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The applicability of front-face excitation-emission fluorescence spectroscopy to compare grape water extracts of two consecutive sampling dates, corresponding with two maturation stages, and subjected to full irrigation and non-irrigation, was carried out. The decomposition of the obtained three way grape samples was initially analyzed by means of parallel factor analysis (PARAFAC). A tentative identification of fluorophores was done by matching PARAFAC score values with HPLC measurements. It was found that the first PARAFAC component was highly correlated with the sum of concentrations of catechin and epicatechin. The decomposition of the three way fluorescence data by linear discriminant analysis (LDA)-PARAFAC and LDA-unfolding partial least squares (U-PLS) allowed to discriminate between the first and the second maturation stage. The incorporation of an additional mode to the data, achieved by a diethyl ether extraction, giving rise to a four-way excitation-emission-solvent-samples data set, allowed to differentiate between irrigated and non-irrigated samples with the same assayed algorithms. As far as we know, the use of four-way data arrays for classification issues has been reported for the first time.

The determination of flavonoid compounds in paprika samples has been performed by liquid chromatography in series diode array and fluorescence detection (LC-DAD-FLD), by means of a pH change to basic medium just before FLD detection. The validation of the method was performed through the establishment of the external standard calibration curves and the analytical figures of merit. Limits of detection ranging from 0.006 to 0.02 mg L(-1) and 0.007 to 0.09 mg L(-1) were achieved using DAD and FLD detection, respectively. The experimental conditions to carry out the hydrolysis procedure to obtain flavonoid aglycones from flavonoid glycosides have been optimized applying an experimental design and the response surface methodology. The final conditions selected were 2.5M HCl during 45 min at 85°C. The repeatability of this procedure was assayed and relative standard deviation (RSD) values for concentration of quercetin and luteolin compounds were lower than 2%. The quantification of quercetin, luteolin and kaempferol compounds was carried out in less than 6 min in paprika samples by means of the external standard calibration. The analytes were extracted with methanol and the extracts were previously subjected to a cleanup procedure to extend the use of the chromatographic column.

Phenanthrene is the most released polycyclic aromatic hydrocarbon into the environment by anthropogenic action. Because of the absorption and biotransformation pathways, this compound is metabolized and the most abundant metabolites are hydroxylated derivatives, such as 1-, 2-, 3-, 4- and 9-hydroxyphenanthrene, which are excreted through biological fluids, included mammals milk. For the resolution and quantitation of co-eluted analytes, elution time-emission fluorescence matrices were analysed with different second-order calibration algorithms: n-way and unfolded partial least squares, both coupled with residual bilinearization (N-PLS/RBL and U-PLS/RBL), and multivariate curve resolution-alternative least squares (MCR-ALS). Once optimized the chromatographic parameters, in isocratic mode, the elution time was of 5.5 min. The second-order data were obtained exciting at 250 nm, with an emission range from 330 to 430 nm, each 1 nm, and elution time from 0 to 5.5 min each 5.4 s. The ranges for the second-order multivariate methods in validation samples were from 1.0 to 9.0 ng mL−1 for 1-, 2-, 3- and 4-hydroxyphenanthrene, and from 5.0 to 45.0 ng mL−1 for 9-hydroxyphenanthrene. Root mean square errors of prediction between 0.45 and 1.82 ng mL−1 (relative errors of prediction 7–22%) were obtained. The optimized procedures were applied in the analysis of human breast milk and in whole and semi-skimmed commercial cow milk. N-PLS/RBL and U-PLS/RBL algorithms show satisfactory results for the five metabolites with recoveries ranging between 82% and 115%.

