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.

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.

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.

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.