High endoreduplication after drought-related conditions in haploid but not diploid mosses.

BACKGROUND AND AIMS: Endoreduplication, the duplication of the nuclear genome without mitosis, is a common process in plants, especially in angiosperms and mosses. Accumulating evidence supports the relationship between endoreduplication and plastic responses to stress factors. Here, we investigated the level of endoreduplication in Ceratodon (Bryophyta), which includes the model organism Ceratodon purpureus. METHODS: We used flow cytometry to estimate the DNA content of 294 samples from 67 localities and found three well-defined cytotypes, two haploids and one diploid, the haploids corresponding to C. purpureus and Ceratodon amazonum, and the diploid to Ceratodon conicus, recombination occurring between the former two. KEY RESULTS: The endoreduplication index (EI) was significantly different for each cytotype, being higher in the two haploids. In addition, the EI of the haploids was higher during the hot and dry periods typical of the Mediterranean summer than during spring, whereas the EI of the diploid cytotype did not differ between seasons. CONCLUSIONS: Endopolyploidy may be essential in haploid mosses to buffer periods of drought and to respond rapidly to desiccation events. Our results also suggest that the EI is closely related to the basic ploidy level, but less so to the nuclear DNA content as previously suggested.

The inversion of multiresponse partial least squares models, a useful tool to improve analytical methods in the framework of analytical quality by design.

Analytical Quality by Design (AQbD) is the adaptation of Quality by Design (QbD) when it is applied to the development of an analytical method. The main idea is to develop the analytical method in such a way that the desired quality of the Critical Quality Attributes (CQAs), stated via the analytical target profile (ATP), is maintained while allowing some variation in the Control Method Parameters (CMPs). The paper presents a general procedure for selecting factor levels in the CMPs to achieve the desired responses, characterized by the CQAs, when liquid chromatographic methods are to be used for the simultaneous determination of several analytes. In such a case, the CMPs are usually the composition of the ternary mobile phase, its flow rate, column temperature, etc., while typical CQAs refer to the quality of the chromatograms in terms of the resolution between each pair of consecutive peaks, initial and final chromatographic time, etc. The analytical target profile in turn defines the desired characteristics for the CQAs, the reason for the whole approach. The procedure consists of four steps. The first is to construct a D-optimal combined design (mixture-process design) to select the domain and levels of the CMPs. The second step is to fit a PLS2 model to predict the analytical responses expressed in the ATP (the good characteristics of the chromatogram) as a function of the CMPs. The third step is the inversion of the PLS2 model to obtain the conditions necessary to obtain the preset ATP in the corresponding CQAs. The inversion is performed computationally in order to estimate the Pareto front of these responses, namely, a set of experimental conditions to perform the chromatographic determination for which the desired critical quality attributes are met. The fourth final step is to obtain the Method Operable Design Region (MODR), that is, the region where the CMPs can vary while maintaining the quality of the CQAs. The procedure has been applied to some cases involving different analytes, all of which are regulated by the European Union due to their toxicity to human health, namely five bisphenols and ten polycyclic aromatic hydrocarbons.

Procedure to explore a ternary mixture diagram to find the appropriate gradient profile in liquid chromatography with fluorescence detector. Application to determine four primary aromatic amines in napkins.

The purpose of this work is to develop a tool to search for a gradient profile with ternary or binary mixtures in liquid chromatography, that can provide well-resolved chromatograms in the shortest time for multianalyte analysis. This approach is based exclusively on experimental data and does not require a retention time model of the compounds to be separated. The methodology has been applied for the quantification of four primary aromatic amines (PAAs) using HPLC with fluorescence detector (FLD). Aniline (ANL), 2,4-diaminotoluene (TDA), 4,4'-methylenedianiline (MDA) and 2-aminobiphenyl (ABP) have been selected since their importance in food contact materials (FCM). In order to achieve that, partial least squares (PLS) models have been fitted to relate CMP (control method parameters) and CQA (critical quality attributes). Specifically, PLS models have been fitted using 30 experiments for each one of the four CQA (resolution between peaks and total elution time), considering 33 predictor variables (the composition of the methanol and acetonitrile in the mobile phase and the time of each one of the 11 isocratic segments of the gradient). These models have been used to predict new candidate gradients, and then, some of those predictions (the ones with resolutions above 1.5, in absolute value, and final time lower than 20 min) have been experimentally validated. Detection capability of the method has been evaluated obtaining 1.8, 189.4, 28.8 and 3.0 µg L-1 for ANL, TDA, MDA and ABP, respectively. Finally, the application of chemometric tools like PARAFAC2 allowed the accurate quantification of ANL, TDA, MDA and ABP in paper napkins in the presence of other interfering substances coextracted in the sample preparation process. ANL has been detected in the three napkins analysed in quantities between 33.5 and 619.3 µg L-1, while TDA is present in only two napkins in quantities between 725.9 and 1908 µg L-1. In every case, the amount of PAAs found, exceeded the migration limits established in European regulations.

