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Analytical and Bioanalytical Chemistry (v.400, #10)

Self-assessment and controlled examination in analytical chemistry by use of the EChemTest by Miltiades I. Karayannis; Constantinos E. Efstathiou (pp. 3181-3185).

is Professor emeritusof the University of Ioannina (Chem. Dept., 1979–2001). He received his BSc. from the Chemistry Department of the Aristotelian University of Thessaloniki (1958) and his PhD from University of Bonn (1965). He worked at Brookhaven National Lab. (1966–68), New York University (1967–1968), and University of Athens (1970–1979). His research is on electrochemistry, kinetic methods of analysis, spectroscopic methods, chemical- and biosensors, FIA, environmental analysis and chromatography. He is the author of two books (Chemical Instrumentation and Treatment Evaluation and Presentation of Analytical Data), co-author of the textbook ‘Analytical Chemistry’ and author of more than 150 original scientific publications. received his BSc and PhD from the University of Athens, Greece, in 1971 and 1976, respectively. He is Professor of Chemistry and he is currently the Director of the Analytical Chemistry Laboratory of the Department of Chemistry in the University of Athens. He has about 100 scientific publications on topics including electroanalytical techniques, kinetic methods of analysis, chemometrics and application of microcomputers in the automation of analytical measurements. He is author and co-author of books on analytical chemistry, electroanalytical techniques, chemical instrumentation and chemometrics.

Ericka A. Pestana, Sandor Belak, Adama Diallo, John R. Crowther and Gerrit J. Viljoen (Eds.): Early, rapid and sensitive veterinary molecular diagnostics—real time PCR applications by Mara Mirasoli (pp. 3187-3188).

Robert L. Wixom, Charles W. Gehrke (Eds.): Chromatography. A science of discovery by Jason W. Coym (pp. 3189-3190).

Laser-induced breakdown spectroscopy (LIBS) by Jagdish P. Singh; José R. Almirall; Mohamad Sabsabi; Andrzej W. Miziolek (pp. 3191-3192).

is a Research Professor and Associate Director-Laser Diagnostics of Institute for Clean Energy Technology, Mississippi State University. He received his MS and Ph.D. degree in Physics from Banaras Hindu University, India, in 1974 and 1980. He was a Post-doctoral Research Associate at McMaster University, Canada, and Kansas State University. Prof. Singh has authored one book on LIBS and over 150 peer-reviewed scientific publications presented over 160 papers and supervised over 20 PhD and MS students in Applied Physics. He is a Fellow of OSA and LASSI. His research interest includes Laser Spectroscopy, Optical Fiber Sensors, Laser Diagnostics for Combustion, and Hazardous Waste Management. is a Professor in the Department of Chemistry and Biochemistry and Director of the International Forensic Research Institute at Florida International University. He was a practicing forensic scientist at the Miami-Dade Police Department Crime Laboratory for 12 years where he testified in over 100 criminal cases in state and federal courts prior to his academic appointment at FIU in 1998. Professor Almirall has authored one book and over 90 peer-reviewed scientific publications in the field of analytical and forensic chemistry, presented over 400 papers and workshops, and mentored over 30 PhD and MS students in forensic chemistry research. His research interests include the development of analytical chemistry tools such as LIBS for use in forensic science and improvements in the value of trace evidence, in particular. is the leader of the Optical Diagnostics Group at the National Research Council of Canada (NRCC) in Boucherville, Canada. His research interests include the industrial exploitation of laser-based spectroscopic techniques such as LIBS, LIFS, RELIBS, and LA, and modeling of the micro-plasma generated by laser ablation. The R&D activities range from fundamental studies, via the development of prototype devices up to the transfer of laser measurement systems into industrial applications for online inspection tasks. He holds 11 patents and has published more than 330 scientific papers in refereed international journals, conference proceedings, conferences, reports, and contributions to two books, covering fundamental aspects and industrial applications of the laser-induced plasma. is a senior Research Physicist at the US Army Research Laboratory. He has led the application of LIBS to the security and protection areas, as well as fostered the development of new technology (broadband high-resolution spectrometer, microwave enhancement) and the adaptation of chemometrics for spectral data processing. He has organized a number of conferences including the LIBS 2002 (2nd International) Conference and he founded the NASLIBS (North American Symposium on LIBS) series. He holds a Ph.D. from the University of California, Berkeley, is a Fellow of the Optical Society of America, and has published three books including "LIBS, Fundamentals and Applications," A.W. Miziolek, V. Palleschi, and I. Schechter, eds., Cambridge University Press, 2006.

Kinetic model of atomic and molecular emissions in laser-induced breakdown spectroscopy of organic compounds by Qianli Ma; Paul J. Dagdigian (pp. 3193-3205).

A kinetic model previously developed to predict the relative intensities of atomic emission lines in laser-induced breakdown spectroscopy has been extended to include processes related to CN and C₂ molecular emissions. Simulations with this model were performed to predict the relative excited-state populations. The results from the simulations are compared with experimentally determined excited-state populations from 1,064 nm laser irradiation of organic residues on aluminum foil. The model reasonably predicts the relative intensity of the molecular emissions. Significantly, the model reproduces the vastly different temporal profiles of the atomic and molecular emissions. The latter are found to extend to much longer times after the laser pulse, and this appears to be due to the increasing concentration of the molecules versus time. From the simulations, the important processes affecting the CN and C₂ concentrations are identified. Figure Molecular emission in laser-induced breakdown spectroscopy of organic compounds

Keywords: Laser-induced breakdown spectroscopy; Plasmas; Laser ablation; Kinetic model

Comparison of single and double-pulse excitation during the earliest stage of laser induced plasma by L. Nagli; M. Gaft; I. Gornushkin (pp. 3207-3216).

This paper deals with comparison of single-pulse (SP) and double-pulse (DP) excited plasmas during their earliest phase of life, from 0 to 500 ns. The samples are Si and Al and the irradiance per pulse is approximately 20 GW cm⁻² under ambient conditions. It was found that at the beginning of a plasma lifespan, Si III and Al III ions were excited even under the SP excitation conditions and became much more abundant under DP excitation. The lifetime of doubly-ionized species was found to be very short, and after a delay of 200–300 ns they were not detected. The DP plasma differs from the SP plasma by the intensity of continuum radiation and by the width of emission lines, which are substantially narrower in the DP plasma. The intensity of light emitted from singly and, especially, doubly-ionized species is higher compared with neutral atoms; whereas the decay time of both neutral and ionized species is longer. An excitation mechanism for the DP plasma is proposed enabling qualitative and a quantitative explanation of the experimental data.

Keywords: Laser induced plasma; Single ionization; Double ionization; Collision dominated plasma model

Effect of atmosphere on collinear double-pulse laser-induced breakdown spectroscopy by Andrew J. Effenberger Jr.; Jill R. Scott (pp. 3217-3227).

Double-pulse laser-induced breakdown spectroscopy (DP-LIBS) has been shown to enhance LIBS spectra. Several researchers have reported significant increases in signal-to-noise and/or spectral intensity compared to single-pulse (SP) LIBS. In addition to DP-LIBS, atmospheric conditions can also increase sensitivity. Thus, in this study, a collinear DP-LIBS scheme was used along with manipulation of the atmospheric conditions. The DP-LIBS scheme consisted of an initial 45-mJ pulse at 1,064-nm fired into a sample contained in a controlled atmospheric/vacuum chamber. A second analytical 45-mJ pulse at 1,064-nm was then fired 0 to 200 μs after and along the same path of the first pulse. Ar, He, and air at pressures ranging from atmospheric pressure to 1 Torr are introduced during DP-LIBS and SP-LIBS experiments. For a brass sample, significant increases in the spectral intensities of Cu and Zn lines were observed in DP-LIBS under Ar compared to DP-LIBS in air. It was also found that Cu and Zn lines acquired with SP-LIBS in Ar are nearly as intense as DP-LIBS in air. While collinear DP-LIBS is effective for increasing the sensitivity for some reduced atmospheres (i.e., Ar and air at 630 to 100 Torr and He at 300 Torr), the enhanced spectral intensity ultimately dropped off as the pressure was reduced below 10 Torr for all atmospheric compositions in the experimental arrangement used in this study. At all pressures of air and Ar, the plasma temperature remained rather constant with increased inter-pulse delays; however, the plasma temperature was more variable for different He gas pressures and inter-pulse delays. Figure Illustration of the dependence of DP-LIBS signal intensity with changes in buffer gas composition

Keywords: DP-LIBS; Helium; Argon; Air; Reduced pressure

Doubly ionized ion emission in laser-induced breakdown spectroscopy in air by M. Gaft; L. Nagli; I. Gornushkin; Y. Groisman (pp. 3229-3237).

The emission from doubly ionized species in laser-induced plasmas has not been properly investigated before since most analytical measurements were made at relatively long delays. This work proves that doubly ionized species, such as boron (B) III and iron (Fe) III, can exist during the first 150–200 ns of the plasma lifetime in plasmas produced in air by typical lasers with irradiances of 10⁹–10¹¹ W/cm². The emission from these ions was detected using both the double- and single-pulse excitations. The sum of the second ionization potential and the energy of corresponding excited states is approximately 30 eV. The presence of doubly charged ions in the early plasma was additionally confirmed by computer simulations using a collision-dominated plasma model. The emission from doubly ionized species may be used for analytical purpose. For example, in the spectrum from a B–Fe ore, the B III analytical line at 206.6 nm is free from Fe spectral interference thus enabling the online laser-induced breakdown spectroscopy sorting of ores into three products with high, medium, and low B₂O₃ contents.

