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Analytical and Bioanalytical Chemistry (v.394, #8)

The need for research in forensic science by Ruth Waddell Smith; Victoria L. McGuffin (pp. 1985-1986).

is Assistant Professor of Forensic Chemistry in the Forensic Science Program in the School of Criminal Justice at Michigan State University. Her research interests include the application of new and emerging analytical techniques for characterization of forensic evidence and the development of chemometric procedures for association and discrimination of samples of forensic interest. is Professor of Chemistry at Michigan State University. Her primary research interests are in the thermodynamic and kinetic basis of chromatography and electrophoresis. She is also interested in the application of chromatography and electrophoresis to problems of forensic and environmental significance.

Current issues in forensic science higher education by Lawrence Quarino; Thomas A. Brettell (pp. 1987-1993).

is an Associate Professor at Cedar Crest College in Allentown, PA and is the Director of both the undergraduate and graduate forensic science programs. Prior to his academic appointment, he worked as a forensic science practitioner for 15 years with both the New York City Medical Examiner’s Office and the New Jersey State Police. He currently serves as a commissioner on the Forensic Science Educational Programs Accreditation Commission (FEPAC) and is a diplomate with the American Board of Criminalistics. is a retired Director of the New Jersey State Police Forensic Science Laboratory System, is Assistant Professor of Chemistry at Cedar Crest College, with primary teaching responsibilities focused on analytical and forensic chemistry. His interests include developing laboratory exercises in forensic chemistry and seeking innovative methods that will help students better understand analytical chemistry. Furthermore, he maintains an active research group in which undergraduate and master’s students examine various applications of analytical chemistry to current forensic science issues.

Forensic applications of ambient ionization mass spectrometry by Demian R. Ifa; Ayanna U. Jackson; Giuseppe Paglia; R. Graham Cooks (pp. 1995-2008).

This review highlights and critically assesses forensic applications in the developing field of ambient ionization mass spectrometry. Ambient ionization methods permit the ionization of samples outside the mass spectrometer in the ordinary atmosphere, with minimal sample preparation. Several ambient ionization methods have been created since 2004 and they utilize different mechanisms to create ions for mass-spectrometric analysis. Forensic applications of these techniques—to the analysis of toxic industrial compounds, chemical warfare agents, illicit drugs and formulations, explosives, foodstuff, inks, fingerprints, and skin—are reviewed. The minimal sample pretreatment needed is illustrated with examples of analysis from complex matrices (e.g., food) on various substrates (e.g., paper). The low limits of detection achieved by most of the ambient ionization methods for compounds of forensic interest readily offer qualitative confirmation of chemical identity; in some cases quantitative data are also available. The forensic applications of ambient ionization methods are a growing research field and there are still many types of applications which remain to be explored, particularly those involving on-site analysis. Aspects of ambient ionization currently undergoing rapid development include molecular imaging and increased detection specificity through simultaneous chemical reaction and ionization by addition of appropriate chemical reagents.

Keywords: Forensics/toxicology; Mass spectrometry plasma mass spectrometry; Ambient analysis; Illicit drugs; Explosives; Counterfeit pharmaceuticals

Forensic analysis of dyed textile fibers by John V. Goodpaster; Elisa A. Liszewski (pp. 2009-2018).

Textile fibers are a key form of trace evidence, and the ability to reliably associate or discriminate them is crucial for forensic scientists worldwide. While microscopic and instrumental analysis can be used to determine the composition of the fiber itself, additional specificity is gained by examining fiber color. This is particularly important when the bulk composition of the fiber is relatively uninformative, as it is with cotton, wool, or other natural fibers. Such analyses pose several problems, including extremely small sample sizes, the desire for nondestructive techniques, and the vast complexity of modern dye compositions. This review will focus on more recent methods for comparing fiber color by using chromatography, spectroscopy, and mass spectrometry. The increasing use of multivariate statistics and other data analysis techniques for the differentiation of spectra from dyed fibers will also be discussed. Figure MIP image of red cotton fiber

Keywords: Forensic science; Textile fibers; Fiber dyes; Trace evidence

Characterization of smokeless powders using nanoelectrospray ionization mass spectrometry (nESI-MS) by Gwynyth Scherperel; Gavin E. Reid; Ruth Waddell Smith (pp. 2019-2028).

Smokeless powder is one of the most common types of explosives used in civilian ammunition and, hence, its detection and identification is of great forensic value. Based on comparison of physical properties, extraction yield in methanol, and the spectra obtained using nanoelectrospray ionization and multistage tandem mass spectrometry (MS/MS) in a quadrupole ion trap mass spectrometer, a method was developed to identify and differentiate unburned smokeless powders from different brands of ammunition. The mass spectrometry method was optimized for the simultaneous detection of the organic stabilizers commonly present in smokeless powders: methyl centralite, ethyl centralite, and diphenylamine. All but two of the powders were differentiated; however, the two that were not differentiated were produced by the same manufacturer. Gunshot residue from the cartridges was deposited on cotton cloth and collision-induced dissociation MS/MS was used to identify low levels of ethyl centralite in the residue, despite the presence of contaminants.

Keywords: Smokeless powder; Gunshot residue; Forensic analysis; Mass spectrometry

Interpretation of laser desorption mass spectra of unexpected inorganic species found in a cosmetic sample of forensic interest: fingernail polish by Emily O’Neill; Danielle Harrington; John Allison (pp. 2029-2038).

