras proteins. The luminescence intensity of the Tb–norfloxacin complex is influenced by nucleoside phosphates as well as by inorganic phosphates. Real-time kinetics of the GTPase activity of wild-type Ras and Ras mutants can be monitored online. The effect of a GTPase activating protein as well as of a downstream effector (Ras-binding domain of human Raf-1) on the GTPase activity of different Ras mutants is examined. In contrast to other methods, this assay does not require the use of radioactively labeled substrates or chromatographic separation steps. Moreover, the application of fluorescently labeled GTP substrates which often interfere with enzymatic activity can be avoided. This in vitro assay can serve as a model system for the screening of regulators affecting the GTPase activity of Ras proteins. Figure The emission of the lanthanide complex Tb(III)-norfloxacin is influenced by nucleoside phosphates as well as by inorganic phosphates. Ras proteins display a specific GTPase activity which converts protein-bound GTP to GDP and phosphate, the latter being released. The Ras activity can be monitored by a significant decrease in luminescence intensity of Tb(III)-norfloxacin owing to the strong quenching effect induced by the enzymatically hydrolyzed phosphate anions. This luminescent assay enables the monitoring of real-time kinetics of the GTPase activity of Ras proteins and Ras mutants and a fast screening of their regulators.
Keywords: GTPase activity; Ras proteins; Fluorescence assay; Luminescence; Terbium–norfloxacin; Phosphate probe

Fluorescence properties of selected benzo[c]phenantridine alkaloids and studies of their interaction with CT DNA by Jana Urbanová; Přemysl Lubal; Iva Slaninová; Eva Táborská; Petr Táborský (997-1002).
The spectral, especially fluorescence properties, of seven selected quaternary benzo[c]phenantridine alkaloids (sanguinarine, chelerythrine, chelirubine, sanguirubine, chelilutine, sanguilutine, and macarpine) were studied in presence and in absence of double-stranded DNA. This study has proved dramatic differences in fluorescence emission of all studied alkaloids in presence of calf thymus DNA in comparison to fluorescence of free alkaloids. The most remarkable are changes in emission spectra of macarpine, chelirubine, and sanguirubine. Association constants (logK) for interaction of all studied alkaloids with CT DNA were calculated.
Keywords: Benzo[c]phenantridine alkaloids; Sanguinarine; Chelirubine; Sanguirubine; Chelilutine; Macarpine; Association constants

Fluorescence study of the dynamic interaction between E1(145–162) sequence of hepatitis GB virus C and liposomes by Maria Jesús Sánchez-Martín; José Manuel Amigo; Montserrat Pujol; Isabel Haro; M. Asunción Alsina; M. Antonia Busquets (1003-1010).
The physicochemical characterization of the peptide sequence E1(145–162) corresponding to the structural protein E1 of the hepatitis G virus was done by studying its interaction with model membranes. Small unilamellar vesicles (SUVs) of dimyristoylphosphatidylglycerol or dimyristoylphosphatidylcholine were chosen as mimetic membranes. Peptide incorporation and location in the phospholipid bilayer was investigated by fluorescence anisotropy with SUVs labeled with diphenylhexatriene (DPH) or trimethylammonium–DPH. The addition of the peptide E1(145–162) showed significant changes in the anisotropy values of the probe located at the air/water interface. These results indicate that the peptide E1(145–162) preferably interacts with the lipid surface without penetrating inside the bilayer. A series of fluorescence experiments based on tryptophan peptide fluorescence were modeled by means of multivariate curve resolution-alternating least squares (MCR-ALS) algorithm to further study the peptide interaction with bilayers at different temperatures. The preliminary results obtained with MCR-ALS showed how the peptide concentration decay is directly linked to the appearance of a new specie, which corresponds to the lipid-peptide binding. These results provide useful information for the design of synthetic immunopeptides that can be incorporated into a liposomal system with potential to promote a direct delivery of the membrane-incorporated immunogen to the immunocompetent cells, thus increasing the immuno response from the host.
Keywords: Hepatitis GB virus C; HGV/GBV-C; MCR-ALS; Tryptophan fluorescence; Liposomes; Synthetic peptides; Anisotropy

The photo-induced luminescence properties of Egyptian blue, Han blue and Han purple were investigated by means of near-infrared digital imaging. These pigments emit infrared radiation when excited in the visible range. The emission can be recorded by means of a modified commercial digital camera equipped with suitable glass filters. A variety of visible light sources were investigated to test their ability to excite luminescence in the pigments. Light-emitting diodes, which do not emit stray infrared radiation, proved an excellent source for the excitation of luminescence in all three compounds. In general, the use of visible radiation emitters with low emission in the infrared range allowed the presence of the pigments to be determined and their distribution to be spatially resolved. This qualitative imaging technique can be easily applied in situ for a rapid characterisation of materials. The results were compared to those for Egyptian green and for historical and modern blue pigments. Examples of the application of the technique on polychrome works of art are presented.
Keywords: Archeometry/fine arts; Fluorescence/luminescence; Egyptian blue; Han blue/Han purple; Imaging; Infrared