Keywords: Plasma; Double ionization; Boron; Iron; Online; Sorting

Spatial and temporal variations of electron temperatures and densities from EUV-emitting lithium plasmas by R. W. Coons; S. S. Harilal; M. Polek; A. Hassanein (pp. 3239-3246).

Planar slabs of pure Li were irradiated with 1.064 nm, 6 ns Nd:YAG laser pulses. Determination of plasma densities at both the earliest times of plasma formation and near the target surface was performed using Nomarski interferometry. The plasma parameters at later times were evaluated using optical emission spectroscopy. The space- and time-dependent electron densities and temperatures of the plasma were determined from their Stark broadening and the relative intensities of the spectral lines, respectively. The advantages and disadvantages of both of these techniques are evaluated and discussed.

Keywords: EUV lithography; Laser-produced plasma; Plasma diagnostics; Emission spectroscopy

Nonlinear mapping technique for data visualization and clustering assessment of LIBS data: application to ChemCam data by J. Lasue; R. C. Wiens; T. F. Stepinski; O. Forni; S. M. Clegg; S. Maurice (pp. 3247-3260).

ChemCam is a remote laser-induced breakdown spectroscopy (LIBS) instrument that will arrive on Mars in 2012, on-board the Mars Science Laboratory Rover. The LIBS technique is crucial to accurately identify samples and quantify elemental abundances at various distances from the rover. In this study, we compare different linear and nonlinear multivariate techniques to visualize and discriminate clusters in two dimensions (2D) from the data obtained with ChemCam. We have used principal components analysis (PCA) and independent components analysis (ICA) for the linear tools and compared them with the nonlinear Sammon’s map projection technique. We demonstrate that the Sammon’s map gives the best 2D representation of the data set, with optimization values from 2.8% to 4.3% (0% is a perfect representation), together with an entropy value of 0.81 for the purity of the clustering analysis. The linear 2D projections result in three (ICA) and five times (PCA) more stress, and their clustering purity is more than twice higher with entropy values about 1.8. We show that the Sammon’s map algorithm is faster and gives a slightly better representation of the data set if the initial conditions are taken from the ICA projection rather than the PCA projection. We conclude that the nonlinear Sammon’s map projection is the best technique for combining data visualization and clustering assessment of the ChemCam LIBS data in 2D. PCA and ICA projections on more dimensions would improve on these numbers at the cost of the intuitive interpretation of the 2D projection by a human operator. Figure Sammon’s map showing the best 2D representation of a set of LIBS spectra.

Keywords: ChemCam; Laser-induced breakdown spectroscopy (LIBS); Multivariate analysis; Sammon’s map; Mars Science Laboratory; Geological samples

A PLS model based on dominant factor for coal analysis using laser-induced breakdown spectroscopy by Jie Feng; Zhe Wang; Logan West; Zheng Li; Weidou Ni (pp. 3261-3271).

Thirty-three bituminous coal samples were utilized to test the application of laser-induced breakdown spectroscopy technique for coal elemental concentration measurement in the air. The heterogeneity of the samples and the pyrolysis or combustion of coal during the laser–sample interaction processes were analyzed to be the main reason for large fluctuation of detected spectra and low calibration quality. Compared with the generally applied normalization with the whole spectral area, normalization with segmental spectral area was found to largely improve the measurement precision and accuracy. The concentrations of major element C in coal were determined by a novel partial least squares (PLS) model based on dominant factor. Dominant C concentration information was taken from the carbon characteristic line intensity since it contains the most-related information, even if not accurately. This dominant factor model was further improved by inducting non-linear relation by partially modeling the inter-element interference effect. The residuals were further corrected by PLS with the full spectrum information. With the physical-principle-based dominant factor to calculate the main quantitative information and to partially explicitly include the non-linear relation, the proposed PLS model avoids the overuse of unrelated noise to some extent and becomes more robust over a wider C concentration range. Results show that RMSEP in the proposed PLS model decreased to 4.47% from 5.52% for the conventional PLS with full spectrum input, while R ² remained as high as 0.999, and RMSEC&P was reduced from 3.60% to 2.92%, showing the overall improvement of the proposed PLS model.

Keywords: Laser-induced breakdown spectroscopy; Bituminous coal; Dominant factor; Partial least squares

Elemental misinterpretation in automated analysis of LIBS spectra by Waldemar Hübert; Georg Ankerhold (pp. 3273-3278).

In this work, the Stark effect is shown to be mainly responsible for wrong elemental allocation by automated laser-induced breakdown spectroscopy (LIBS) software solutions. Due to broadening and shift of an elemental emission line affected by the Stark effect, its measured spectral position might interfere with the line position of several other elements. The micro-plasma is generated by focusing a frequency-doubled 200 mJ pulsed Nd/YAG laser on an aluminum target and furthermore on a brass sample in air at atmospheric pressure. After laser pulse excitation, we have measured the temporal evolution of the Al(II) ion line at 281.6 nm (4s 1 S-3p 1 P) during the decay of the laser-induced plasma. Depending on laser pulse power, the center of the measured line is red-shifted by 130 pm (490 GHz) with respect to the exact line position. In this case, the well-known spectral line positions of two moderate and strong lines of other elements coincide with the actual shifted position of the Al(II) line. Consequently, a time-resolving software analysis can lead to an elemental misinterpretation. To avoid a wrong interpretation of LIBS spectra in automated analysis software for a given LIBS system, we recommend using larger gate delays incorporating Stark broadening parameters and using a range of tolerance, which is non-symmetric around the measured line center. These suggestions may help to improve time-resolving LIBS software promising a smaller probability of wrong elemental identification and making LIBS more attractive for industrial applications. Figure Misinterpretation of a LIBS analysis due to the temporal spectral evolution of a red-shifted aluminum line inclenced by the Stark effect

Keywords: Laser-induced breakdown spectroscopy; Stark effect; Elemental analysis

Direct analysis of powder samples using transversely excited atmospheric CO₂ laser-induced gas plasma at 1 atm by Ali Khumaeni; Zener Sukra Lie; Hideaki Niki; Koo Hendrik Kurniawan; Efendy Tjoeng; Yong Inn Lee; Kazuyoshi Kurihara; Yoji Deguchi; Kiichiro Kagawa (pp. 3279-3287).

A novel method for the direct and sensitive analysis of powder samples has been developed by utilizing the characteristics of a transversely excited atmospheric (TEA) CO₂ laser. In this study, a powder sample was placed in a container and covered by a metal mesh; the metal mesh functions to control the blowing-off of the powder. The container was then perpendicularly attached on a metal surface. When a TEA CO₂ laser (1.5 J, 200 ns) was focused on the metal surface, a large hemispherical gas plasma (radius of around 8 mm) with long emission lifetime (several tens of microseconds) was produced without ablating the metal surface. The high-speed expansion force of the gas plasma samples the powder covered by the metal mesh and fine powder particles are sent into the gas plasma region to be dissociated and excited. Sensitive semi-quantitative analysis was made on organic powder samples such as powdered rice, starch, seaweed (agar), and supplements. The detection limit of heavy metals of Cr in powdered mineral supplement was approximately 0.55 mg/kg.

Keywords: Laser-induced breakdown spectroscopy (LIBS); Laser-induced gas plasma; Transversely excited atmospheric CO₂ laser (TEA CO₂ laser); Direct analysis of powder sample; Food powder sample

Discrimination of biological and chemical threat simulants in residue mixtures on multiple substrates by Jennifer L. Gottfried (pp. 3289-3301).

The potential of laser-induced breakdown spectroscopy (LIBS) to discriminate biological and chemical threat simulant residues prepared on multiple substrates and in the presence of interferents has been explored. The simulant samples tested include Bacillus atrophaeus spores, Escherichia coli, MS-2 bacteriophage, α-hemolysin from Staphylococcus aureus, 2-chloroethyl ethyl sulfide, and dimethyl methylphosphonate. The residue samples were prepared on polycarbonate, stainless steel and aluminum foil substrates by Battelle Eastern Science and Technology Center. LIBS spectra were collected by Battelle on a portable LIBS instrument developed by A3 Technologies. This paper presents the chemometric analysis of the LIBS spectra using partial least-squares discriminant analysis (PLS-DA). The performance of PLS-DA models developed based on the full LIBS spectra, and selected emission intensities and ratios have been compared. The full-spectra models generally provided better classification results based on the inclusion of substrate emission features; however, the intensity/ratio models were able to correctly identify more types of simulant residues in the presence of interferents. The fusion of the two types of PLS-DA models resulted in a significant improvement in classification performance for models built using multiple substrates. In addition to identifying the major components of residue mixtures, minor components such as growth media and solvents can be identified with an appropriately designed PLS-DA model. Figure Laser - induced breakdown spectroscopy (LIBS) is a promising technique under development for the detection of hazardous materials. LIBS offers the potential for real-time chemical and biological threat detection in the field for military and civilian first responders.

Keywords: LIBS; PLS-DA; Biological warfare agents; Chemical warfare agents; Residue detection

Comparative measurements of mineral elements in milk powders with laser-induced breakdown spectroscopy and inductively coupled plasma atomic emission spectroscopy by W. Q. Lei; J. El Haddad; V. Motto-Ros; N. Gilon-Delepine; A. Stankova; Q. L. Ma; X. S. Bai; L. J. Zheng; H. P. Zeng; J. Yu (pp. 3303-3313).