Method operable design region obtained with a partial least squares model inversion in the determination of ten polycyclic aromatic hydrocarbons by liquid chromatography with fluorescence detection.

A chromatographic method with the Analytical Quality by Design (AQbD) methodology is developed for the simultaneous determination by HPLC-FLD of ten PAHs (naphthalene, phenanthrene, anthracene, fluoranthene, pyrene, chrysene, benzo[a]anthracene, perylene, benzo[b]fluoranthene, and benzo[a]pyrene), widely spread in the environment. The construction of the Method Operable Design Region (MODR) is conducted, for the first time, via the inversion of a multiresponse Partial Least Squares (PLS2) model, which is needed to maintain the correlations among the Critical Method Parameters (CMP), among the Critical Quality Attributes (CQA), and the covariance between one another. The five CMP considered were the composition of the mobile phase (water, methanol, acetonitrile), flow rate, and column temperature. The eight CQA were linked to resolution between peaks recorded in the same emission wavelength (greater than 1.4) and the total time (less than 15 minutes). By systematic use of experimental design and parallel coordinates plots to explore the Pareto optimal front obtained with the PLS2 model inversion, the computed MODR is formed by convex combinations of eight specific settings of Critical Method Parameters that have a mobile phase with percentages of water between 37 and 38 %, of methanol from 13 and 22 %, and of acetonitrile between 41 and 49 %, together with a flow rate between 1.47 and 1.50 mL min-1, and column temperature between 41.9 and 44.0 °C in their adequate combinations. All the chromatographic peaks are well resolved, with total time varying between 12.96 and 15.66 min inside the estimated MODR and the analytical method is accurate with CCß between 0.9 and 7.0 µg L-1 with probability of both false positive and false negative equal to 0.05.

A new approach based on inversion of a partial least squares model searching for a preset analytical target profile. Application to the determination of five bisphenols by liquid chromatography with diode array detector.

The paper shows a procedure for selecting the control method parameters (factors) to obtain a preset 'analytical target profile' when a liquid chromatographic technique is going to be carried out for the simultaneous determination of five bisphenols (bisphenol-A, bisphenol-S, bisphenol-F, bisphenol-Z and bisphenol-AF), some of them regulated by the European Union. The procedure has three steps. The first consists of building a D-optimal combined design (mixture-process design) for the control method parameters, which are the composition of the ternary mobile phase and its flow rate. The second step is to fit a PLS2 model to predict six analytical responses (namely, the resolution between each pair of consecutive peaks, and the initial and final chromatographic time) as a function of the control method parameters. The third final step is the inversion of the PLS2 model to obtain the conditions needed for attaining a preset analytical target profile. The computational inversion of the PLS2 prediction model looking for the Pareto front of these six responses provides a set of experimental conditions to conduct the chromatographic determination, specifically 22% of water, mixed with 58% methanol and 20% of acetonitrile, keeping the flow rate at 0.66 mL min-1. These conditions give a chromatogram with retention times of 2.180, 2.452, 2.764, 3.249 and 3.775 min for BPS, BPF, BPA, BPAF and BPZ, respectively, and excellent resolution among all the chromatographic peaks. Finally, the analytical method is validated under the selected experimental conditions, in terms of trueness and precision. In addition, the detection capability for the five bisphenols were: 596, 334, 424, 458 and 1156 µg L-1, with probabilities of false positive and of false negative equal to 0.05.

Procedure to build a signal transfer set, independent of the target analytes, between a portable fluorimeter based on light-emitting diodes and a master fluorimeter.