ATR and transmission analysis of pigments by means of far infrared spectroscopy by Elsebeth L. Kendix; Silvia Prati; Edith Joseph; Giorgia Sciutto; Rocco Mazzeo (1023-1032).
In the field of FTIR spectroscopy, the far infrared (FIR) spectral region has been so far less investigated than the mid-infrared (MIR), even though it presents great advantages in the characterization of those inorganic compounds, which are inactive in the MIR, such as some art pigments, corrosion products, etc. Furthermore, FIR spectroscopy is complementary to Raman spectroscopy if the fluorescence effects caused by the latter analytical technique are considered. In this paper, ATR in the FIR region is proposed as an alternative method to transmission for the analyses of pigments. This methodology was selected in order to reduce the sample amount needed for analysis, which is a must when examining cultural heritage materials. A selection of pigments have been analyzed in both ATR and transmission mode, and the resulting spectra were compared with each other. To better perform this comparison, an evaluation of the possible effect induced by the thermal treatment needed for the preparation of the polyethylene pellets on the transmission spectra of the samples has been carried out. Therefore, pigments have been analyzed in ATR mode before and after heating them at the same temperature employed for the polyethylene pellet preparation. The results showed that while the heating treatment causes only small changes in the intensity of some bands, the ATR spectra were characterized by differences in both intensity and band shifts towards lower frequencies if compared with those recorded in transmission mode. All pigments' transmission and ATR spectra are presented and discussed, and the ATR method was validated on a real case study.
Keywords: Far infrared (FIR) spectroscopy; Attenuated total reflectance (ATR); Transmission; Inorganic compounds; Polyethylene (PE) pellets

Spectroscopic analysis of works of art using a single LIBS and pulsed Raman setup by I. Osticioli; N. F. C. Mendes; S. Porcinai; A. Cagnini; E. Castellucci (1033-1041).
A nanosecond pulsed laser setup has been optimized to perform laser-induced breakdown spectroscopy (LIBS) and pulsed Raman spectroscopy measurements in the field of cultural heritage. Three different samples of artistic/architectural interest with different typologies have been analyzed. The results from the two techniques allowed the identification of the materials used in their manufacture or contaminating them, probably coming from atmospheric pollution and biological activity. No sampling and sample preparation was required before the measurements, and no visual or structural damage was observed. Depth profiling using LIBS was performed in one of the samples, providing elemental information along the different layers composing the object and covering its surface. The quality of the results and the rather short time needed for the measurements and for switching between techniques confirmed the instrument’s capabilities and specificity for dealing with objects of artistic or historical interest.
Keywords: Pulsed Raman spectroscopy; LIBS spectroscopy; Frescoes; Terra-cotta; Bronze head; Cultural heritage

Towards the differentiation of non-treated and treated corundum minerals by ion-beam-induced luminescence and other complementary techniques by H. Calvo del Castillo; N. Deprez; T. Dupuis; F. Mathis; A. Deneckere; P. Vandenabeele; T. Calderón; D. Strivay (1043-1058).
Differentiation of treated and non-treated gemstones is a chief concern for major jewellery import companies. Low-quality corundum specimens coming from Asia appear to be often treated with heat, BeO or flux in order to enhance their properties as precious minerals. A set of corundum samples, rubies and sapphires from different origins, both treated and non-treated has been analysed at the Centre Européen d’Archéométrie, with ion-beam-induced luminescence (IBIL) and other complementary techniques such as Raman, proton-induced X-ray emission (PIXE), and proton-induced gamma-ray emission (PIGE). IBIL, also known as ionoluminescence, has been used before to detect impurities or defects inside synthetic materials and natural minerals; its use for the discrimination of gemstone simulants or synthetic analogues has been elsewhere discussed (Cavenago-Bignami Moneta, Gemología, Tomo I Piedras preciosas, perlas, corales, marfil. Ediciones Omega, Barcelona, 1991). PIXE has been frequently applied in the archaeometric field for material characterisation and provenance studies of minerals (Hughes, Ruby & sapphire. RWH Publishing, Fallbrook, 1997; Calvo del Castillo et al., Anal Bioanal Chem 387:869–878, 2007; Calligaro et al., NIM-B 189:320–327, 2002) and PIGE complements the elemental analysis by detecting light elements in these materials such as—and lighter than—sodium that cannot be identified with the PIXE technique (Sanchez et al., NIM-B 130:682–686, 1997; Emmett et al., Gems Gemology 39:84–135, 2003). The micro-Raman technique has also been used complementarily to ion beam analysis techniques for mineral characterisation (Novak et al., Appl Surf Sci 231–232:917–920, 2004). The aim of this study is to provide new means for systematic analysis of corundum gemstone-quality mineral, alternative to the traditional gemmologic methods; for this purpose, a Spanish jewellery import company supplied us with a number of natural corundum samples coming from different places (part of them treated as explained above). The PIXE elemental concentrations of the samples showed large quantities of calcium and lead in some cases that can be linked to treatment with fluxes or lead oxide. The plot of the chromium and iron concentration grouped the samples in various aggregates that corresponded to the different types of corundum analysed. Micro-Raman complemented the PIXE analysis corroborating the presence of lead oxides but the use of the PIGE technique was not successful for the detection of beryllium due to the low cross section of the nuclear reaction chosen for its identification. IBIL was capable of distinguishing between treated and non-treated samples of the same type based on the luminescent features of the materials.
Keywords: Corundum; Ruby; Sapphire; IBIL; PIXE; PIGE; Micro-Raman