Mineral elements contained in commercially available milk powders, including seven infant formulae and one adult milk, were analyzed with inductively coupled plasma atomic emission spectrometry (ICP-AES) and laser-induced breakdown spectroscopy (LIBS). The purpose of this work was, through a direct comparison of the analytical results, to provide an assessment of the performance of LIBS, and especially of the procedure of calibration-free LIBS (CF-LIBS), to deal with organic compounds such as milk powders. In our experiments, the matrix effect was clearly observed affecting the analytical results each time laser ablation was employed for sampling. Such effect was in addition directly observed by determining the physical parameters of the plasmas induced on the different samples. The CF-LIBS procedure was implemented to deduce the concentrations of Mg and K with Ca as the internal reference element. Quantitative analytical results with CF-LIBS were validated with ICP-AES measurements and nominal concentrations specified for commercial milks. The obtained good results with the CF-LIBS procedure demonstrate its capacity to take into account the difference in physical parameters of the plasma in the calculation of the concentrations of mineral elements, which allows a significant reduction of the matrix effect related to laser ablation. We finally discuss the way to optimize the implementation of the CF-LIBS procedure for the analysis of mineral elements in organic materials.

Keywords: LIBS; CF-LIBS; ICP-AES; Matrix effect; Milk powder

Quantitative analysis of slurry sample by laser-induced breakdown spectroscopy by Krishna K. Ayyalasomayajula; Vivek Dikshit; Fang Yu Yueh; Jagdish P. Singh; Laura T. Smith (pp. 3315-3322).

Laser-induced breakdown spectroscopy (LIBS) has been employed for the analysis of slurry samples. Quantitative analysis of slurry samples is crucial and challenging. The problems associated with slurry samples include splashing, surface turbulence, and the difficulties of obtaining reproducible samples due to sedimentation. The LIBS analysis has achieved limited success due to inherent disadvantages when applied to slurry samples. In order to achieve improved measurement precision and accuracy, a spin-on-glass sampling method was evaluated. Five elements (Al, Ca, Fe, Ni, and Si) were examined in five slurry simulants containing varying amounts of each ion. Three calibration models were developed by using univariate calibration, multiple linear regression, and partial least square regression. LIBS analysis results obtained from the partial least square regression model were determined to be the best fit to results obtained from inductively coupled plasma optical emission spectroscopy analysis.

Keywords: Laser-induced breakdown spectroscopy; Slurry; Multivariate analysis; Nuclear waste; Coating

Development of a LIBS assay for the detection of Salmonella enterica serovar Typhimurium from food by Cleon Barnett; Courtneé Bell; Komal Vig; A. C. Akpovo; Lewis Johnson; Shreekumar Pillai; Shree Singh (pp. 3323-3330).

Laser-induced breakdown spectroscopy (LIBS) is used for the identification of the presence of hazardous bacteria in food. In this study, our main focus was centered on the identification of S. enterica serovar Typhimurium, a Gram-negative foodborne pathogen, in various liquids such as milk, chicken broth, and brain heart infusion due to the infection being most prevalent in raw meat and dairy products. A Nd:YAG laser of operating wavelength 266 nm was used to obtain the spectra from the artificially inoculated liquid samples. A series of experiments were performed to determine the effectiveness of LIBS to discriminate the bacteria from the background liquids. These results are compared with competing modern molecular methods of detection which include polymerase chain reaction (PCR) and quantitative real-time PCR. In addition to analyzing S. enterica serovar Typhimurium, another common Gram-negative, Escherichia coli, as well as Gram-positive pathogen, Staphlycoccus auerus, were used to determine the specificity of the LIBS technique.

Keywords: Laser-induced breakdown spectroscopy; LIBS; PCR; qPCR

Laser-induced breakdown spectroscopy for polymer identification by Sylvain Grégoire; Marjorie Boudinet; Frédéric Pelascini; Fabrice Surma; Vincent Detalle; Yves Holl (pp. 3331-3340).

This study aims at differentiating several organic materials, particularly polymers, by laser induced breakdown spectroscopy. The goal is to apply this technique to the fields of polymer recycling and cultural heritage conservation. We worked with some usual polymers families: polyethylene (PE), polypropylene (PP), polyoxymethylene, (POM), poly(vinyl chloride), polytetrafluoroethylene, polyoxyethylene (POE), and polyamide for the aliphatic ones, and poly(butylene terephthalate), acrylonitrile–butadiene–styrene, polystyrene, and polycarbonate for the aromatic ones. The fourth harmonic of a Nd:YAG laser (266 nm) in ambient air at atmospheric pressure was used. A careful analysis of the C₂ Swan system (0,0) band in polymers containing no C–C (POM), few C–C (POE), or aromatic C–C linkages led us to the conclusion that the C₂ signal might be native, i.e., the result of direct ablation from the sample. With use of these results, aliphatic and aromatic polymers could be differentiated. Further data treatments, such as properly chosen line ratios, principal component analysis, and partial least squares regression, were evaluated. It was shown that many polymers could be separated, including PE and PP, despite their similar chemical structures. Figure LIBS analysis for cultural heritage conservation

Keywords: Laser induced breakdown spectroscopy (LIBS); Polymer recycling; Cultural heritage; Molecular signal; Chemometrics; Principal component analysis; Partial least squares

Characterization and forensic analysis of soil samples using laser-induced breakdown spectroscopy (LIBS) by Sarah C. Jantzi; José R. Almirall (pp. 3341-3351).

A method for the quantitative elemental analysis of surface soil samples using laser-induced breakdown spectroscopy (LIBS) was developed and applied to the analysis of bulk soil samples for discrimination between specimens. The use of a 266 nm laser for LIBS analysis is reported for the first time in forensic soil analysis. Optimization of the LIBS method is discussed, and the results compared favorably to a laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) method previously developed. Precision for both methods was <10% for most elements. LIBS limits of detection were <33 ppm and bias <40% for most elements. In a proof of principle study, the LIBS method successfully discriminated samples from two different sites in Dade County, FL. Analysis of variance, Tukey’s post hoc test and Student’s t test resulted in 100% discrimination with no type I or type II errors. Principal components analysis (PCA) resulted in clear groupings of the two sites. A correct classification rate of 99.4% was obtained with linear discriminant analysis using leave-one-out validation. Similar results were obtained when the same samples were analyzed by LA-ICP-MS, showing that LIBS can provide similar information to LA-ICP-MS. In a forensic sampling/spatial heterogeneity study, the variation between sites, between sub-plots, between samples and within samples was examined on three similar Dade sites. The closer the sampling locations, the closer the grouping on a PCA plot and the higher the misclassification rate. These results underscore the importance of careful sampling for geographic site characterization.

Keywords: LIBS; Laser-induced breakdown spectroscopy; Soil; Forensics; Criminalistics; Heterogeneity

Standoff detection of explosives: critical comparison for ensuing options on Raman spectroscopy–LIBS sensor fusion by J. Moros; J. A. Lorenzo; J. J. Laserna (pp. 3353-3365).

In general, any standoff sensor for the effective detection of explosives must meet two basic requirements: first, a capacity to detect the response generated from only a small amount of material located at a distance of several meters (high sensitivity) and second, the ability to provide easily distinguishable responses for different materials (high specificity). Raman spectroscopy and laser-induced breakdown spectroscopy (LIBS) are two analytical techniques which share similar instrumentation and, at the same time, generate complementary data. These factors have been taken into account recently for the design of sensors used in the detection of explosives. Similarly, research on the proper integration of both techniques has been around for a while. A priori, the different operational conditions required by the two techniques oblige the acquisition of the response for each sensor through sequential analysis, previously necessary to define the proper hierarchy of actuation. However, such an approach does not guarantee that Raman and LIBS responses obtained may relate to each other. Nonetheless, the possible advantages arising from the integration of the molecular and elemental spectroscopic information come with an obvious underlying requirement, simultaneous data acquisition. In the present paper, strong and weak points of Raman spectroscopy and LIBS for solving explosives detection problems, in terms of selectivity, sensitivity, and throughput, are critically examined, discussed, and compared for assessing the ensuing options on the fusion of the responses of both sensing technologies.

Keywords: Raman spectroscopy; LIBS; Standoff sensor; Explosives; Fusion

Quantitative determination of element concentrations in industrial oxide materials by laser-induced breakdown spectroscopy by B. Praher; R. Rössler; E. Arenholz; J. Heitz; J. D. Pedarnig (pp. 3367-3375).

Calibration-free laser-induced breakdown spectroscopy (CF-LIBS) method is employed for quantitative determination of oxide concentrations in multi-component materials. Industrial oxide materials from steel industry are laser ablated in air, and the optical plasma emission is collected by spectrometers and gated detectors. The temperature and electron number density of laser-induced plasma are determined from measured LIBS spectra. Emission lines of aluminium (Al), calcium (Ca), iron (Fe), manganese (Mn), magnesium (Mg), silicon (Si), titanium (Ti), and chromium (Cr) of low self-absorption are selected, and the concentration of oxides CaO, Al₂O₃, MgO, SiO₂, FeO, MnO, TiO₂, and Cr₂O₃ is calculated by CF-LIBS analysis. For all sample materials investigated, we find good match of calculated concentration values (C _CF) with nominal concentration values (C _N). The relative error in oxide concentration, e _r = |C _CF − C _N|/C _N, decreases with increasing concentration and it is e _r ≤ 100% for concentration C _N ≥ 1 wt.%. The CF-LIBS results are stable against fluctuations of experimental parameters. The variation of laser pulse energy over a large range changes the error by less than 10% for major oxides (C _N ≥ 10 wt.%). The results indicate that CF-LIBS method can be employed for fast and stable quantitative compositional analysis of multi-component materials.