The need of performing "in situ" analytical determinations together with the availability of high-power deep UV-LEDs have led to the use of fluorescence spectroscopy. However, it is necessary to register excitation-emission matrices (EEM) to obtain three-way data which can be decomposed using parallel factor analysis for enabling the unequivocal identification of the analytes. In this context, the feasibility of transferring EEM between a portable fluorimeter based on LEDs and a master fluorimeter based on a xenon source has been recently reported without losing analytical quality. To build the transfer function, the signals of the same N samples must be recorded in the portable and in the master fluorimeter. In literature, these samples always contained the target analytes so the EEM signal transfer methodology is very limited in practice. Therefore, the challenge is to search for a set of samples whose EEM enable to perform the signal transfer without previously knowing the target analytes. The aim of this work is the design of a procedure to build N mixtures of P fluorophores so the N EEM would be optimal for the signal transfer. Five criteria have been defined a priori to identify the quality of a transfer set made up of N EEM. Then, a procedure has been designed to obtain the n mixtures of the P fluorophores "in silico" using the Pareto front of the optimal solutions and a desirability function to choose the desired N EEM. The procedure has been used to find five mixtures of the three chosen fluorophores for the signal transfer (coumarin 120, DL-Tyrosine and DL-Tryptophan) which are chemically different from the analytes of interest (enrofloxacin and flumequine) and are contained in a different matrix. These two analytes are antibiotics which have maximum residue limits set in the EU legislation in force. The correlation coefficients between the experimental reference spectra and the PARAFAC spectral loadings of the data registered with the master fluorimeter were greater than or equal to 0.999 in all cases. On the other hand, the correlation coefficients obtained with the portable fluorimeter ranged from 0.900 to 0.950 once the procedure was applied to the two antibiotics. Therefore, the unequivocal identification of the analytes was ensured.

Improvement in the identification and quantification of UV filters and additives in sunscreen cosmetic creams by gas chromatography/mass spectrometry through three-way calibration techniques.

The simultaneous determination of 2,6-di-tert-butyl-4-methyl-phenol (BHT), benzophenone (BP), benzophenone-3 (BP3) and diisobutyl phthalate (DiBP) in seven sunscreen creams was carried out by gas chromatography/mass spectrometry (GC/MS) using DiBP-d4 as internal standard. The content of BP3, which is a UV filter, must not exceed 6% (w/w) in cosmetic products according to Regulation (EU) 2017/238 and the use of DiBP in cosmetic products shall be prohibited according to Regulation (EC) No 1223/2009. The conclusions obtained with the univariate standard methodology in the identification of the analytes contained in the creams were wrong. However, a calibration based on PARAFAC or PARAFAC2 decompositions, where the samples of the prediction set were projected on the model obtained previously with the calibration set, enabled the unequivocal identification and quantification of the analytes even in the presence of interferents not considered in the calibration model. The PARAFAC2 decomposition was used to overcome the shifts in the retention time of BP and BP3. These three-way calibration techniques are needed to avoid false negative results. The method had not proportional or constant bias. The presence of BHT was detected in the seven sunscreen creams analysed at an amount of 6.48 10-2%, 8.53 10-2%, 1.70 10-4%, 1.11 10-4%, 2.51 10-3%, 3.20 10-5% and 6.35 10-3%. The concentrations of DiBP found in four creams were 3.49 10-2%, 3.19 10-2%, 3.26 10-2% and 2.51 10-2%. On the other hand, BP was only detected in two of the cosmetic creams analysed at an amount of 7.84 10-3% and 1.04 10-2%. In addition, BP3 was detected in six of the creams at an amount of 4.73%, 3.49%, 4.94 10-3%, 1.98 10-3%, 6.62 10-1% and 1.73%. Therefore, none of the cosmetic creams contained BP3 in an amount higher than 6%.

Kinetic models of migration of melamine and formaldehyde from melamine kitchenware with data of liquid chromatography.

European legislation has established a specific migration limit (SML) of 15 mg kg-1 for formaldehyde and 2.5 mg kg-1 for melamine. Formaldehyde resins are used in the manufacture of melamine kitchenware. Formaldehyde is listed in group 1 of the IARC list of carcinogenic compounds. To determine the quantity of formaldehyde and melamine as potential migrants from different types of melamine kitchenware (glass, mug, cutlery, big cup and bowl), a HPLC-DAD method has been implemented. This method is an alternative to the ones proposed in technical guidelines to determine formaldehyde by UV-vis spectrophotometry and melamine by HPLC. The final objective was to fit the migration kinetic curves of these two analytes in melamine kitchenware. After the method was validated, decision limit (CCα) and detection capability (CCß) were calculated for both analytes, when the probabilities of false positive (α) and false negative (ß) were fixed at 0.05; being CCß 0.269 mg L-1 and 0.311 mg L-1 for melamine and formaldehyde respectively. CCα and CCß were also calculated at the SML of both analytes. The migration testing were conducted with simulant B (3% acetic acid (w/v) in aqueous solution), the conditions of each exposure being 70 °C for 2 h. The quantities of melamine and formaldehyde found in the third exposure of the total kitchenware analysed were between 0.21 and 1.09 mg L-1 and between 0.55 and 3.86 mg L-1, respectively. Migration kinetic curves were built for each type of kitchenware with the data of sixteen consecutive migration cycles (70 °C each 30 min). The SML for melamine was surpassed in the mug, in the big cup and in the bowl after eleven, thirteen and one cycles, respectively. When more cycles were carried out in the mug, the values of the accumulated quantity of formaldehyde and melamine were 15.30 and 6.79 mg L-1, respectively, after thirty-two cycles. Both concentrations exceeded the corresponding SML.