HUMANN-based system to identify benzimidazole fungicides using multi-synchronous fluorescence spectra: An ensemble approach by Carmen Paz Suárez Araujo; Patricio García Báez; Álvaro Sánchez Rodríguez; José Juan Santana Rodríguez (1059-1072).
In this paper, we approach, using neural computation and ensemble systems, a pattern classification problem in fluorescence spectrometry, the resolution of difficult multi-fungicide mixtures (overlapping), specifically the benzimidazole fungicides, benomyl, carbendazim, thiabendazole and fuberidazole. These fungicides are compounds of an important environmental interest. Because of this, from an analytical point of view, it is interesting to develop sensitive, selective and simple methods for their determination. Fluorescence spectrometry has proven to be a sensitive and selective technique for determination of many compounds of environmental interest, but in some cases it is not enough. HUMANN is a hierarchical, unsupervised, modular, adaptive neural net with high biological plausibility, which has shown to be suitable for identification of these fungicides and organochlorinated compounds of environmental interest. We propose two modular artificial intelligent systems, with a structure of pre-processing and processing stage, a multi-input HUMANN-based system, using multi-fluorescence spectra as input to the system, and a HUMANN-ensemble system. We analyze the optimal configuration of inputs and the ensemble in order to obtain better results. We study such figures as precision and sensitivity of the method. Our proposal is a smart, flexible and effective complementary method, which allows reducing the analytical and/or computational complexity of the analysis. Figure Stages in identification of benzimidazole fungicides
Keywords: Unsupervised artificial neural network; HUMANN; Benzimidazole fungicides; Fluorescence spectrometry; Environment; Ensemble system

Determination of diquat by flow injection–chemiluminescence by J. L. López-Paz; M. Catalá-Icardo; B. Antón-Garrido (1073-1079).
A simple, economic, sensitive and rapid method for the determination of the pesticide diquat was described. This new method was based on the coupling of flow injection analysis methodology and direct chemiluminescent detection; to the authors’ knowledge, this approach had not been used up to now with this pesticide. It was based on its oxidation with ferricyanide in alkaline medium; significant improvements in the analytical signal were achieved by using high temperatures and quinine as sensitiser. Its high throughput (144 h−1), together with its low limit of detection (2 ng mL−1), achieved without need of preconcentration steps, permitted the reliable quantification of diquat over the linear range of (0.01–0.6) μg mL−1 in samples from different origins (river, tap, mineral and ground waters), even in the presence of a 40-fold concentration of paraquat, a pesticide commonly present in the commercial formulations of diquat. Figure Quartz luminometer cell
Keywords: Chemiluminescence; Flow injection; Diquat; Pesticides; Water

Chlamydomonas reinhardtii genetic variants as probes for fluorescence sensing system in detection of pollutants by V. Scognamiglio; D. Raffi; M. Lambreva; G. Rea; A. Tibuzzi; G. Pezzotti; U. Johanningmeier; M. T. Giardi (1081-1087).
The unicellular green alga Chlamydomonas reinhardtii is employed here for the setup of a biosensor demonstrator based on multibiomediators for the detection of herbicides. The detection is based on the activity of photosystem II, the multienzymatic chlorophyll–protein complex located in the thylakoid membrane that catalyzes the light-dependent photosynthetic primary charge separation and the electron transfer chain in cyanobacteria, algae, and higher plants. Several C. reinhardtii mutants modified on the D1 photosystem II protein are generated by site-directed mutagenesis and experimentally tested for the development of a biosensor revealing the modification of the fluorescence parameter (1 − V J) in the presence of herbicides. The A250R, A250L, A251C, and I163N mutants are highly sensitive to the urea and triazine herbicide classes; the newly generated F255N mutant is shown to be especially resistant to the class of urea. It follows that the response of the multibiomediators is associated to a particular herbicide subclass and can be useful to monitor several species of pollutants.
Keywords: Chlamydomonas reinhardtii ; Site-directed mutagenesis; Biomediator; Biosensor; Pollutants; Fluorescence

