Analytica Chimica Acta (v.611, #1)

Contents (iii-iv).

Novel analytical methods for the determination of steroid hormones in edible matrices by H. Noppe; B. Le Bizec; K. Verheyden; H.F. De Brabander (1-16).
This paper reviews recently published multi-residue chromatographic methods for the determination of steroid hormones in edible matrices. After a brief introduction on steroid hormones and their use in animal fattening, the most relevant EU legislation regarding the residue control of these substances is presented. An overview of multi-residue analytical methods, covering sample extraction and purification as well as chromatographic separation and different detection methods, being in use for the determination of steroid hormones (estrogens, gestagens and androgens), is provided to illustrate common trends and method variability. Emphasis was laid on edible matrices and more specifically on meat, liver, kidney, fat and milk. Additionally, the possibilities of novel analytical approaches are discussed. The review also covers specific attention on the determination of natural steroids. Finally, the analytical possibilities for phytosterols, naturally occurring steroid analogues of vegetable origin and a specific group of steroid hormones with a hemi-endogenous status are highlighted.
Keywords: Steroid hormones; EU-criteria; Residue analysis; Edible matrices; Gas chromatography; Liquid chromatography; Mass spectrometry; Isotope ratio MS; Phytosterols;

Current development in microfluidic immunosensing chip by Terence G. Henares; Fumio Mizutani; Hideaki Hisamoto (17-30).
This review accounts for the current development in microfluidic immunosensing chips. The basic knowledge of immunoassay in relation to its microfluidic material substrate, fluid handling and detection mode are briefly discussed. Here, we mainly focused on the surface modification, antibody immobilization, detection, signal enhancement and multiple analyte sensing. Some of the clinically important currently implemented on the microfluidic immunoassay chips are C-reactive protein (CRP), prostate specific antigen (PSA), ferritin, vascular endothelial growth factor (VEGF), myoglobin (Myo), cardiac troponin T (cTnT), cardiac troponin I (cTnI), and creatine kinase-cardiac muscle isoform (CK-MB). The emerging microfludic immunosensor technology may be a promising prospect that can propel the improvement of clinical and medical diagnosis.
Keywords: Antibody; Antigen; ELISA; Immunoassay; Microfluidics;

NMR and Chemometric methods: A powerful combination for characterization of Balsamic and Traditional Balsamic Vinegar of Modena by R. Consonni; L.R. Cagliani; F. Benevelli; M. Spraul; E. Humpfer; M. Stocchero (31-40).
This work presents the capability of NMR spectroscopy combined with Chemometrics in predicting the ageing of Balsamic and Traditional Balsamic Vinegar of Modena. The need of an analytical method is an important requirement for both research oriented and commercial evaluation of these very valuable products. 1H NMR spectroscopy, based on the advantage of rapid sample analysis without any manipulation or derivatization, is here proposed as a valid tool to describe Balsamic and Traditional Balsamic Vinegar of Modena. For this purpose, 72 reliable samples, were divided into three different groups according to their ageing process: young (<12 years), old (>12 and <25 years) and extra old (>25 years). Hierarchical Projection to Latent Structures Discriminant Analysis (PLS-DA) allowed us to characterize the ageing process. Variables showing the largest VIP (Variable Importance in the Projection) were extracted from PLS-DA model, thus shedding lights onto the role played by specific compounds in this complex ageing process. Two robust classification models, were built by PLS-DA and Naïve Bayes classifier and compared to prove the accuracy of the representation on both training and test sets. The predictions obtained for 41 “unknown” vinegar samples with these both methods gave more than 80% agreement among them.
Keywords: Traditional Balsamic Vinegar of Modena; Principal component analysis; Projection to Latent Structures Discriminant Analysis; Naïve Bayes; 1H NMR; Vinegar; Ageing process;