Keywords: Calibration-free laser-induced breakdown spectroscopy (CF-LIBS); Multi-element analysis; Slag; Industrial oxides

Can the provenance of the conflict minerals columbite and tantalite be ascertained by laser-induced breakdown spectroscopy? by Russell S. Harmon; Katrina M. Shughrue; Jeremiah J. Remus; Michael A. Wise; Lucille J. East; Richard R. Hark (pp. 3377-3382).

Conflict minerals is a term applied to ores mined in conditions of armed conflict and human rights abuse. Niobium and tantalum are two rare metals whose primary natural occurrence is in the complex oxide minerals columbite and tantalite, the ore of which is commonly referred to as coltan. The illicit export of coltan ore from the Democratic Republic of the Congo is thought to be responsible for financing the ongoing civil conflicts in this region. Determining the chemical composition of an ore is one of the means of ascertaining its provenance. Laser-induced breakdown spectroscopy (LIBS) offers a means of rapidly distinguishing different geographic sources for a mineral because the LIBS plasma emission spectrum provides the complete chemical composition (i.e., “chemical fingerprint”) of any material in real time. To test this idea for columbite–tantalite, three sample sets were analyzed. Partial least squares discriminant analysis (PLSDA) allows correct sample-level geographic discrimination at a success rate exceeding 90%.

Keywords: Laser-induced breakdown spectroscopy; LIBS; Conflict minerals; Columbite–tantalite; Coltan; PLSDA

Quantifying mRNA levels across tissue sections with 2D-RT-qPCR by Michael Armani; Michael A. Tangrea; Benjamin Shapiro; Michael R. Emmert-Buck; Elisabeth Smela (pp. 3383-3393).

Measurement of mRNA levels across tissue samples facilitates an understanding of how genes function and what their roles are in disease. Quantifying low-abundance mRNA requires a workflow that preserves spatial information, isolates RNA, and performs reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR). This is complex because these steps are typically performed in three separate platforms. In the present study, we describe two-dimensional RT-qPCR (2D-RT-qPCR), a method that quantifies RNA across tissues sections in a single integrated platform. The method uses the grid format of a multi-well plate to macrodissect tissue sections and preserve the spatial location of the RNA; this also eliminates the need for physical homogenization of the tissue. A new lysis and nucleic acid purification protocol is performed in the same multi-well plate, followed by RT-qPCR. The feasibility 2D-RT-qPCR was demonstrated on a variety of tissue types. Potential applications of the technology as a high-throughput tissue analysis platform are discussed.

Keywords: Tissue lysis; RNA purification; Gene expression mapping; Tissue section; PCR

Kinetic analyses and performance of a colloidal magnetic nanoparticle based immunoassay dedicated to allergy diagnosis by Bruno Teste; Frédéric Kanoufi; Stéphanie Descroix; Pascal Poncet; Thomas Georgelin; Jean-Michel Siaugue; Jan Petr; Anne Varenne; Marie-Claire Hennion (pp. 3395-3407).

In this paper, we demonstrate the possibility to use magnetic nanoparticles as immunosupports for allergy diagnosis. Most immunoassays used for immunosupports and clinical diagnosis are based on a heterogeneous solid-phase system and suffer from mass-transfer limitation. The nanoparticles’ colloidal behavior and magnetic properties bring the advantages of homogeneous immunoassay, i.e., species diffusion, and of heterogeneous immunoassay, i.e., easy separation of the immunocomplex and free forms, as well as analyte preconcentration. We thus developed a colloidal, non-competitive, indirect immunoassay using magnetic core–shell nanoparticles (MCSNP) as immunosupports. The feasibility of such an immunoassay was first demonstrated with a model antibody and described by comparing the immunocapture kinetics using macro (standard microtiter plate), micro (microparticles) and nanosupports (MCSNP). The influence of the nanosupport properties (surface chemistry, antigen density) and of the medium (ionic strength, counter ion nature) on the immunocapture efficiency and specificity was then investigated. The performances of this original MCSNP-based immunoassay were compared with a gold standard enzyme-linked immunosorbent assay (ELISA) using a microtiter plate. The capture rate of target IgG was accelerated 200-fold and a tenfold lower limit of detection was achieved. Finally, the MCSNP-based immunoassay was successfully applied to the detection of specific IgE from milk-allergic patient’s sera with a lower LOD and a good agreement (CV < 6%) with the microtiter plate, confirming the great potential of this analytical platform in the field of immunodiagnosis.

Keywords: Allergy diagnosis; IgE; Colloidal immunoassay; Kinetic reaction; Magnetic core–shell nanoparticles

Determination of steroid hormones in blood by GC–MS/MS by Martin Hansen; Naja W. Jacobsen; Frederik K. Nielsen; Erland Björklund; Bjarne Styrishave; Bent Halling-Sørensen (pp. 3409-3417).

This paper presents the development, optimization and validation of a methodology to determine nine key steroid hormones (viz. pregnenolone, progesterone, dehydroepiandrosterone, androstenedione, testosterone, dihydrotestosterone, estrone, 17α-estradiol and 17β-estradiol) expressed in the steroidogenesis in biological fluids. The analytical method allows for the determination of steroid hormones in blood plasma and serum down to 0.08–0.16 ng/mL for estrogens, 0.20–0.36 ng/mL for androgens and 0.36–0.43 ng/mL for progestagens. These limits of detection were obtainable using a two-step solid-phase clean-up for fractionation and elimination of interfering lipids (fatty acids, phospholipids, glycerides and sterols) from the steroid hormones. The accuracy of the method was 50–112% in the range 0.10 to 2.00 ng/mL. Figure

Keywords: Plasma; Serum; Solid-phase extraction; Gas chromatography; Lipid separation; Biological samples

Development of a high-throughput G4-FID assay for screening and evaluation of small molecules binding quadruplex nucleic acid structures by Eric Largy; Florian Hamon; Marie-Paule Teulade-Fichou (pp. 3419-3427).

G4-FID (G-quadruplex fluorescent intercalator displacement) is a simple and fast method that allows to evaluate the affinity of a compound for G-quadruplex DNA and its selectivity towards duplex DNA. This assay is based on the loss of fluorescence of thiazole orange (TO) upon competitive displacement from DNA by a putative ligand. We describe here the development of a high-throughput version of this assay performed in 96-well microplates, and fully transposable to 384-well microplates. The test was calibrated with a set of G-quadruplex ligands characterized for their ability to bind quadruplex within a large range of affinity. The comparison of the results obtained in microplates and in cuvettes was conducted indicating a full agreement. Additionally, the spectral range of the test was enlarged using two other fluorescent on/off probes whose absorption are red-shifted (TO-PRO-3) and blue-shifted (Hoechst 33258) as compared to that of TO. These labels enable to screen a large diversity of compounds with various optical properties, which was exemplified by evaluation of affinity and selectivity of the porphyrin TMPyP4 that could not be evaluated previously. Altogether, our study demonstrates that the HT-G4-FID assay offers the possibility to label a large variety of G-quadruplexes of biological interest and should enable screening of collections of putative G4-ligands of high structural diversity. It thus represents a powerful tool to bring into light new ligands able to discriminate between quadruplexes of different structures. Figure High-throughput screening and evaluation of quadruplex nucleic acid ligands

Keywords: G-quadruplex DNA; FID assay; High-throughput; Screening; Fluorescence; G-quadruplex ligands

Quantification of clomiphene metabolite isomers in human plasma by rapid-resolution liquid chromatography–electrospray ionization–tandem mass spectrometry by Boian Ganchev; Georg Heinkele; Reinhold Kerb; Matthias Schwab; Thomas E. Mürdter (pp. 3429-3441).

Since the 1960s, clomiphene citrate is used for ovulation induction. Since nonresponse to clomiphene therapy is still not well understood, interindividual variability of clomiphene metabolism has been considered to be a plausible explanation. Therefore, a comprehensive, rapid, sensitive, and specific analytical method for the quantification of (E)- and (Z)-isomers of clomiphene and their putative N-desethyl, N,N-didesethyl, 4-hydroxy, and 4-hydroxy-N-desethyl metabolites, and the N-oxides in human plasma has been newly developed, using HPLC-tandem mass spectrometry and stable isotope-labeled internal standards. All standards other than the parent drug were synthesized in our laboratory. Following protein precipitation analytes were separated on a ZORBAX Eclipse plus C18 1.8 μm column with a gradient of 0.1% formic acid in water and 0.1% formic acid in acetonitrile and detected on a triple quadrupole mass spectrometer with positive electrospray ionization in the multiple reaction monitoring mode. Lower limit of quantification for metabolites ranged from 0.06 ng/mL for clomiphene-N-oxides to 0.3 ng/mL for (E)-N-desethylclomiphene. The assay was validated according to FDA guidelines. Plasma levels of clomiphene and its metabolites were measured in two women after single-dose treatment with clomiphene. Figure Structures of clomiphene isomers and overlay of MRM-chromatograms of clomiphene and its metabolites

Keywords: Clomiphene; Metabolites; Plasma; HPLC; Mass spectrometry

Development of bovine serum albumin certified reference material by Liqing Wu; Bin Yang; Jiaming Bi; Jing Wang (pp. 3443-3449).

