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Analytical and Bioanalytical Chemistry (v.403, #4)

Euroanalysis XVI—challenges in modern analytical chemistry by Slavica Ražić (pp. 899-901).

is a professor in the Department of Analytical Chemistry of the Faculty of Pharmacy of the University of Belgrade. She served as the Head of the Institute of Analytical Chemistry for two terms in the last decade. Her main research interests are analysis of environmental and biological samples, method development, and monitoring trace and minor elements by use of atomic absorption and emission spectrometry (F–AAS, F–AES, GF–AAS, ICP–AES, and ICP–MS) combined with contemporary sample-preparation techniques and chemometric methods of analysis. Elucidation of the bioavailability, mobility, distribution, and accumulation of elemental species in different environmental compartments, and their affect on human health, is a constant analytical challenge. Her current research interests were recently extended to GC–MS analysis of active organic compounds in plants important in phytopharmacy. Since 2002, she has chaired the Division of the Analytical Chemistry of the Serbian Chemical Society (SCS) and also served as its representative in the Division of Analytical Chemistry of the European Association of Chemical and Molecular Sciences (EuCheMS). Slavica Ražić is a member of the editorial board of the Journal of the Serbian Chemical Society and is a subeditor of Analytical Chemistry.

Some people and places important in the history of analytical chemistry in Serbia by D. Thorburn Burns; Snežana Bojović (pp. 903-908).

Burns MRIA FRSE has been Emeritus Professor of Analytical Chemistry in the Queen’s University of Belfast since 1999, currently a Visiting Senior Research Fellow in the School of Chemistry and actively involved with the research and publication carried out by the Government Chemist Programme in LGC. He is Head of the Study Group History in the Division of Analytical Chemistry of EuCheMS and has published 12 books and 420 papers including studies of the history of analytical chemistry in the host countries of EUROANALYSIS and of Robert Boyle in particular. taught Organic Chemistry at the High Pedagogic School in Belgrade from 1975-1987. Since 1987 she has lectured on the History of Chemistry and Chemistry Didactics in the Faculty of Chemistry at the University of Belgrade and from 1990 to postgraduate students on the History of Sciences in Serbia to postgraduate students in the Faculty of Chemistry and in Department of History and Philosophy of Natural Sciences and Technology. She has published 10 books and 60 papers mainly on the history of chemistry and education in Serbia.

Characterization of in vitro metabolic profiles of cinitapride obtained with liver microsomes of humans and various mammal species using UHPLC and chemometric methods for data analysis by Helena Marquez; Joan Albertí; Miquel Salvà; Javier Saurina; Sonia Sentellas (pp. 909-916).

An ultra-high performance liquid chromatographic method has been utilized to obtain metabolic profiles of cinitapride with liver microsomes of humans and various mammal species such as rats, mice, mini pigs, dogs, and monkeys. Metabolites have been generated by incubation of cinitapride in the presence of microsomes using nicotinamide adenine dinucleotide phosphate as a cofactor. Incubation times from 15 to 60 min have been assayed. Cinitapride and its metabolites have been separated by reversed-phase C₁₈ mode using ammonium formate aqueous solution (pH 6.5) and acetonitrile as the components of the mobile phase. Concentrations of metabolites in the incubated samples have resulted in an excellent source of multivariate data to be used to extract metabolic information. Statistic parameters and principal component analysis have been used to compare the in vitro metabolism of humans with the other species.

Keywords: Cinitapride; Metabolic profiling; Liver microsomes; Humans; Mammals; UHPLC; Chemometrics; Principal component analysis

Sensitive and rapid amperometric magnetoimmunosensor for the determination of Staphylococcus aureus by Berta Esteban-Fernández de Ávila; María Pedrero; Susana Campuzano; Vanessa Escamilla-Gómez; José M. Pingarrón (pp. 917-925).

The preparation and characteristics of a disposable amperometric magnetoimmunosensor, based on the use of functionalized magnetic beads (MBs) and gold screen-printed electrodes (Au/SPEs), for the specific detection and quantification of Staphylococcal protein A (ProtA) and Staphylococcus aureus (S. aureus) is reported. An antiProtA antibody was immobilized onto ProtA-modified MBs, and a competitive immunoassay involving ProtA antigen labelled with HRP was performed. The resulting modified MBs were captured by a magnetic field on the surface of tetrathiafulvalene-modified Au/SPEs and the amperometric response obtained at −0.15 V vs the silver pseudo-reference electrode of the Au/SPEs after the addition of H₂O₂ was used as transduction signal. The developed methodology showed very low limits of detection (1 cfu S. aureus/mL of raw milk samples), and a good selectivity against the most commonly involved foodborne pathogens originating from milk. These features, together with a short analysis time (2 h), the simplicity, and easy automation and miniaturization of the required instrumentation make the developed methodology a promising alternative in the development of devices for on-site analysis. Figure Schematic display of the developed S. aureus amperometric magnetoimmunosensor

Keywords: Electrochemical magnetoimmunosensor; Protein A; Staphylococcus aureus ; Raw milk

Miniaturized extraction methods of triclosan from aqueous and fish roe samples. Bioconcentration studies in zebrafish larvae (Danio rerio) by R. Gonzalo-Lumbreras; J. Sanz-Landaluze; J. Guinea; C. Cámara (pp. 927-937).

Triclosan, an antibacterial and antifungal agent, is widely used in household and personal care products, processed foods and food packaging, etc., and thus also released into the environment. Triclosan is acutely and chronically toxic to aquatic organisms and bioaccumulates in fish tissue. Here, we propose a new miniaturized triclosan extraction method for aqueous and fish roe samples, based on the use of a vortex mixer and an ultrasonic probe, respectively, and useful for triclosan determination by gas chromatography coupled to a micro electron capture detector. Different solvents for extraction and sorbents for clean-up purposes were tested. Multivariate optimization of the variables affecting ultrasonic extraction (ultrasound radiation amplitude, sonication time, sample temperature, and the ratio of sample amount and extracting volume) was carried out. Solvent extraction using ethyl acetate and further clean-up with mixed bed cartridges with two layers of Florisil® and Florisil® impregnated with 10% sulfuric acid only for fish roe samples was finally selected. Extraction efficiencies of up to 95% and 90%, and detection limits of 0.165 ng ml⁻¹ and 2.7 ng g⁻¹ for aqueous and fish roe samples were obtained, respectively. The optimized method was used in bioconcentration studies with zebrafish larvae (Danio rerio), as an alternative method to the Organization for Economic Cooperation and Development technical guideline 305. Bioconcentration values, BCF = 2,630 and 2,018 at exposure concentrations of 30 and 3 μg L⁻¹, respectively, were assessed. These results are in agreement with those reported in the literature, showing the feasibility of the method for estimation of toxicokinetic parameters and bioconcentration factors using zebrafish larvae instead of adult fishes, reducing considerable animal testing, as suggested by the European legislation.

