Analytica Chimica Acta (v.685, #2)

This paper reports an automated analytical method for rapid determination of plutonium isotopes (239Pu and 240Pu) in environmental solid extracts. Anion exchange chromatographic columns were incorporated in a sequential injection (SI) system to undertake the automated separation of plutonium from matrix and interfering elements. The analytical results most distinctly demonstrated that the crosslinkage of the anion exchanger is a key parameter controlling the separation efficiency. AG 1-×4 type resin was selected as the most suitable sorbent material for analyte separation. Investigation of column size effect upon the separation efficiency revealed that small-sized (2 mL) columns sufficed to handle up to 50 g of environmental soil samples. Under the optimum conditions, chemical yields of plutonium exceeded 90% and the decontamination factors for uranium, thorium and lead ranged from 103 to 104. The determination of plutonium isotopes in three standard/certified reference materials (IAEA-375 soil, IAEA-135 sediment and NIST-4359 seaweed) and two reference samples (Irish Sea sediment and Danish soil) revealed a good agreement with reference/certified values. The SI column-separation method is straightforward and less labor intensive as compared with batch-wise anion exchange chromatographic procedures. Besides, the automated method features low consumption of ion-exchanger and reagents for column washing and elution, with the consequent decrease in the generation of acidic waste, thus bearing green chemical credentials.
Keywords: Plutonium; Automation; Environmental samples; Anion exchange chromatography; Sequential injection; Inductively coupled plasma mass spectrometry;

Review: Authentication and traceability of foods from animal origin by polymerase chain reaction-based capillary electrophoresis by Roberto Rodríguez-Ramírez; Aarón F. González-Córdova; Belinda Vallejo-Cordoba (120-126).
This work presents an overview of the applicability of PCR-based capillary electrophoresis (CE) in food authentication and traceability of foods from animal origin. Analytical approaches for authenticating and tracing meat and meat products and fish and seafood products are discussed. Particular emphasis will be given to the usefulness of genotyping in food tracing by using CE-based genetic analyzers.
Keywords: Food authentication and traceability; Food DNA; Capillary electrophoresis;

Design of experiments on 135 cloned poplar trees to map environmental influence in greenhouse by Rui Climaco Pinto; Hans Stenlund; Magnus Hertzberg; Torbjörn Lundstedt; Erik Johansson; Johan Trygg (127-131).
To find and ascertain phenotypic differences, minimal variation between biological replicates is always desired. Variation between the replicates can originate from genetic transformation but also from environmental effects in the greenhouse. Design of experiments (DoE) has been used in field trials for many years and proven its value but is underused within functional genomics including greenhouse experiments. We propose a strategy to estimate the effect of environmental factors with the ultimate goal of minimizing variation between biological replicates, based on DoE. DoE can be analyzed in many ways. We present a graphical solution together with solutions based on classical statistics as well as the newly developed OPLS methodology.In this study, we used DoE to evaluate the influence of plant specific factors (plant size, shoot type, plant quality, and amount of fertilizer) and rotation of plant positions on height and section area of 135 cloned wild type poplar trees grown in the greenhouse. Statistical analysis revealed that plant position was the main contributor to variability among biological replicates and applying a plant rotation scheme could reduce this variation.
Keywords: Design of experiments; Poplar; Green house; Functional genomics; Orthogonal projections to latent structures (OPLS); Multivariate analysis;