When analytes containing color are irradiated with a pulsed UV laser in the ion source of a mass spectrometer, molecules such as dyes or pigments absorb energy, resulting in their desorption and ionization. This method, laser desorption mass spectrometry (LDMS), has been used successfully to analyze colorants of forensic interest in a wide variety of materials. Here, we present and interpret the most complex of such spectra obtained to date from a sample of fingernail polish. Interpretation of the spectrum provides a unique opportunity to characterize the laser desorption mass spectra of some unexpected inorganic materials found in cosmetics, such as “broken glass”, cyanide compounds, and heavy metals. Also, the possibility of a useful forensic database of LDMS spectra of fingernail polishes is considered. Figure

Keywords: Forensic science; Laser desorption; Mass spectrometry; Fingernail polish; Pigments; Spectral interpretation; Cosmetics

Detection of illicit substances in fingerprints by infrared spectral imaging by Ping Hei Ronnie Ng; Sarah Walker; Mark Tahtouh; Brian Reedy (pp. 2039-2048).

FTIR and Raman spectral imaging can be used to simultaneously image a latent fingerprint and detect exogenous substances deposited within it. These substances might include drugs of abuse or traces of explosives or gunshot residue. In this work, spectral searching algorithms were tested for their efficacy in finding targeted substances deposited within fingerprints. “Reverse” library searching, where a large number of possibly poor-quality spectra from a spectral image are searched against a small number of high-quality reference spectra, poses problems for common search algorithms as they are usually implemented. Out of a range of algorithms which included conventional Euclidean distance searching, the spectral angle mapper (SAM) and correlation algorithms gave the best results when used with second-derivative image and reference spectra. All methods tested gave poorer performances with first derivative and undifferentiated spectra. In a search against a caffeine reference, the SAM and correlation methods were able to correctly rank a set of 40 confirmed but poor-quality caffeine spectra at the top of a dataset which also contained 4,096 spectra from an image of an uncontaminated latent fingerprint. These methods also successfully and individually detected aspirin, diazepam and caffeine that had been deposited together in another fingerprint, and they did not indicate any of these substances as a match in a search for another substance which was known not to be present. The SAM was used to successfully locate explosive components in fingerprints deposited on silicon windows. The potential of other spectral searching algorithms used in the field of remote sensing is considered, and the applicability of the methods tested in this work to other modes of spectral imaging is discussed.

Keywords: FTIR imaging; Spectral library searching; Fingerprints; Explosives; Drugs of abuse

Association and discrimination of diesel fuels using chemometric procedures by Lucas J. Marshall; John W. McIlroy; Victoria L. McGuffin; Ruth Waddell Smith (pp. 2049-2059).

Five neat diesel samples were analyzed by gas chromatography-mass spectrometry and total ion chromatograms as well as extracted ion profiles of the alkane and aromatic compound classes were generated. A retention time alignment algorithm was employed to align chromatograms prior to peak area normalization. Pearson product moment correlation coefficients and principal components analysis were then employed to investigate association and discrimination among the diesel samples. The same procedures were also used to investigate the association of a diesel residue to its neat counterpart. Current limitations in the retention time alignment algorithm and the subsequent effect on the association and discrimination of the diesel samples are discussed. An understanding of these issues is crucial to ensure the accuracy of data interpretation based on such chemometric procedures.

Keywords: Chemometrics; Pearson product moment correlation; Principal components analysis; Diesel fuel; Gas chromatography-mass spectrometry

Comparison of differential mobility spectrometry and mass spectrometry for gas chromatographic detection of ignitable liquids from fire debris using projected difference resolution by Yao Lu; Ping Chen; Peter B. Harrington (pp. 2061-2067).

The significance of forensic arson analysis accelerates the applications of new technologies in this area. Based on the previously reported application of differential mobility spectrometry (DMS) as a detection method for gas chromatography (GC) in arson analysis, the performances of DMS and mass spectrometry (MS) were compared using a novel chemometric tool, projected difference resolutions (PDRs). The PDR results show that one-way mass spectra data exhibit higher resolution than DMS data, while total ion chromatograms from GC–DMS show higher resolution than that from GC/MS for differentiating seven kinds of ignitable liquids. Combining the information from both chromatography and spectra, two-way data always have higher resolution than one-way data for these two detection methods, and GC/MS would exhibit better performance than GC–DMS according to the minimum resolution value. To verify the PDR results, a fuzzy rule-building expert system was applied for classifying these seven kinds of ignitable liquids from fire debris based on GC–DMS and GC/MS data, respectively. The prediction accuracies were consistent with PDR results, which proved that PDR is a powerful tool in comparing the performances of different analysis methods for pattern recognition.

Keywords: Differential mobility spectrometry; Mass spectrometry; Projected difference resolution; Two-way Classification; Ignitable liquids

Non-invasive detection of superimposed latent fingerprints and inter-ridge trace evidence by infrared spectroscopic imaging by Rohit Bhargava; Rebecca Schwartz Perlman; Daniel C. Fernandez; Ira W. Levin; Edward G. Bartick (pp. 2069-2075).

Current latent print and trace evidence collecting technologies are usually invasive and can be destructive to the original deposits. We describe a non-invasive vibrational spectroscopic approach that yields latent fingerprints that are overlaid on top of one another or that may contain trace evidence that needs to be distinguished from the print. Because of the variation in the chemical composition distribution within the fingerprint, we demonstrate that linear unmixing applied to the spectral content of the data can be used to provide images that reveal superimposed fingerprints. In addition, we demonstrate that the chemical composition of the trace evidence located in the region of the print can potentially be identified by its infrared spectrum. Thus, trace evidence found at a crime scene that previously could not be directly related to an individual, now has the potential to be directly related by its presence in the individual-identifying fingerprints.

