Determination of marker pteridines in urine by HPLC with fluorimetric detection and second-order multivariate calibration using MCR-ALS.

A liquid chromatographic method has been developed, in combination with the multivariate curve resolution-alternating least squares algorithm (MCR-ALS), for the simultaneous determination of marker pteridines in urine samples. A central composite design has been applied to optimize the factors influencing the separation (buffer concentration, buffer pH, flow rate, oven temperature, mobile-phase composition). A set of 15 calibration samples were randomly prepared, in a concentration range of 0.5-10.5 ng mL(-1) for neopterin, biopterin, and pterin; 4.0-8.0 ng mL(-1) for xanthopterin; and 0.5-4.5 ng mL(-1) for isoxanthopterin. The validation was carried out with fortified urine samples from healthy adults. The optimized conditions were a mobile-phase composition of 10 mM citric buffer at pH 5.44 and acetonitrile (94.5/5.5, v/v), a flow rate of 1.0 mL min(-1), and an oven temperature of 25 °C. The detection system consisted of a fast-scanning spectrofluorimeter, which allows obtaining of second-order data matrices containing the fluorescence intensity as a function of retention time and emission wavelength. In this work, MCR-ALS was used to cope with coeluting interferences, on account of the second-order advantage inherent to this algorithm which, in addition, is able to handle data sets deviating from trilinearity, like the high-performance liquid chromatography data analyzed in the present report. The developed approach enabled us to determine five pteridines, some of them with overlapped profiles, reducing the experimental time and reagent consumption. Ratio values for pteridines/creatinine in urine, for infected children with different pathologies, are reported in this work.

Solving matrix-effects exploiting the second order advantage in the resolution and determination of eight tetracycline antibiotics in effluent wastewater by modelling liquid chromatography data with multivariate curve resolution-alternating least squares and unfolded-partial least squares followed by residual bilinearization algorithms I. Effect of signal pre-treatment.

The effect of piecewise direct standardization (PDS) and baseline correction approaches was evaluated in the performance of multivariate curve resolution (MCR-ALS) algorithm for the resolution of three-way data sets from liquid chromatography with diode-array detection (LC-DAD). First, eight tetracyclines (tetracycline, oxytetracycline, chlorotetracycline, demeclocycline, methacycline, doxycycline, meclocycline and minocycline) were isolated from 250 mL effluent wastewater samples by solid-phase extraction (SPE) with Oasis MAX 500 mg/6 mL cartridges and then separated on an Aquasil C18 150 mm x 4.6mm (5 microm particle size) column by LC and detected by DAD. Previous experiments, carried out with Milli-Q water samples, showed a considerable loss of the most polar analytes (minocycline, oxitetracycline and tetracycline) due to breakthrough. PDS was applied to overcome this important drawback. Conversion of chromatograms obtained from standards prepared in solvent was performed obtaining a high correlation with those corresponding to the real situation (r2 = 0.98). Although the enrichment and clean-up steps were carefully optimized, the sample matrix caused a large baseline drift, and also additive interferences were present at the retention times of the analytes. These problems were solved with the baseline correction method proposed by Eilers. MCR-ALS was applied to the corrected and uncorrected three-way data sets to obtain spectral and chromatographic profiles of each tetracycline, as well as those corresponding to the co-eluting interferences. The complexity of the calibration model built from uncorrected data sets was higher, as expected, and the quality of the spectral and chromatographic profiles was worse.

Solving matrix effects exploiting the second-order advantage in the resolution and determination of eight tetracycline antibiotics in effluent wastewater by modelling liquid chromatography data with multivariate curve resolution-alternating least squares and unfolded-partial least squares followed by residual bilinearization algorithms II. Prediction and figures of merit.