Keywords: Miniaturization; Triclosan; Bioconcentration; Zebrafish larvae; OCDE 305

Ultrasensitive determination of human growth hormone (hGH) with a disposable electrochemical magneto-immunosensor by V. Serafín; N. Úbeda; L. Agüí; P. Yáñez-Sedeño; J. M. Pingarrón (pp. 939-946).

In this paper, an electrochemical magneto-immunosensor for the detection of human growth hormone (hGH) is described for the first time. The immunosensor involves the use of tosyl-activated magnetic microparticles (TsMBs) to covalently immobilize a monoclonal mAbhHG antibody. A sandwich-type immunoassay with a secondary pAbhGH antibody and anti-IgG labelled with alkaline phosphatase (anti-IgG-AP) was employed. TsMBs–mAbhGH–hGH–pAbhGH–anti-IgG-AP conjugates were deposited onto the surface of a screen-printed gold electrode using a small neodymium magnet, and electrochemical detection was performed by square-wave voltammetry upon the addition of 4-aminophenyl phosphate as the AP substrate. All the variables involved in the preparation of immunoconjugates and in the immunoassay protocol were optimized. A calibration curve for hGH was constructed with a linear range between 0.01 and 100 ng/mL (r = 0.998) and a limit of detection of 0.005 ng/mL. This value is nearly three orders of magnitude lower than that obtained using surface plasmon resonance (Treviño et al., Talanta 78:1011–1016, 2009). Furthermore, good repeatability, with RSD = 3% (n = 10) at the 1-ng/mL hGH level, was obtained. Cross-reactivity studies with other hormones demonstrated good selectivity. The magneto-immunosensor was applied to the analysis of human serum spiked with hGH at the 4- and 0.1-ng/mL levels. Mean recoveries of 96 ± 6% and 99 ± 2%, respectively, were obtained.

Keywords: Human growth hormone; Immunosensor; Electrochemical; Tosyl-activated magnetic microparticles; Screen-printed electrodes

Simultaneous analysis of plasma and CSF by NMR and hierarchical models fusion by Agnieszka Smolinska; Joram M. Posma; Lionel Blanchet; Kirsten A. M. Ampt; Amos Attali; Tinka Tuinstra; Theo Luider; Marek Doskocz; Paul J. Michiels; Frederic C. Girard; Lutgarde M. C. Buydens; Sybren S. Wijmenga (pp. 947-959).

Because cerebrospinal fluid (CSF) is the biofluid which interacts most closely with the central nervous system, it holds promise as a reporter of neurological disease, for example multiple sclerosis (MScl). To characterize the metabolomics profile of neuroinflammatory aspects of this disease we studied an animal model of MScl—experimental autoimmune/allergic encephalomyelitis (EAE). Because CSF also exchanges metabolites with blood via the blood–brain barrier, malfunctions occurring in the CNS may be reflected in the biochemical composition of blood plasma. The combination of blood plasma and CSF provides more complete information about the disease. Both biofluids can be studied by use of NMR spectroscopy. It is then necessary to perform combined analysis of the two different datasets. Mid-level data fusion was therefore applied to blood plasma and CSF datasets. First, relevant information was extracted from each biofluid dataset by use of linear support vector machine recursive feature elimination. The selected variables from each dataset were concatenated for joint analysis by partial least squares discriminant analysis (PLS-DA). The combined metabolomics information from plasma and CSF enables more efficient and reliable discrimination of the onset of EAE. Second, we introduced hierarchical models fusion, in which previously developed PLS-DA models are hierarchically combined. We show that this approach enables neuroinflamed rats (even on the day of onset) to be distinguished from either healthy or peripherally inflamed rats. Moreover, progression of EAE can be investigated because the model separates the onset and peak of the disease. Figure Graphical representation of Hierarchical Models Fusion applied to concatenated plasma and CSF datasets.

Keywords: EAE; Multiple sclerosis; Metabolomics; Data fusion; Classification; Variable selection

Simultaneous online SPE-LC-MS/MS quantification of six widely used synthetic progestins in human plasma by Christina Moser; Daniela Zoderer; Gerhard Luef; Markus Rauchenzauner; Ludwig Wildt; Andrea Griesmacher; Christoph Seger (pp. 961-972).

Co-administration of synthetic progestin containing hormonal contraceptives (HCs) and antiepileptic drugs (AEDs) is a common clinical situation which needs specific considerations due to drug interactions. Several studies have demonstrated that lamotrigine plasma levels are significantly decreased during co-medication with HCs, and that this interaction is associated with increased seizure frequency in most of the cases. Additionally, an increase in contraceptive failure and unintended pregnancy could be observed during co-medication. Hence, monitoring of progestin plasma levels in patients with AED co-medication is of interest. A rapid and reliable online solid-phase extraction-high performance liquid chromatography–tandem mass spectrometry (online SPE-LC-MS/MS) method using gradient elution in the LC domain was established and validated for the simultaneous quantitative determination of gestodene, dienogest, drospirenone, etonogestrel, cyproterone acetate, and levonorgestrel in human plasma. The online SPE-LC-MS/MS method covered a quantification concentration range of 5–100 ng/ml for dienogest, 1–100 ng/ml for etonogestrel and 2–100 ng/ml for all other analytes. Stable isotope-labeled internal standards were used for analyte quantification based on selected reaction monitoring experiments. Inter- and intra-assay precision and accuracy were determined from quality control (QC) samples at the lower limits of quantification and at low, medium, and high concentration levels within the calibration range. Inter-assay reproducibility at the QC levels was better than 10% (relative standard deviation, RSD), accuracy at these levels ranged from −3.7% to 11.3%. Total extraction efficiency, tested at three concentrations, ranged from 92.5% to 106.4%. Matrix interferences were excluded by post-column infusion experiments. To prove the applicability of the assay in clinical cohorts, a sample set (n = 298) stemming from study patients under AED/oral HC co-medication was screened for progestin plasma levels. This method has to be considered a research-use-only assay and must not be used for diagnostic or therapeutic purposes, since it did not undergo formal performance evaluation in the sense of the IVD directive (98/79/EG) of the European Community.