Fluorescence determination of cochineal in strawberry jam in the presence of carmoisine as a quencher by means of four-way PARAFAC decomposition.

The determination of cochineal (E-120) in strawberry jam was carried out in the presence of carmoisine (E-122) using the four-way PARAFAC decomposition and excitation-emission fluorescence matrices. In the measured conditions, there was no fluorescence signal for carmoisine due to a strong quenching effect and this colorant also led to a decrease of the fluorescence signal of cochineal. The European Union has fixed a maximum residue level, MRL, for cochineal in jam (100 mg kg-1). Therefore, the addition of other food colorant (carmoisine) in the jam could lead to false compliant decisions. The four-way PARAFAC decomposition avoided false compliant decisions caused by the quenching effect. Cochineal was unequivocally identified. Detection capability (CCß) was 0.72 mg L-1 for probabilities of false positive and false negative fixed at 0.05. Cochineal was detected in the jam (104.63 mg kg-1) above the MRL. This amount was compared with the one obtained using a HPLC/DAD method.

Determination of cochineal and erythrosine in cherries in syrup in the presence of quenching effect by means of excitation-emission fluorescence data and three-way PARAFAC decomposition.

The simultaneous determination of two food colorants (cochineal (E-120) and erythrosine (E-127)) was achieved by means of excitation-emission fluorescence matrices and three-way PARAFAC decomposition together with the use of a calibration set that contained binary mixtures of both analytes. In the measured conditions, the amount of cochineal present in the sample affected the fluorescence signal of erythrosine since cochineal caused a quenching effect in the fluorescence of the other food additive. However, the signal of cochineal was not affected by the presence of erythrosine. A calibration line for erythrosine was built for each different concentration level of cochineal. The slopes of these regressions were different depending on the amount of quencher, whereas the intercepts were statistically equal to 0 at a 95% confidence level. The quantification of erythrosine was possible using the regression "amount of cochineal" versus "the slope of the calibration line for erythrosine". Using this procedure, the mean of the absolute values of the relative errors in prediction for mixtures of both colorants were 5.86% (n = 10) for cochineal and 4.17% (n = 10) for erythrosine. Both analytes were unequivocally identified by the correlation between the pure spectra and the PARAFAC excitation and emission spectral loadings. Pitted cherries in syrup were analyzed. Cochineal and erythrosine were detected in those cherries at a concentration of 185.05 mg kg-1 and 10.76 mg kg-1, respectively. These concentration values were statistically equal to the ones obtained with a HPLC/DAD method.

The behaviour of Tenax as food simulant in the migration of polymer additives from food contact materials by means of gas chromatography/mass spectrometry and PARAFAC.

The migration of benzophenone (BP), an antioxidant (2,6-di-tert-butyl-4-methyl-phenol (BHT)) and three plasticizers (diisobutyl phthalate (DiBP), bis(2-ethylhexyl) adipate (DEHA) and diisononyl phthalate (DiNP)) from different food contact materials into Tenax as food simulant was studied. The packaging materials analysed were: polyethylene (PE) and polyvinyl chloride (PVC) cling-films, paper bread bag, brown paper popcorn bag intended to be heated in a microwave oven and polypropylene (PP) coffee capsules. The analysis was carried out using PARAFAC and PARAFAC2 decompositions and gas chromatography/mass spectrometry (GC/MS), being DiBP-d4 the internal standard. Tenax has been used as food simulant for specific migration of dry foodstuffs according to Commission Regulation (EU) 10/2011. PARAFAC and PARAFAC2 decompositions enabled the unequivocal identification and quantification of all the analytes despite some of the m/z ratios of the coeluting interferents were shared with the analytes. Otherwise, the presence of the analytes could not have been ensured according to the EU legislation in force. BHT, DiBP and DEHA were contained in the Tenax blanks in some of the analyses. The amount of BP and DiBP migrated from the PVC film was 83.53 µg L-1 and 31.30 µg L-1, respectively; whereas 71.62 µg L-1 of BP and 27.45 µg L-1 of DiBP migrated from the PP coffee capsules. None of the analytes were detected above the capability of detection in the non-spiked migration samples of the rest of the food contact materials analysed. The efficiency of Tenax as an adequate food simulant has also been studied through the values of its adsorption capability which were different depending on the analytes and the materials. In the spiked migration samples, these values ranged from 25.33% to 99.37%.