Usefulness of cyclodextrin media for the determination of α-cypermethrin by photochemically induced fluorescence: analytical applications to natural waters by Moussa Mbaye; Mame Diabou Gaye Seye; Atanasse Coly; Alphonse Tine; Jean-Jacques Aaron (1089-1098).
The photochemically induced fluorescence (PIF) spectral properties of α-cypermethrin in organic solvents (hexane, dichloromethane, acetonitrile, ethanol) and in cyclodextrin aqueous solutions (β-CD and 2-hydroxypropyl-β-CD, 2-HP-β-CD) were investigated. The photolysis kinetics of α-cypermethrin were evaluated in the various media. The PIF signal was found to be significantly enhanced in the CD media relative to the organic solvents. The stoichiometry and the formation constants of the α-cypermethrin inclusion complexes formed with the CDs were determined. The analytical performances of the PIF method were improved in the presence of HP-β-CD relative to the other media, and a CD-enhanced PIF analytical method was developed. The limits of detection and limits of quantification ranged, respectively, between 6 and 98 ng/mL and between 24 and 343 ng/mL, depending on the medium. Application to the analysis of tap water and Senegal natural water samples collected close to agricultural areas and spiked with α-cypermethrin yielded satisfactory recoveries going from about 77% to 98%. An interference study of foreign species, including pesticides and inorganic ions likely to be present in natural waters, was also carried out. Figure Photolysis reaction of α-cypermethrin in presence of HP-β-CD
Keywords: α-Cypermethrin; Photochemically induced fluorescence; Cyclodextrins; Inclusion complexes; Water analysis

Toxicological study of pesticides in air and precipitations of Paris by means of a bioluminescence method by S. Trajkovska; M. Mbaye; M. D. Gaye Seye; J. J. Aaron; M. Chevreuil; H. Blanchoud (1099-1106).
A detailed toxicological study on several pesticides, including chlorothalonil, cyprodynil, dichlobénil, pendimethaline, trifluraline, and α-endosulfan, present at trace levels in air and total atmospheric precipitations of Paris is presented. The pesticides contained in the atmospheric samples, collected during sampling campaigns in February–March 2007, are identified and quantified by a high-performance liquid chromatographic (HPLC)-UV detection method. The toxicity measurements are performed by means of the Microtox® bioluminescence method, based on the evaluation of the bioluminescence inhibition of the Vibrio fischeri marine bacteria at two exposure times to the pesticide solutions. The specific toxicity, corresponding to the particular toxicity of the compound under study and represented by the EC50 parameter, is determined for these pesticides. Also, the global toxicity, which is the toxicity of all micro-pollutants present in the sample under study, is estimated for the extracts of air and atmospheric precipitation (rainwater) samples. The specific toxicities strongly vary with the nature of the pesticide, the EC50 parameter values being comprised between 0.17 and 0.83 mg/mL and 0.15 and 0.66 mg/mL, respectively, for exposure times of 5 and 15 min. The importance of the atmospheric samples’ global toxicity and the respective contribution of the toxic potency of the various pesticides contained in these samples are discussed. Figure Passive sampling device for rainwater, located on the roof of Paris 6-Paris 7 universities (Jussieu campus, Paris 5th district)
Keywords: Air sample pollution; Atmospheric precipitation pollution; Pesticide traces; Toxicity measurements; Bioluminescence method

Focus on RNA analysis by Sapna Deo (1107-1108).
has been an Assistant Professor of Bioanalytical Chemistry in the Department of Chemistry and Chemical Biology at Indiana University–Purdue University, Indianapolis, since 2005. She is author and co-author of over 55 scientific publications and several patents and an editor of the book “Photoproteins in Bioanalysis”. Dr Deo’s research interest is in the development of novel bioanalytical techniques for detection of microRNAs and RNA molecules, based on luminescence detection, for application in diagnosis and pathogen detection. Other areas of research interest in Dr Deo’s laboratory include development of molecular probes for biosensing and bioimaging applications. Dr Deo’s research is funded by the National Science Foundation and the National Institutes of Health.