Keywords: LC-MS/MS; SRM; Drug monitoring; Progestins; Antiepileptics

DESI-MS²: a rapid and innovative method for trace analysis of six cytostatic drugs in health care setting by Giovanni Fabrizi; Marzia Fioretti; Lucia Mainero Rocca; Roberta Curini (pp. 973-983).

With the aim of establishing exposure levels for hospital personnel preparing and administering cytostatic drugs (CDs), here, we present an innovative screening method based on the use of the desorption electrospray ionization (DESI) interface coupled with a hybrid quadrupole linear ion trap mass spectrometer. A rapid, simple, and sensitive procedure was developed for the simultaneous surface monitoring of cyclophosphamide, dacarbazine, methotrexate, vincristine, gemcitabine, and cytarabine. Since analytes were in the solid state, a novel approach based on the use of passive samplers was combined with the direct analysis of wipes. A PTFE-printed glass slide was used as a passive sampler, while hydrophobic centers of Swiffer® cloths were judged extremely efficient as wipe samplers. After the sampling period, the CD collectors were directly processed with the DESI-MS system without any further treatment. MS/MS confirmatory analysis was conducted using selected reaction monitoring in the positive ion mode and detection limits were evaluated. Values were at the picograms per square millimeter levels on the passive collector and at the picograms per square centimeter levels for the wipe ones. Direct determination on solid-state samples combined with mass spectrometry selectivity provided a powerful tool so far unapplied to occupational hygiene. Figure DESI interface detail (spray emitter and MS inlet) with 3D spacefill structures of the six cytostatic drugs analyzed

Keywords: DESI; Mass spectrometry; Cytostatic drugs analysis; Occupational exposure; Environmental monitoring

Surface-enhanced vibrational spectroscopy of B vitamins: what is the effect of SERS-active metals used? by A. Kokaislová; P. Matějka (pp. 985-993).

Surface-enhanced Raman scattering (SERS) spectroscopy and surface-enhanced infrared absorption (SEIRA) spectroscopy are analytical tools suitable for the detection of small amounts of various analytes adsorbed on metal surfaces. During recent years, these two spectroscopic methods have become increasingly important in the investigation of adsorption of biomolecules and pharmaceuticals on nanostructured metal surfaces. In this work, the adsorption of B-group vitamins pyridoxine, nicotinic acid, folic acid and riboflavin at electrochemically prepared gold and silver substrates was investigated using Fourier transform SERS spectroscopy at an excitation wavelength of 1,064 nm. Gold and silver substrates were prepared by cathodic reduction on massive platinum targets. In the case of gold substrates, oxidation–reduction cycles were applied to increase the enhancement factor of the gold surface. The SERS spectra of riboflavin, nicotinic acid, folic acid and pyridoxine adsorbed on silver substrates differ significantly from SERS spectra of these B-group vitamins adsorbed on gold substrates. The analysis of near-infrared-excited SERS spectra reveals that each of B-group vitamin investigated interacts with the gold surface via a different mechanism of adsorption to that with the silver surface. In the case of riboflavin adsorbed on silver substrate, the interpretation of surface-enhanced infrared absorption (SEIRA) spectra was also helpful in investigation of the adsorption mechanism.

Keywords: Surface-enhanced Raman scattering; Surface-enhanced infrared absorption; Electrochemical deposition; B-group vitamins

Systematic bottom-up approach for flavonoid derivative screening in plant material using liquid chromatography high-resolution mass spectrometry by László Abrankó; Juan F. García-Reyes; Antonio Molina-Díaz (pp. 995-1006).

In this work, a systematic comprehensive screening procedure has been proposed for the detection of multiclass flavonoid derivatives by liquid chromatography high-resolution mass spectrometry (LC-HRMS). The procedure is based on the combined use of accurate mass measurements and in-source fragmentation obtained with a liquid chromatography time-of-flight mass spectrometry instrument. The method relies on automated screening of selected diagnostic ions based on an exact mass database. The included diagnostic ions represent theoretical combinations of aglycones and typical glycan part constituents of flavonoid derivatives (i.e., various saccharide units and acyl moieties). The proposed identification protocol is following a systematic evaluation of the obtained positive hits from the diagnostic ions database according to a “bottom-up” approach that is thoroughly discussed. The main benefit of the proposed bottom-up protocol resides in the fact that untargeted flavonoid derivatives can be detected and tentatively identified without the need for any preliminary knowledge on the sought compound. In addition to information on the nature of the (1) aglycone and the (2) glycan part, further indication of (3) sugar unit distribution and information on (4) the type of the glycosidic bonds can also be attained. Selected examples of plant extracts demonstrate the potential of the proposed LC-HRMS approach for the systematic screening of flavonoids. A broad variety of compounds were tentatively identified including both anthocyanins and non-anthocyanin flavonoids having various glycan moieties such as mono-, di-, and triglycosides with varying distributions and linkage types of carbohydrate moieties (O-glycosides, C-glycosides, O,C-glycosides).

Keywords: High-resolution mass spectrometry; Non-target analysis; Time-of-flight mass spectrometry; Profiling

Investigation of the chemical composition–antibacterial activity relationship of essential oils by chemometric methods by Dragoljub L. Miladinović; Budimir S. Ilić; Tatjana M. Mihajilov-Krstev; Nikola D. Nikolić; Ljiljana C. Miladinović; Olga G. Cvetković (pp. 1007-1018).

