Florentina Cañada-Cañada

Analysis of antibiotics in fish samples

The use of antibiotics in food-producing animals has generated considerable interest because the widespread administration of these drugs may lead to the development of resistant human pathogens. A large increase in the demand for seafood products has occurred in the last century. This has led to a concomitant increase in high-intensity aquaculture methods, characterized by high stock density and volume, and the heavy use of formulated feeds containing antibiotics, among other substances. Therefore, accurate and sensitive determination of antibiotic residues is now a necessity. In order to protect human health, the European Union and other regulatory authorities worldwide have established maximum residue limits (MRL) for antibiotic residues in animal products entering the human food chain. This paper reviews the most recent methods for analysis of antibiotic residues in fish.

Second-order multivariate calibration procedures applied to high-performance liquid chromatography coupled to fast-scanning fluorescence detection for the determination of fluoroquinolones

Different second-order multivariate calibration algorithms, namely parallel factor analysis (PARAFAC), N-dimensional partial least-squares (N-PLS) and multivariate curve resolution-alternating least-squares (MCR-ALS) have been compared for the analysis of four fluoroquinolones in aqueous solutions, including some human urine samples (additional four fluoroquinolones were simultaneously determined by univariate calibration). Data were measured in a short time with a chromatographic system operating in the isocratic mode. The detection system consisted of a fast-scanning spectrofluorimeter, which allows one to obtain second-order data matrices containing the fluorescence intensity as a function of retention time and emission wavelength. The developed approach enabled us to determine eight analytes, some of them with overlapped profiles, without the necessity of applying an elution gradient, and thus significantly reducing both the experimental time and complexity. The study was employed for the discussion of the scopes of the applied second-order chemometric tools. The quality of the proposed technique coupled to each of the evaluated algorithms was assessed on the basis of the figures of merit for the determination of fluoroquinolones in the analyzed water and urine samples. Univariate calibration of four analytes led to limits of detection in the range 20-40 ng mL(-1) and root mean square errors for the validation samples in the range 30-60 ng mL(-1) (corresponding to relative prediction errors of 3-8%). The ranges for second-order multivariate calibration (using PARAFAC and N-PLS) of the remaining four analytes were: limit of detection, 2-8 ng mL(-1), root mean square errors, 3-50 ng mL(-1) and relative prediction errors, 1-5%.

Nonlinear Four-Way Kinetic-Excitation-Emission Fluorescence Data Processed by a Variant of Parallel Factor Analysis and by a Neural Network Model Achieving the Second-Order Advantage : Malonaldehyde Determination in Olive Oil Samples

Four-way data were obtained by recording the kinetic evolution of excitation-emission fluorescence matrices for the product of the Hantzsch reaction between the analyte malonaldehyde and methylamine. The reaction product, 1,4-disubstituted-1,4-dihydropyridine-3,5-dicarbaldehyde, is a highly fluorescent compound. The nonlinear nature of the kinetic fluorescence data has been demonstrated, and therefore the four-way data were processed with parallel factor analysis combined with a nonlinear pseudounivariate regression, based on a quadratic polynomial fit, and also with a recently introduced neural network methodology, based on the combination of unfolded principal component analysis, residual trilinearization, and radial basis functions. The applied chemometric strategies are not only able to adequately model the nonlinear data but also to successfully determine malonaldehyde in olive oil samples. This is possible since the experimentally recorded four-way data, modeled with the above-mentioned advanced chemometric approaches, permit the achievement of the second-order advantage. This allows us to predict the analyte concentration in a complex background, in spite of the nonlinear behavior and in the presence of uncalibrated interferences. The present work is a new example of the use of higher-order data for the resolution of a complex nonlinear system, successfully employed in the context of food chemical analysis.

Enhanced MCR-ALS modeling of HPLC with fast scan fluorimetric detection second-order data for quantitation of metabolic disorder marker pteridines in urine

This work presents the development of a liquid chromatographic method based on modeling entire fast scan fluorimetric detection second-order data with the multivariate curve resolution alternating least squares algorithm, for the simultaneous determination of five marker pteridines in urine samples. The modeling strategy involves the building of a single MCR-ALS model composed of matrices augmented in the spectral mode, i.e. time profiles remain invariant while spectra may change from sample to sample. This approach allowed us to separate and determine the whole analytes at once. The developed approach enabled us to determine five of the most important metabolic disorder marker pteridines: biopterin, neopterin, isoxanthopterin, pterin and xanthopterin, three of them presenting emission spectra with the same emission wavelength maxima. In addition, some of these analytes present overlapped time profiles. As a consequence of using the entire data sets, a considerable reduction of the data processing experimental time can be achieved. Results are compared with a previous strategy in which data were split in five different regions, and information about the figures of merit of the new strategy compared with the previously reported strategy is reported.