A comparative study of calibration transfer methods for determination of gasoline quality parameters in three different near infrared spectrometers by Claudete Fernandes Pereira; Maria Fernanda Pimentel; Roberto Kawakami Harrop Galvão; Fernanda Araújo Honorato; Luiz Stragevitch; Marcelo Nascimento Martins (41-47).
This work presents a comparative study of calibration transfer among three near infrared spectrometers for determination of naphthenes and RON (Research Octane Number) in gasoline. Seven transfer methods are compared: direct standardization (DS), piecewise direct standardization (PDS), orthogonal signal correction (OSC), reverse standardization (RS), piecewise reverse standardization (PRS), slope and bias correction (SBC) and model updating (MU). Two pre-treatment procedures, namely standard normal variate (SNV) and multiplicative scatter correction (MSC), are also investigated. The choice of an appropriate number of transfer samples for each technique, as well as the effect of window size in PDS/PRS and OSC components, are discussed. A broad set of gasoline samples representative of the Northeastern states of Brazil is employed in the investigation. The results show that the use of calibration transfer yields prediction errors comparable to those obtained with complete recalibration of the secondary instrument. Overall, the results point to RS as the best method for the analytical problem under consideration. When storage and/or physical transportation of transfer samples are impractical, MU is more appropriate. The comprehensive investigation carried out in the present work will be of value for practitioners involved in networks of fuel monitoring.
Keywords: Calibration Transfer; Standardization methods; Chemometrics; Near infrared spectrometry; Gasoline;

Co-elution of cationic methylated arsenic, e.g. arsenobetaine may interfere with the determination of arsenite on the Hamilton PRP-X100 anion-exchange column using a phosphate buffer isocratically. Therefore, a sample pre-treatment method with self-packed AG MP-50 cation-exchange cartridges was proposed, which enables the arsenite determination in samples containing arsenobetaine on a PRP-X100 column using a phosphate buffer (pH 5.6) isocratically. Methylated arsenic, including dimethylarsinic acid, trimethylarsine oxide, tetramethylarsonium ion, arsenobetaine and arsenocholine, with concentrations below 1000 μg As L−1, may be completely retained in the AG MP-50 cartridge without any changes of arsenite, arsenate and monomethylarsonic acid speciation. Such retention was independent of the pH and matrix. It is proposed to be based on hydrophobic interaction. With the help of AG MP-50 cartridges, 11 arsenic species were detected in fish (DORM-2), mussels (BCR-477) and red algae (Porphyra tenera) in 10 min on the PRP-X100 column using a phosphate buffer isocratically. Arsenite was the only minor species (up to 0.9%) among all water extractable arsenic species in fish, mussel and red algae.
Keywords: Arsenite; PRP-X100 anion-exchange column; Arsenic speciation;

Nanostructured titania-based solid-phase microextraction (SPME) fibers were fabricated through the in situ oxidation of titanium wires with H2O2 (30%, w/w) at 80 °C for 24 h. The obtained SPME fibers possess a ∼1.2 μm thick nanostructured coating consisting of ∼100 nm titania walls and 100–200 nm pores. The use of these fibers for headspace SPME coupled with gas chromatography with electron capture detection (GC–ECD) resulted in improved analysis of dichlorodiphenyltrichloroethane (DDT) and its degradation products. The presented method to detect DDT and its degradation products has high sensitivity (0.20–0.98 ng L−1), high precision (relative standard deviation R.S.D. = 9.4–16%, n  = 5), a wide linear range (5–5000 ng L−1), and good linearity (coefficient of estimation R 2  = 0.991–0.998). As the nanostructured titania was in situ formed on the surface of a titanium wire, the coating was uniformly and strongly adhered on the titanium wire. Because of the inherent chemical stability of the titania coating and the mechanical durability of the titanium wire substrate, this new SPME fiber exhibited long life span (over 150 times).
Keywords: Solid-phase microextraction fiber; Nano-titania coating; Titanium wire; In situ fabrication; Pesticides;