Within olive oils, extra virgin olive oil is the highest quality and, in consequence, the most expensive one. Because of that, it is common that some merchants attempt to take economic advantage by mixing it up with other less expensive oils, like olive oil or olive pomace oil. In consequence, the characterization and authentication of extra virgin olive oils is a subject of great interest, both for industry and consumers. This paper reports the potential of front-face total fluorescence spectroscopy combined with second-order chemometric methods for the detection of extra virgin olive oils adulteration with other olive oils. Excitation-emission matrices (EEMs) of extra virgin olive oils and extra virgin olive oils adulterated with olive oils or with olive pomace oils were recorded using front-face fluorescence spectroscopy. The full information content in these fluorescence images was analyzed with the aid of unsupervised parallel factor analysis (PARAFAC), PARAFAC supervised by linear discriminant analysis (LDA-PARAFAC), and discriminant unfolded partial least-squares (DA-UPLS). The discriminant ability of LDA-PARAFAC was studied through the tridimensional plots of the canonical vectors, defining a surface separating the established categories. For DA-UPLS, the discriminant ability was established through the bidimensional plots of predicted values of calibration and validation samples, in order to assign each sample to a given class. The models demonstrated the possibility of detecting adulterations of extra virgin olive oils with percentages of around 15% and 3% of olive and olive pomace oils, respectively. Also, UPLS regression was used to quantify the adulteration level of extra virgin olive oils with olive oils or with olive pomace oils.

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.

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.

An off-line photoirradiation LC fluorimetric method to determine tetrahydrobiopterin (BH4), by photogeneration of biopterin (BIO), is described, as an alternative way to the chemical oxidation procedure. To minimize the uncontrolled BH4 oxidation, due to environmental oxygen, an antioxidant, dithiothreitol (DTT), was used. The acidity of the medium, as well as the presence of hydrogen peroxide, affects the rate of the photoreaction and the nature of the obtained fluorescent photoproducts. The best conditions were achieved by irradiation in hydrochloric acid (0.2 M) medium, in presence of 100 mM hydrogen peroxide, and using an irradiation time of 20 min. The method was tested in the analysis of serum samples containing BH4, and recoveries between 89 and 105% were found. Also, the proposed method allows the resolution of BH4 and BIO, in the same sample, by injection of non-irradiated and irradiated sample aliquots in the chromatographic system.

This manuscript presents the development of a new photochemically induced fluorescence flow-injection system, combined with solid-surface adsorption, for the determination of the benzoylurea pesticide linuron. The determination is carried out by measuring the fluorescence intensity of a photoproduct, formed by on-line irradiation with ultraviolet light, and retention on a solid matrix filling the flow-cell. The procedure is developed in the presence of two surfactants: sodium dodecyl sulphate and hexadecyltrimethylammonium chloride, which are critical for the detection of the fluorescence emission in the solid surface. The possible interactions between the linuron photoproduct, the micellar medium employed and the solid support (C18 silica gel) are discussed. Parameters related to the analytical signal and to the FIA manifold are optimized. Finally, the feasibility of the developed method was tested by the determination of linuron in both environmental and drinking water samples. Determinations at μg L−1 level were accomplished after a solid-phase pre-concentration procedure.

A high performance liquid chromatography (HPLC) method has been developed for the simultaneous determination of four fluoroquinolones. The studied compounds have been enoxacin (ENO), norfloxacin (NOR), ofloxacin (OFLO) and enrofloxacin (ENRO). An isocratic elution method, using a mixture of tetrahydrofuran (8%) and phosphate buffer (pH 3.00, 30.0mM, 92%) as mobile phase, has been developed. Fluorimetric detection, exciting at 277nm, and multiemission scan (407nm for ENO, 444nm for both NOR and ENRO and 490nm for OFLO) has been used. Detection limits of 500, 14.7, 25.2 and 15.0ngmL(-1) for ENO, NOR, OFLO and ENRO, respectively, have been obtained. The proposed method has been satisfactorily applied to analyze NOR, OFLO and ENRO in human urine and serum samples.

A widely employed compound for honey treatment, sulfathiazole (ST), was determined in commercial honey samples, employing a combination of photochemically induced fluorescence excitation-emission matrices (EEMs) and chemometric processing of the recorded second-order data. Parallel Factor Analysis (PARAFAC) and Self-Weighted Alternating Trilinear Decomposition (SWATLD) methods were used for calibration. An appropriately designed calibration with a set of standards composed of 18 samples, coupled to the use of the second-order advantage offered by the applied chemometric techniques, allowed quantitation of sulfathiazole in spiked commercial honey samples. No previous separation or sample pretreatment steps were required. The results were compared with other calibration methods such as N-PLS and PLS-1 that produced good results on synthetic samples but not on the investigated commercial honey samples.