The antibacterial effects of Thymus vulgaris (Lamiaceae), Lavandula angustifolia (Lamiaceae), and Calamintha nepeta (Lamiaceae) Savi subsp. nepeta var. subisodonda (Borb.) Hayek essential oils on five different bacteria were estimated. Laboratory control strain and clinical isolates from different pathogenic media were researched by broth microdilution method, with an emphasis on a chemical composition–antibacterial activity relationship. The main constituents of thyme oil were thymol (59.95%) and p-cymene (18.34%). Linalool acetate (38.23%) and β-linalool (35.01%) were main compounds in lavender oil. C. nepeta essential oil was characterized by a high percentage of piperitone oxide (59.07%) and limonene (9.05%). Essential oils have been found to have antimicrobial activity against all tested microorganisms. Classification and comparison of essential oils on the basis of their chemical composition and antibacterial activity were made by utilization of appropriate chemometric methods. The chemical principal component analysis (PCA) and hierachical cluster analysis (HCA) separated essential oils into two groups and two sub-groups. Thyme essential oil forms separate chemical HCA group and exhibits highest antibacterial activity, similar to tetracycline. Essential oils of lavender and C. nepeta in the same chemical HCA group were classified in different groups, within antibacterial PCA and HCA analyses. Lavender oil exhibits higher antibacterial ability in comparison with C. nepeta essential oil, probably based on the concept of synergistic activity of essential oil components.

Keywords: Chemometrics; Essential oil; Antibacterial activity

A novel application of microwave-assisted extraction of polyphenols from brewer’s spent grain with HPLC-DAD-MS analysis by Manuela M. Moreira; Simone Morais; Aquiles A. Barros; Cristina Delerue-Matos; Luís F. Guido (pp. 1019-1029).

This paper reports a novel application of microwave-assisted extraction (MAE) of polyphenols from brewer’s spent grains (BSG). A 2⁴ orthogonal composite design was used to obtain the optimal conditions of MAE. The influence of the MAE operational parameters (extraction time, temperature, solvent volume and stirring speed) on the extraction yield of ferulic acid was investigated through response surface methodology. The results showed that the optimal conditions were 15 min extraction time, 100 °C extraction temperature, 20 mL of solvent, and maximum stirring speed. Under these conditions, the yield of ferulic acid was 1.31 ± 0.04% (w/w), which was fivefold higher than that obtained with conventional solid–liquid extraction techniques. The developed new extraction method considerably reduces extraction time, energy and solvent consumption, while generating fewer wastes. HPLC-DAD-MS analysis indicated that other hydroxycinnamic acids and several ferulic acid dehydrodimers, as well as one dehydrotrimer were also present, confirming that BSG is a valuable source of antioxidant compounds. Figure Response surface of FA yield (Y, %, w/w) as a function of extraction time (X ₁) and the other variables studied: a solvent volume (X ₃); b temperature (X ₂)

Keywords: Brewer’s spent grain; Polyphenols; Ferulic acid; Microwave-assisted extraction; HPLC-DAD-MS analysis

Application of gas-diffusion microextraction to the analysis of free and bound acetaldehyde in wines by HPLC–UV and characterization of the extracted compounds by MS/MS detection by Manuel P. Cruz; Inês M. Valente; Luís M. Gonçalves; José A. Rodrigues; Aquiles A. Barros (pp. 1031-1037).

In wines, the presence of high levels of acetaldehyde (AA) not only is responsible for undesirable characteristic odours but can also cause health adverse effects. Such sensorial activity of AA can be overcome by adding sulphites during winemaking, due to the formation of adducts between AA and sulphites, which lower the sensorial impact of AA. Nevertheless, bound AA can be released during wine storage; therefore, the knowledge of its total amount can be important to estimate the long-term wine quality. The proposed methodology is based on the extraction of AA from wines using gas-diffusion microextraction and determination by liquid chromatography. Free and bound forms of AA could be differentiated and determined using an alkaline hydrolysis step to dissociate the sulphites–AA adducts. This methodology was successfully applied to different wine types, with free AA values ranging between 5 and 26 mg L⁻¹ and total form between 154 and 906 mg L⁻¹. Bound AA was above 90% of the total content determined for all samples analysed, and higher amounts were obtained for white wines (around 98%). Other carbonyl compounds were also identified in the extracts using mass spectrometry. Figure Simplified scheme of the extraction procedure for the analysis of free and total acetaldehyde

Keywords: Acetaldehyde; HPLC; Gas-diffusion microextraction (GDME); Wine; Sulphites; Adducts

Study of the naphthalene-2,3-dicarboxaldehyde pre-column derivatization of biogenic mono- and diamines in mixture and fluorescence−HPLC determination by Anastasia Zotou; Maria Notou (pp. 1039-1048).

A new fluorescence−HPLC method was developed for the simultaneous determination of eight biogenic monoamines (histamine, methylamine, tyramine, ethylamine, propylamine, tryptamine, 2-phenylethylamine, isoamylamine) and two biogenic diamines (putrescine, cadaverine) in the presence of heptylamine as the internal standard. The amines were pre-column derivatized with naphthalene-2,3-dicarboxaldehyde in the presence of cyanide ion as the nucleophile. The effect of the derivatization reaction conditions on the reaction yield was investigated. The derivatives were separated on an Inertsil ODS-3 column (250 × 4mm i.d., 5 μm) using gradient elution and detected fluorimetrically at excitation and emission wavelengths of 424 and 494 nm, respectively. Limits of detection between 0.002 and 0.4 ng, injected on-column (10-μL loop), were achieved. The within- and between-day relative standard deviations ranged between 0.2−3.4% and 0.3−4.8%, respectively. The utility of the method in assaying biogenic mono- and diamine mixtures in Greek cheeses is demonstrated. Ultrasound-assisted liquid−liquid extraction was applied prior to derivatization. Figure Reaction of primary amines with NDA, dependence of biogenic amines' derivatization yield on the reaction pH and chromatogram of a NDA-derivatized cheese sample

Keywords: Biogenic amines; Cheese; Derivatization; Fluorescence; HPLC; Naphthalene-2,3-dicarboxaldehyde

An improved and fast UHPLC-PDA methodology for determination of L-ascorbic and dehydroascorbic acids in fruits and vegetables. Evaluation of degradation rate during storage by Vítor Spínola; Berta Mendes; José S. Câmara; Paula C. Castilho (pp. 1049-1058).