Keywords: Latent fingerprints; Trace evidence; Infrared spectroscopy; IR; FT-IR; Spectroscopic imaging; Chemical imaging

Forensic analysis of anthraquinone, azo, and metal complex acid dyes from nylon fibers by micro-extraction and capillary electrophoresis by Amy R. Stefan; Christopher R. Dockery; Alexander A. Nieuwland; Samantha N. Roberson; Brittany M. Baguley; James E. Hendrix; Stephen L. Morgan (pp. 2077-2085).

The extraction and separation of dyes present on textile fibers offers the possibility of enhanced discrimination between forensic trace fiber evidence. An automated liquid sample handling workstation was programmed to deliver varying solvent combinations to acid-dyed nylon samples, and the resulting extracts were analyzed by an ultraviolet/visible microplate reader to evaluate extraction efficiencies at different experimental conditions. Combinatorial experiments using three-component mixture designs varied three solvents (water, pyridine, and aqueous ammonia) and were employed at different extraction temperatures for various extraction durations. The extraction efficiency as a function of the three solvents (pyridine/ammonia/water) was modeled and used to define optimum conditions for the extraction of three subclasses of acid dyes (anthraquinone, azo, and metal complex) from nylon fibers. The capillary electrophoresis analysis of acid dye extracts is demonstrated using an electrolyte solution of 15 mM ammonium acetate in acetonitrile/water (40:60, v/v) at pH 9.3. Excellent separations and discriminating diode array spectra are obtained even for dyes of similar color. Figure Capillary electropherogram of three acid dyes extracted from nylon 6,6 thread

Keywords: Forensic analysis; Nylon fibers; Extraction of acid dyes; Capillary electrophoresis

Microextraction, capillary electrophoresis, and mass spectrometry for forensic analysis of azo and methine basic dyes from acrylic fibers by Amy R. Stefan; Christopher R. Dockery; Brittany M. Baguley; Brandi C. Vann; Alexander A. Nieuwland; James E. Hendrix; Stephen L. Morgan (pp. 2087-2094).

Designed experiments based on a simplex mixture design were employed to explore the effects of three solvent components (water, formic acid, and aqueous acetic acid), extraction time, and extraction temperature for the automated microextraction of basic (cationic) dyes from acrylic fibers. Extractions were conducted by an automated liquid handling system, and dye extraction was evaluated using a UV/visible microplate reader. Highest extraction efficiency for two subclasses of basic dyes (methine and azo) from acrylic fibers was achieved with an extraction solvent containing 88% formic acid/12% water. Cationic dyes were analyzed by capillary electrophoresis using a 45 mM ammonium acetate buffer in acetonitrile–water at pH 4.7. The utility of microextraction combined with capillary electrophoresis–mass spectrometry for analysis of extracts from trace fibers was demonstrated by the detection and characterization of three basic dyes extracted from a 2-mm length of single acrylic fiber.

Keywords: Forensic analysis; Acrylic fibers; Extraction of basic dyes; Capillary electrophoresis

Automated extraction of direct, reactive, and vat dyes from cellulosic fibers for forensic analysis by capillary electrophoresis by C. R. Dockery; A. R. Stefan; A. A. Nieuwland; S. N. Roberson; B. M. Baguley; J. E. Hendrix; S. L. Morgan (pp. 2095-2103).

Systematic designed experiments were employed to find the optimum conditions for extraction of direct, reactive, and vat dyes from cotton fibers prior to forensic characterization. Automated microextractions were coupled with measurements of extraction efficiencies on a microplate reader UV–visible spectrophotometer to enable rapid screening of extraction efficiency as a function of solvent composition. Solvent extraction conditions were also developed to be compatible with subsequent forensic characterization of extracted dyes by capillary electrophoresis with UV–visible diode array detection. The capillary electrophoresis electrolyte successfully used in this work consists of 5 mM ammonium acetate in 40:60 acetonitrile–water at pH 9.3, with the addition of sodium dithionite reducing agent to facilitate analysis of vat dyes. The ultimate goal of these research efforts is enhanced discrimination of trace fiber evidence by analysis of extracted dyes. Figure Fitted absorbance response surface for extraction of a direct dye, C. I. yellow 58, using a ternary solvent system.

Keywords: Forensic analysis; Cotton fibers; Extraction of direct, reactive and vat dyes; Capillary electrophoresis

Gradient HPLC separation of dehydroepiandrosterone (DHEA) from its metabolites and biological congeners: role of tetrahydrofuran in the chromatographic mechanism by András Gergely; Péter Horváth; György Szász; Gábor Veress (pp. 2105-2109).

A three-step gradient reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed for the separation of dehydroepiandrosterone (DHEA), its sulfate ester (DHEA-S), its three C7-oxidized metabolites (7αOH-DHEA, 7βOH-DHEA, 7-keto-DHEA), and its biosynthetic congeners (androstenedione, testosterone, estradiol, pregnenolone). This new method allows the quantitative characterization of DHEA metabolism and biosynthetic transformation under given physiological, pathological, or therapeutically influenced circumstances. Tetrahydrofuran probably acts as a proton acceptor coadsorbent, while isopropanol behaves as a proton donor during the separation of testosterone, estradiol, and the stereoisomers of 7-OH-DHEA. Figure Optimized gradient RP-HPLC results in full separation of DHEA from its biosynthetic congeners and metabolites

Keywords: Dehydroepiandrosterone (DHEA); 7-DHEA derivatives; DHEA biosynthetic congeners; DHEA metabolites; Gradient RP-HPLC

Patterning of 293T fibroblasts on a mica surface by Sen Hou; Xin-Xin Li; Xiao-Yu Li; Xi-Zeng Feng; Li Guan; Yan-Lian Yang; Chen Wang (pp. 2111-2117).