We present the development process for National Institute of Metrology (NIM) bovine serum albumin (BSA) certified reference material (CRM). Each CRM unit contains about 200 mg of purified BSA. The moisture, ignition residue, molecular weight, and high-performance liquid chromatography (HPLC) purity were analyzed and mass spectrometry based protein identification was carried out to ensure the material was BSA. Both amino acid based isotope dilution mass spectrometry (IDMS) and a purity deduction method were selected for value assignment. The certified value was the average of the IDMS and the purity deduction result. HPLC purity analysis was used to examine the homogeneity and stability of solid BSA CRM. Fifteen units were selected for between-bottle homogeneity examination and seven subsamples from the same bottle were selected for within-bottle homogeneity examination. Statistics showed the CRM passed both the between-bottle and the within-bottle homogeneity examination. The CRM stability under storage conditions (-20 °C) was tested for 18 months and no trend was observed. Uncertainties from the balance, amino acid purity, hydrolysis, method reproducibility, homogeneity, and stability were taken into account in uncertainty evaluation. The final certified value of NIM BSA CRM is (0.963±0.038) g/g.

Keywords: Certified reference material; Bovine serum albumin; Isotope dilution mass spectrometry; Protein quantitation

Highly selective and sensitive enrichment of phosphopeptides via NiO nanoparticles using a microwave-assisted centrifugation on-particle ionization/enrichment approach in MALDI-MS by Nazim Hasan; Hui-Fen Wu (pp. 3451-3462).

The strategy to concentrate phosphopeptides has become a critical issue for mapping protein phosphorylation sites, which are well known as posttranslational modifications in proteomics. In this study, we propose a simple and highly sensitive method for phosphopeptide enrichment on NiO nanoparticles (NPs) from a trypsin predigested phosphoprotein complex solution in a microwave oven. Furthermore, this technique was combined with centrifugation on-particle ionization/enrichment of phosphopeptides and phosphopeptides were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Weak magnetism of these NPs and a positive surface charge effect at low pH accomplished rapid and selective phosphopeptide enrichment within 30s. Trypsin-digested products of phosphoproteins such as α-casein and β-casein, human blood serum, nonfat milk, and egg white were also investigated to explore their phosphopeptide enrichment from complex samples by this approach. The results demonstrate that NiO NPs exhibit good affinity to trace the phosphopeptides even in the presence of 30 times higher molar concentration of complex solution of non-phosphopeptide proteolytic predigested bovine serum albumin. The detection limits of NiO NPs for α-casein and β-casein were 2.0 × 10^–9 M, with good signal-to-noise ratio in the mass spectrum. NiO NPs were found to be effective and selective for enrichment of singly and multiply phosphorylated peptides at a trace level in complex samples in a microwave oven. The cost of preparing NiO NPs is low, the NiO NPs are thermally stable, and therefore, they hold great promise for use in phosphopeptide enrichment. Graphical Abstract Phosphoprotein tryptic digest product and NiO NPs

Keywords: Nickel oxide nanoparticles; Phosphopeptide enrichment; Microwaves; Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A deuterium-labelling mass spectrometry–tandem diode-array detector screening method for rapid discovery of naturally occurring electrophiles by Xiaoyu Zhang; Liping Luo; Zhongjun Ma (pp. 3463-3471).

Because electrophiles regulate many signalling pathways in cells, by modifying cysteine residues in proteins, they have a wide range of biological activity. In this study, a deuterium-labelling mass spectrometry–tandem diode-array detector (MS–DAD) screening method was established for rapid discovery of naturally occurring electrophiles. Glutathione (GSH) was used as a probe and incubated with natural product extracts. To distinguish different types of electrophile, incubation was performed in two reaction solvents, H₂O and D₂O. Ten types of naturally occurring electrophile were chosen, on the basis of their properties, to undergo the screening assay. By using this screening method, we successfully discovered the bioactive electrophile 4-hydroxyderricin in an ethanol extract of Angelica keiskei. This electrophile had potent NAD(P)H:quinone oxidoreductase 1 (NQO1)-inducing activity at a concentration of 20 μmol L⁻¹.

Keywords: Bioactive compound screening; Deuterium-labelling; Electrophiles; Natural products

Dried blood spot UHPLC-MS/MS analysis of oseltamivir and oseltamivircarboxylate—a validated assay for the clinic by Gero P. Hooff; Roland J. W. Meesters; Jeroen J. A. van Kampen; Nick A. van Huizen; Birgit Koch; Asmar F. Y. Al Hadithy; Teun van Gelder; Albert D. M. E. Osterhaus; Rob A. Gruters; Theo M. Luider (pp. 3473-3479).

The neuraminidase inhibitor oseltamivir (Tamiflu®) is currently the first-line therapy for patients with influenza virus infection. Common analysis of the prodrug and its active metabolite oseltamivircarboxylate is determined via extraction from plasma. Compared with these assays, dried blood spot (DBS) analysis provides several advantages, including a minimum sample volume required for the measurement of drugs in whole blood. Samples can easily be obtained via a simple, non-invasive finger or heel prick. Mainly, these characteristics make DBS an ideal tool for pediatrics and to measure multiple time points such as those needed in therapeutic drug monitoring or pharmacokinetic studies. Additionally, DBS sample preparation, stability, and storage are usually most convenient. In the present work, we developed and fully validated a DBS assay for the simultaneous determination of oseltamivir and oseltamivircarboxylate concentrations in human whole blood. We demonstrate the simplicity of DBS sample preparation, and a fast, accurate and reproducible analysis using ultra high-performance liquid chromatography coupled to a triple quadrupole mass spectrometer. A thorough validation on the basis of the most recent FDA guidelines for bioanalytical method validation showed that the method is selective, precise, and accurate (≤15% RSD), and sensitive over the relevant clinical range of 5–1,500 ng/mL for oseltamivir and 20–1,500 ng/mL for the oseltamivircarboxylate metabolite. As a proof of concept, oseltamivir and oseltamivircarboxylate levels were determined in DBS obtained from healthy volunteers who received a single oral dose of Tamiflu®.

Keywords: Ultra high-performance liquid chromatography (UHPLC); Mass spectrometry (MS); Dried blood spot (DBS); Validation; Tamiflu®; Oseltamivir-(carboxylate)

Drugs of abuse screening in urine as part of a metabolite-based LC-MSⁿ screening concept by Dirk K. Wissenbach; Markus R. Meyer; Daniela Remane; Anika A. Philipp; Armin A. Weber; Hans H. Maurer (pp. 3481-3489).

Today, immunoassays and several chromatographic methods are in use for drug screening in clinical and forensic toxicology and in doping control. For further proof of the authors’ new metabolite-based liquid chromatography-mass spectrometry (LC-MSⁿ) screening concept, the detectability of drugs of abuse and their metabolites using this screening approach was studied. As previously reported, the corresponding reference library was built up with MS² and MS³ wideband spectra using a LXQ linear ion trap with electrospray ionization in the positive mode and full scan information-dependent acquisition. In addition to the parent drug spectra recorded in methanolic solution, metabolite spectra were identified after protein precipitation of urine from rats after administration of the corresponding drugs and added to the library. This consists now of data of over 900 parent compounds, including 87 drugs of abuse, and of over 2,300 metabolites and artifacts, among them 436 of drugs of abuse. Recovery, process efficiency, matrix effects, and limits of detection for selected drugs of abuse were determined using spiked human urine, and the resulting data have been acceptable. Using two automatic data evaluation tools (ToxID and SmileMS), the intake of 54 of the studied drugs of abuse could be confirmed in urine samples of drug users after protein precipitation and LC separation. The following drugs classes were covered: stimulants, designer drugs, hallucinogens, (synthetic) cannabinoids, opioids, and selected benzodiazepines. The presented LC-MSⁿ method complements the well-established gas chromatography-mass spectroscopy procedure in the authors’ laboratory.

Keywords: Urine; Screening; LC-MS; Library; Metabolite; Drugs of abuse

Monitoring of the non-steroid anti-inflammatory drug indomethacin: development of immunochemical methods for its purification and detection by Nir Skalka; Alex Krol; Haim Schlesinger; Miriam Altstein (pp. 3491-3504).

The present research focused on the development of an immunoassay and an immunochemical sol–gel-based immunoaffinity purification (IAP) method for purification and detection of the non-steroid anti-inflammatory drug (NSAID) indomethacin (IMT). A polyclonal antibody (Ab) for IMT was generated, and two sensitive microplate assays for the detection of IMT were developed (termed OV and HRP formats), based on the enzyme-linked immunosorbent assay (ELISA) method. The limits of detection of the assays were 15 ± 1.25 ng mL⁻¹ (n = 50) and 12 ± 0.17 ng mL⁻¹ (n = 4) for the OVA and HRP formats, respectively. The Abs exhibited slight cross-reactivity with other NSAIDs. The Abs were also used to develop a sol–gel-based IAP method for clean-up and concentration of IMT. Several sol–gel formats with various amounts of antibodies were examined; the best and most reproducible format was at a TMOS:HCl molar ratio of 1:6 in which 120 μL of IMT Abs was entrapped. The binding capacity under these conditions was ca. 100 to 250 ng of IMT with very low non-specific binding (less than 5% of the applied amount). The sol–gel IAP method, combined with solid-phase extraction, successfully eliminated serum interference to a degree that enabled analysis of spiked serum samples by ELISA. The method was also found to be fully compatible with subsequent chemical analytical methods, such as liquid chromatography followed by mass spectrometry. The approaches developed in this study form a basis for analysis of IMT in biological samples in order to monitor their pharmacokinetic properties, and may be further used to study population exposure to IMT, and to monitor the occurrence of IMT contamination in water samples.