An on-line flow injection spectrofluorimetric method for the direct determination of aluminium in water samples is described. The method is based on the reaction of aluminium with N-o-vanillidine–2-amino-p-cresol (OVAC) in acidic medium at pH 4.0 to form a water-soluble complex. The excitation and emission wavelengths were 423.0 and 553.0 nm, respectively, at which the OVAC–Al complex gave the maximum fluorescence intensity at pH 4.0 in a 50% methanol–50% water medium at 50 °C. An interference from fluoride ions was minimised by the addition of Be2+. Other ions were found not to interfere at the concentrations likely to be found in natural waters. The proposed methods were validated in terms of linearity, repeatability, detection limit, accuracy and selectivity. Under these conditions, the calibration was linear up to 1000 μg L−1 (r  = 0.999). The limit of detection (3σ) for the determination of Al(III) was 0.057 μg L−1 and the precision for multiple determinations of 3 ng mL−1 Al(III) prepared in ultra-pure water was found to be 0.62% (n  = 10).The Schiff base ligand could be used to determine ultra-trace aluminium from natural waters. Analysis of environmental certified reference materials showed good agreement with the certified values. The procedure was found to be equally applicable to both freshwater and saline solutions, including seawater.
Keywords: Flow injection; Aluminium determination; N-o-vanillidine–2-amino-p-cresol; Spectrofluorimetry; Seawater; River water;

Currently, two common techniques for nanomolar-level phosphate measurements in seawater are magnesium-induced co-precipitation (MAGIC) and long-path liquid-waveguide capillary cell (LWCC) spectrophotometry. These techniques have been applied in the open ocean, and our understanding of phosphate distributions in oligotrophic subtropical gyres is based on those data. However, intercomparison of these methods has not previously been performed at nanomolar levels. Here, we report experimental results directly comparing the MAGIC and LWCC techniques. We also evaluated the impact of various commonly employed filters on phosphate determinations, as well as interferences from dissolved organic phosphorus (DOP) and arsenate. Our results find agreement between these methods at phosphate concentrations <100 nM. We found that filter selection is important for accurate determinations of phosphate, and that DOP hydrolysis affects both techniques similarly. Finally, we demonstrate the advantage of combining MAGIC preconcentration and LWCC spectrophotometry for analysis of very low nanomolar concentrations.
Keywords: Nanomolar; Phosphate; Seawater;

Raman global illumination and near-infrared (NIR) mapping instruments were used to chemically image pharmaceutical granules obtained by the wet granulation process in order to determine whether the API was mixed with the major excipient or granulates on its own. The granules were randomly distributed onto a microscope slide and an average area of about 3.5 mm × 3.5 mm, covering 50–100 granules, was analyzed by both instruments. Light microscopy images of the separated granules were collected before the spectroscopic data acquisition. Both Raman and NIR signals of API and major excipient (mannitol) were easily detected by both techniques which allowed the chemical structure of the granules to be characterised. Most of the granules were found to contain both API and mannitol but pure mannitol and a few pure API granules were also identified. Raman global illumination was found to provide a comprehensive insight into chemical structure of the granules being able to more clearly determine the API in comparison with NIR mapping. Owing to the differences in shapes of the particles and reflection characteristics, visual microscopy and methods based on reflection can be potentially useful for analyzing this particular formulation.
Keywords: Raman; Near-infrared; Chemical imaging; Pharmaceutical granules; Wet granulation;

Demonstration of multi-analyte patterning using piezoelectric inkjet printing of multiple layers by Melissa S. Hasenbank; Thayne Edwards; Elain Fu; Richard Garzon; T. Fettah Kosar; Michael Look; Afshin Mashadi-Hossein; Paul Yager (80-88).
Patterning substrates with biological reagents is a critical component of biosensor development. Many applications require multi-analyte patterning capabilities, with a need to deposit several species reproducibly with a high degree of precision. We demonstrate a piezoelectric inkjet printing system that is capable of creating sub-millimeter (down to 150 μm) patterns of aqueous and nonaqueous reagents with precise placement for biosensor applications. The size, shape, and density of the patterns may be modified by simple adjustments of the patterning parameters. Using this system, two methods of multi-analyte protein patterning for use in biosensor assays are demonstrated. The first method involves the deposition of multiple proteins directly onto a gold substrate. Specific binding of an antibody to the deposited antigen is demonstrated, although nonspecific adsorption of the antibody may limit the utility of this simple method in quantitative biosensor applications. A second, more sophisticated multi-analyte patterning method involves two sequential patterning steps, consisting of an initial deposition onto gold of a mixed thiol layer to provide oriented binding capabilities in a nonfouling background and a second deposition of multiple biotinylated proteins. Highly specific antibody binding to this patterned multi-analyte surface was demonstrated, with minimal nonspecific adsorption to the surrounding regions. Thus, this method produces high-quality, localized, and customizable sub-millimeter patterns in a nonfouling background for multi-analyte bioassay development.
Keywords: Inkjet printing; Protein patterning; Thiol patterning; Multi-analyte;

