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The latest advances in both theory and experimental procedures on third-order/four-way and fourth-order/five-way calibration methods are discussed. This report is focused on excitation-emission (fluorescence and phosphorescence) matrices generation, employing different variables as the third data mode (time retention in chromatography, pH gradient, fluorescence/phosphorescence lifetime, kinetics, or other chemical treatments). Fully capitalizing on the second-order advantage, it has been possible to develop appealing analytical applications in spite of the complexity of the data. Extraction of the significant chemical information about the system under study as well as the individual abundance of the contributing constituents after proper higher-order data decomposition has allowed to analytical researchers performing quantitative analysis of complex samples. The experimental works reported up to the present are introduced and discussed in order to illustrate concepts. Throughout this work, the analytical benefits achieved by modeling third- and fourth-order data are exposed, attempting to contribute to the ongoing debate in the chemometric community regarding the existence and the true nature of the third-order advantage.

The present tutorial aims to review the most frequently reported criteria for the calculation of the limits of detection (LOD) and quantification (LOQ) in univariate calibration, summarizing their fundamentals, advantages, and limitations. The current criteria for estimating LOD and LOQ are based on diverse theoretical and/or empirical assumptions and require different amounts of experimental data, making the calculation rather complex in some cases. Moreover, alternative forms for calculating LOD/LOQ frequently lead to dissimilar results. This scenario might worsen in the case of complex analytical systems. Throughout this tutorial, different forms of calculating LOD/LOQ are illustrated using previously reported experimental datasets in the environmental chemistry field as examples. The influence of the sample matrix during the estimation of LOD/LOQ parameters is investigated through one calibration approache. The discrepancies in the obtained results with different criteria for the calculation of LOD/LOQ are highlighted. Finally, general guidelines and recommendations regarding experimental and data processing issues are proposed, aiming to promote fair criteria for the comparison of different analytical methodologies in terms of prediction ability and detection capability.

An introduction to multi-way calibration based on second- and higher-order data generation and processing is provided, with emphasis on practical experimental aspects. After a discussion concerning a proper nomenclature scheme, a suitable classification of the obtainable data, and the general features of the available algorithms and their underlying models, a series of examples is discussed in detail, with the purpose of illustrating the great potentiality of the field for the analytical community. Emphasis is directed toward the most popular multi-way data, i.e., second-order or matrix data, which can be conveniently measured in a variety of instruments. Third-order data are being increasingly studied and are also discussed, along with the less explored field of fourth-order data. The estimation of figures of merit, which analysts need to report during method development, is now sufficiently mature to be provided for the general audience. Fil: Escandar, Graciela Monica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Química Rosario; Argentina Fil: Goicoechea, Hector Casimiro. Universidad Nacional del Litoral; Argentina Fil: Muñoz de la Peña, A.. Universidad de Extremadura; España Fil: Olivieri, Alejandro Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Química Rosario; Argentina

This paper presents a study regarding the acquisition and analytical utilization of four and three-way data, acquired by following the excitation?emission fluorescence matrices at different elution times, in a fast liquid chromatographic HPLC procedure. This kind of data were implemented for first time for quantitative purposes, and applied to the determination of two fluoroquinolones in tap water samples, as a model to show the potentiality of the proposed strategy of four-way data generation. The data were modeled with three well-known algorithms: PARAFAC, U-PLS/RTL and MCR-ALS, the latter conveniently adapted to model third-order data. The second-order advantage was exploited when analyzing samples containing uncalibrated interferences. PARAFAC and MCR-ALS were the algorithms that better exploited the second-order advantage when no peak time shifts occurred among samples. On the other hand, when the quadrilinearity was lost due to the occurrence of temporal shifts, MCR-ALS furnished the better results. Relative error of prediction (REP%) obtained were 9.9% for ofloxacin and 14.0% for ciprofloxacin. In addition, a significant enhancement in the analytical figures of merit was observed when going from second- to third-order data (reduction of ca. 70% in LODs). Fil: Alcaraz, Mirta Raquel. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina Fil: Siano, Gabriel German. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina Fil: Culzoni, Maria Julia. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina Fil: Muñoz de la Peña, Arsenio. Department Of Analytical Chemistry, Faculty Of Sciences; España Fil: Goicoechea, Hector Casimiro. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina

A new spectrofluorimetric method has been developed for the quantitative determination of the anti-inflammatory flufenamic and meclofenamic acids in urine samples. The method is based on second-order multivariate calibration applying parallel factor analysis (PARAFAC). The fluorescence excitation–emission matrices (EEMs) of these compounds are highly overlapped, precluding analyte direct determination without performing a previous separation procedure. The use of a micellar medium allowed for both fluorescence enhancement and analyte discrimination from the biological background. The calibration solutions were prepared in water, with concentrations in the ranges from 0.15 to 4.00 μg ml−1 for flufenamic acid and from 0.25 to 6.00 μg ml−1 for meclofenamic acid. The use of the second-order calibration method, in the standard addition mode, was required to avoid internal filter interference effects from the urine background. The measured EEMs of the two analytes as analytical signals allowed their simultaneous determination in human urine samples, in the therapeutic ranges from ca. 40 to 150 mg l−1. The proposed procedure was validated by comparing the obtained results with a capillary electrophoresis method, with satisfactory results for the assayed method.

Solid-phase extraction (SPE) (reversed-phase and mixed-mode) and nylon membranes were investigated as solid matrices for obtaining fluorescence signals from adsorbed triamterene (TA). Although a significant emission was found in all investigated surfaces, solid-phase extraction systems yielded the best results. The analytical figures of merit obtained under the best experimental conditions, using nonpolar/strong cation mixed phase MP1 discs and 5 ml of extracting sample, were: linear calibration range from 1.7 to 21 ng ml−1 (the lowest value corresponds to the quantitation limit), relative standard deviation, 1.3% (n = 5) at a level of 12.7 ng ml−1, and limit of detection, 0.6 ng ml−1 (calculated according to Clayton's definition, taking into account false positive and false negative errors). An approach for the spectrofluorimetric determination of triamterene in pharmaceutical formulations was successfully applied. In addition, a simple and rapid method for the separation and fluorimetric determination of triamterene and its main metabolite (hydroxytriamterene sulfate) in urine was developed and rendered very satisfactory results, requiring in this case the use of reversed-phase C18 solid-phase extraction.

Multivariate methods comprise of a group of chemometric tools allowing the analysis of different analytical data, i.e., spectroscopic, chromatographic obtained from multichannel detector systems. Second-way data are widely used in analytical applications in combination with multivariate calibration methods, but three- and higher-way data are yet not as widely applied. In complex biological samples, the employment of the three-way data is of special interest, as they may be combined with methods that exploit the second-order advantage allowing calculating individual concentrations of the analytes of interest in the presence of unknown interferences in untreated samples. A very sensitive and selective method is proposed, by coupling photoinduced fluorescence and multivariate analysis of the three-way data excitation-emission fluorescence matrices (EEMs), of the photoproducts obtained from UV irradiation of three fluoroquinolones: enoxacin (ENO), norfloxacin (NOR) and ofloxacin (OFLO). The application of a previous photoirrradiation process allows the determination of mixtures of ENO, NOR and OFLO, in urine samples at biological levels without sample pretreatments. The resolution ability of N-way partial least squares (N-PLS), parallel factor analysis (PARAFAC) and self weighted alternating trilinear decomposition (SWATLD), is compared with partial least squares (PLS) and unfolded-PLS (U-PLS), in the analysis of ENO, NOR and OFLO in human urine samples.

A novel approach is presented for the spectrofluorimetric determination of the powerful anticonvulsant carbamazepine and its main metabolite in human serum. The strategy consists in the support of both compounds on a nylon membrane, and their subsequent determination through a solid-surface fluorescence methodology combined with a suitable chemometric analysis. The novelty of the present method lies in the fact that while carbamazepine does not fluoresce neither in solution nor supported on a variety of surfaces, significant emission signals are observed when it is supported on the nylon matrix, a property which has not been previously exploited by analysts. Multivariate calibration analysis was performed on three-way excitation-emission matrix data. The algorithms applied were: parallel factor analysis (PARAFAC), self-weighted alternating trilinear decomposition (SWATLD) and N-way partial least-squares regression (N-PLS). The results were compared with two-way calibration data analysed with partial least-squares regression (PLS-1). The methodology is highly specific, and it appears to be suitable for the routine monitoring of serum concentrations in patients receiving chronic therapy. In addition, the technique was satisfactorily applied to the determination of carbamazepine in pharmaceutical formulations.