Keywords: Indomethacin; ELISA; Immunoaffinity chromatography; Sol–gel; Pharmaceutical residues; Residue monitoring; Serum monitoring

Development and validation of a rapid LC-ESI-MS/MS method for quantification of fluoxetine and its application to MS binding assays by Marielle Hess; Georg Höfner; Klaus Theodor Wanner (pp. 3505-3515).

In the present study, a rapid and sensitive LC-ESI-MS/MS method for quantification of (S)-fluoxetine as a native marker in mass spectrometry (MS) binding assays addressing the human serotonin transporter (hSERT) was developed and validated. The concept of MS binding assays based on mass spectrometric quantification of a nonlabeled marker recently introduced by us represents a promising alternative to conventional radioligand binding without the drawbacks inherently connected with radioisotope labeling. For high-performance liquid chromatography (HPLC), a 20 × 2-mm RP-18 column with a mobile phase composed of acetonitrile and ammonium bicarbonate buffer (5 mmol L⁻¹, pH 9.5) at a ratio of 80:20 (v/v) and a flow rate of 800 μL min⁻¹ in an isocratic mode were used, resulting in a chromatographic cycle time of 60 s. Employing [²H₅]fluoxetine as internal standard enabled ESI-MS/MS quantification of (S)-fluoxetine between 3 nmol L⁻¹ and 50 pmol L⁻¹ (LLOQ) in matrix obtained from binding experiments without the need of any sample preparation. Validation of the method showed that linearity, intra-, and inter-batch accuracy as well as precision meet the requirements of the FDA guidance for bioanalytical method validation. Considering sensitivity and speed, the established method is clearly superior to those published for biological matrices so far. Furthermore, the method was transferred to other RP-18 columns of different lengths and respective validation experiments demonstrated its versatility and chromatographic robustness. Finally, the newly developed method was successfully applied to MS binding assays for hSERT. The affinity determined for (S)-fluoxetine in saturation experiments was in good agreement with literature data obtained in respective radioligand binding assays.

Keywords: LC-MS/MS; Method development; Validation; Fluoxetine; Binding assay; Serotonin transporter (SERT)

Enantioselective analysis of zopiclone and its metabolites in plasma by liquid chromatography/tandem mass spectrometry by Milena Araújo Tonon; Valquíria A. P. Jabor; Pierina Sueli Bonato (pp. 3517-3525).

A high-performance liquid chromatographic method with triple-quadrupole mass spectrometry detection (LC-MS-MS) was developed and validated for the first time for the simultaneous quantification of zopiclone and its metabolites in rat plasma samples. The analytes were isolated from rat plasma by liquid–liquid extraction and separated using a chiral stationary phase based on an amylose derivative, Chiralpak ADR-H column, and ethanol–methanol–acetonitrile (50:45:5, v/v/v) plus 0.025% diethylamine as the mobile phase, at a flow-rate of 1.0 mL min⁻¹. Moclobemide was used as the internal standard. The developed method was linear over the concentration range of 7.5–500 ng mL⁻¹. The mean absolute recoveries were 74.6 and 75.7; 61.6 and 56.9; 72.5, and 70.7 for zopiclone enantiomers, for N-desmethyl zopiclone enantiomers and for zopiclone-N-oxide enantiomers, respectively, and 75.9 for the internal standard. Precision and accuracy were within acceptable levels of confidence (<15%). The method application in a pilot study of zopiclone kinetic disposition in rats showed that the levels of (+)-(S)-zopiclone were always higher than those of (−)-R-zopiclone. Higher concentrations were also observed for (+)-(S)-N-desmethyl zopiclone and (+)-(S)-N-oxide zopiclone, confirming the stereoselective disposition of zopiclone.

Keywords: Zopiclone; LC-MS-MS; Metabolites; Kinetic disposition

Comparison of quartz and Teflon filters for simultaneous collection of size-separated ultrafine aerosol particles and gas-phase zero samples by Jevgeni Parshintsev; Jose Ruiz-Jimenez; Tuukka Petäjä; Kari Hartonen; Markku Kulmala; Marja-Liisa Riekkola (pp. 3527-3535).

In this research, the two most common filter media, quartz and Teflon, were tested to obtain information about the possible adsorption of gas-phase compounds onto filters during long sample collection of atmospheric aerosols. Particles of nanometer-size for off-line chemical characterization were collected using a recently introduced differential mobility analyzer for size separation. Samples were collected at an urban site (Helsinki, SMEARIII station) during spring 2010. Sampling time was 4 to 10 days for particles 50, 40, or 30 nm in diameter. Sample air flow was 4 L/min. The sampling setup was arranged so that two samples were obtained for each sampling period almost simultaneously: one containing particles and adsorbed gas-phase compounds and one containing adsorbed gas-phase compounds only. Filters were extracted and analyzed for the presence of selected carboxylic acids, polyols, nitrogen-containing compounds, and aldehydes. The results showed that, in quartz filter samples, gas-phase adsorption may be responsible for as much as 100% of some compound masses. Whether quartz or Teflon, simultaneous collection of gas-phase zero samples is essential during the whole sampling period. The dependence of the adsorption of gas-phase compounds on vapor pressure and the effect of adsorption on the deposited aerosol layer are discussed.

Keywords: Atmospheric aerosols; Differential mobility analyzer; Filter; Teflon; Quartz

Determination of 19 volatile organic compounds in wastewater effluents from different treatments by purge and trap followed by gas-chromatography coupled to mass spectrometry by Nieves Barco-Bonilla; Patricia Plaza-Bolaños; José Luis Fernández-Moreno; Roberto Romero-González; Antonia Garrido Frenich; José Luis Martínez Vidal (pp. 3537-3546).

A rapid and simple methodology based on purge and trap with gas-chromatography coupled to triple quadrupole mass spectrometry has been developed for the analysis of 19 volatile organic compounds (VOCs) in wastewater (WW) effluents from four different treatments. The determination was carried out in the raw WW effluents, which were not submitted to any pre-treatment (e.g., filtration). A matrix effect study was also performed, concluding that solvent calibration was adequate to quantify VOCs in WW effluent samples containing a variety of suspended particulate matter. Adequate validation parameters were obtained with recovery values in the range 73–124% and precision values lower than 24%. Limits of quantification were established at 0.1 μg L^-1 for all VOCs. The proposed method was applied to the analysis of WW samples, detecting chloroform and toluene at concentrations ranging from 0.1 to 4.80 μg L^-1.

Keywords: Wastewater; Volatile organic compounds; Purge and trap; Matrix effect; GC-MS

Direct chiral liquid chromatography determination of aryloxyphenoxypropionic herbicides in soil: deconvolution tools for peak processing by V. Guillén-Casla; J. Magro-Moral; N. Rosales-Conrado; L. V. Pérez-Arribas; M. E. León-González; L. M. Polo-Díez (pp. 3547-3560).

In this paper, the enantiomeric separation of two aryloxyphenoxypropionic esters (fluazifop-butyl and quizalofop-ethyl) and a safener herbicide (mefenpyr-diethyl), which is widely used for protecting crop plants, has been studied by direct liquid chromatography (LC) with UV detection on an α₁-acid glycoprotein as chiral stationary phase. Optimization of separation conditions was done by factorial experimental design. Experimental factors and ranges selected were propanol (5–10%), phosphate buffer pH (6.5–7.0), and column temperature (15–25 °C). Responses were expressed in terms of enantioresolution (R _s) and adjusted retention time of the second eluted enantiomer (t _r2′). The chemometric method used to explore data was response surface analysis. Multiple response analyses were carried out to determine the combination of experimental factors which simultaneously optimize experimental responses. Under optimum conditions for enantioseparation of each herbicide, partially overlapped or fully resolved enantiomers were obtained. Deconvolution tools were employed as an integration method to fit chromatographic data and to achieve a more precise enantiomeric ratio (ER) and enantiomeric fraction (EF) values. Applicability of both direct chiral LC and peak deconvolution methods was evaluated in spiked soil samples at different R/S enantiomeric ratios. Acceptable and reproducible recoveries between 71% and 96% with precision in the range 1–6% were achieved for herbicide-spiked levels from 0.50 to 9.0 μg g^–1. In addition, parameters such as R _s, ER, and EF were calculated and compared with values obtained using the common valley drop integration method. Figure Experimental and deconvolved chromatograms obtained for quizalofop-ethyl at two different experimental conditions"

Keywords: HPLC; Chiral analysis; Pesticides; Chemometrics; Peak deconvolution; Soil

Evaluation of different adsorbents for large-volume pre-concentration for analyzing atmospheric Persistent Organic Pollutants at trace levels by Pasquale Avino; Giuseppe Cinelli; Ivan Notardonato; Mario Vincenzo Russo (pp. 3561-3571).