Phylogenetic analyses of Chlamydia psittaci ompA gene sequences from captive blue-fronted Amazon parrots (Amazona aestiva) with hepatic disease in Brazil.

Chlamydia psittaci was detected in 152 (72%) blue-fronted Amazon parrots (Amazona aestiva, parrot from the Psittacidae family) out of a population of 212 that died during 2009-2011 in a wildlife rescue and rehabilitation centre in Minas Gerais, Brazil, following rescue from illegal wildlife trafficking. The macroscopic changes observed in these animals were hepatomegaly with multifocal white foci visible at the serosal surfaces of the liver, and extending into the parenchyma, and splenomegaly. The microscopic lesions observed in the liver included multifocal to coalescing miliary necrosis of hepatocytes with infiltration by heterophils, lymphocytes and plasma cells. In the spleen, loss of the normal architecture and infiltration by macrophages and plasma cells were observed. Stained tissue sections (Gimenez technique) revealed small round clusters suggestive of C. psittaci (reticulate bodies) in the cytoplasm of macrophages from the liver and spleen. Nine sequences of segments of the ompA gene, obtained from different individuals, were randomly selected for sequencing. The phylogenetic analyses showed that all strains clustered with genotype A, which is the most virulent genotype for birds. This genotype is involved in mortality of psittacines, is easily transmitted in captivity and represents a problem for successful rehabilitation. The results indicate the necessity to improve biosecurity in triage and to provide individual personal protection for professionals and caretakers.

Chlamydia psittaci a été détectée chez 152 (72 %) amazones à front bleu (Amazona aestiva, perroquet de la famille des Psittacidés) sur un total de 212 individus rescapés du trafic illégal et décédés en 2009 et 2011 dans un centre de sauvetage et de réhabilitation de la faune sauvage à Minas Gerais (Brésil). Les modifications macroscopiques observées sur ces oiseaux étaient une hépatomégalie avec des foyers blancs multifocaux visibles sur les surfaces séreuses du foie et s'étendant dans le parenchyme, et une splénomégalie. Les lésions microscopiques observées dans le foie comprenaient une nécrose miliaire multifocale à coalescente des hépatocytes avec infiltration d'hétérophiles, de lymphocytes et de plasmocytes. Dans la rate, une perte de l'architecture normale et l'infiltration de macrophages et de plasmocytes ont été observées. La coloration de coupes de tissus (technique de Gimenez) a révélé de petites grappes rondes évoquant C. psittaci (corps réticulés) dans le cytoplasme des macrophages du foie et de la rate. Neuf produits segmentés d'une partie du gène ompA, obtenus de différents individus, ont été sélectionnés de manière aléatoire pour le séquençage. Les analyses phylogénétiques ont montré que toutes les souches se regroupaient dans le génotype A, qui est le plus virulent pour les oiseaux. Ce génotype est responsable de cas de mortalité chez les psittacidés et se transmet facilement en captivité, ce qui représente un risque pour la réussite des opérations de réhabilitation. Au vu de ces résultats, les auteurs soulignent la nécessité d'améliorer la biosécurité lors du tri des animaux dans les centres de soins et de fournir une protection individuelle aux professionnels et aux gardiens.