This study provides a versatile validated method to determine the total vitamin C content, as the sum of the contents of L-ascorbic acid (L-AA) and dehydroascorbic acid (DHAA), in several fruits and vegetables and its degradability with storage time. Seven horticultural crops from two different origins were analyzed using an ultra-high-performance liquid chromatographic–photodiode array (UHPLC-PDA) system, equipped with a new trifunctional high strength silica (100% silica particle) analytical column (100 mm × 2.1 mm, 1.7 μm particle size) using 0.1% (v/v) formic acid as mobile phase, in isocratic mode. This new stationary phase, specially designed for polar compounds, overcomes the problems normally encountered in HPLC and is suitable for the analysis of large batches of samples without L-AA degradation. In addition, it proves to be an excellent alternative to conventional C18 columns for the determination of L-AA in fruits and vegetables. The method was fully validated in terms of linearity, detection (LOD) and quantification (LOQ) limits, accuracy, and inter/intra-day precision. Validation experiments revealed very good recovery rate of 96.6 ± 4.4% for L-AA and 103.1 ± 4.8 % for total vitamin C, good linearity with r 2-values >0.999 within the established concentration range, excellent repeatability (0.5%), and reproducibility (1.6%) values. The LOD of the method was 22 ng/mL whereas the LOQ was 67 ng/mL. It was possible to demonstrate that L-AA and DHAA concentrations in the different horticulture products varied oppositely with time of storage not always affecting the total amount of vitamin C during shelf-life. Locally produced fruits have higher concentrations of vitamin C, compared with imported ones, but vegetables showed the opposite trend. Moreover, this UHPLC-PDA methodology proves to be an improved, simple, and fast approach for determining the total content of vitamin C in various food commodities, with high sensitivity, selectivity, and resolving power within 3 min of run analysis.

Keywords: Ascorbic acid; Dehydroascorbic acid; Total vitamin C; Ultra-high-performance liquid chromatography; Validation; Fruits and vegetables

Dispersive liquid–liquid microextraction coupled to liquid chromatography for thiamine determination in foods by Pilar Viñas; Ignacio López-García; María Bravo-Bravo; Marisol Briceño; Manuel Hernández-Córdoba (pp. 1059-1066).

A miniaturized dispersive liquid–liquid microextraction (DLLME) procedure coupled to liquid chromatography (LC) with fluorimetric detection was evaluated for the preconcentration and determination of thiamine (vitamin B₁). Derivatization was carried out by chemical oxidation of thiamine with 5 × 10⁻⁵ M ferricyanide at pH 13 to form fluorescent thiochrome. For DLLME, 0.5 mL of acetonitrile (dispersing solvent) containing 90 μL of tetrachloroethane (extraction solvent) was rapidly injected into 10 mL of sample solution containing the derivatized thiochrome and 24% (w/v) sodium chloride, thereby forming a cloudy solution. Phase separation was carried out by centrifugation, and a volume of 20 μL of the sedimented phase was submitted to LC. The mobile phase was a mixture of a 90% (v/v) 10 mM KH₂PO₄ (pH 7) solution and 10% (v/v) acetonitrile at 1 mL min⁻¹. An amide-based stationary phase involving a ligand with amide groups and the endcapping of trimethylsilyl was used. Specificity, linearity, precision, recovery, and sensitivity were satisfactory. Calibration graph was carried out by the standard additions method and was linear between 1 and 10 ng mL⁻¹. The detection limit was 0.09 ng mL⁻¹. The selectivity of the method was judged from the absence of interfering peaks at the thiamine elution time for blank chromatograms of unspiked samples. A relative standard deviation of 3.2% was obtained for a standard solution containing thiamine at 5 ng mL⁻¹. The esters thiamine monophosphate and thiamine pyrophosphate can also be determined by submitting the sample to successive acid and enzymatic treatments. The method was applied to the determination of thiamine in different foods such as beer, brewer’s yeast, honey, and baby foods including infant formulas, fermented milk, cereals, and purees. For the analysis of solid samples, a previous extraction step was applied based on an acid hydrolysis with trichloroacetic acid. The reliability of the procedure was checked by analyzing a certified reference material, pig’s liver (CRM 487). The value obtained was 8.76 ± 0.2 μg g⁻¹ thiamine, which is in excellent agreement with the certified value, 8.6 ± 1.1 μg g⁻¹.

Keywords: Dispersive liquid–liquid microextraction; Liquid chromatography; Fluorimetric derivatization; Thiamine; Thiamine phosphate; Foods

Measurement of benzene concentration in urban air using passive sampling by Sylwia Król; Bożena Zabiegała; Jacek Namieśnik (pp. 1067-1082).

The concentration of benzene in urban air in the Tri-City area of Poland (Gdańsk–Sopot–Gdynia, and Tczew) was assessed using diffusive passive samplers (Radiello). Samples were collected during a four-year monitoring campaign (2007–2010) at selected monitoring stations managed by the Agency of Regional Air Quality Monitoring in the Gdańsk Metropolitan Area (ARMAAG) Foundation. The performance of the passive samplers was investigated in a field study that measured the benzene concentration in urban air. The results obtained by the Radiello samplers were compared with the results obtained using an on-line monitor (Chrompack CP 7001). Statistical analysis of the results obtained by the two different techniques (passive and on-line) was performed by a linear regression method (Student’s t-test). The influence of temperature fluctuations on the uptake rate behavior of the passive samplers was also investigated.

Keywords: Passive sampling; Benzene; Radiello; Atmospheric air; Sampling rate; Monitoring

A flow injection analyser conductometric coupled system for the field analysis of free dissolved CO₂ and total dissolved inorganic carbon in natural waters by Valter Martinotti; Marcella Balordi; Giovanni Ciceri (pp. 1083-1093).