This paper investigates the performance of some adsorbents, Carbopack B, Tenax-GC, and XAD-2, in a SPE and GC analytical method for sampling and determining some persistent organic pollutants such as benzene, toluene, o-, m-, and p-xylenes, naphthalene, anthracene, fluorene, fluoranthene, benzo(i,k)fluorene, pyrene and benzo(a)pyrene, aldrin, dieldrin, endrin, endosulfan, and PCB congeners (nos. 1, 15, 44, 77, and 209). Adsorbents evaluated in this study are Carbopack B, Tenax-GC, and XAD-2. Before applying the analytical method to air samples, it was widely investigated in laboratory: the sampler is constituted by a glass pyrex vial home-filled with 300 mg (sampling section) and 50 mg of adsorbent material (backup section). The re-extraction is performed by CS₂ (1–2 mL) and analysis is performed by GC-FID and GC-ECD. The evaluation of breakthrough volumes and desorption efficiencies shows the XAD-2 performance in the enrichment of different organic species present in atmosphere at trace levels (ppt) to be more advantageous than the other two materials in terms of analytical and technical parameters. One of the advantages is the high volume of sampled air with high concentration factor and limited loss of analytes (breakthrough volumes are higher than 5,000 L g⁻¹ for high-boiling compounds and higher than 400 L g⁻¹ for low-boiling solutes). Another advantage is the possibility of easy and speed re-extraction of analytes using small volumes of solvent (a few milliliters). The recoveries are about 100% with a RSD ≤ 2.3 for low-boiling compounds, and between 77% and 109% with a RSD ≤ 5.7% for high-boiling species. The XAD-2 adsorbent was applied to real air samples collected in different polluted areas (urban, industrial, rural, and remote locations) demonstrating the wide application of such methodology in various environmental situation. Figure Scheme of the glass pyrex vial sampler

Keywords: Adsorbent; XAD-2; Enrichment; GC analysis; Atmospheric pollution; POPs

Novel cyanobacterial bioreporters of phosphorus bioavailability based on alkaline phosphatase and phosphate transporter genes of Anabaena sp. PCC 7120 by M. Ángeles Muñoz-Martín; Pilar Mateo; Francisco Leganés; Francisca Fernández-Piñas (pp. 3573-3584).

There is heterogeneity in the way cyanobacteria respond to P starvation and subsequently how they adapt to environments with low or fluctuating P concentrations. In this study, we have fused the promoterless lux operon luxCDABE to the promoter regions of Anabaena sp. PCC 7120 phoA genes putatively encoding alkaline phosphatases, phoA (all2843) and phoA-like (alr5291) and to the promoter region of one operon putatively encoding a high affinity phosphate transporter pst1 (all4575-4572). The self-bioluminescent strains constructed in this way, Anabaena AP (phoA promoter), Anabaena AP-L (phoA-like promoter), and Anabaena PST (pst1 promoter) have been used to study the expression of these genes in response to P starvation and P re-feeding with inorganic and organic phosphate sources. Our data showed that the pst1 promoter was activated at much higher level than the phoA-like promoter following P starvation; however, we did not observe activation of the phoA promoter. The P re-feeding experiments revealed that both strains, Anabaena (A.) PST and A. AP-L could be used as novel bioreporters of P availability in environmental samples. Both strains were used to estimate bioavailable P in environmental samples (fresh- and wastewaters) with a wide range of soluble P concentrations. The results indicated that most of the P in the water samples was in chemical forms available to the cyanobacterium; however there were some differences in the estimates given by both strains as A. PST appeared to be more adequate for the samples with the lowest P load while A. AP-L gave similar or even higher values of P concentrations than those chemically measured in samples with higher P load. Figure Schematic representation of the cellular elements involved in the Pho regulon in most cyanobacteria. The micrograph shows a filament of an Anabaena P-bioavailability bioreporter where P-responses are coupled to a luminescent signal

Keywords: Cyanobacterium; Environmental samples; Phosphorus bioavailability; Phosphorus starvation; Self-luminescent bioreporters

Smart thorium and uranium determination exploiting renewable solid-phase extraction applied to environmental samples in a wide concentration range by Jessica Avivar; Laura Ferrer; Montserrat Casas; Víctor Cerdà (pp. 3585-3594).

A smart fully automated system is proposed for determination of thorium and uranium in a wide concentration range, reaching environmental levels. The hyphenation of lab-on-valve (LOV) and multisyringe flow injection analysis (MSFIA), coupled to a long path length liquid waveguide capillary cell, allows the spectrophotometric determination of thorium and uranium in different types of environmental sample matrices achieving high selectivity and sensitivity levels. Online separation and preconcentration of thorium and uranium is carried out by means of Uranium and TEtraValents Actinides resin. The potential of the LOV–MSFIA makes possible the full automation of the system by the in-line regeneration of the column and its combination with a smart methodology is a step forward in automation. After elution, thorium(IV) and uranium(VI) are spectrophotometrically detected after reaction with arsenazo-III. We propose a rapid, inexpensive, and fully automated method to determine thorium(IV) and uranium(VI) in a wide concentration range (0–1,200 and 0–2,000 μg L^-1 Th and U, respectively). Limits of detection reached are 5.9 ηg L^-1 of uranium and 60 ηg L^-1 of thorium. Different water sample matrices (seawater, well water, freshwater, tap water, and mineral water), and a channel sediment reference material which contained thorium and uranium were satisfactorily analyzed with the proposed method.

Keywords: Smart; Uranium; Thorium; UTEVA; Environmental samples

Accurate mass measurements and ultrahigh-resolution: evaluation of different mass spectrometers for daily routine analysis of small molecules in negative electrospray ionization mode by Nuria Cortés-Francisco; Cintia Flores; Encarnación Moyano; Josep Caixach (pp. 3595-3606).

Six mass spectrometers based on different mass analyzer technologies, such as time-of-flight (TOF), hybrid quadrupole-TOF (Q-TOF), orbitrap, Fourier transform ion cyclotron resonance (FT-ICR), and triple quadrupole (QqQ), installed at independent laboratories have been tested during a single day of work for the analysis of small molecules in negative electrospray ionization (ESI) mode. The uncertainty in the mass measurements obtained from each mass spectrometer has been determined by taking the precision and accuracy of replicate measurements into account. The present study is focused on calibration processes (before, after, and during the mass measurement), the resolving power of the mass spectrometers, and the data processing for obtaining elemental formulae. The mass range between m/z 100 and 600 has been evaluated with a mix of four standards. This mass range includes small molecules usually detected in food and environmental samples. Negative ESI has been tested as there is almost no data on accurate mass (AM) measurements in this mode. Moreover, it has been used because it is the ESI mode for analysis of many compounds, such as pharmaceutical, herbicides, and fluorinated compounds. Natural organic matter has been used to demonstrate the significance of ultrahigh-resolution in complex mixtures. Sub-millidalton accuracy and precision have been obtained with Q-TOF, FT-ICR, and orbitrap achieving equivalent results. Poorer accuracy and precision have been obtained with the QqQ used: 11 mDa root-mean-square error and 6–11 mDa standard deviation. Some advice and requirements for daily AM routine analysis are also discussed here. Figure Mass errors of the six mass spectrometers in the mass range of the study (expressed as the mean and the standard deviation of n=10 replicates).

Keywords: Ultrahigh-resolution; Accurate mass measurements; Molecular formulae determination; Small organic compounds; TOF; Orbitrap

Electrospun composite of polypyrrole-polyamide as a micro-solid phase extraction sorbent by Habib Bagheri; Ali Aghakhani; Maryam Akbari; Zahra Ayazi (pp. 3607-3613).

A micro-solid phase extraction technique was developed using a novel polypyrrole-polyamide nanofiber sheet, fabricated by electrospinning method. The applicability of the new nanofiber sheet was examined as an extracting medium to isolate malathion as a model pesticide from aqueous samples. Solvent desorption was subsequently performed in a microvial, and an aliquot of extractant was injected into gas chromatography–mass spectrometry. Various parameters affecting the electrospinning process including monomer concentration, polyamide content, applied voltage, and electrospinning time were examined. After fabricating the most suitable preparation conditions, influential parameters on the extraction and desorption processes were optimized. The developed method proved to be rather convenient and offers sufficient sensitivity and good reproducibility. The limit of detection (S/N = 3) and limit of quantification (S/N = 10) of the method under optimized conditions were 50 and 100 ng L⁻¹, respectively. The relative standard deviation at concentration level of 1 ng mL⁻¹ was 2% (n = 3). The calibration curve of analyte showed linearity in the range of 0.1–1 ng mL⁻¹ (R ² = 0.9975). The developed method was successfully applied to tap and Zayanderood river water samples, while the relative recovery percentages of 98% and 96% were obtained, respectively. The whole procedure showed to be conveniently applicable and quite easy to be manipulated. Figure Scheme of the electrospinning setup used for preparation of nanofibers-based polypyrrole-polyamide sorbent

Keywords: Polypyrrole-polyamide composite; Electrospinning; Nanofiber sheet; Gas chromatography-mass spectrometry; Micro-solid phase extraction; Malathion

High-speed counter-current chromatographic separation of phytosterols by Markus Schröder; Walter Vetter (pp. 3615-3623).

Phytosterols are bioactive compounds which occur in low concentrations in plant oils. Due to their beneficial effects on human health, phytosterols have already been supplemented to food. Commercial phytosterol standards show insufficient purity and/or are very expensive. In this study, we developed a high-speed counter-current chromatography (HSCCC) method for the fractionation and analysis of a commercial crude β-sitosterol standard (purity ∼60% according to supplier). Different solvent systems were tested in shake-flask experiments, and the system n-hexane/methanol/aqueous silver nitrate solution (34/24/1, v/v/v) was finally used for HSCCC fractionation. About 50 mg phytosterols was injected and distributed into 57 fractions. Selected fractions were condensed and re-injected into the HSCCC system. This measure provided pure sitostanol (>99%) and β-sitosterol (∼99%), as well as a mixture of campesterol and stigmasterol without further phytosterols. An enriched HSCCC fraction facilitated the mass spectrometric analysis of further 11 minor phytosterols (after trimethylsilylation). It was also shown that the commercial product contained about 0.3% carotinoids which eluted without delay into an early HSCCC fraction and which were separated from the phytosterols. Figure Separation of a phytosterol mixture by means of counter-current chromatography provided pure phytosterols

Keywords: Counter-current chromatography; Lipids; Sterols; Phytosterols; Sitostanol; β-Sitosterol

Cell membrane chromatography competitive binding analysis for characterization of α_1A adrenoreceptor binding interactions by Hui Du; Jing Ren; Sicen Wang; Langchong He (pp. 3625-3633).