A new powerful algorithm (unfolded-partial least squares followed by residual bilinearization (U-PLS/RBL)) was applied for first time on second-order liquid chromatography with diode array detection (LC-DAD) data and compared with a well-known established method (multivariate curve resolution-alternating least squares (MCR-ALS)) for the simultaneous determination of eight tetracyclines (tetracycline, oxytetracycline, meclocycline, minocycline, metacycline, chlortetracycline, demeclocycline and doxycycline) in wastewaters. Tetracyclines were pre-concentrated using Oasis Max C18 cartridges and then separated on a Thermo Aquasil C18 (150 mm x 4.6mm, 5 microm) column. The whole method was validated using Milli-Q water samples and both univariate and multivariate analytical figures of merit were obtained. Additionally, two data pre-treatment were applied (baseline correction and piecewise direct standardization), which allowed to correct the effect of breakthrough and to reduce the total interferences retained after pre-concentration of wastewaters. The results showed that the eight tetracycline antibiotics can be successfully determined in wastewaters, the drawbacks due to matrix interferences being adequately handled and overcome by using U-PSL/RBL.

Photoinduced electron transfer fluorometric Hg(II) chemosensor based on a BODIPY armed with a tetrapod receptor.

From the great variety of BODIPY based-chemosensors able to determine Hg(2+), only a small portion has been applied to its determination in environmental and/or biological samples. The lack of studies on the analytical performance of the latter sensors makes interesting the development of investigations oriented to their possible analytical applications. The synthesis of a BODIPY derivative armed with a tetrapod receptor is described. The procedure is based on a previous publication, and the modifications performed to improve the synthesis include alternative procedures with different objectives, as the consecution of a multigram synthesis, improving the low yields of some of the previously proposed procedure steps, simplifying the experimental steps, achieving the desired purity requirements for use with analytical purposes, and enriching the characterization of the implied structures. The characteristics of its selectivity towards Hg(2+) have been investigated, and the OFF-ON fluorometric response, based on a photo-electron transfer (PET) mechanism, served as the base for the development of a method able to determine Hg(2+) in environmental waters at ng mL(-1) levels. The intrinsic fluorescence of the BODIPY core is inhibited and the probe exhibits a weak fluorescence (i.e. "OFF" state due to the deactivating PET effect). Upon complexation, Hg(2+) interacts with the lone-pair electrons on the nitrogen atoms of the receptor moiety so that the electronic transfer from the receptor to the photo-excited fluorophore is slowed down or switched off (i.e. "ON" state due to the suppression of the deactivating PET effect by coordination of the analyte to the probe). Regarding the complex photostability in aqueous solution, it is mandatory to conduct the experiments at darkness due to its photodegradation. The stoichiometry studies indicated a 1:2 relationship for the BODIPY-Hg(2+) complex. The high selectivity towards mercuric ions is considerably influenced by pH, being necessary to conduct the experiments in a pH value higher than 6. Calibration samples were prepared by adding appropriate amounts of Hg(2+) between 20.0-120.0 ng mL(-1), at a constant BODIPY concentration of 1 µmol L(-1). After agitating for 5 min at darkness, phosphate buffer (pH=7.50) was added, and it was diluted to the mark with water. Fluorescence measurements were carried out at 18 °C, exciting at 515 nm, and obtaining fluorescence emission at 538 nm. The method has been satisfactory applied to Hg(2+) determination in environmental water samples.

Chemometric strategies for enhancing the chromatographic methodologies with second-order data analysis of compounds when peaks are overlapped.

This overview covers the different chemometric strategies linked to chromatographic methodologies that have been used and presented in the recent literature to cope with problems related to incomplete separation, the presence of unexpected components in the sample, matrix effect and changes in the analytical signal due to pre-treatment of sample. Among the different chemometric strategies it focuses on pre-treatment of data to correct background and time shift of chromatographic peaks and the use of second-order algorithms to cope with overlapping peaks from analytes or from analytes and interferences in liquid chromatography coupled to diode array, fast-scanning fluorescence spectroscopy and mass spectrometry detectors. Finally the review presents the strategies used to deal with changes in the analytical response as result of matrix effect in liquid and gas chromatography, as well as the use of standardization strategies to correct modifications in the analytical signal as a consequence of sample pre-treatment in liquid chromatography.