A flow injection analyser coupled with a gas diffusion membrane and a conductometric microdetector was adapted for the field analysis of natural concentrations of free dissolved CO₂ and dissolved inorganic carbon in natural waters and used in a number of field campaigns for marine water monitoring. The dissolved gaseous CO₂ presents naturally, or that generated by acidification of the sample, is separated by diffusion using a hydrophobic semipermeable gas porous membrane, and the permeating gas is incorporated into a stream of deionised water and measured by means of an electrical conductometric microdetector. In order to make the system suitable and easy to use for in-field measurements aboard oceanographic ships, the single components of the analyser were compacted into a robust and easy to use system. The calibration of the system is carried out by using standard solutions of potassium bicarbonate at two concentration ranges. Calibration and sample measurements are carried out inside a temperature-constant chamber at 25 °C and in an inert atmosphere (N₂). The detection and quantification limits of the method, evaluated as 3 and 10 times the standard deviation of a series of measurements of the matrix solution were 2.9 and 9.6 μmol/kg of CO₂, respectively. Data quality for dissolved inorganic carbon was checked with replicate measurements of a certified reference material (A. Dickson, Scripps Institution of Oceanography, University of California, San Diego), both accuracy and repeatability were −3.3% and 10%, respectively. Optimization, performance qualification of the system and its application in various natural water samples are reported and discussed. In the future, the calibration step will be operated automatically in order to improve the analytical performance and the applicability will be increased in the course of experimental surveys carried out both in marine and freshwater ecosystems. Considering the present stage of development of the method, it can only be applied for studying of the carbon cycle in oxic environments.

Keywords: Flow injection analysis; Free dissolved CO₂ ; Total dissolved inorganic carbon; Seawater; Carbon capture and storage; CO₂ baseline fluxes monitoring

Usefulness of a PARAFAC decomposition in the fiber selection procedure to determine chlorophenols by means SPME-GC-MS by Rocío Morales; M. Cruz Ortiz; Luis A. Sarabia (pp. 1095-1107).

In this work, a procedure based on solid-phase microextraction and gas chromatography coupled with mass spectrometry is proposed to determine chlorophenols in water without derivatization. The following chlorophenols are studied: 2,4-dichlorophenol; 2,4,6-trichlorophenol; 2,3,4,6-tetrachlorophenol and pentachlorophenol. Three kinds of SPME fibers, polyacrylate, polydimethylsiloxane, and polydimethylsiloxane/divinylbenzene are compared to identify the most suitable one for the extraction process on the basis of two criteria: (a) to select the equilibrium time studying the kinetics of the extraction, and (b) to obtain the best values of the figures of merit. In both cases, a three-way PARAllel FACtor analysis decomposition is used. For the first step, the three-way experimental data are arranged as follows: if I extraction times are considered, the tensor of data, X, of dimensions I × J × K is generated by concatenating the I matrices formed by the abundances of the J m/z ions recorded in K elution times around the retention time for each chlorophenol. The second-order property of PARAFAC (or PARAFAC2) assesses the unequivocal identification of each chlorophenol, as consequence, the loadings in the first mode estimated by the PARAFAC decomposition are the kinetic profile. For the second step, a calibration based on a PARAFAC decomposition is used for each fiber. The best figures of merit were obtained with PDMS/DVB fiber. The values of decision limit, CCα, achieved are between 0.29 and 0.67 μg L⁻¹ for the four chlorophenols. The accuracy (trueness and precision) of the procedure was assessed. This procedure has been applied to river water samples. Figure PARAFAC decomposition in the fiber selection in an SPME procedure when an interferent is present

Keywords: Chlorophenols; Gas chromatography; Mass spectrometry; Solid-phase microextraction; PARAFAC; River water samples

Comparing different means of signal treatment for improving the detection power in HPLC-ICP-MS by Simon Prikler; Denis Pick; Jürgen W. Einax (pp. 1109-1116).

The purpose of detecting trace concentrations of analytes often is hindered by occurring noise in the signal curves of analytical methods. This is also a problem when different arsenic species (inorganic As(III) and As(V) as well as organic dimethylarsinic acid and arsenobetaine) are to be determined in food and feeding stuff by HPLC-ICP-MS, which is the basis of this work. In order to improve the detection power, methods of signal treatment may be applied. We show a comparison of convolution with Gaussian distribution curves, Fourier transform, and wavelet transform. It is illustrated how to estimate decisive parameters for these techniques. All methods result in improved limits of detection. Furthermore, applying baselines and evaluating peaks thoroughly is facilitated. However, there are differences. Convolution with Gaussian distribution curves may be applied, but Fourier transform shows better results of improvement. The best of the three is wavelet transform, whereby the detection power is improved by factors of about 6. Figure Signal-to-noise ratios for As concentrations of 2.4 μg L⁻¹ in the original chromatograms (black), after convolution with Gaussian distribution curves (red), after de-noising with FT (yellow), and with WT (green)

Keywords: Gaussian convolution; Fourier transform; Wavelet transform; Limit of detection; Signal-to-noise ratio; Arsenic species

Chemometric approach for development, optimization, and validation of different chromatographic methods for separation of opium alkaloids by J. Acevska; G. Stefkov; R. Petkovska; S. Kulevanova; A. Dimitrovska (pp. 1117-1129).

The excessive and continuously growing interest in the simultaneous determination of poppy alkaloids imposes the development and optimization of convenient high-throughput methods for the assessment of the qualitative and quantitative profile of alkaloids in poppy straw. Systematic optimization of two chromatographic methods (gas chromatography (GC)/flame ionization detector (FID)/mass spectrometry (MS) and reversed-phase (RP)–high-performance liquid chromatography (HPLC)/diode array detector (DAD)) for the separation of alkaloids from Papaver somniferum L. (Papaveraceae) was carried out. The effects of various conditions on the predefined chromatographic descriptors were investigated using chemometrics. A full factorial linear design of experiments for determining the relationship between chromatographic conditions and the retention behavior of the analytes was used. Central composite circumscribed design was utilized for the final method optimization. By conducting the optimization of the methods in very rational manner, a great deal of excessive and unproductive laboratory research work was avoided. The developed chromatographic methods were validated and compared in line with the resolving power, sensitivity, accuracy, speed, cost, ecological aspects, and compatibility with the poppy straw extraction procedure. The separation of the opium alkaloids using the GC/FID/MS method was achieved within 10 min, avoiding any derivatization step. This method has a stronger resolving power, shorter analysis time, better cost/effectiveness factor than the RP-HPLC/DAD method and is in line with the “green trend” of the analysis. The RP-HPLC/DAD method on the other hand displayed better sensitivity for all tested alkaloids. The proposed methods provide both fast screening and an accurate content assessment of the six alkaloids in the poppy samples obtained from the selection program of Papaver strains. Figure

Keywords: Chemometry; Design of experiments; GC/FID/MS; HPLC/DAD; Opium poppy alkaloids

Advantages of a programmed temperature vaporizer inlet and parallel factor analysis in the determination of triazines in the presence of non-intentionally added substances by gas chromatography by L. Rubio; L. A. Sarabia; A. Herrero; M. C. Ortiz (pp. 1131-1143).