Controllable cell growth on the defined areas of surfaces is important for potential applications in biosensor fabrication and tissue engineering. In this study, controllable cell growth was achieved by culturing 293 T fibroblast cells on a mica surface which had been patterned with collagen strips by a microcontact printing (μCP) technique. The collagen area was designed to support cell adhesion and the native mica surface was designed to repel cell adhesion. Consequently, the resulting cell patterns should follow the micro-patterns of the collagen. X-ray photoelectron spectroscopy (XPS), water contact angle (WCA) measurement, atomic-force microscope (AFM) observation, and force-curve measurement were used to monitor property changes before and after the collagen adsorption process. Further data showed that the patterned cells were of good viability and able to perform a gene-transfection experiment in vitro. This technique should be of potential applications in the fields of biosensor fabrication and tissue engineering. Figure Controllable cells growth has been achieved by culturing 293T fibroblast cells on the mica surface which had been patterned with collagen strips by microcontact printing (µCP) technique

Keywords: Cell adhesion; Microcontact printing; Collagen; Gene transfection; Mica

Development of a metabonomic approach based on LC-ESI-HRMS measurements for profiling of metabolic changes induced by recombinant equine growth hormone in horse urine by Fanny Kieken; Gaud Pinel; Jean-Philippe Antignac; Fabrice Monteau; Anne Christelle Paris; Marie-Agnès Popot; Yves Bonnaire; Bruno Le Bizec (pp. 2119-2128).

Despite the worldwide existing regulation banning the use of the recombinant equine growth hormone (reGH) as growth promoter, it is suspected to be used in horseracing to improve performances. Various analytical methods previously developed to screen for its misuse have encountered some limitations in terms of detection timeframes, in particular during the first days following reGH administration. A novel strategy involving the characterization of global metabolomic fingerprints in urine samples of non-treated and reGH-treated horses by liquid chromatography–electrospray–high-resolution mass spectrometry (LC-ESI-HRMS) is described and assessed in this paper in order to develop a new screening tool for growth hormone abuse in horseracing. The strategy involves a limited sample preparation of the urine samples and the use of appropriate software for data processing and analysis. As preliminary work, reproducibility of both sample preparation and mass spectrometry (MS) measurements was evaluated in order to demonstrate the reliability of the method. Application of the developed protocol on two horses demonstrated the suitability of the developed strategy and preliminary results showed significant modifications of the metabolome after treatment with reGH.

Keywords: Recombinant equine growth hormone; Metabolomic; Fingerprints; Chemometrics; Urine; LC-ESI-HRMS

Iron oxide/tantalum oxide core–shell magnetic nanoparticle-based microwave-assisted extraction for phosphopeptide enrichment from complex samples for MALDI MS analysis by Hong-Yi Lin; Wei-Yu Chen; Yu-Chie Chen (pp. 2129-2136).

A new type of metal-oxide-coated magnetic nanoparticles (NPs)—tantalum-oxide-coated magnetic iron oxide (Fe₃O₄@Ta₂O₅) NPs—which are used as affinity probes for selectively trapping phosphopeptides from complex samples, is demonstrated in this study. In this approach, phosphopeptide enrichment was achieved by incubating the NPs with sample solutions under microwave heating within 1 min. The NP–target species conjugates were readily isolated from samples by magnetic separation followed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometric analysis. When using human serum as the sample, phosphorylated fibrinopeptide-A-derived ions are the only ions observed in the MALDI mass spectra after enrichment by the Fe₃O₄@Ta₂O₅ NPs. Furthermore, only phosphopeptides appear in the MALDI mass spectra after using the affinity probes to selectively trap target species from the tryptic digest of a cell lysate and milk sample. The results demonstrated that the Fe₃O₄@Ta₂O₅ NPs have the capability of selectively trapping phosphorylated peptides from complex samples. The detection limit of this approach for a phosphopeptide (FQpSEEQQQTEDELQDK) was ~10 fmol. Figure For the first time, tantalum oxide-coated magnetic iron oxide (Fe₃O₄@Ta₂O₅) NPs were demonstrated as suitable affinity-probes for selectively trapping phosphopeptides from complex samples. To shorten the analysis time, phosphopeptide enrichment was achieved by incubating the NPs with sample solutions under microwave-heating within 1 min. MALDI MS was employed for characterization of the species trapped by the NPs.

Keywords: Tantalum oxide; Magnetic nanoparticles; Phosphopeptides; MALDI MS

Determination of enzyme activity inhibition by FTIR spectroscopy on the example of fructose bisphosphatase by M. López-Sánchez; M. J. Ayora-Cañada; A. Molina-Díaz; M. Siam; W. Huber; G. Quintás; S. Armenta; B. Lendl (pp. 2137-2144).

A mid-infrared enzymatic assay for label-free monitoring of the enzymatic reaction of fructose-1,6-bisphosphatase with fructose 1,6-bisphosphate has been proposed. The whole procedure was done in an automated way operating in the stopped flow mode by incorporating a temperature-controlled flow cell in a sequential injection manifold. Fourier transform infrared difference spectra were evaluated for kinetic parameters, like the Michaelis–Menten constant (K _M) of the enzyme and V _max of the reaction. The obtained K _M of the reaction was 14 ± 3 g L⁻¹ (41 μM). Furthermore, inhibition by adenosine 5′-monophosphate (AMP) was evaluated, and the K _M ^App value was determined to be 12 ± 2 g L⁻¹ (35 μM) for 7.5 and 15 μM AMP, respectively, with V _max decreasing from 0.1 ± 0.03 to 0.05 ± 0.01 g L⁻¹ min⁻¹. Therefore, AMP exerted a non-competitive inhibition.