A device for membrane assisted solvent extraction from an aqueous sample to an organic solvent within a micro-vial compatible with a chromatography auto-sampler was used to extract trace amounts of seven polycyclic aromatic hydrocarbons from seawater. The device consisted in an assembly of a volumetric flask containing the sample and a micro-vial containing the organic solvent by means of a screw stopper in which the septum was replaced by a sized piece of a membrane. Extraction conditions (nature of the organic solvent, extraction time, presence of ethanol in the donor phase, ionic content of the donor phase, characteristics of the membrane and volumes of donor and acceptor phases) were studied in order to find the conditions for maximum extraction. Analytical performance characteristics have also been established. The extraction efficiency was between 12.5 and 23%, which implies an enrichment factor value above 40. The repeatability and reproducibility were in the range of 8.6–10.0% and 13–19%, respectively. Detection limits were in the range of 24–39 ng L−1. Nine seawater samples have been studied. Most of the concentrations were under the limits of detection. Naphthalene and phenanthrene contents have been determined in a sample using the method of standard additions, and concentrations 100 and 91 ng L−1, respectively.
Keywords: Membrane assisted solvent extraction; Gas chromatography–mass spectrometry; Polycyclic aromatic hydrocarbons; Seawater;

Ionic liquid–salt aqueous two-phase flotation (ILATPF) is a novel, green, non-toxic and sensitive samples pretreatment technique. ILATPF coupled with high-performance liquid chromatography (HPLC) was developed for the analysis of chloramphenicol, which combines ionic liquid aqueous two-phase system (ILATPS) based on imidazolium ionic liquid (1-butyl-3-methylimidazolium chloride, [C4mim]Cl) and inorganic salt (K2HPO4) with solvent sublation. In ILATPF systems, phase behaviors of the ILATPF were studied for different types of ionic liquids and salts. The sublation efficiency of chloramphenicol in [C4mim]Cl–K2HPO4 ILATPF was influenced by the types of salts, concentration of K2HPO4 in aqueous solution, solution pH, nitrogen flow rate, sublation time and the amount of [C4mim]Cl. Under the optimum conditions, the average sublation efficiency is up to 98.5%. The mechanism of ILATPF contains two principal processes. One is the mechanism of IL–salt ILATPS formation, the other is solvent sublation. This method was practical when applied to the analysis of chloramphenicol in lake water, feed water, milk, and honey samples with the linear range of 0.5–500 ng mL−1. The method yielded limit of detection (LOD) of 0.1 ng mL−1 and limit of quantification (LOQ) of 0.3 ng mL−1. The recovery of CAP was 97.1–101.9% from aqueous samples of environmental and food samples by the proposed method. Compared with liquid–liquid extraction, solvent sublation and ionic liquid aqueous two-phase extraction, ILATPF can not only separate and concentrate chloramphenicol with high sublation efficiency, but also efficiently reduce the wastage of IL. This novel technique is much simpler and more environmentally friendly and is suggested to have important applications for the concentration and separation of other small biomolecules.
Keywords: Ionic liquid; Aqueous two-phase flotation; Solvent sublation; Chloramphenicol; Environmental; Food;

Molecularly imprinted polymer (MIP) has been synthesized by precipitation polymerization using ciprofloxacin (CIP) as template for the analysis of fluoroquinolone antibiotics (FQs). This MIP material was packed as sorbent in a device for microextraction by packed sorbent (MEPS) combined with liquid chromatography–tandem mass spectrometry (LC–MS/MS) for the analysis of selected FQs drugs including CIP, norfloxacin (NOR) and ofloxacin (OFLO) in municipal wastewater samples. In comparison to the new MIP-MEPS procedure, the target compounds were also determined by solid-phase extraction (MISPE) using the new molecular imprinted polymer material to validate the new MIP-MEPS method. The ability of the MIP for molecular recognition of CIP, NOR and OFLO was proved in presence of structurally different environmental relevant substances such as quinolones (Qs), flumequine (FLU), di(methyl)phthalate (DMP), technical 4-nonylphenol (NP), caffeine, Galaxolide®, Tonalid®, di(butyl)phthalate (DBP), Triclosan, bisphenol-A (BPA), carbamazepine, di(ethylhexyl)phthalate (DEHP), estradiol and octocrylene. The analysis of wastewater samples revealed the high selectivity of the synthesized polymer which was able to recognize and retain the target analytes by both extraction methods, the offline SPE with MIP material and the semi-automated MEPS packed with MIP material.
Keywords: Microextraction by packed sorbents; Molecularly imprinted polymer; Solid phase extraction; Fluoroquinolones; Wastewater; HPLC–MS/MS;

