Multivariate calibration-assisted high-performance liquid chromatography with dual UV and fluorimetric detection for the analysis of natural and synthetic sex hormones in environmental waters and sediments.

A green method is reported based on non-sophisticated instrumental for the quantification of seven natural and synthetic estrogens, three progestagens and one androgen in the presence of real interferences. The method takes advantage of: (1) chromatography, allowing total or partial resolution of a large number of compounds, (2) dual detection, permitting selection of the most appropriate signal for each analyte and, (3) second-order calibration, enabling mathematical resolution of incompletely resolved chromatographic bands and analyte determination in the presence of interferents. Consumption of organic solvents for cleaning, extraction and separation are markedly decreased because of the coupling with MCR-ALS (multivariate curve resolution/alternating least-squares) which allows the successful resolution in the presence of other co-eluting matrix constituents. Rigorous IUPAC detection limits were obtained: 6-24 ng L(-1) in water, and 0.1-0.9 ng g(-1) in sediments. Relative prediction errors were 2-10% (water) and 1-8% (sediments).

Optimization of pancreatic trypsin extraction in PEG/citrate aqueous two-phase systems.

Enzyme extraction using aqueous two-phase systems (ATPS) has been increasingly used as a primary recovery technique which integrates the clarification, concentration and partial purification of important biomolecules from their natural source in a single step. The goal of this work was to optimize the extraction of trypsin from pancreas homogenate with polyethylene glycol and sodium citrate (PEG/NaCit) ATPS by using the tools of experimental design. The variables NaCl concentration - added inert salt -, the top/bottom phase volume ratio - Vr - and the biomass loaded into the system - in percentage - were selected as the main factors in the trypsin extraction. The yield (%) and the purification factor of trypsin were considered the responses to be optimized. The central composite design and the response surface analysis proved to be suitable tools for a quick and efficient study. As a result, the optimal extraction conditions in PEG3350/NaCit system were 3.34% wt/wt for NaCl concentration, a biomass load which represented 9.30% wt/wt of the total ATPS mass and 6.37 top/bottom volume ratio giving a purification factor of 2.55 and a yield of 99.7% in top phase.

Liquid chromatography with diode array detection and multivariate curve resolution for the selective and sensitive quantification of estrogens in natural waters.

Following the green analytical chemistry principles, an efficient strategy involving second-order data provided by liquid chromatography (LC) with diode array detection (DAD) was applied for the simultaneous determination of estriol, 17ß-estradiol, 17α-ethinylestradiol and estrone in natural water samples. After a simple pre-concentration step, LC-DAD matrix data were rapidly obtained (in less than 5 min) with a chromatographic system operating isocratically. Applying a second-order calibration algorithm based on multivariate curve resolution with alternating least-squares (MCR-ALS), successful resolution was achieved in the presence of sample constituents that strongly coelute with the analytes. The flexibility of this multivariate model allowed the quantification of the four estrogens in tap, mineral, underground and river water samples. Limits of detection in the range between 3 and 13 ng L(-1), and relative prediction errors from 2 to 11% were achieved.

Spectrofluorimetric study of estrogen-cyclodextrin inclusion complexes in aqueous systems.

For the first time, the spectrofluorimetric properties of estrone (E1), 17ß-estradiol (E2), estriol (E3), and 17α-ethinylestradiol (EE2) are studied in aqueous solutions after the addition of native and derivative cyclodextrins. In contrast to previous reports, the behavior of the systems is analysed in the absence of organic solvents able to modify the guest-host interaction. The significant differences between the obtained association constants with those reported in solvent mixtures are shown and discussed. In order to evaluate the influence of both the estrogen structure and the presence of cyclodextrin substituents on the inclusion phenomenon, fluorescent and acid-base behaviors of the systems are compared and discussed. The interaction of estrogens with micellar media formed by selected surfactants is also studied. It is demonstrated that estrogen-cyclodextrin complexes in aqueous solution are useful for improving fluorimetric detection limits and, since cyclodextrins are non-toxic and mitigate most of the solubility problems which require the use of organic solvents, the studied complexes are excellent candidates for extraction, separation, pre-concentration and removal processes maintaining the principles of the green analytical chemistry.