Keywords: Fourier transform infrared spectroscopy; Sequential injection analysis; Enzyme; Inhibition kinetics; Fructose-1,6-bisphosphatase

In vitro amyloid Aβ_1-42 peptide aggregation monitoring by asymmetrical flow field-flow fractionation with multi-angle light scattering detection by Diana C. Rambaldi; Andrea Zattoni; Pierluigi Reschiglian; Raffaella Colombo; Ersilia De Lorenzi (pp. 2145-2149).

Self-assembly of the 42-amino-acid-long amyloid peptide Aβ_1-42 into insoluble fibrillar deposits in the brain is a crucial event in the pathogenesis of Alzheimer's disease. The fibril deposition occurs through an aggregation process during which transient and metastable oligomeric intermediates are intrinsically difficult to be accurately monitored and characterised. In this work, the time-dependent Aβ_1-42 aggregation pattern is studied by asymmetrical flow field-flow fractionation with on-line multi-angle light scattering detection. This technique allows separating and obtaining information on the molar mass (M _r) and size distribution of both the early-forming soluble aggregates and the late prefibrillar and fibrillar species, the latter having very high M _r. Preliminary results demonstrate that unique information on the dynamic aggregation process can be obtained, namely on the M _r and size of the forming aggregates as well as on their formation kinetics.

Keywords: Flow field-flow fractionation; Multi-angle light scattering detection; ß-amyloid peptide aggregation

Surface plasmon resonance immunosensor for cortisol and cortisone determination by Marco Frasconi; Monica Mazzarino; Francesco Botrè; Franco Mazzei (pp. 2151-2159).

In this paper, we present a surface-plasmon-resonance-based immunosensor for the real-time detection of cortisol and cortisone levels in urine and saliva samples. The method proposed here is simple, rapid, economic, sensitive, robust, and reproducible thanks also to the special features of the polycarboxylate-hydrogel-based coatings used for the antibody immobilization. The sensor surface displays a high level of stability during repeated regeneration and affinity reaction cycles. The immunosensor shows high specificity for cortisol and cortisone; furthermore, no significant interferences from other steroids with a similar chemical structure have been observed. The suitability of the hydrogel coating for the prevention of nonspecific binding is also investigated. A good correlation is noticed between the results obtained by the proposed method and the reference liquid chromatography/tandem mass spectrometry method for the analysis of cortisol and cortisone in urine and saliva samples. Standard curves for the detection of cortisol and cortisone in saliva and urine are characterized by a detection limit less than 10 μg l⁻¹, sufficiently sensitive for both clinical and forensic use. Application of a newly developed SPR immunosensor for the measurement of cortisol in anti-doping analysis

Keywords: Cortisol; Cortisone; SPR; Immunosensor; Antidoping

Use of Fourier transform infrared spectroscopy and chemometrics to discriminate clinical isolates of bacteria of the Burkholderia cepacia complex from different species and ribopatterns by Carla Patrícia Coutinho; Isabel Sá-Correia; João Almeida Lopes (pp. 2161-2171).

A methodology for the discrimination of Burkholderia cepacia complex (Bcc) clinical isolates at the species level and at the ribopattern level using Fourier transform infrared (FTIR) spectroscopy and chemometrics analysis was assessed in this study. Different Bcc sequential isolates collected at the Santa Maria Hospital (HSM), in Portugal, from clinically infected cystic fibrosis (CF) patients were previously classified by established molecular methods at the species level and differentiated at the strain level, based on their ribopatterns. A set of 185 of these isolates, representing four different Bcc species (Burkholderia cepacia, Burkholderia cenocepacia (recA lineages III-A and III-B), Burkholderia multivorans and Burkholderia stabilis), was analyzed by FTIR and results were processed with chemometric methods. Ten reference strains of these species were used to test the FTIR method. The discrimination at the species level led to misclassification error rates of 10% and 32% for the HSM isolates and reference strains, respectively, clearly indicating that the FTIR classification method was unable to generalize results for the reference strains. Infrared spectra of HSM isolates were further analyzed in terms of the discrimination according to the ribopattern. Results showed misclassification error rates of 4%, 2%, and 8% for B. cepacia, B. cenocepacia III-A, and B. cenocepacia III-B ribopatterns, respectively. These results demonstrated good FTIR spectroscopy discrimination capacity at the ribopattern level, for the HSM isolates but showed difficulty at the species level, especially when the reference strains were included. Remarkably, this methodology was found to discriminate isolates belonging to the same species and ribopattern that were collected from the same patient during prolonged colonization, opening the door to the identification of chemical modifications resulting from adaptation strategies to the CF lung stressing environment, in particular to aggressive and prolonged antibiotic therapy.

Keywords: Burkholderia cepacia complex; Fourier transform infrared spectroscopy; Chemometrics; Cystic fibrosis; Bacteria identification

Electrochemical immunoassay using quantum dot/antibody probe for identification of cyanobacterial hepatotoxin microcystin-LR by Hye-Weon Yu; Jinwook Lee; Sungyoun Kim; Giang Huong Nguyen; In S. Kim (pp. 2173-2181).

The presence of cyanobacterial hepatotoxins such as microcystin-LR poses health threats to humans due to their potential for causing severe physiological effects when contaminated drinking water is ingested. Here, the electrochemical detection of microcystin-LR is explored using a quantum dot/antibody (QD/Ab) probe for nanoparticle-based amplification and direct electrochemical transduction. The immunological recognition of microcystin-LR using the QD/Ab probe was amplified and converted to an electrochemical signal by measuring the cadmium ions released from QD based on square wave stripping voltammetry under optimized electrochemical factors. Whereas a qualitative analysis for microcystin-LR was achieved using the specific peak potential of the anodic voltammogram at −0.6 ± 0.05 V, concentration of the toxin was quantified based on the charge density of the anodic peak; a dynamic range of 0.227 to 50 μg/L and limit of detection of 0.099 μg/L were obtained with high sensitivity. The extracted microcystin-LR from Microcystis aeruginosa was estimated as 1,944 μg/g of dried weight of the microorganism.