Trends in microRNA detection by Kyle A. Cissell; Sapna K. Deo (1109-1116).
MicroRNAs (miRNAs) are short, ~22 nucleotide length RNAs that perform gene regulation. Recently, miRNA has been shown to be linked with the onset of cancer and other diseases based on miRNA expression levels. It is important, therefore, to understand miRNA function as it pertains to disease onset; however, in order to fully understand miRNA’s role in a disease, it is necessary to detect the expression levels of these small molecules. The most widely used miRNA detection method is Northern blotting, which is considered as the standard of miRNA detection methods. This method, however, is time-consuming and has low sensitivity. This has led to an increase in the amount of detection methods available. These detection methods are either solid phase, occurring on a solid support, or solution phase, occurring in solution. While the solid-phase methods are adaptable to high-throughput screening and possess higher sensitivity than Northern blotting, they lack the ability for in vivo use and are often time-consuming. The solution-phase methods are advantageous in that they can be performed in vivo, are very sensitive, and are rapid; however, they cannot be applied in high-throughput settings. Although there are multiple detection methods available, including microarray technology, luminescence-based assays, electrochemical assays, etc., there is still much work to be done regarding miRNA detection. The current gaps of miRNA detection include the ability to perform multiplex, sensitive detection of miRNA with single-nucleotide specificity along with the standardization of these new methods. Current miRNA detection methods, gaps in these methods, miRNA therapeutic options, and the future outlook of miRNA detection are presented here.
Keywords: miRNA; Bioprobe; RNAi

MicroRNA detection by microarray by Wei Li; Kangcheng Ruan (1117-1124).
MicroRNAs (miRNAs) are a class of small noncoding RNAs ∼22 nt in length that regulate gene expression and play fundamental roles in multiple biological processes, including cell differentiation, proliferation and apoptosis as well as disease processes. The study of miRNA has thus become a rapidly emerging field in life science. The detection of miRNA expression is a very important first step in miRNA exploration. Several methodologies, including cloning, northern blotting, real-time RT-PCR, microRNA arrays and ISH (in situ hybridization), have been developed and applied successfully in miRNA profiling. This review discusses the main existing microRNA detection technologies, while emphasizing microRNA arrays.
Keywords: MicroRNA; MicroRNA detection; MicroRNA microarray; MicroRNA labeling

Individual transfer ribonucleic acids (tRNAs) in a complex mixture can be identified by the matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) detection of their signature digestion products. Signature digestion products are endonuclease digestion products whose mass-to-charge value is unique thus corresponding to only a single tRNA. To improve the effectiveness of this approach, we have expanded the applicable endonucleases and examined the use of multiple endonucleases for tRNA identification. The purine specific endonucleases RNase T1 and RNase TA generate the largest number of predicted signature digestion products. Experimentally, MALDI-MS analysis of endonuclease digests from Escherichia coli and Bacillus subtilis finds that any two endonucleases used in combination increases tRNA identification by about 25% over the number identified with a single endonuclease. Using three endonucleases, RNase T1, RNase A, and RNase TA, further improves the number of tRNAs identified by 10–15% over those found with two endonucleases. Limitations in the MALDI-MS approach for complex mixtures were revealed in this study, suggesting that the direct MALDI-MS analysis of signature digestion products is more effective for organisms having 30 or less unique tRNAs. Figure Signature digestion products for tRNACys
Keywords: Transfer RNA; Isoaccepting tRNAs; Signature digestion product; Ribonuclease digestion; RNase T1; RNase A; RNase TA; MALDI-MS

Competitive binding of cadmium by plant thiols: an electrochemical study assisted by multivariate curve resolution by Rui Gusmão; Cristina Ariño; José Manuel Díaz-Cruz; Miquel Esteban (1137-1145).
Multivariate curve resolution with alternating least squares (MCR-ALS) has been applied to voltammetric data obtained from analysis of the competitive binding of cysteine (Cys) and cysteine–glycine (Cys-Gly) by Cd(II) as a first approach towards mixtures of phytochelatins and related compounds in natural media. From different starting points, the possibilities of formation of mixed complexes and/or displacements between ligands are investigated. Analysis of the resulting unitary voltammograms and concentration profiles of the resolved components by MCR-ALS suggests that the strongest ligand (Cys-Gly) is able to displace the weakest (Cys) from its metal complexes, whereas this does not happen in the opposite direction. On the other hand, no evidence of Cd mixed-ligand complexes was found. Figure Differential pulse polarograms measured in the independent titrations of 1 × 10-5 mol L-1 Cys, 1 × 10-5 mol L-1 Cys-Gly, and a mixture of Cys-Gly (0.5 × 10-5 mol L-1) and Cys (1 × 10-5 mol L-1) with Cd2+, at TRIS-HNO3 buffer (0.1 mol L-1 and PH 7.5) in the presence of 0.1 mol L-1 KNO3
Keywords: Plant thiols; Cadmium; Competitive binding; Multivariate curve resolution with alternating least squares; Differential pulse voltammetry; ESI-MS