Ternary mixtures of fluoroquinolones, with a 7-piperazinyl substituted group have been simultaneously determined in human urine samples by application of a multivariate calibration partial least squares (PLS) model. The calibration set was designed with 15 urine samples containing different concentrations of the three fluoroquinolones and 16 blank urine samples. The concentration range for the fluoroquinolones were up to 25 ng ml −1 for norfloxacin (NOR), 80 ng ml −1 for ofloxacin (OFLO) and 300 ng ml −1 for enoxacin (ENO). The method is based on the native fluorescence emission of these compounds in sodium dodecyl sulfate (SDS) medium, at pH 4.0, when exciting at 277 nm. A selection of the emission wavelength range used for the analysis was made for each component. Intraday and interday precision values were determined. Figures of merit as selectivity, sensitivity, limit of detection (LOD) and analytical sensitivity were also calculated. Using the standard addition methodology, five urine samples from five different persons, fortified with three concentration levels of the fluoroquinolones, were analyzed. The limits of detection in urine were 10.0, 0.5 and 0.8 ng ml −1 for ENO, NOR and OFLO, respectively.

The separation and determination of five herbicides, including propanil and the phenylureas diuron, isoproturon, linuron and neburon, has been performed by an HPLC method, using photochemically-induced fluorescence detection. The non-fluorescent herbicides were transformed into fluorescent compounds by post-column photochemical reaction. A 60:40 (v/v) acetonitrile-buffer solution of potassium phosphate dibasic (pH 7, 0.01 M) was used for the chromatographic elution to separate propanil, linuron and neburon. The overlapping of isoproturon and diuron peaks, in the selected conditions, was resolved by changing the initial movil phase composition to 50:50 (v/v) methanol-buffer solution of potassium phosphate dibasic (pH 7, 0.01 M). The procedure was applied with satisfactory results to the analysis of these herbicides in Guadiana river water samples (Badajoz, Spain), allowing the detection of herbicide residues in the order of mug l(-1), by using a solid-phase extraction (SPE) pre-concentration step.

The net analyte signal (NAS)-based method HLA/GO, modification of the original hybrid linear analysis (HLA) method, has been used to determine carbendazim, fuberidazole and thiabendazole in water samples. This approach was used after a solid-phase extraction (SPE) step, using the native fluorescence emission spectra of real samples, previously standardized by piecewise direct standardization (PDS). The results obtained show that the modification of HLA performs in a similar way that partial least-squares method (PLS-1). The NAS concept was also used to calculate multivariate analytical figures of merit such as limit of detection, selectivity, sensitivity and analytical sensitivity (g 1 ). With this purpose, blanks of methanol and ternary mixtures, with the target analyte at low concentration and the other two ranging according to the calibration matrix, were used, with different results. Detection limits calculated in the last way are more realistic and show the influence of the other components in the sample. Selectivity for carbendazim is higher than the corresponding values for fuberidazole and thiabendazole, whereas sensitivity, as well as the values obtained for their detection limits, are lower for carbendazim, followed by thiabendazole and fuberidazole. Results obtained by modification of HLA vary in the same way that the ones obtained by PLS-1. # 2003 Elsevier Science B.V. All rights reserved.

Leucovorin (LV) and methotrexate (MTX) were determined in human blood serum samples by using a model based in the net analytical signal concept. The calibration method used is a variation of the original hybrid linear analysis (HLA) method, developed by Goicoechea and Olivieri (HLA/GO). The calibration set was composed by nine serum samples with different amounts of LV and MTX in the range of 0–10 gm l − 1 . The selection of the optimum wavelength range involved the calculation of the net analyte signal regression plot for each test sample, in conjunction with the calculation of the minimum error indicator. Relative errors of prediction (REP, %) of 3.0 and 5.3% were calculated for LV and MTX, respectively. Only two factors were necessary to optimize the proposed HLA/GO model. Sensitivity, selectivity, analytical sensitivity and limit of detection of the proposed procedure were calculated. Detection limits of 0.34 and 0.93 gm l − 1 for LV and MTX were determined. The proposed model was tested in the analysis of serum samples, without previous separation steps, obtaining recovery values between 96 and 99%, and between 92 and 103% for LV and MTX, respectively. © 2002 Elsevier Science B.V. All rights reserved.