Keywords: Biosensor; Microcystin-LR; Quantum-dot/antibody probe; Nanoparticle-based amplification; Direct electrochemical transduction

Improvement of the traceability of reference materials certified for purity: evaluation of a semi-preparative high-performance liquid chromatography approach by Thierry Le Goff; Steve Wood (pp. 2183-2192).

A semi-preparative high-performance liquid chromatography process was evaluated as a tool to quantitatively determine the purity or percentage mass fraction content (% m/m) of organic compounds. The method is simple and does not require the identification and subsequent quantitation of organic-related structure impurities. A protocol was developed and tested on four reference materials certified for purity from 95% m/m to 99.3% m/m. Comparing the purity results of each certified reference material using the new approach with their respective certified values showed no significant analytical bias. Semi-preparative high-performance liquid chromatography has proved the potential to be a primary method directly traceable to mass with an uncertainty statement written down also in terms of mass with expanding uncertainty ranging from 0.8% to 1.3% m/m compared to 0.3 to 2.0% m/m for the certified purity values at the 95% confidence interval.

Keywords: Certified reference material; Purity; Semi-preparative HPLC

A new example of reversal of the order of migration of enantiomers, as a function of cyclodextrin concentration and pH, by cyclodextrin-modified capillary zone electrophoresis: enantioseparation of 6,6′-dibromo-1,1′-binaphthyl-2,2′-diol by H. Krajian; N. Mofaddel; D. Villemin; P. L. Desbène (pp. 2193-2201).

Enantioseparation of 6,6′-dibromo-1,1′-binaphthyl-2,2′-diol (DBBD) by cyclodextrin-modified capillary zone electrophoresis (CD-CZE) was studied using the three native α, β, and γ cyclodextrins, the three hydroxypropylated cyclodextrins (2-hydroxypropyl-α, β, and γ), heptakis-2,6-di-O-methyl-β-CD (DM-β-CD), and heptakis-2,3,6-tri-O-methyl-β-cyclodextrin (TM-β-CD). First, the acidity constants of DBBD were determined using capillary electrophoresis, before performing enantioseparation. The influence of the concentrations of the studied cyclodextrins on the enantioseparation was explored and the experimental optimal concentrations were determined and compared to the theoretical optimal concentrations. Moreover, the apparent complexation constants between each studied cyclodextrin and the two DBBD enantiomers were evaluated using a non-linear curve fitting method and three linear plotting methods (x-reciprocal, y-reciprocal and double reciprocal). For TM-β-CD, the order of migration of the enantiomers of DBBD reversed as a function of TM-β-CD concentration. The influence of the nature of methylated cyclodextrin derivatives (methyl-β-CD (M-β-CD) and DM-β-CD) was then studied. Inversion of the order of migration of the enantiomers of DBBD was observed for DM-β-CD, whereas the S enantiomer of DBBD always migrated first for M-β-CD.

Keywords: 6,6′-Dibromo-1,1′-binaphthyl-2,2′-diol; CD-CZE; Order of migration of enantiomers; Enantioseparation; Methylated cyclodextrins

Development and validation of a sensitive and selective method using GC/MS-MS for quantification of 5-fluorouracil in hospital wastewater by Jean-Ulrich Mullot; Sara Karolak; Anne Fontova; Bruno Huart; Yves Levi (pp. 2203-2212).

Pollution of the environment by pharmaceuticals is a subject of growing scientific and societal concern. However, few quantitative data have been reported concerning hospital wastewater contamination. Among the different molecules used at hospital, antineoplastic drugs appear to be of special interest, and 5-fluorouracil (5-FU) can be considered as a key compound of this therapeutic class. To monitor this pharmaceutical in hospital wastewater, a highly specific and selective method was developed using gas chromatography tandem mass spectrometry after solid-phase extraction. This sensitive method (limit of quantification = 40 ng L⁻¹) was then applied to assess sewage contamination of a middle-size hospital with oncology service located in Paris, France. Native 5-FU was detectable in 12 of the 14 analysed samples. In positive samples, concentration range was measured from 0.09 to 4.0 μg L⁻¹. Finally, a predicting model for the hospital wastewater concentrations is presented, and results of this model are discussed.

Keywords: 5-Fluorouracil; Hospital; Wastewater; Predicted concentrations; GC/MS/MS

Quantitation and identity confirmation of residues of quinolones in tilapia fillets by LC-ESI-MS-MS QToF by Jonas Augusto Rizzato Paschoal; Felix Guillermo Reyes Reyes; Susanne Rath (pp. 2213-2221).

A method for simultaneous determination of flumequine (FLM), oxolinic acid (OXO), sarafloxacin (SAR), danofloxacin (DAN), enrofloxacin (ENR), and ciprofloxacin (CIP) in tilapia (Orechromis niloticus) fillets, using liquid chromatography-tandem mass spectrometry (LC-ESI-MS-MS QToF) is presented. The quinolones were extracted from the food matrix with a solution of 10% trichloroacetic acid-methanol (80:20 v/v) with ultrasonic assistance. Clean-up of the extract solution was performed by using polymeric solid-phase extraction cartridges. The LC separation was carried out on an octadecyl hybrid silica column (C₁₈, 150 mm × 3 mm, 5 μm). The column temperature was set at 30 °C, and gradient elution (0.2 mL min⁻¹) was performed using water and acetonitrile, both containing 0.1% of acetic acid, as mobile phase components. The analytes were ionized using electrospray in the positive polarity mode. The following analytical results were obtained: linearity was about 0.99 for all the quinolones; intra and inter-assay precision (RSD%) were lower than 12.7 and 20%, respectively; and recoveries were from 89 to 112%. The quantitation limits were below the maximum residue limits established for the analytes. The method is suitable for the determination of quinolone residues in fish fillets and the QToF technique made it possible to obtain m/z ratios with less than 10 ppm of error for each analyte.