The carbamates are a well-known thermosensible pesticides class, which are highly prone to degradation via fragmentation and/or rearrangement mechanisms leading to a difficult direct gas chromatography (GC) analysis, i.e., without derivatization. In this paper, spermine and thiabendazole both at 1 mg/mL were highlighted as efficient analyte protectants to improve the direct and simultaneous analysis of 16 carbamates both in solvent and green vegetable matrices. These two molecules were compared in mixture or in combination with three well-known efficient analyte protectants 3-ethoxy-1,2-propanediol, d-sorbitol, and l-gulonic acid-γ-lactone. The potential benefits were investigated in GC hyphenated to mass spectrometry (GC–MS) with two injection modes: programmable temperature vaporizing injector in a solvent split mode (PTV-SSI) and on-column injection (OCI). It was shown that the combined effect of the five protective agents led to the best sensitivity improvement with limits of detection between 0.1–0.4 and 0.03–0.1 μg/kg and limits of quantification between 0.3–1.1 and 0.1–0.5 μg/kg for PTV-SSI and OCI mode, respectively. The correlation coefficients from the analyzed 1–500 μg/kg range were all >0.999 both in the solvent and matrices studied. The recoveries of carbamates from three spiked matrices over five replicates at 20 and 100 µg/kg were in the range 90–107% with relative standard deviation (RSD) equal to 2–7% for PTV-SSI and 92–107% with an RSD equal to 1–6% for OCI. The use of spermine and thiabendazole with other analyte protectants shows very efficient partial or total reduction of breakdown of the most sensitive carbamates such as the N-sulfenylated ones.
Keywords: Pesticides; Carbamates; GC–MS; On-column injection; Analyte protectants

Automated on-line in-tube solid-phase microextraction coupled with HPLC/MS/MS for the determination of butyrophenone derivatives in human plasma by Takeshi Kumazawa; Koichi Saeki; Isao Yanagisawa; Seisaku Uchigasaki; Chika Hasegawa; Hiroshi Seno; Osamu Suzuki; Keizo Sato (1161-1170).
This paper describes a fully automated on-line method combining in-tube solid-phase microextraction (SPME) in which sample clean-up and enrichment are conducted through an open tubular fused-silica capillary column and high-performance liquid chromatography (HPLC)/tandem mass spectrometry (MS/MS) detection for the determination of six butyrophenone derivatives (moperone, floropipamide, haloperidol, spiroperidol, bromperidol, and pimozide) in human plasma samples. The six butyrophenones were extracted by repeatedly aspirating and dispensing plasma sample solutions on a DB-17 capillary column (60 cm × 0.32 mm i.d., film thickness 0.25 µm). The analytes retained on the inner surface of the capillary column were then eluted into an acetonitrile-rich mobile phase using a gradient separation technique. Extraction efficiencies ranged from 12.7% to 31.8% for moperone, spiroperidol, and pimozide, and from 1.08% to 4.86% for floropipamide, haloperidol, and bromperidol. The regression equations for all compounds showed excellent linearity, ranging from 0.05 to 50 ng/0.1 mL of plasma, except for moperone and spiroperidol (0.01 to 50 ng/0.1 mL). The limits of detection and quantification in plasma for each drug were 0.03–0.2 and 0.1–0.5 ng/mL, respectively. The intra- and inter-day coefficients of variation for all compounds in plasma were not greater than 13.7%.
Keywords: In-tube SPME; Butyrophenone; HPLC; MS/MS

A sensitive, specific and efficient high-performance liquid chromatography-tandem mass spectrometry assay for the simultaneous determination of vincristine and actinomycin-D in human dried blood spots is presented. Dried blood spots were punched out of a collection paper with a 0.25-in.-diameter punch. The analytes were extracted from the punched-out disc using sonication during 15 min in a mixture of acetonitrile–methanol–water (1:1:1, v/v/v) containing the internal standard vinorelbine. Twenty-microlitre volumes were injected onto the HPLC system. Separation was achieved on a 50 × 2.1 mm ID Xbridge C18 column using elution with 1 mM ammonium acetate–acetonitrile (70:30, v/v) adjusted to pH 10.5 with ammonia and run in a gradient with methanol at a flow rate of 0.4 mL/min. HPLC run time was 6 min. The assay quantifies vincristine from 1 to 100 ng/mL and actinomycine-D from 2 to 250 ng/mL using a blood sample obtained by a simple finger prick. Validation results demonstrate that vincristine and actinomycin-D can be accurately and precisely quantified in human dried blood spots with the presented method. The assay can now be used to support clinical pharmacologic studies with vincristine and actinomycin-D.
Keywords: Dried blood spot; Filter paper; Collection cards; Vincristine; Actinomycin-D; Liquid chromatography; Mass spectrometry; Electrospray ionisation; ESI-LS-MS/MS