The simultaneous determination of the antitubercular drugs rifampicin, pyrazinamide, isoniazid and the acetylisoniazid metabolite has been accomplished by LC, using a C-18 analytical column. The assayed drugs are usually administered together in the treatment of tuberculosis. Creatinine was also included in the chromatographic determination, in order to establish the curve of excretion of the drugs in urine. The chromatographic method uses a gradient flow in three steps, in conjunction with a programmed diode array photometric detection. In a 0.02 M potassium dihydrogen phosphate pH 7.0 buffer, a 5% (v/v) content of methanol for 1 min, a 8% (v/v) content of methanol for 3.4 min, and a 75% (v/v) content of methanol for 4 min were used. At 4.5 min, the wavelength value of detection was changed from 254 to 475 nm. Creatinine, acetylisoniazid, isoniazid and pyrazinamide were eluted in the first 4.5 min and rifampicin before 8 min. The method has been satisfactorily applied to the determination of the drugs in urine samples and in pharmaceuticals. The proposed LC method is simple, and a short time, less than 8 min is necessary for compounds elution.

Mixtures of amoxycillin and clavulanic acid were determined by using kinetic data in combination with partial least-squares multivariate calibration. The reaction of oxidation of these compounds with cerium(IV) in sulfuric acid medium has been monitored fluorimetrically. To follow the kinetics of the reaction, the stopped-flow mixing technique was used. Partial least-squares calibration of the kinetic data allowed the resolution of the analytes investigated in the concentration ranges between 0 and 4 μg ml−1. The method was applied satisfactorily to several pharmaceutical formulations, including Clavucid, Augmentine, Pangamox, Eupeclanic and Clamoxyl. The results obtained were validated by using an HPLC method. The percentages of recovery range from 91 to 105% for amoxycillin and from 78 to 117% for clavulanic acid, respectively.

The influence of different factors on the room-temperature phosphorescence (RTP) emission from the inclusion complex between α-cyclodextrin (α-CD) and 6-bromo-2-naphthol (BN) was analyzed. Although RTP signals are detected even in aerated solutions, an efficient enhancement of the phosphorescence emission (about 13 times) is obtained when the solutions of the complex are deaerated with nitrogen bubbling, while quenching is produced when sodium sulfite is used for deoxygenation. Association constants of the 1:1 and 2:1 α-CD–BN complexes have been evaluated by molecular absorption and fluorimetric methods. Exciting at 287 nm, the RTP phosphorescence emission showed two maxima located at 500 and 535 nm and a shoulder at 577 nm. The RTP emission increases with the irradiation time of the sample by the xenon lamp of the fluorimeter, until it achieves a constant value after around 10 min of irradiation. The addition of organic molecules such as alcohols and bromoalcohols as a third component of the system produces an enhancement of the RTP emission smaller than that obtained in the absence of them. The calibration graph for BN was linear for the range of concentrations between 0.4 and 2.0 μg ml −1 with a detection limit of 0.26 μg ml −1 , with relative standard deviation (RSD) ( n =6) of 4%, for 1.6 μg ml −1 . The solution was transparent, and there was no precipitation.

The spectral characteristics of the inclusion of 1-naphthalenacetamide in beta-cyclodextrin have been studied by luminescence methods. The addition of bromoalcohols or bromoalkanes produces a decrease of the fluorescence of the inclusion complex, giving rise to a new band corresponding to phosphorescence emission. The emission intensity is much higher in the presence of bromoalkanes than in the presence of bromoalcohols. The deoxygenation of the solutions is achieved by flowing nitrogen, by the addition of sodium sulphite and by a combination of both methods, which results in the most effective approach. The phosphorescence emission is produced in the turbid suspension formed in the presence of the bromoalkane. The formation of microcrystals seems necessary to obtain phosphorescence emission from the ternary system.