Determination of marker pteridins and biopterin reduced forms, tetrahydrobiopterin and dihydrobiopterin, in human urine, using a post-column photoinduced fluorescence liquid chromatographic derivatization method

A liquid chromatographic method for the simultaneous analysis of marker pteridins and biopterin reduced forms, in urine samples is proposed. A Zorbax Eclipse XDB-C18 column was used for the chromatographic separation, using a 98/2 (v/v), citrate buffer (pH 5.5)-acetonitrile mobile phase, in isocratic mode. A post-column photoderivatization was carried out with an on-line photoreactor, located between a diode array detector (DAD) and a fast scanning fluorescence detector (FSFD). Neopterin (NEO), biopterin (BIO), pterin (PT) and dihydrobiopterin (BH2) were determined by measuring native fluorescence, using the photoreactor in OFF-mode, and tetrahydrobiopterin (BH4) was determined by measuring of the induced fluorescence of the generated photoproducts, using the photoreactor in ON-mode. In addition, Creatinine (CREA), as a reference of metabolites excrection in urine, was simultaneously determined using the DAD detector. Detection limits were 0.2, 13.0, 0.3, 0.3 and 3.5 ng mL(-1), for NEO, BH2, BIO, PT and BH4, respectively, and 0.4 microg mL(-1) for CREA. Ratio values for NEO/CREA, PT/CREA, BH4/CREA, BH2/CREA, NEO/BIO and BIO(total)/CREA, in urine samples, of healthy children and adults, phenylketonuric children and infected mononucleosis children, are reported. A comparative study, about the mean values obtained for each of the compounds, by the present procedure and by the classical iodine oxidation method (Fukushimas method), has been performed, in urine samples belonging to healthy volunteers. The values obtained were BH4/CREA: 0.41, BH2/CREA: 0.31 and BIO(total)/CREA: 0.73, by the proposed method, and BH4/CREA: 0.35, BH2/CREA: 0.20 and BIO(total)/CREA: 0.48, by iodine oxidation method.

Separation and determination of 11 marker pteridines in human urine by liquid chromatography and fluorimetric detection.

A simple liquid chromatographic method has been developed to achieve the complete separation and determination of a wide range of pteridinic compounds and creatinine (CREA) in urine samples, in just one run. The influences of mobile phase composition and buffer pH have been studied. The optimized mobile phase was composed of a Tris-HCl buffer (15 mmol/L) at pH 6.10 solution (eluent A) and a Tris-HCl buffer (15 mmol/L) at pH 6.40 solution (eluent B), in gradient mode. Analytes were determined by fluorimetric detection, exciting at 272 nm, and measuring the fluorescence emission at three wavelengths, 410, 445 and 465 nm. CREA, as a reference of metabolites excretion in urine, was determined by photometric detection at 230 nm. Pteridines detection limits varied from 0.2 to 6.1 ng/mL, and 0.2 g/mL for CREA. Calculated precision values expressed as RSD (%) varied from 1.1 to 5.9. Two different oxidation procedures for urine samples were optimized. The neopterin/biopterin ratios found were 0.98 and 0.86 for adults and children, respectively, by means of the alkaline iodide/iodine oxidation and 0.45 and 0.57 using neutral KMnO(4) oxidation.

A chemometric sensor for determining sulphaguanidine residues in honey samples

The inclusion complex of sulphaguanidine (SGN) in beta-cyclodextrin has been investigated. To avoid the problem of the low solubility of beta-cyclodextrin in water, solutions of beta-cyclodextrin in urea have been used. A 1:1 stoichiometry and an association constant of 450M(-1) have been established for the complex. A new spectrofluorimetric method has been developed for the determination of SGN residues in honey samples. This sulphonamide is widely employed for honey treatment. The method for the determination is based on second-order multivariate calibration, applying parallel factor analysis (PARAFAC). No previous separation or samples pre-treatment were required. The calibration solutions were prepared in water, with concentrations in the range from 0.02 to 0.20mugmL(-1) for SGN. The use of the second-order calibration method in the standard addition mode, using the excitation-emission matrices (EEMs) as analytical signal, allowed its determination in honey samples, even in the presence of interferences, with satisfactory results. The proposed procedure was validated by comparing the obtained results with a HPLC method, with satisfactory results for the assayed method.

Improvement in protein separation in Barretts esophagus samples using two-dimensional capillary electrophoresis analysis in presence of cyclodextrins as buffer additives

In two-dimensional capillary electrophoresis (2DCE) components are separated based on their size and hydrophobicity. A preliminary run separates analytes in the first capillary based on size (CSE). Following that, fractions are electrokinetically transferred across an interface into a second capillary, where components are further resolved according to hydrophobicity. In order to succeed in this analysis, two orthogonal methods should be selected for the different modes. The transfers from the first to the second capillary must be efficient in order to reduce tailing effects and lost of resolution. We report a new method to improve the resolution with our 2DCE instrumentation using CD doped buffers. When methyl beta cyclodextrin (mbetaCD) is added to the 2DCE interface buffer a stacking effect is described in the transfers from the first to the second dimension. In addition to that, changes in retention times are observed when proteins form complex with CDs helping in the separation. Protein fingerprints were obtained from BE homogenates using this method in presence of methyl beta cyclodextrin (mbetaCD). Within-day and between-day precision has been studied in order to establish the reproducibility of the methodology proposed.

Rapid ultrasensitive chemometrics-fluorescence methodology to quantify fluoroquinolones antibiotics residues in surface water

A sensitive method for the determination of fluoroquinolones in surface waters at trace concentration level is presented. The proposed two-step methodology consists in a solid-phase extraction using C-18 membranes followed measurement of the emission molecular fluorescence spectra over extracted membrane without elution of the analytes. Membrane background signal was removed by the used of chemometrics calculations, in addition chemometrics was as well used for the direct and simultaneous determination of the studied compounds. The method was optimized for the analysis of three fluoroquinolones: enoxacin (ENO), norfloxacin (NOR) and ofloxacin (OFLO). The fluorescence of these compounds increase drastically when they are in the membrane, thus with this method low concentrations are possible to be determined, as the concentration in which these compounds appear in surface water. Limits of detection at the ng•L–1 level were estimated for ENO, NOR and OFLO.