Se detectó Chlamydia psittaci en 152 (72%) amazonas frentiazules (Amazona aestiva, loro de la familia Psittacidae) de un total de 212 que murieron durante 2009­2011 en un centro de rescate y rehabilitación de fauna silvestre de Minas Gerais, Brasil, tras haber sido rescatadas del tráfico ilegal. Los cambios macroscópicos que se observaron en estos animales fueron hepatomegalia con focos blancos multifocales visibles en las superficies serosas del hígado y que se extendían hacia el parénquima, y esplenomegalia. Las lesiones microscópicas observadas en el hígado consistieron en necrosis miliar multifocal a coalescente de hepatocitos con infiltración de heterófilos, linfocitos y células plasmáticas. En el bazo, se observó pérdida de la arquitectura normal y infiltración de macrófagos y células plasmáticas. Cortes de tejido teñidos (con la técnica de Giménez) revelaron pequeños racimos redondos que sugerían la presencia de C. psittaci (cuerpos reticulados) en el citoplasma de macrófagos del hígado y del bazo. A partir de distintos individuos, se escogieron aleatoriamente nueve segmentos del gen ompA para ser secuenciados. Los análisis filogenéticos mostraron que todas las cepas correspondían al genotipo A, que es el más virulento para las aves. Este genotipo está involucrado en la mortalidad de psitácidas, se transmite fácilmente en cautiverio y supone un riesgo para el éxito de la rehabilitación. Los resultados indican la necesidad de mejorar la bioseguridad en el triaje y de procurar protección personal individual a profesionales y cuidadores.

Effect of the cleaning procedure of Tenax on its reuse in the determination of plasticizers after migration by gas chromatography/mass spectrometry.

This paper presents the simultaneous determination of a UV stabilizer (benzophenone (BP)) together with four plasticizers (butylated hydroxytoluene (BHT), diisobutyl phthalate (DiBP), bis(2-ethylhexyl) adipate (DEHA) and diisononyl phthalate (DiNP)) in Tenax by gas chromatography/mass spectrometry and PARAFAC, using DiBP-d4 as internal standard. Regulation (EU) No. 10/2011 establishes Tenax as food simulant E for testing specific migration from plastics into dry foodstuffs. This simulant must be cleaned before its use to eliminate impurities. Tenax is expensive, so its reuse would save costs. A two-way ANOVA was used to study some parameters affecting the cleaning and the extraction of Tenax. The most adequate conditions were chosen taking the values of the coefficient of variation and the average recovery rates of spiked Tenax samples into account. A study to determine if some analytes remain in Tenax when it is reused and the effect that the cleaning procedure may have in the adsorption capability of Tenax was proposed. This study led to the conclusion that Tenax could not be reused in this multiresidue determination. All the analytes were unequivocally identified in all the stages of this work and trueness was verified at a 95% confidence level in all cases. A calibration based on PARAFAC provided the following values of capability of detection (CCß): 2.28 µg L-1 for BHT, 10.57 µg L-1 for BP, 7.87 µg L-1 for DiBP, 3.04 µg L-1 for DEHA and 124.8 µg L-1 for DiNP, with the probabilities of false positive and false negative fixed at 0.05. The migration of the analytes from a printed paper sample into Tenax was also studied. The presence of BHT in the food simulant was confirmed and the amount released of this analyte from the paper was 2.56 µg L-1.

Easy-to-use procedure to optimise a chromatographic method. Application in the determination of bisphenol-A and phenol in toys by means of liquid chromatography with fluorescence detection.

Legal limits for phenol and bisphenol-A (BPA) in toys are 15 and 0.1 mg L-1 respectively. The latest studies show that in Europe the content of BPA, which reaches our bodies through different contact routes, in no cases exceed legal limits. But it is true that the effects caused by continued intake of this analyte for a long time and other possible processes that could increase their migration, are still under consideration by the health agencies responsible. A multiresponse optimization using a D-optimal design for simultaneously optimising two experimental factors (temperature and flow) at three levels and one (mobile phase composition) at four levels, in the determination by means of HPLC-FLD is proposed in this work. The D-optimal design allows ones to reduce the experimental effort from 36 to 11 experiments guaranteeing the quality of the estimates. The model fitted is validated and, after the responses are estimated in the whole experimental domain, the experimental conditions that maximize peak areas and minimize retention times for both analytes are chosen by means of a Pareto front. In this way, the sensitivity and the time of the analysis have been improved with this optimization. Decision limit and capability of detection at the limits obtained were 33.9 and 66.1 µg L-1 for phenol and 25.6 and 50.0 for BPA µg L-1 respectively when the probabilities of false negative and false positive were fixed at 0.05. The procedure has been successfully applied to determine phenol and BPA in different samples (toys, clinical serum bags and artificial tears). The simulants HCl 0.07 M and water were used for the analysis of toys. The quantity of phenol found in serum bags and in artificial tears ranged from 15 to 600 µg L-1. No BPA has been found in the objects analysed. In addition, this work incorporates computer programmes which implement the procedure used (COOrdinates parallel plot and Pareto FROnt, COO-FRO) such that it can be used in any other chromatographic optimization.

Migration test of Bisphenol A from polycarbonate cups using excitation-emission fluorescence data with parallel factor analysis.