Multi-wavelength detectors offer improved detection capabilities for liquid chromatographic methods, but require multivariate approaches to utilise all the available information. The photodiode-array detector in high-performance liquid chromatography (HPLC) generates a three-dimensional data matrix, which is conventionally presented as an isometric projection or a contour plot. In this work, a new graphical technique is described for improving the quantitative results obtained from HPLC, using the available spectrochromatographic information in both the time and wavelength domains. The technique consists of performing cross-sections through the data matrix to obtain the maximum analytical information for each of the analytes. Hence, the resolution of overlapping peaks and the sensitivity in the determination are optimised. In order to demonstrate the validity and simplicity of the approach, the method has been applied to the resolution of synthetic mixtures of iprodione, procymidone and chlorothalonil. Also, the method has been satisfactorily applied to the simultaneous determination of the pesticides in environmental groundwater samples.

The resolution of binary mixtures of nalidixic acid (NA) and 7-hydroxymethylnalidixic acid (OH–NA) has been accomplished by partial least squares (PLS) and principal component regression (PCR) multivariate calibration. The method of determination is based on the fluorescence emission of these compounds in the presence of γ-cyclodextrin (γ-CD). The formation of the inclusion compounds gives rise to an increase of the fluorescence emission compared to aqueous solution. The total luminescence information of the compounds has been used to optimize the spectral data set to perform the calibration. A comparison between the predictive ability of three multivariate calibration methods, PLS-1, PLS-2 and PCR, on three spectral data sets, excitation, emission and synchronous spectra has been performed. The PLS-1 method, applied to the emission spectra, has been selected as optimum. The proposed method has been applied to the simultaneous determination of NA and OH–NA in urine. Recovery values from urine samples containing (NA) and (OH–NA) range from 91 to 103% (mean 97%), and from 92 to 105% (mean 99%), respectively.

The simultaneous determination of salicylic acid and diflunisal in human serum has been accomplished by synchronous fluorimetry, in combination with partial least-squares multivariate calibration. The total luminescence information of the analytes has been used to optimize the spectral data set for the calibration, by analysis of the three-dimensional excitation-emission matrices. The synchronous spectrum, maintaining a constant difference of Deltalambda = 128 nm between the emission and excitation wavelengths, has been selected as optimum to perform the determination. The method is based on the fluorescence of these compounds in chloroform containing 1% (v/v) acetic acid. Serum samples are treated with trichloroacetic acid to remove the proteins, and both analytes are extracted into chloroform-1% (v/v) acetic acid prior to the determination. For concentrations ranging from 60-240 mug ml(-1) of each drug, analytical recoveries range from 96% to 103% for salicylic acid and from 97% to 105% for diflunisal.

The oxidation reactions of methotrimeprazine and thioridazine by iron(III) were used to develop kinetic methods based on measurements of the formation rate of the corresponding coloured phenothiazinyl free radicals. The calibration graphs were linear over the range 0.8-60 microg/ml for methotrimeprazine and 1.1-60 microg/ml for thioridazine. The relative standard deviation was 1.1-3.4%. The method was used to determine the analytes in commercially available pharmaceutical preparations. The different absorption maxima of the oxidation products formed and also the different, high initial rates of the reactions for both analytes allow their simultaneous determination by using a stopped-flow module coupled to a diode-array spectrophotometric detector. Reaction rate and absorbance increment measurements of the kinetic curves obtained were used to resolve mixtures of both phenothiazines by using the proportional-equation method and establishing three different equation systems that allowed the results obtained in each case to be compared. Mixtures of methotrimeprazine and thioridazine in ratios between 8:1 and 1:8 were satisfactorily resolved.

Room temperature phosphorescence (RTP) from 2-naphthyl-oxy-acetic acid (NOA) and 1-naphthyl-acetic acid (NAA), with stabilization by use of beta-cyclodextrin (beta-CD) as a host system, has been examined. 2-Bromoethanol and 2,3-dibromopropanol have been evaluated as external heavy atom perturbers to enhance the rate of intersystem crossing and, consequently, populating the triplet state for phosphorescence emission. The deoxygenation of the solutions was achieved chemically by use of sodium sulphite. The spectral characteristics of the phosphorescence emission from these relatively polar compounds and the optimization of the chemical variables involved are reported. The role of the bulkiness of the bromoalcohol employed, in comparison with the unoccupied space of the interior of the cyclodextrin cavity by the guest, is an important factor in the attainment of an effective RTP emission, and should be taken into account in the selection of the appropriate external heavy atom for the observation of RTP from other organic molecules of interest by this approach. 2,3-Dibromopropanol seems a more adequate bromoalcohol than 2-bromoethanol for the observation of RTP emission in the systems investigated.