A new high α_1A adrenoreceptor (α_1AAR) expression cell membrane chromatography (CMC) method was developed for characterization of α_1AAR binding interactions. HEK293 α_1A cell line, which expresses stably high levels of α_1AAR, was used to prepare the stationary phase in the CMC model. The HEK293 α_1A/CMC-offline-HPLC system was applied to specifically recognize the ligands which interact with the α_1AAR, and the dissociation equilibrium constants (K _D) obtained from the model were (1.87 ± 0.13) × 10⁻⁶ M for tamsulosin, (2.86 ± 0.20) × 10⁻⁶ M for 5-methylurapidil, (3.01 ± 0.19) × 10⁻⁶ M for doxazosin, (3.44 ± 0.19) × 10⁻⁶ M for terazosin, (3.50 ± 0.21) × 10⁻⁶ M for alfuzosin, and (7.57 ± 0.31) × 10⁻⁶ M for phentolamine, respectively. The competitive binding study between tamsulosin and terazosin indicated that the two drugs interacted at the common binding site of α_1AAR. However, that was not the case between tamsulosin and oxymetazoline. The results had a positive correlation with those from radioligand binding assay and indicated that the CMC method combined modified competitive binding could be a quick and efficient way for characterizing the drug–receptor interactions. Figure Chromatograms of mixed standard solution using an HEK293 α_1A/CMC-offline-HPLC method

Keywords: HEK293 α_1A adrenoreceptor; Cell membrane chromatography; Modified competitive binding; Dissociation equilibrium constant; Binding site

High-performance liquid chromatography with photodiode array detection for determination of nobiletin content in the brain and serum of mice administrated the natural compound by Daisuke Saigusa; Masatoshi Shibuya; Daisuke Jinno; Hiroyuki Yamakoshi; Yoshiharu Iwabuchi; Akihito Yokosuka; Yoshihiro Mimaki; Akira Naganuma; Yasushi Ohizumi; Yoshihisa Tomioka; Tohru Yamakuni (pp. 3635-3641).

We recently demonstrated that nobiletin, a citrus flavonoid, exhibits anti-dementia action in animals. However, no determination methods for the content of nobiletin with beneficial action in the brain of nobiletin-administered animals have been developed, nor has its pharmacokinetics been revealed completely. Here, we established the high-performance liquid chromatography/photodiode array detection method for nobiletin determination using Bond Elut C18 SPE cartridges for extraction, where the calibration curve was linear over 0.025–10 ng, with coefficient of variation of less than 6.76%. This method enabled us to determine pharmacokinetic parameters of nobiletin given intraperitoneally or per os in the brain of mice.

Keywords: High-performance liquid chromatography; Photodiode array; Nobiletin; Brain; Dementia; Pharmacokinetics

Identification of phenolic compounds in aqueous and ethanolic rooibos extracts (Aspalathus linearis) by HPLC-ESI-MS (TOF/IT) by Ihsan Iswaldi; David Arráez-Román; Inmaculada Rodríguez-Medina; Raúl Beltrán-Debón; Jorge Joven; Antonio Segura-Carretero; Alberto Fernández-Gutiérrez (pp. 3643-3654).

Rooibos (Aspalathus linearis) is a rich source of polyphenols and used to make a mild-tasting tea containing no caffeine, is low in tannins compared to green or black teas, and has antioxidant and antimutagenic/antitumoral properties. In vivo results show that rooibos has beneficial effects upon the lipid profile by decreasing serum triglycerides and cholesterol. In this sense, we have developed a simple and rapid method to separate and characterize simultaneously the polyphenolic compounds in aqueous and ethanolic rooibos extracts using high-performance liquid chromatography coupled to electrospray ionization time-of-flight mass spectrometry (HPLC-ESI-TOF-MS) and ion trap multiple mass spectrometry (HPLC-ESI-IT-MS²). The phenolic compounds were separated on a C₁₈ column (4.6 × 150 mm, 1.8 μm) with 1% formic acid in water/acetonitrile 90:10 v/v and acetonitrile as mobile phases. The accuracy mass data generated by TOF-MS together with the fragmentation pattern obtained by IT-MS² experiments confirmed the presence of 25 and 30 phenolic compounds in the aqueous and ethanolic extracts, respectively.

Keywords: Rooibos tea; Polyphenols; High-performance liquid chromatography (HPLC); Mass spectrometry (MS)

Development of an ultrasensitive immunochromatography test to detect nicotine metabolites in oral fluids by Jesus M. Gonzalez; Michael W. Foley; Natalie M. Bieber; Peter A. Bourdelle; R. Sam Niedbala (pp. 3655-3664).

Passive exposure to tobacco smoke causes a variety of illnesses ranging from allergic responses to cancer. Assessment of exposure to second-hand tobacco smoke (SHS), particularly among vulnerable populations enables intervention and prevention of future disease. A minimally invasive oral fluids-based onsite test to detect such exposure would create a valuable tool for researchers and clinicians. Here we describe the development of a test that uses an inexpensive reader that utilizes a CMOS image sensor to reliably quantify a reporter signal and determine nicotine exposure. The rapid lateral flow test consists of a nitrocellulose strip with a control line containing goat anti-rabbit IgG, used as an internal standard, and a test line containing BSA–cotinine conjugate. To run the test, diluted sample containing antibodies against cotinine, the major metabolite of nicotine, is mixed with protein A–gold nanoparticles and placed on the sample pad. As the sample runs up to the nitrocellulose pad, antibodies in the running buffer bind to available cotinine. If cotinine is absent, the antibodies will bind to the BSA–cotinine derivative immobilized on the test line, resulting in an intense purple–red band. The concentration of cotinine equivalents in the sample can be estimated from interpretation of the test line. In this article we describe the effect of different cotinine derivatives, oral fluid pretreatment, and application and running buffers on assay sensitivity. The test can reliably detect as little as 2 ng mL⁻¹ cotinine equivalents. The assay is sensitive, simple, rapid, inexpensive, and easily implementable in point-of-care facilities to detect second-hand smoke exposure.

Keywords: Second-hand smoke; Lateral flow; Oral fluid; Assay; Antibody; Gold aggregation

Optimization of polymerization parameters for the sorption of oseltamivir onto molecularly imprinted polymers by Yajun Yang; Jianyong Li; Yurong Liu; Jiyu Zhang; Bing Li; Xuepeng Cai (pp. 3665-3674).

Molecularly imprinted polymers (MIPs) are tailor-made polymers with high selectivity for a given analyte, or group of structurally related compounds. The influence of the process parameters (the moles of functional monomer and cross-linker, the selection of functional monomer and solvent) on the preparation of oseltamivir (OS)-imprinted polymers was investigated. A mathematical method for uniform design to optimize these selected parameters and to increase the MIP selectivity for template molecules was applied. The optimal conditions to synthesize MIP were 0.69 mmol 30% acrylamide (AA) + 70% 4-Vinylpyridine (4-VP) and 5.0 mmol ethylene glycol dimethacrylate (EGDMA) copolymerized in 5 ml toluene in the presence of 0.1 mmol OS. MIP showed high affinity and selectivity for separation of the template molecule from other compounds. In the present study, we have established an effective LC-MS/MS method to identify and quantify OS with good sensitivity, accuracy and precision. Figure A Complexation; B polymerization and template removal; C MIP has high affinity to template molecule; D MIP has some affinity to template molecule analog; E MIP has no affinity to other chemicals with different type from template molecule. a Template molecule; b template molecule analog; c other chemicals

Keywords: Molecularly imprinted polymer (MIP); Polymerization optimization; Selectivity; Uniform design; Oseltamivir; LC-MS/MS

Evaluation and evidence of natural gangliosides with two unsaturated bonds in the ceramide structure obtained by a combination of MALDI-MS and NMR spectroscopy by Raine Garrido Arteaga; Roberto Carlos Veloso Pita; Miguel Antonio López López; José Antonio González Labaut; María del Carmen Rodríguez Montero; Hermán Vélez Castro; José Alberto Cremata Alvarez (pp. 3675-3680).

Gangliosides are membrane-associated glycosphingolipids. N-Acetyl GM3 and N-glycolyl GM3 are two tumor-associated antigens expressed in cancer tissues such as melanoma and mammalian cancer. In order to use these antigens in GM3-based vaccines for patients with early stage cancer, the synthetic version is recommended to avoid the risk of animal virus transmission from the source. However, the isolation of natural gangliosides is of comparative value for the structural characterization. The structures of N-acetyl and N-glycolyl GM3 extracted from dog and horse erythrocytes were evaluated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nuclear magnetic resonance techniques; additionally, the natural N-acetyl ganglioside was compared to a synthetic one. In addition to the main compound with C24:0 fatty acid chain, a minor component with an additional unsaturation in the ceramide chain was detected, in both the dog and the horse gangliosides. This paper shows spectroscopic evidence of the aforementioned compounds.

Keywords: Ganglioside; Spectroscopy; Mass spectrometry; NMR

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Analytical and Bioanalytical Chemistry (v.400, #10)