Production and certification of NIST Standard Reference Material 2372 Human DNA Quantitation Standard by Margaret C. Kline; David L. Duewer; John C. Travis; Melody V. Smith; Janette W. Redman; Peter M. Vallone; Amy E. Decker; John M. Butler (1183-1192).
Modern highly multiplexed short tandem repeat (STR) assays used by the forensic human-identity community require tight control of the initial amount of sample DNA amplified in the polymerase chain reaction (PCR) process. This, in turn, requires the ability to reproducibly measure the concentration of human DNA, [DNA], in a sample extract. Quantitative PCR (qPCR) techniques can determine the number of intact stretches of DNA of specified nucleotide sequence in an extremely small sample; however, these assays must be calibrated with DNA extracts of well-characterized and stable composition. By 2004, studies coordinated by or reported to the National Institute of Standards and Technology (NIST) indicated that a well-characterized, stable human DNA quantitation certified reference material (CRM) could help the forensic community reduce within- and among-laboratory quantitation variability. To ensure that the stability of such a quantitation standard can be monitored and that, if and when required, equivalent replacement materials can be prepared, a measurement of some stable quantity directly related to [DNA] is required. Using a long-established conventional relationship linking optical density (properly designated as decadic attenuance) at 260 nm with [DNA] in aqueous solution, NIST Standard Reference Material (SRM) 2372 Human DNA Quantitation Standard was issued in October 2007. This SRM consists of three quite different DNA extracts: a single-source male, a multiple-source female, and a mixture of male and female sources. All three SRM components have very similar optical densities, and thus very similar conventional [DNA]. The materials perform very similarly in several widely used gender-neutral assays, demonstrating that the combination of appropriate preparation methods and metrologically sound spectrophotometric measurements enables the preparation and certification of quantitation [DNA] standards that are both maintainable and of practical utility. Figure NIST Standard Reference Material (SRM) 2372 Human Quantitation Standard
Keywords: Certified reference material (CRM); Decadic attenuance; Forensic; Human identity; Interlaboratory comparison; Short tandem repeat (STR) multiplex assay; Standard Reference Material (SRM); UV/visible absorbance spectrophotometry

Simultaneous on-line size and chemical analysis of gas phase and particulate phase of cigarette mainstream smoke by Thomas Adam; John McAughey; Conor McGrath; Christoph Mocker; Ralf Zimmermann (1193-1203).
This paper describes the combined set-up of on-line chemical analysis of gas phase by single-photon ionisation/resonance enhanced multiphoton ionisation–time-of-flight mass spectrometry (SPI/REMPI-TOFMS) and on-line particle size analysis by differential electrical mobility particle spectrometry (DMS 500) for the investigation of fresh cigarette mainstream smoke. SPI is well suited for the investigation of a great variety of organic species, whereas REMPI is highly sensitive for aromatic compounds. Gas phase measurements of filtered and unfiltered smoke are possible with the SPI/REMPI-TOFMS in order to determine the influence of the presence of particles on the chemical composition of the gas phase. Initial results are shown for the characterisation and comparison of three pure Virginia tobacco research cigarettes having filter ventilations of 0%, i.e. no filter ventilation, 35% and 70% ventilation. The three cigarette types are smoked under two different smoking regimes, a standard regime using puff parameters equivalent to the conventional International Standard Organisation regime and a more intense smoking regime. For the gas phase, qualitative puff-by-puff resolved yields of three selected compounds (acetaldehyde, phenol and styrene) are shown and compared. For particulate matter, particle number, count median diameter and total surface area are illustrated on a puff-by-puff basis. Yields of the chemicals analysed, puff number and surface area are in good agreement with the intensity of the smoking regime and the dilution of smoke by filter ventilation. However, gaseous compounds are influenced differently, depending whether an absolute particle filter is present or not, i.e. they can be totally removed (phenol), partially removed (styrene) or not affected (acetaldehyde). For particle analysis, the count median diameter decreases from puff to puff and is strongly dependent on the smoking regime and ventilation rate. Thereby, 0% ventilated cigarettes smoked under the intense regime result in the smallest count median diameters of ca. 180 nm, whereas 70% ventilated cigarettes smoked with a standard regime lead to the largest values of up to 280 nm. As particle diameter increases, particle number decreases as a consequence of increasing time for particle coagulation.
Keywords: Photoionisation; Electrical mobility particle spectrometry; Cigarette smoke; Tobacco smoke; Gas phase; Particulate phase; Particle diameter