The inclusion complex of sulphaguanidine (SGN) in β-cyclodextrin has been investigated. To avoid the problem of the low solubility of β-cyclodextrin in water, solutions of β-cyclodextrin in urea have been used. A 1:1 stoichiometry and an association constant of 450 M−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 0.20 μg mL−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.

The combination of unfolded-partial least-squares (U-PLS) with a recently proposed separate procedure, known as residual trilinearization (RTL), has been successfully employed for four-way data calibration. The chemometric method employs the evolution of excitation–emission matrices (EEMs) with time, for the resolution of folic acid–methotrexate mixtures, in human serum samples. The fluorogenic products monitored correspond to the oxidation of the studied analytes with potassium permanganate, in slightly acidic medium. The reaction is developed in 7 min and followed using a fast-scanning spectrofluorimeter, capable of recording each complete EEM in 12 s. This allows the acquisition of 10 successive EEMs, at different reaction times, during the development of the oxidation reaction, given rise to the four-way data set employed. The procedure, which had been previously reported for urine determination, is extended to serum analysis in this work. The combination of U-PLS/RTL is providing enhanced predictive results in comparison with standard methods as PARAFAC and N-PLS, in the presence of human serum, where significant unexpected components and or inner filter effects may occur.

The hydrolysis of the pesticide quinalphos in basic medium was kinetically followed and the measurement of the reaction rates allowed us to develop two kinetic-fluorimetric methods. In one of them the mixing of the reagents was directly performed in the measurement cell and, in the another one, the stopped-flow mixing technique was used. The reaction was completed in 100 s after the reactants were mixed and it allowed the simple application of the proposed methods to routine analyses of the pesticide. The sensitivity of the methods was very high, being the detection limits 50 and 140 ng mL −1 for the manual procedure and the stopped-flow mixing technique, respectively. Both methods were compared using regression with uncertainties in both axes. The effect of the presence of several pesticides in the determination was tested. A solid-phase extraction process was also developed for the application of the methods to diverse waters samples. The proposed kinetic-fluorimetric methods were applied to the determination of quinalphos in drinking water, well water and river water, with very satisfactory results.

The stopped flow mixing technique has been used to study the kinetic determination of dipyridamole by means of micellar-stabilized room temperature phosphorescence (RTP). This mixing system diminishes the time required for the deoxygenation of the micellar medium by sodium sulfite. The phosphorescence enhancers thallium (I) nitrate, sodium dodecyl sulfate (SDS), and sodium sulfite were optimized to obtain maximum sensitivity and selectivity. A pH value of 10.6 was selected as adequate for phosphorescence development. The kinetic curve of dipyridamole phosphorescence was scanned at λ ex =303 nm and λ em =616 nm. Then, the intensity at 10 s, and the maximum slope of phosphorescence development, for an interval time of 1 s, were measured. Two determination approaches: intensity and rate methods, were proposed. The calibration graphs were linear for the concentration range from 50 to 400 ng ml −1 . The detection limits, according to Clayton et al., Anal. Chem. 59 (1987) 2506, were 21.5 and 37.5 ng ml −1 , for intensity and initial rate measurements, respectively. By applying the error propagation theory, the detection limits were 19.0 and 33.0 ng ml −1 , for intensity and initial rate measurements, respectively. Two commercial formulations (persantin and asasantin) were analyzed by both proposed methodologies. Adequate recovery values were obtained in both cases.

The dependence of the absorption and fluorescence spectra of 1-hydroxy-2-carboxy-anthraquinone on pH and Hammett acidity have been studied. This compound exhibits phototautomerism in its uncharged and its singly-charged anionic species in aqueous media. Its ground state (pK(a)) and lowest excited singlet-state (pK(a)( *)) dissociation constants have been determined by absorptiometric and fluorimetric titrations and the assignment of the pK(a) and pK( *)(a) values to the equilibria concerned has been carefully considered.