Screening of fibrillogenesis inhibitors of β2-microglobulin: Integrated strategies by mass spectrometry capillary electrophoresis and in silico simulations by Luca Regazzoni; Raffaella Colombo; Laura Bertoletti; Giulio Vistoli; Giancarlo Aldini; Massimo Serra; Marina Carini; Roberto Maffei Facino; Sofia Giorgetti; Monica Stoppini; Gabriele Caccialanza; Ersilia De Lorenzi (153-161).
The challenging search of ligands for the amyloidogenic protein β2-microglobulin led us to set up an integrated strategy that combines analytical techniques and molecular modelling. Using a chemical library composed of 90 sulphonated molecules and a novel MS screening approach, we initially single out a few new binders. To check for anti-amyloid activity, the best hit obtained was thoroughly studied by docking analysis, affinity and refolding experiments by capillary electrophoresis and in vitro fibrillogenesis Thioflavin T test. Correlative analysis of the overall results obtained from the MS screening led to develop an equation able to identify the key factors of the affinity for β2-microglobulin and to predict the affinity for novel derivatives. The proposed equation was then used for a virtual screening of a large compound database. Studies on the new hit thus retrieved confirm the predictive potential of both the equation on affinity and of docking analysis on anti-amyloid activity.
Keywords: β2-Microglobulin; Mass spectrometry; Capillary electrophoresis; Affinity screening; Molecular modeling; Amyloidosis;

The determination of 14 N-nitrosamines by a supersonic molecular beam electron ionization mass spectrometer equipped with triple quadruple analyzer, GC/SMB/EI/QQQ/MS is presented. The supersonic molecular beam electron ionization ion source allows the elucidation of the molecular ion of 13 out of the 14 examined nitrosamines (except for diphenylnitrosamine which was degraded before the analysis). It was possible to use the molecular ions of all the nitrosamines as the parent ions for multiple reactions monitoring mode, which in turn allows significant increase of specificity and lowering of the method limit of detection of the higher molecular weight nitrosamines. The instrumental LOD for different N-nitrosamines was 1–5 pg injection−1. The proposed method was exemplified by analysis of N-nitrosamines and N-nitrosatables in rubber teats according to the British Standard BS EN 12868:1999.
Keywords: N-nitrosamines; Supersonic molecular beam; Soft ionization;

Are antibiotic screening approaches sufficiently adequate? A proficiency test by Bjorn J.A. Berendsen; Mariël G. Pikkemaat; Linda (A)A.M. Stolker (170-175).
A proficiency test including the screening analysis of antibiotics in beef using cryogenicly minced materials was organized by RIKILT in 2009. The test included blank beef samples and beef samples spiked with either flumequine or a combination of lincomycin and spectinomycin around the maximum residue limits . The suitability of the materials was demonstrated with a homogeneity and a stability study. This study showed that cryogenically minced spiked muscle material is suited for proficiency tests aiming at the screening and the confirmatory analysis.Of the 26 participants, 23 carried out their in-house screening approach involving microbial, biochemical or instrumental methods, or a combination of these to cover the broad range of antibiotic groups. The false negative rate was 73% for microbial methods, 50% for biochemical and 22% for instrumental methods. These results indicate that substantial effort is needed to improve screening approaches and that more regular proficiency tests are needed to reveal the shortcomings in the currently applied screening methods.
Keywords: Antibiotics; Proficiency test; Screening assays; Microbiological; Biochemical; Instrumental;