Bisphenol A (BPA) is one of the most largely produced chemical in the world; it is used to make plastics and epoxy resins. The endocrine disruptor potential of BPA is well known, but recent researches suggest a relationship between chronic exposure to BPA, genotoxic activity and epigenetic modifications. The main source of exposure to BPA includes food contact materials (FCM). Thus simple and robust test methods are needed to improve the migration test of BPA. In this work, a non-separative, easy, fast and inexpensive spectrofluorimetric method based on the second order calibration of excitation-emission fluorescence matrices (EEMs) was proposed for the determination of BPA. For the first time, molecular fluorescence was used to identify unequivocally and quantify BPA. Trilinearity of the data tensor guarantees the uniqueness of the solution obtained through parallel factor analysis (PARAFAC), so one factor of the decomposition matches up with BPA even if other fluorophores are in the test sample. The effect of four experimental factors of the procedure on the figures of merit and the unequivocally identification was investigated by means of a D-optimal design and PARAFAC calibration. The method is linear and accurate in the range 0-720µgL-1. The decision limit CCα and detection capability CCß are 6.63µgL-1 and 18.85µgL-1 respectively (with probabilities of false positive and false negative fixed at 0.05). Finally the proposed method was applied to carry out a migration test from two polycarbonate cups, using 3% (w/v) acetic acid in aqueous solution as food simulant. The migrated amount of BPA was found to be 688.7µgL-1 (n=5) for the first cup and 710.5µgL-1 (n=4) for the second one, above the specific migration limit set by EFSA (European Food Safety Authority).

A new multiresponse optimization approach in combination with a D-Optimal experimental design for the determination of biogenic amines in fish by HPLC-FLD.

A new strategy to approach multiresponse optimization in conjunction to a D-optimal design for simultaneously optimizing a large number of experimental factors is proposed. The procedure is applied to the determination of biogenic amines (histamine, putrescine, cadaverine, tyramine, tryptamine, 2-phenylethylamine, spermine and spermidine) in swordfish by HPLC-FLD after extraction with an acid and subsequent derivatization with dansyl chloride. Firstly, the extraction from a solid matrix and the derivatization of the extract are optimized. Ten experimental factors involved in both stages are studied, seven of them at two levels and the remaining at three levels; the use of a D-optimal design leads to optimize the ten experimental variables, significantly reducing by a factor of 67 the experimental effort needed but guaranteeing the quality of the estimates. A model with 19 coefficients, which includes those corresponding to the main effects and two possible interactions, is fitted to the peak area of each amine. Then, the validated models are used to predict the response (peak area) of the 3456 experiments of the complete factorial design. The variability among peak areas ranges from 13.5 for 2-phenylethylamine to 122.5 for spermine, which shows, to a certain extent, the high and different effect of the pretreatment on the responses. Then the percentiles are calculated from the peak areas of each amine. As the experimental conditions are in conflict, the optimal solution for the multiresponse optimization is chosen from among those which have all the responses greater than a certain percentile for all the amines. The developed procedure reaches decision limits down to 2.5 µg L-1 for cadaverine or 497 µg L-1 for histamine in solvent and 0.07 mg kg-1 and 14.81 mg kg-1 in fish (probability of false positive equal to 0.05), respectively.

Migration kinetics of primary aromatic amines from polyamide kitchenware: Easy and fast screening procedure using fluorescence.

Primary aromatic amines, PAAs, and their derivatives constitute a health risk and control of their migration from food contact materials is the subject of permanent attention by the authorities. 25.1% of notifications made by Rapid Alert System for Food and Feed in the European Union between 2010 and 2015 concerned PAAs, polyamide cooking utensils being a common source. It is thus useful to have fast and efficient analytical methods for their control. In this work a non-separative, easy, fast and inexpensive spectrofluorimetric method based on the second order calibration of excitation-emission fluorescence matrices (EEMs) was proposed for the determination of aniline (ANL), 2,4-diaminotoluene (2,4-TDA) and 4,4'-methylenedianiline (4,4'-MDA) in polyamide cooking utensils. The procedure made it possible to identify unequivocally each analyte. Trilinearity of the data tensor guarantees the uniqueness of the solution obtained through parallel factor analysis (PARAFAC), so the factors of the decomposition match up with the analytes. The three analytes were unequivocally identified by the correlation between the pure spectra and the PARAFAC excitation and emission spectral loadings. The recovery percentages found were, 82.6%, 112.7% and 84.4% for ANL, 2,4-TDA and 4,4'-MDA respectively. The proposed method was applied to carry out a migration test from polyamide cooking utensils, using a 3% (w/v) acetic acid in aqueous solution as food simulant. Detectable levels of 4,4'-MDA were found in food simulant from some of the investigated cooking utensils. Finally, a kinetic model for the migration of 4,4'-MDA has been fitted to experimental data obtained in the migration test. Thanks to the selectivity of PARAFAC calibration, which greatly simplifies sample treatment avoiding the use of toxic solvents, the developed method follows most green analytical chemistry principles.