Keywords: Quinolones; Fish; Tilapia; LC-MS-MS QToF

Development of an enzyme-linked immunosorbent assay for metolcarb residue analysis and investigation of matrix effects from different agricultural products by Jing-Wei Sun; Bing Liu; Yan Zhang; Shuo Wang (pp. 2223-2230).

The development of a direct competitive enzyme-linked immunosorbent assay based on polyclonal antibodies for N-methylcarbamate insecticide metolcarb is described. Two new haptens for the metolcarb were designed and synthesized. Both haptens were conjugated with keyhole limpet hemocyanin to form the immunogens. Four rabbits were immunized with the immunogens for production of polyclonal antibodies against metolcarb. Antisera titers were tested on the homologous coating antigens using a noncompetitive indirect enzyme-linked immunosorbent assay. The high titer antisera were used to develop the direct competitive enzyme-linked immunosorbent assay for the detection of metolcarb. The antibody–antigen combination with the highest selectivity for metolcarb was further optimized and its tolerance to changes in chemical conditions (ionic strength, pH value, and organic solvent) was studied. Under optimum conditions, the sensitivity and the limit of detection were determined to be 22 μg L⁻¹ and 1.2 μg L⁻¹ respectively. Determination of metolcarb in fruit juices and vegetables was accomplished by simple, rapid, and efficient extraction methods. Recoveries of metolcarb from spiked samples ranged from 80.5% to 109.5%. Validation of the developed immunosorbent assay was conducted by comparison of results from high-performance liquid chromatography. The correlation between the data obtained using developed immunosorbent assay and high-performance liquid chromatography was high (R ² = 0.9884). Therefore, the developed immunosorbent assay in this study was suitable for the rapid quantitative determination of metolcarb in agricultural products.

Keywords: Metolcarb; Enzyme-linked immunosorbent assay; Polyclonal antibodies; Matrix effects

Determination of dimethyl fumarate and other potential allergens in desiccant and antimould sachets by J. Pablo Lamas; Lucia Sanchez-Prado; Jorge Regueiro; Maria Llompart; Carmen Garcia-Jares (pp. 2231-2239).

A method for the determination of dimethyl fumarate (DMF), benzothiazole (BT) and tert-butylphenol (TBP) in desiccant and antimould agents employed for protecting consumer products from humidity and mould has been developed. The method is based on ultrasound-assisted extraction (UAE) followed by GC-MS analysis. Parameters that could affect the extraction of the compounds have been optimised using a multivariate approach. In the final conditions, the extraction is performed using only 0.5 or 1 mL ethyl acetate and applying ultrasound energy for 5 min. Simultaneous extractions could also be carried out in 5 min without losing efficiency. The method was validated showing good linearity (R ² >0.995). Both intra- and inter-day precisions were studied at several concentration levels, being satisfactory in all cases (RSD <10%). Recovery was evaluated in four real desiccant samples at different compound concentrations, ranging between 87% and 109%. Limits of detection and quantification were in the low nanogramme per gramme level, thus allowing the determination of DMF at concentrations well below the limit established by the recent EU Directive (0.1 μg/g). The proposed procedure was applied to the determination of the target compounds in several desiccant and antimould samples. Although most of them were simply labelled as “silica gel”, more than 70% of the tested samples contained high amounts of DMF, many of them at the high microgram per gramme level. Many samples also showed the presence of the other two potential allergens. These results demonstrate that the content of the “desiccant” sachets and tablets in consumer products does not usually belong with the label of the desiccant, and hence, the high risk of exposition to the powerful allergen DMF and other potentially harmful chemicals through consumer goods should be a matter of concern.

Keywords: Dimethyl fumarate; Benzothiazol; tert-Butylphenol; Ultrasound-assisted extraction; GC-MS; Desiccant; Antimould agent; Allergy

Rapid analysis of organic farming insecticides in soil and produce using ultra-performance liquid chromatography/tandem mass spectrometry by Dariusz Drożdżyński; Jolanta Kowalska (pp. 2241-2247).

A new method for the analysis of three ecological insecticides, namely azadyrachtin, spinosad (sum of spinosyn A and spinosyn D) and rotenone, in produce and soil samples is presented. Investigated compounds are one of the most significant insecticides authorized for organic farming crop protection in many countries. Extraction of the pesticides from plant and soil matrices was performed by using a modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) method. The method entailed a single extraction of the investigated compounds with acidified acetonitrile followed by a dispersive solid-phase extraction cleanup step prior to the final determination by reverse-phase ultra-performance liquid chromatography/tandem quadrupole mass spectrometry (UPLC-MS/MS). Validation studies were carried out on cabbage, tomato and soil samples. Recoveries of the spiked samples were in the range between 67% and 108%, depending on the matrix and the spiking level. Relative standard deviations for all matrix–compound combinations did not exceed 12%. The limits of quantification were ≤0.01 mg kg⁻¹ in all cases, except for azadirachtin. The developed method was applied to the analysis of real samples originating from organic farming production.

Keywords: Ecological insecticides; Soil; Food; QuEChERS; UPLC-MS/MS

Chromatographic evaluation of polymers imprinted with analogs of chloramphenicol and application to selective solid-phase extraction by Christina Schirmer; Hans Meisel (pp. 2249-2255).