An evanescent wave infrared chemical sensor for the sensitive and selective detection of copper ions in aqueous solutions is described. Because copper ions have no vibrational features, a band-shifting technique was utilized to produce the analytical signal. To enhance the sensitivity of the detection process, a three-step procedure was employed to prepare acidified tris(2-aminoethyl)amine (ATAA) phase on an evanescent wave sensing element. This sensing phase has a chemical structure similar to that of ethylenediamine tetraacetic acid (EDTA), a common chelating agent for metal ions. After formation of complex with copper ions, the shifts in the absorption bands of the ATAA phase were used for quantitation. An additional four sensing phases having chemical structures related to that of EDTA were synthesized to compare their performances for detection of copper ions. The synthetic sensing phases are highly stable in water and insensitive to changes in solutions at pH greater than 4. ATAA was the most sensitive of the phases tested, probably because of the accessibility and flexibility of the functional groups in the ATAA phase. To explore these systems in greater detail and to optimize detection, the effects of parameters such as the buffer concentration, the pH of the sample solution, and the matrix effect on response time and linearity of detection were examined. The analytical signals for copper ions were similar – and highly selective – when the pH of the solution was between 5 and 6.5. For a detection time of 5 min, these signals were linear for concentrations up to 200 μM with a detection limit ca. 3 μM.
Keywords: Infrared; Chemical sensor; Optical sensor; Copper ions; Complexation;

The synthesis and characterization of a novel polymethacylate polymer with covalently linked Al(III)-tetraphenylporphyrin (Al(III)-TPP) groups is reported. The new polymer is examined as a potential macromolecular ionophore for the preparation of polymeric membrane-based potentiometric and optical fluoride selective sensors. To prepare the polymer, an Al(III) porphyrin monomer modified with a methacrylate functionality is synthesized, allowing insertion into a polymethacrylate block copolymer (methyl methacrylate and decyl methacrylate) backbone. The resulting polymer can then be incorporated, along with appropriate additives, into conventional plasticized poly(vinyl chloride) films for testing electrochemical and optical fluoride response properties. The covalent attachment of the Al(III)-TPP ionophore to the copolymer matrix provides potentiometric sensors that exhibit significant selectivity for fluoride ion with extended lifetimes (compared to ion-selective membrane electrodes formulated with conventional free Al(III)-TPP structure). However, quite surprisingly, the attachment of the ionophore to the polymer does not eliminate the interaction of Al(III)-TPP structures to form dimeric species within the membrane phase in the presence of fluoride ion. Such interactions are confirmed by UV/visible spectroscopy of the blended polymeric films. Use of the new polymer-Al(III)-TPP conjugates to prepare optical fluoride sensors by co-incorporating a lipophilic pH indicator (4′,5′-dibromofluorescein octadecyl ester; ETH7075) is also examined and the resulting optical sensing films are shown to exhibit excellent selectivity for fluoride, with the potential for prolonged operational lifetime.
Keywords: Fluoride sensors; Aluminum(III)-porphyrins; Polymer-tetraphenylporhyrin conjugates; Dimer–monomer equilibrium;