Dealing with the ubiquity of phthalates in the laboratory when determining plasticizers by gas chromatography/mass spectrometry and PARAFAC.

Determining plasticizers and other additives migrated from plastic materials becomes a hard task when these substances are already present in the laboratory environment. This work dealt with this drawback in the multiresidue determination of four plasticizers (2,6-di-tert-butyl-4-methyl-phenol (BHT), diisobutyl phthalate (DiBP), bis(2-ethylhexyl) adipate (DEHA) and diisononyl phthalate (DiNP)) and a UV stabilizer (benzophenone (BP)) by gas chromatography/mass spectrometry (GC/MS) using DiBP-d4 as internal standard. The ubiquity of DiBP by a non-constant leaching process in the laboratory was detected, which could not guarantee the achievement of a trustworthy quantification. To handle this, the assessment of the level of DiBP in solvent blanks having fixed the probabilities of false non-compliance (α) and false compliance (ß) at 0.01 was performed. On the other hand, another special case was that of DiNP, in whose chromatogram finger peaks appear because of an array of possible C9 isomers. PARAFAC, used for the identification and quantification of all the substances, is a useful chemometric tool that enabled a more reliable determination of this analyte since no peak areas were considered but chromatographic and spectral loadings. Since phthalates may migrate from rubber latex items, an evaluation of the existence of matrix effects on the determination of the five analytes was conducted prior to an extraction with hexane from a dummy for infants. As matrix effects were present, the quantification of the compounds under study was performed following the standard addition method using PARAFAC sample loadings as response variable. As a result, the presence of BHT was confirmed, being its concentration equal to 37.87µgL(-1). Calibrations based on PARAFAC yielded the following values for the decision limit (CCα): 1.16µgL(-1) for BHT, 1.34µgL(-1) for BP, 1.84µgL(-1) for DEHA and 51.42µgL(-1) for DiNP(for α=0.05 and two replicates).

Standard addition method based on four-way PARAFAC decomposition to solve the matrix interferences in the determination of carbamate pesticides in lettuce using excitation-emission fluorescence data.

The simultaneous determination of two carbamate pesticides (carbaryl and carbendazim) and of the degradation product of carbaryl (1-naphthol) in iceberg lettuce was achieved by means of PARAFAC decomposition and excitation-emission fluorescence matrices. A standard addition method for a calibration based on four-way data was applied using different dilutions of the extract from iceberg lettuce as a fourth way that provided the enough variation of the matrix to carry out the four-way analysis. A high fluorescent overlapping existed between the three analytes and the fluorophores of the matrix. The identification of two fluorescent matrix constituents through the four-way model enabled to know the matrix contribution in each dilution of the extract. This contribution was subtracted from the previous signals and a subsequent three-way analysis was carried out with the tensors corresponding to each dilution. The PARAFAC decomposition of these resulting tensors showed a CORCONDIA index equal to 99%. For the identification of the analytes, the correlation between the PARAFAC spectral loadings and the reference spectra has been used. The trueness of the method, in the concentration range studied, was guaranteed because there was neither constant nor proportional bias according to the appropriate hypothesis tests. The best recovery percentages were obtained with the data from the most diluted extract, being the results: 127.6% for carbaryl, 125.55% for carbendazim and 87.6% for 1-naphthol. When the solvent calibration was performed, the decision limit (CCα) and the capability of detection (CCß) values, in x0=0, were 2.21 and 4.38 µg L(-1) for carbaryl, 4.87 and 9.64 µg L(-1) for carbendazim; and 3.22 and 6.38 µg L(-1) for 1-naphthol, respectively, for probabilities of false positive and false negative fixed at 0.05. However, these values were 5.30 and 10.49 µg L(-1) for carbaryl, 18.05 and 35.73 µg L(-1) for carbendazim; and 1.92 and 3.79 µg L(-1) for 1-naphthol, respectively, when the matrix-matched calibration using the most diluted extract was carried out in the recovery study.