Most liquid chromatography (LC) mass spectrometric (MS) methods used for routine monitoring of lipophilic marine toxins focus on the analysis of the 13 toxins that are stated in European Union legislation. However, to date over 200 lipophilic marine toxins have been described in the literature. To fill this gap, a screening method using LC coupled to high resolution (HR) orbitrap MS (resolution 100 000) for marine lipophilic toxins has been developed. The method can detect a wide variety of okadaic acid (OA), yessotoxin (YTX), azaspiracid (AZA) and pectenotoxin (PTX) group toxins. To build a library of toxins, shellfish and algae samples with various toxin profiles were obtained from Norway, Ireland, United Kingdom, Portugal and Italy. Each sample extract was analyzed with and without collision induced dissociation fragmentation. Based on their mass and specific fragmentation pattern, 85 different toxins were identified comprising 33 OA, 26 YTX, 18 AZA and 8 PTX group toxins. A major complication of full scan HRMS is the huge amount of data generated (file size), which restricts the possibility of a fast search. A software program called metAlign was used to reduce the orbitrap MS data files. The 200-fold reduced data files were screened using an additional software tool for metAlign: ‘Search_LCMS’. A search library was constructed for the 85 identified toxins. The library contains information about compound name, accurate mass, mass deviation (<5 ppm), retention time (min) and retention time deviation (<0.2 min). An important feature is that the library can easily be exchanged with other instruments as the generated metAlign files are not brand-specific. The developed screening procedure was tested by analyzing a set of known positive and blank samples, processing them with metAlign and searching with Search_LCMS. A toxin profile was determined for each of the contaminated samples. No toxins were found in the blank sample, which is in line with the results obtained for this sample in the routine monitoring program (rat bioassay and tandem LC–MS).
Keywords: Metalign; Lipophilic marine toxins; Okadaic acid; Azaspiracids; Yessotoxins; Pectenotoxins;

Highly routinely reproducible alignment of 1H NMR spectral peaks of metabolites in huge sets of urines by Amerigo Beneduci; Giuseppe Chidichimo; Giuseppe Dardo; Gabriele Pontoni (186-195).
A method to obtain high reproducibility of 1H NMR chemical shift of peaks of biofluid metabolites, by simple acidification with HCl is evaluated. Biofluid 1H NMR analysis is indeed spoiled by a strong chemical shift dependence of metabolite peaks on parameters such as ionic strength, concentration of some earth alkali cations and, mostly, on pH of samples. The resulting chemical shift variations, as large as 0.1 ppm, generate misalignments of homogeneous peaks, artifacts and misinterpretations.Reproducible alignment is essential in 1H NMR based metabonomics, where peak misalignments prevent even very wide bins (i.e., 0.04 ppm, as elsewhere proposed) from being used to integrate spectral data for multivariate statistical analysis. Here is demonstrated that routine acidification with HCl to 1.2 ≤ pH ≤ 2.0 ensures highly reproducible peak alignment of urine 1H NMR spectra. In this respect, simple inspection of citrate peaks in the urine can be used to measure pH, as it will be extensively discussed, in that at such low pH they show no dependency on other urine components as reported at higher pH. Under these conditions, in as many as 493 urine samples, in which concentrations of Ca2+, Mg2+, K+, Na+, Cl, phosphate, and creatinine and ionic strength measured by means of well standardized conventional procedures, showed very wide ranges, peaks align within a SD always lower than 0.002 ppm, thus allowing the use of integration bins at least five times narrower than 0.04 ppm.
Keywords: 1H NMR; Urine metabolite; Metabonomics; Peak alignment; pH; Inorganic composition;