Real-time immuno-PCR assay for detecting PCBs in soil samples by Han-Yu Chen; Hui-Sheng Zhuang (1205-1211).
A fast and robust assay, based on immuno-polymerase chain reaction (IPCR) techniques, was developed for the detection of polychlorinated biphenyls (PCBs) in soil samples. Real-time IPCR (rt-IPCR) is a powerful technique that combines enzyme-linked immunosorbent assay (ELISA) with the specificity and sensitivity of PCR. In our assay, indirect ELISAs based on immobilization of PCB37 hapten–ovalbumin conjugates was used for evaluation of the immune response. The effect of optimal reagent concentrations to reduce background fluorescence was also investigated. Using the optimized assay, the linear sensitivity range of the assay covered more than six orders of magnitude, and the minimum detection limits reached 5 fg ml–1 antigen. Rt-IPCR was tested for its cross-reactivity profiles using four selected congeners and four Aroclor products. The assays were highly specific for congeners but less specific for Aroclor1242. We took four soil samples to validate the method, and the results were confirmed by gas chromatography/mass spectrometry (GC/MS). The rt-IPCR results for soil samples correlated well with the concentrations of PCBs obtained by GC/MS (r = 0.99, n = 6). These data indicate that this highly specific, sensitive, and robust assay can be modified for detecting PCB compounds in the environment.
Keywords: Immunoassays; Real-time-immuno-polymerase-chain-reaction; Polychlorinated biphenyls limit of detection

Solid phase extraction for removal of matrix effects in lipophilic marine toxin analysis by liquid chromatography-tandem mass spectrometry by Arjen Gerssen; Mairead A. McElhinney; Patrick P. J. Mulder; Ronel Bire; Philipp Hess; Jacob de Boer (1213-1226).
The potential of solid phase extraction (SPE) clean-up has been assessed to reduce matrix effects (signal suppression or enhancement) in the liquid chromatography-tandem mass spectrometry (LC–MS/MS) analysis of lipophilic marine toxins. A large array of ion-exchange, silica-based, and mixed-function SPE sorbents was tested. Polymeric sorbents were found to retain most of the toxins. Optimization experiments were carried out to maximize recoveries and the effectiveness of the clean-up. In LC–MS/MS analysis, the observed matrix effects can depend on the chromatographic conditions used, therefore, two different HPLC methods were tested, using either an acidic or an alkaline mobile phase. The recovery of the optimized SPE protocol was around 90% for all toxins studied and no break-through was observed. The matrix effects were determined by comparing signal response from toxins spiked in crude and SPE-cleaned extracts with those derived from toxins prepared in methanol. In crude extracts, all toxins suffered from matrix effects, although in varying amounts. The most serious effects were observed for okadaic acid (OA) and pectenotoxin-2 (PTX2) in the positive electrospray ionization mode (ESI+). SPE clean-up on polymeric sorbents in combination with the alkaline LC method resulted in a substantial reduction of matrix effects to less than 15% (apparent recovery between 85 and 115%) for OA, yessotoxin (YTX) in ESI and azaspiracid-1 (AZA1), PTX2, 13-desmethyl spirolides C (SPX1), and gymnodimine (GYM) in ESI+. In combination with the acidic LC method, the matrix effects after SPE were also reduced but nevertheless approximately 30% of the matrix effects remained for PTX2, SPX1, and GYM in ESI+. It was concluded that SPE of methanolic shellfish extracts can be very useful for reduction of matrix effects. However, the type of LC and MS methods used is also of great importance. SPE on polymeric sorbents in combination with LC under alkaline conditions was found the most effective method.
Keywords: Lipophilic marine toxins; Solid phase extraction; Shellfish; Matrix effects

Herein, we introduce the fabrication of a micro-perforated elastomeric poly(dimethylsiloxane) (PDMS) mask and employ it for spatially defined surface modification. To fabricate the micro-perforated PDMS mask, high-aspect-ratio micro-pillar arrays having millimeter scale height were first fabricated via direct photopolymerization using a thiol–ene-based UV-curable adhesive. Square pillars (500 × 500 µm) and 200 µm circular pillars with 5 and 12.5 in the aspect ratios, respectively, were successfully fabricated with high pattern fidelity, reaching 2.5mm in height. Next, using the micro-pillar-array platform as a master mold, PDMS prepolymer was cast and polymerized to form an elastomeric PDMS mask having micro-perforation arrays. Alternating hydrophilic and hydrophobic surfaces were successfully obtained by oxidizing PDMS-covered Si wafer with corona discharge. Spatially defined chemical functionalities obtained by selective oxidation and subsequent silanization were clearly distinguished via colorimetric detection methods employing ninhydrin and toluidine reagents. The micro-perforated elastomeric PDMS mask enables selective modification of a surface without utilizing photoreactive chemicals and a photomask.
Keywords: High-aspect-ratio micro-pillar arrays; Thiol–ene adhesive; Micro-perforation array; Elastomeric PDMS mask; Selective surface modification; Microarray platform