To obtain a highly selective material for the antibiotic chloramphenicol, which has several harmful side effects in humans, different molecularly imprinted polymers (MIPs) were prepared. In order to avoid a major traditional drawback associated with MIPs of residual template bleeding, molecules that are structurally related to chloramphenicol were used as templates for polymer synthesis. Chromatographic evaluation indicated that the employed template imparted a significant influence on the recognition properties of the corresponding polymer. A strong retention of chloramphenicol under nonpolar elution conditions (k = 68.03, IF = 17.72) and under aqueous elution conditions (k = 92.44, IF = 1.35) was achieved. After chromatographic evaluation, the MIP was utilized as the recognition sorbent in a solid-phase extraction to determine chloramphenicol using either an organic or aqueous washing solvent. Recoveries of nearly 100% from the chloramphenicol standard solution and nearly 90% from honey samples spiked with chloramphenicol were attained. Furthermore, the applicability of the MIP for sample cleanup was demonstrated.

Keywords: Molecular imprinting; Liquid chromatography; Solid-phase extraction; Chloramphenicol; Honey

Selection of SPE cartridge for automated solid-phase extraction of pesticides from water followed by liquid chromatography-tandem mass spectrometry by Timothy B. Jordan; David S. Nichols; Neil I. Kerr (pp. 2257-2266).

In environmental analyses there is an ever-increasing need to develop simple and sensitive multi-residue methods. In many agricultural regions, there is particular concern of the potential for pesticides to enter rivers and other waterways. This study reports on the development and validation of a multi-residue method of analysis for 30 pesticides in water samples using solid-phase extraction (SPE) followed by LC-MS/MS. The electrospray and MS/MS parameters were optimised for each pesticide, including capillary voltage, collision-induced dissociation voltage, and selection of a precursor ion and two product ions. A variety of SPE sorbents were tested for sample pre-concentration, including numerous polymeric based phases. Bond Elut PPL and Oasis HLB were the only phases capable of retaining the majority of the target analyte classes in a single method. An off-line pre-concentration method using a Gilson Aspec system was optimised using the Bond Elut PPL cartridges, with a concentration factor of 25 producing limits of quantitation in the order of 6–100 ng/L. Excellent linearity (r ² > 0.9), precision (<20%) and recovery (>60%) was obtained for nearly all of the analytes, covering a wide variety of chemical and physical properties. This is the first study to fully validate Bond Elut PPL cartridges for use in multi-residue pesticide analysis.

Keywords: SPE; Bond Elut PPL; Pesticide; LC-MS/MS

Effect of Fusarium oxysporum f. sp. lycopersici on the degradation of humic acid associated with Cu, Pb, and Ni: an in vitro study by Alma Rosa Corrales Escobosa; Julio Alberto Landero Figueroa; J. Félix Gutiérrez Corona; Katarzyna Wrobel; Kazimierz Wrobel (pp. 2267-2276).

The intent of this work was to gain further insight on the fungus-assisted degradation/solubilization of humic acid and the related changes in metal-binding profiles. In the experimental design, Aldrich reagent humic acid (HA) or HA enriched with Cu, Pb, and Ni (HA(Me)) was added to Fusarium oxysporum f. sp. lycopersici cultures in vitro. The cultures were supplied by different carbon- and nitrogen-containing nutrients (glucose, Glc, or glutamate, Glu and ammonium, NH ₄ ⁺ , or nitrate, NO ₃ ⁻ , ions, respectively) in order to examine their possible effect on HA and HA(Me) decomposition. During the first 48 h of fungus growth, gradual acidification to pH 2 was observed in medium containing Glc + NH ₄ ⁺ , while for other cultures, alkalinization to pH 9 occurred and then, the above conditions were stable up to at least 200 h. Size exclusion chromatography (SEC) with UV/Vis detection showed progressive degradation and solubilization of both HA and HA(Me) with the increasing time of fungus growth. However, the molecular mass distributions of HA-related soluble species were different in the presence of metals (HA(Me)) as referred to HA and were also influenced by the composition of growth medium. The solubilization of Pb, Cu, and Ni and their association with HA molecular mass fractions were studied using inductively coupled plasma mass spectrometry (ICP-MS) detection. Under acidic conditions, relatively high concentrations of low-molecular-mass metallic species were found in culture supernatants, while in alkaline media, metal solubilization was generally poorer. In contrast to low pH culture, SEC-ICP-MS results obtained in alkaline supernatants indicated metal binding to degradation products of humic substances of MM > 5 kDa. In summary, the results of this study suggest that fungus-assisted degradation of HA and HA(Me) might be controlled using appropriate N- and C- sources required for fungus growth, which in turn would affect molecular mass distribution of soluble metallic species thus potentially influencing their actual bioaccessibility.

Keywords: Humic acid; Metals; Fusarium oxysporum ; SEC (size exclusion chromatography); ICP-MS (inductively coupled plasma–mass spectrometry)

Time-resolved flow-flash FT-IR difference spectroscopy: the kinetics of CO photodissociation from myoglobin revisited by Michael Schleeger; Christoph Wagner; Michiel J. Vellekoop; Bernhard Lendl; Joachim Heberle (pp. 2277-2277).

Use of spermine and thiabendazole as analyte protectants to improve direct analysis of 16 carbamates by gas chromatography–mass spectrometry in green vegetable matrices by Cédric Przybylski; Véronique Bonnet (pp. 2279-2280).

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Analytical and Bioanalytical Chemistry (v.394, #8)

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Analytical and Bioanalytical Chemistry (v.394, #8)