Liquid chromatographic method development for anabolic androgenic steroids using a monolithic column by R. Muñiz-Valencia; R. Gonzalo-Lumbreras; A. Santos-Montes; R. Izquierdo-Hornillos (103-112).
An isocratic HPLC method for the determination with screening purposes of anabolic androgenic steroids (AASs: fluoxymesterone, boldenone, nortestosterone, metandrostenolone, norethindrone, methyltestosterone and bolasterone), used as growth promoting agents, in finishing pig feed samples has been developed and validated. The separation was achieved by using a reversed-phase Chromolith RP-18e column at controlled temperature, UV-detection at 245 nm and epitestosterone as internal standard. The method development involved optimization of different aqueous-organic mobile phases using methanol or acetonitrile as organic modifiers, flow-rate and temperature. The optimum separation for these compounds was achieved at 40 °C using ultrapure water:acetonitrile (71:29, v/v) as mobile phase and 3 mL min−1 flow-rate, allowing the separation of AASs with baseline resolution in about 15 min. The optimized method was applied to the analysis of AASs in finishing pig feed samples. Prior to HPLC, sample preparation procedure was used by leaching using acetonitrile, saponification in a basic medium and solid-phase extraction using polymeric Abselut Nexus cartridges. Method validation has been carried out according to the European Commission Decision 2002/657/EC. The extraction efficiencies, decision limits (CCα) and detection capabilities (CCβ) for these compounds were in the range 83–96%, 27–37 and 32–47 μg kg−1 range, respectively. The within-laboratory reproducibility at 1, 1.5 and 2 CCβ concentration levels were smaller than 13, 10 and 8%, respectively. Finally, the proposed method was successfully applied to nine different kinds of animal feed.
Keywords: Anabolic androgenic steroids; Growth promoting agents; Animal feed; Monolithic column;

Docosanoic (C22), tetracosanoic (C24) and hexacosanoic (C26) acids are saturated very-long-chain fatty acids (VLCFA) present at trace levels in biosamples. VLCFA can be used as potential biomarkers for the diagnosis of hereditary diseases such as X-linked adrenoleukodystrophy. Because the analytes to be detected are at trace levels, a sensitive fluorimetric liquid chromatographic method was developed to analyze VLCFA in plasma. The method is simple based on extracting VLCFA from plasma with toluene, and the obtained toluene extract was subject to the derivatization of VLCFA with a fluorescent reagent 2-(2-naphthoxy)ethyl-2-(piperidino)ethanesulfonate (NOEPES) without solvent evaporation/replacement. The resulting fluorescent derivatives were monitored by fluorimetric detection (excitation at 225 nm and emission at 360 nm), giving a high sensitivity with the limit of detection about 5.0 nM (S/N = 3, 10 μL injected) of the analytes. Application of the method to the analysis of VLCFA in the plasma of patients with adrenoleukodystrophy proved practical and effective.
Keywords: Very-long-chain free fatty acids; Adrenoleukodystrophy; Biomarker; Human plasma; Fluorimetric liquid chromatography;

A comparison of direct immersion solid-phase microextraction (DI-SPME) and stir bar sorptive extraction (SBSE) coupled to liquid chromatography (HPLC) with fluorimetric detection for the rapid analysis of resveratrol isomers is described. For DI-SPME, a polar Carbowax-template resin (CW/TPR) 50 μm fiber was the most efficient and optimum extraction conditions were 40 °C and an extraction time of 30 min, stirring in the presence of 5% (m/v) sodium chloride and 0.07 M acetate/acetic acid buffer (pH 6). Desorption was carried out using the static mode for 10 min. Linearity was obtained in the 5–150 and 2–150 ng mL−1 ranges for trans- and cis-resveratrol, with detection limits of 2 and 0.5 ng mL−1, respectively. When using SBSE, a polydimethylsiloxane (PDMS) twister provided best extraction by means of a derivatization reaction in the presence of acetic anhydride and potassium carbonate. The same time and temperature were used for the extraction step in the presence of 2.5% (m/v) sodium chloride, and liquid desorption was performed with 150 μL of a 50/50 (v/v) acetonitrile/1% (v/v) acetic acid solution in a desorption time of 15 min. Linearity was now between 0.5 and 50 ng mL−1 for trans-resveratrol with a detection limit of 0.1 ng mL−1, while cis-resveratrol could not be extracted. The proposed methods were successfully applied to determining the resveratrol isomer content of wine, must and fruit juices.
Keywords: Solid-phase microextraction; Stir bar sorptive extraction; Liquid chromatography; Resveratrol; Wine; Fruit juices;