Non-intentionally added substances (NIASs) are usually detected by acquiring mass spectra in full scan mode and then identifying the compounds corresponding to the unexpected peaks. High-resolution mass spectrometry detectors are frequently used, but this does not solve the problem that an NIAS can contribute to the abundance at m/z ratios that correspond to the fragmentation of other molecules. This problem leads to false negatives when identifying compounds, even in target analysis when the maximum permitted tolerances for relative ion abundances (SANCO/10684/2009) are taken into account. In this work, the introduction of different volumes of a test sample onto a GC/MS system that has a programmed temperature vaporizer inlet and is operating in full scan mode provides a data tensor. The proposed approach consists of considering the structure of the matrix of abundances of K m/z ratios acquired at J elution times for each chromatographic peak. Upon concatenating I of such matrices, a three-way tensor X is obtained, which is then decomposed using parallel factor analysis into as many factors as there are substances coeluting, thus providing the mass spectrum and the chromatographic profile for each of them. If the amount of an analyte changes significantly during the I injections, then it can be unequivocally identified. This procedure thus identifies coeluting NIASs, provides information about their mass spectra, and guarantees the identification and quantification of target compounds. In this work, it is used to determine five triazines in the presence of NIASs which match some of the m/z ratios of the triazines and coelute with them. Decision limits (CCα) of between 7.5 and 25.0 μg L⁻¹ were obtained. Figure Fig. A GC/MS system equipped with a PTV inlet provides a structured three-way data set per sample. Using a PARAFAC decomposition of the data tensor is possible to determine target and non-target substances in the sample

Keywords: Parallel factor analysis; Programmed temperature vaporizer; Gas chromatography/mass spectrometry; Non-target analysis; Non-intentionally added substances; Triazines

A design of experiment approach to the sol–gel synthesis of titania monoliths for chromatographic applications by Maguy Abi Jaoudé; Jérôme Randon; Claire Bordes; Pierre Lanteri; Laurence Bois (pp. 1145-1155).

A design of experiement approach is described for the optimization of the microscopic morphology of macro-mesoporous titania monoliths that were elaborated for the chromatographic enrichment of phosphorylated compounds. The monolithic titania gels were formed via an alkoxy-derived sol–gel route in association with a phase separation mechanism. The synthesis was performed at mild temperatures of gelation using starting mixtures of titanium n-propoxide, hydrochloric acid, N-methylformamide, water, and poly (ethylene oxide). The gelation temperature and the chemical compositions of N-methylformamide, water, and poly (ethylene oxide) were chosen as the most relevant experimental factors of the sol–gel process. Using the sizes of the skeletons and macropores as morphological descriptors of the dried porous monoliths, the statistical analyses simultaneously revealed the effects and interactions between the different factors. Crack-free TiO₂ monolithic rods of 8 to 10 cm long with well-defined co-continuous macropores and micro-structured skeletons were obtained after selection of the sol–gel parameters and optimization of the drying and heat-treatment steps of the gels. The bimodal texture of the rods exhibited macropores of 1.5 μm and mesopores centered at 5.2 nm with a total surface area of 140 m² g⁻¹. The ability of the macro-mesoporous titania rods to selectively bind phosphorylated compounds was demonstrated for O-phosphoamino acids (P-Ser, P-Thr, P-Tyr).

Keywords: Titania; Monolithic rod; Sol–gel synthesis; Design of experiments; Phosphate enrichment; O-phosphoamino acids

Improving selectivity in gas chromatography by using chemically modified multi-walled carbon nanotubes as stationary phase by Andrea Speltini; Daniele Merli; Daniele Dondi; Giorgio Paganini; Antonella Profumo (pp. 1157-1165).

Amino-terminated alkyl MWCNTs (MWCNTs-R-NH₂), synthesized by chemical modification of the nanotube skeleton by nucleophilic substitution with 2,2′-(ethylenedioxy)diethylamine, were successfully used as stationary phases for gas chromatographic separation of esters and chloroaromatics. The presence of alkyl chains with polar embedded groups made the functionalized MWCNTs (f-MWCNTs) a mixed-mode GC separation material able to interact in different ways with the analytes. Compared with non-functionalized MWCNTs (nf-MWCNTs), MWCNTs-R-NH₂ had higher selectivity, enhanced resolution, and optimum retention behaviour, and they were proved to perform better than the commercial stationary phase Porapak QS (PQS), claimed to be suitable for similar applications. The so-prepared stationary phase was used for analysis of a synthetic mixture containing different classes of analytes, viz. esters, ketones, alcohols, alkanes, and aromatic hydrocarbons, and finally used for investigation of similar real matrices. In particular, the constituents of a commercial paint thinner were determined by direct injection of the sample, with good reproducibility (inter-day precision RSDs from 5 to 19%). Two unknown samples of commercial white spirit were also analysed for determination of the aromatic hydrocarbon content, and their composition was profiled on the basis of the different compounds identified.

Keywords: Functionalized carbon nanotubes; gas chromatography; packed column; paint thinner; selectivity; stationary phase

Characterization of 40 kDa poly(ethylene glycol) polymers by proton transfer reaction QTOF mass spectrometry and ¹H-NMR spectroscopy by Joan Malmstrøm (pp. 1167-1177).