Determination of the nitrogen content of nitrocellulose from smokeless gunpowders and collodions by alkaline hydrolysis and ion chromatography by María López-López; Jose María Ramiro Alegre; Carmen García-Ruiz; Mercedes Torre (196-203).
In this work, a method to determine the nitrogen content of nitrocellulose from gunpowders and collodions is proposed. A basic hydrolysis of nitrocellulose with 1.0% (m/v) NaOH at 150 °C during 30 min was carried out for nitrocellulose from gunpowders (after its previous isolation by a protocol optimized by our research group) and from collodion samples. The concentration of nitrate and nitrite ions in the hydrolysate was determined by ion chromatography with suppression and conductimetric detection. The nitrogen content of nitrocellulose was calculated from the values of the concentration of both ions. The quantitative method was evaluated in terms of selectivity, sensitivity, robustness, limits of detection and quantification, and precision, measured as repeatability and intermediate precision. These parameters were good enough to demonstrate the validity of the method and its applicability to the determination of the nitrogen content of nitrocellulose contained in different types of gunpowders (single- and double-base gunpowders, manufactured from 1944 to 1997) and in commercial collodion samples. For gunpowders, the nitrogen content determined with the optimized method was compared with the values reported by the official label of the ammunition (obtained by a digestion/titration method) and errors, by defect, ranging from 1% to 15.2% (m/m) were calculated. The highest errors were obtained for the oldest gunpowders and could be attributed to the loss of nitro groups in the nitrocellulose molecule during aging. For collodion samples, errors could not be calculated since the real nitrogen content for these samples was not given in the label. In addition, the analysis time (2 h for nitrocellulose isolation, 1.5 h for nitrocellulose hydrolysis, and 0.2 h for chromatographic separation) was about 10 times lower than in the digestion/titration method nowadays used for gunpowder samples.
Keywords: Nitrogen content; Nitrocellulose; Gunpowder; Ion chromatography; Alkaline hydrolysis;

A fast HPLC method with diode-array absorbance detector and fluorescence detector for the analysis of 19 phenolic acids, flavan-3-ols, flavones, flavonols and caffeine in different types of samples was developed. Using a C18 reverse-phase fused-core column separation of all compounds was achieved in less than 5 min with an overall sample-to-sample time of 10 min. Evaluation of chromatographic performance revealed excellent reproducibility, resolution, selectivity and peak symmetry. Limits of detection for all analyzed compounds ranged from 0.5 to 211 μg L−1, while limits of quantitation ranged between 1.5 and 704 μg L−1. The developed method was used for the determination of analytes present in different samples, including teas (black, white, green), mate, coffee, cola soft drink and an energetic drink. Concentration of the analyzed compounds occurring in the samples ranged from 0.4 to 314 mg L−1. Caffeine was the analyte found in higher concentrations in all samples. Phytochemical profiles of the samples were consistent with those reported in the literature.
Keywords: Fast separations; Phenolic acids; Flavan-3-ols; Flavones; Flavonols; Caffeine; HPLC; Fused-core;

Layered double hydroxides: A novel nano-sorbent for solid-phase extraction by H. Abdolmohammad-Zadeh; Z. Rezvani; G.H. Sadeghi; E. Zorufi (212-219).
The nickel–aluminum layered double hydroxide (Ni–Al LDH) was synthesized by a simple co-precipitation method with controlled pH and followed by hydrothermal treatment. The obtained nano-structured inorganic material was employed, for the first time, as a new solid-phase extraction (SPE) sorbent for the extraction and pre-concentration of trace levels of fluoride ions from aqueous solutions. An indirect method was used for monitoring of extracted fluoride ions. The method is based on the quenching effect of extracted fluoride ions upon the fluorescence intensity of Al–oxine complex via the forming of AlF6 3−, which was determined spectrofluorometrically at λ em  = 510 nm with excitation at λ ex  = 404 nm. The effect of several parameters such as type of interlayer anion in Ni–Al LDH structure, pH, sample flow rate, elution conditions, amount of nano-sorbent, sample volume and co-existing ions on the extraction efficiency of the analyte were investigated. The results showed that fluoride ions could be retained on the Ni–Al (NO3 ) LDH at pH 6.0 and stripped by 1.2 mL of 3.0 mol L−1 NaOH. In the optimum experimental conditions, the limit of detection (3 s) and enrichment factor were 9.0 ng mL−1 and 50, respectively. The optimized method was successfully applied to the determination of fluoride concentration in various water samples. The results obtained from the proposed method were successfully compared with those provided by standard SPADNS method.
Keywords: Nickel–aluminum layered double hydroxide; Solid phase extraction; Nano-particles; Fluoride; Spectrofluorometry; Water samples;