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.

Science based calibration for the extraction of 'analyte-specific' HPLC-DAD chromatograms in environmental analysis.

Multivariate science based calibration (SBC) has been applied to the resolution of overlapped peaks in liquid chromatography with diode array detection (LC-DAD). Complex river water samples spiked with 11 pharmaceutical substances resulted in poorly resolved chromatograms containing additional peaks from interfering matrix compounds and a change in the background absorbance due to the mobile phase gradient. Applying the present multivariate approach it was possible to resolve all 11 analytes from overlapping peaks, obtaining linear calibration lines (R(2)>0.96). Recovery percentages on spiked samples ranged between 74.6 and 113.5%, which are quite satisfactory taking into account the low concentration ranges considered to 1-7 µg L(-1).

Determination of pharmaceuticals in river water by column switching of large sample volumes and liquid chromatography-diode array detection, assisted by chemometrics: an integrated approach to green analytical methodologies.

An analytical method for the simultaneous determination of nine beta-blockers (sotalol atenolol, nadolol, pindolol, metoprolol, timolol, bisoprolol, propanolol and betaxolol) and two analgesics (paracetamol and phenazone) in river water by liquid chromatography and diode array detection is reported. The method involves a modified precolumn switching methodology replacing the small precolumn with a short C18 liquid chromatography column (50 mm x 4.6 mm, 5 microm particle size), thus allowing the preconcentration of large water sample volumes whereas interferences eluting at the first of the chromatogram were discarded to waste. This approach allowed to preconcentrate 30 mL river water samples, modified with 0.4% MeOH, achieving univariate method detection and determination limits ranged between 0.03 and 0.16 microg L(-1) and between 0.2 and 0.5 microg L(-1), respectively, with precision values lower than 9.4% for spiking levels at the quantitation limits of each analyte and lower than 4.0%, except bisoprolol (8.3%), for higher spiking levels (1.0 microg L(-1) of all analytes). Matrix background was reduced in three way data by a baseline correction following the Eilers methodology, whereas multivariate curve resolution and alternating least squares in combination with the standard addition calibration method, applied to these data, coped with overlapping peak, systematic (additive) and proportional (matrix effect) errors. The method was successfully applied for the determination of the target pharmaceuticals in river water from three places in a river stream with acceptable recoveries and precision values, taking into account the complexity of the analytical problem. The joint statistical test for the slope and the intercept of the linear regression between the nominal concentration values versus those predicted, showed that the region computed contained the theoretically expected values (0) for the intercept and (1) for the slope (at a confidence level of 95%), which indicates the absence of both constant and proportional errors in the predicted concentrations.

Chemometric tools improving the determination of anti-inflammatory and antiepileptic drugs in river and wastewater by solid-phase microextraction and liquid chromatography diode array detection.

An analytical method for the simultaneous determination of seven non-steroidal anti-inflammatory drugs (naproxen, ketoprofen, diclofenac, piroxicam, indomethacin, sulindac and diflunisal) and the anticonvulsant carbamazepine in river and wastewater is reported. The method involves pre-concentration and clean-up by solid-phase microextraction using polydimethylsiloxane/divinylbenzene fibers, followed by liquid chromatography with diode array detection analysis. Owing to the fact that river water samples did not contain interferences and no sensitivity changes due to sample matrix were observed, external calibration was implemented. Standardization was also applied in order to carry out the prediction step by preparing only two diluted standards that were subjected to the pre-concentration step and a set of standards prepared in solvent. For the analysis of wastewater samples, in contrast, it was necessary to implement standard addition calibration in combination with the multivariate curve resolution-alternating least squares (MCR-ALS) algorithm, which allowed us to overcome matrix effect and exploit the second order advantage. Recoveries ranging from 72% to 125% for all pharmaceuticals proved the accuracy of the proposed method in river water samples. On the other hand, wastewater sample recoveries ranged from 83% to 140% for all pharmaceuticals, showing an acceptable performance - considering this sample contains no modeled interferences.