Several electrospray mass spectrometry (ESI-MS) techniques have been described during the past years to enable the characterization work of large poly(ethylene glycol)s (PEGs) and PEGylated proteins. The proton transfer reaction ESI-MS method utilizes amines to charge reduce the electrospray envelope of PEGs, hence PEG molecules are aminated instead of protonated. This method simplifies the mass spectrum of large PEGs, and enables the interpretation of the charge state of the observable envelopes (R ≥ 3,000 (FWHM) measured at the (M + 6H)⁶⁺ ion from 40 K PEG compound 7,324.19). Hence, deconvolution of the MS data can be performed and relative molecular masses of the individual chain lengths of the PEGs can be calculated. However, as the poly-dispersity of PEGs may vary from batch to batch and from sample to sample, it was of interest to examine if the method could distinguish between these kinds of different material. Therefore, sample materials of each intermediate obtained at five synthetic steps during synthesis of a 40 kDa PEG molecule were collected. These four intermediates, starting material and the target molecule were examined by ¹H-NMR spectroscopy and ESI-MS using a proton stripping base. The study revealed that the charge-stripping ESI-MS method is able to differentiate between even small changes in the structure of the polymeric molecules only when the analysis is assisted by ¹H-NMR spectroscopy. A proper characterization of polymer molecules requires besides relative molecular mass, also poly-dispersity and end-group characterization. No end-group information is obtained based on MS data. Examination of the PEG polymers by ¹H-NMR spectroscopy provides the needed information. In addition, the ¹H-NMR spectra clearly distinguishes the examined polymers.

Keywords: Poly(ethylene glycols); PEG; Proton transfer reaction mass spectrometry; Proton NMR spectroscopy; Polymer characterization

Analytical characterization of laser-generated copper nanoparticles for antibacterial composite food packaging by D. Longano; N. Ditaranto; N. Cioffi; F. Di Niso; T. Sibillano; A. Ancona; A. Conte; M. A. Del Nobile; L. Sabbatini; L. Torsi (pp. 1179-1186).

A new type of nanomaterial has been developed as antibacterial additive for food packaging applications. This nanocomposite is composed of copper nanoparticles embedded in polylactic acid, combining the antibacterial properties of copper nanoparticles with the biodegradability of the polymer matrix. Metal nanoparticles have been synthesised by means of laser ablation, a rising and easy route to prepare nanostructures without any capping agent in a liquid environment. As prepared, nanoparticle suspensions have been easily mixed to a polymer solution. The resulting hybrid solutions have been deposited by drop casting, thus obtaining self-standing antibacterial packages. All samples have been characterized by UV–Vis spectroscopy, X-ray photoelectron spectroscopy and electro-thermal atomic absorption spectroscopy. Ion release data have been matched with bioactivity tests performed by Japanese Industrial Standard (JIS) method (JIS Z 2801:2000) against Pseudomonas spp., a very common Gram-negative microbial group able to proliferate in processed food. Online abstract figure Analytical characterization of copper nanoparticles: an XPS spectrum and a TEM image

Keywords: Laser ablation; Copper nanocomposites; Antibacterial packaging; XPS; ETAAS; Pseudomonas spp.

Carbon nanotube enhanced mediator-type biosensor for real-time monitoring of glucose concentrations in fish by Mai Takase; Yohei Yoneyama; Masataka Murata; Kyoko Hibi; Huifeng Ren; Hideaki Endo (pp. 1187-1190).

We have developed a mediator-type biosensor to rapidly monitor blood glucose concentrations in fish, which are an indicator of stress. Glucose oxidase was used to detect glucose concentrations and ferrocene was used to limit the effect of oxygen. We also improved the sensitivity and durability of the sensor for better performance. Single-walled carbon nanotubes were used to enhance sensor sensitivity. Affixing the carbon nanotubes (30 mg ml^-1) to the working electrode increased the sensor sensitivity to 61.9 mM nA^-1 mm^-2, twice the value for the sensor without single-walled carbon nanotubes. A fabricated mediator-type biosensor sensor was used to perform real-time in vivo measurements. The sensor was implanted into the interstitial fluid of a fish eyeball, and detection was transmitted to a personal computer by a wireless potentiostat. Continuous measurement of the glucose concentration was possible for 78 hours. Stress was artificially applied to the fish during the measurement, and the change of blood glucose concentrations were observed. Our proposed sensor is applicable for effectively monitoring stress in free-swimming fish.

Keywords: Mediator-type biosensor; Single-walled carbon nanotube; Continuous in vivo measurement; Poly(ethylene glycol); Fish

An ELIME assay for the rapid diagnosis of coeliac disease by Gianluca Adornetto; Giulia Volpe; Alessia De Stefano; Sonia Martini; Giuseppina Gallucci; Angelo Manzoni; Sergio Bernardini; Marco Mascini; Danila Moscone (pp. 1191-1194).

Coeliac disease (CD) is a gluten-induced autoimmune enteropathy found in genetically susceptible subjects. Because of the high number of undetected cases, rapid and cheaper screening methods are needed. Currently, the CD diagnosis involves the detection of anti-transglutaminase IgA antibodies (anti-tTG IgA) in blood serum through the use of ELISA systems with confirmation by histology of the intestinal mucosa. A new, rapid magneto-electrochemical immunosensor for CD diagnosis has been developed and applied to serum sample analysis. The system uses magnetic beads coated with tTG antigen to detect anti-tTG antibodies in positive serum samples and an alkaline phosphatase-conjugated anti-human IgA as label. An electrochemical readout, using magnetized screen-printed electrodes coupled with a portable instrument, is made after the addition of α-naphtyl phosphate, which is enzymatically converted into the electrochemically active α-naphthol product. The work involved the following considerations: (1) optimization of analytical parameters; (2) recovery evaluation, adding known concentrations of anti-tTG IgA to “blank” sera; (3) analysis of 107 blood serum samples; (4) calculation of the ROC curve, resulting in a cut-off of 1.0 U/ml, 100% of clinical sensitivity and 98.36% of clinical specificity; evaluation of the agreement between electrochemical and ELISA kit values (r ² of 0.943). The system developed could be an useful tool for a correct and rapid CD diagnosis. This method is simple, cheap, rapid, and suitable for screening analyses performed outside of the classical diagnostic laboratory.

Keywords: Coeliac disease; Transglutaminase; Magnetic beads; Electrochemical sensor

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Analytical and Bioanalytical Chemistry (v.403, #4)