A comparative study about advantages and limitations of net analyte signal (NAS)-based methods (NBMs) and partial least squares (PLS) calibration in kinetic analysis has been performed. The different multivariate calibration methods were applied to the determination of binary mixtures of amoxycillin and clavulanic acid, by stopped-flow kinetic analysis. The reactions of oxidation of these compounds with cerium(IV), in sulphuric acid medium, were monitored by following the changes on the fluorescence of the oxidation products, in stopped-flow mode. The differences on the kinetic profiles obtained at λ ex =256 nm and λ em =351 nm, were used to determine mixtures of both compounds by multivariate calibration of the kinetic data, using PLS-1, a modification of hybrid linear analysis (HLA) and net analyte pre-processing combined with classical least squares (NAP/CLS) methods. The NBMs allowed the selection of optimal time data regions by calculating the minimum error indicator function (EIF), improving the results and making NBMs very convenient for the analysis. In addition, the use of the net analyte signal concept allows the calculation of the analytical figures of merit, limit of detection (LOD), sensitivity and selectivity, for each component.

Two multivariate methods, the classical partial least squares (PLS-1) and a modification of the novel hybrid linear analysis (HLA/XS method), are described for the simultaneous determination of rifampicin, pyrazinamide and isoniazid. A calibration set is constructed, using a combined central composite and factorial design (15 objects), in the range of concentrations comprised between 0 and 15 μg/ml for rifampicin and pyrazinamide, and 0–10 μg/ml for isoniazid. The proposed methods are satisfactorily applied to the determination of mixtures of these drugs. The precision expressed as R.S.D. (%) is calculated for both models. HLA/XS and PLS optimized calibrations are tested and compared in the analysis of several pharmaceutical formulations. The calculation of the net analytical signal using a modified HLA/XS method, allows us the determination of several figures of merit, as selectivity, sensitivity, analytical sensitivity and limit of detection of the proposed multivariate calibration. PLS-1 and HLA/XS calibration show similar ability to determine rifampicin and pyrazinamide but slightly worse results for isoniazid are obtained when using HLA/XS.

Partial least-squares (PLS-1) multivariate calibration of spectrophotometric data have been applied to the determination of theophylline in blood serum. After sample deproteinization and alkalinization, the electronic absorption spectrum (in the 265–302 nm region) is processed using a calibration design which includes a series of serum pools spiked with theophylline. The concentrations lay within the therapeutic range (0.0–35.0 μg ml −1 ). All statistical indicators (prediction in validation sets, precision and accuracy) are reasonably good. A comparison of this method with an immunofluorescent polarization technique revealed no significant differences in their prediction abilities. Studies in the presence of triglycerides, bilirubin, hemoglobin and caffeine showed that only the latter was able to interfere, as with other theophylline monitoring techniques.

The inclusional complexation between γ-cyclodextrin and acenaphthene in aqueous solution was investigated by spectroscopic techniques. A 1:1 complex was detected and its formation constant was determined. After adding bromoalcohols such as 2,3-dibromopropane-1-ol or 2-bromoethanol both a decrease of the fluorescence and an enhancement of the room-temperature phosphorescence (RTP) of acenaphthene were observed. The RTP signals from acenaphthene included in γ-cyclodextrin were optimized. The apparent formation constant of the ternary γ-cyclodextrin-acenaphthene-2,3-dibromopropane-1-ol complex was determined from RTP data. On the other hand, the inclusion complex formed between γ-cyclodextrin, acenaphthene and 2-bromoethanol was found to be very labile and its equilibrium constant could not be determined with accuracy. Large RTP signals were obtained in both ternary systems, even in the absence of sodium sulfite as deoxygenant agent. This fact would indicate that in these complexes the acenaphthene is protected from the action of quenchers such oxygen.

Because of the capacity of the diode array detector (DAD) in high performance liquid chromatography (HPLC), three-dimensional data can be easily generated in analytical laboratories. This allows new possibilities for the identification and quantification of solutes eluting from a chromatographic column. This paper deals with the application of a computer aided technique in the resolution of overlapping peaks by Partial Least Squares (PLS) calibration. This consists in performing cross-sections through the three-dimensional (A, λ, t) data matrix to increase the analytical information for each analyte. In this way, the sensitivity in the determination is maximized. The application of this technique in combination with the PLS-1 method offers a greater ability to resolve overlapped chromatographic peaks, and greater accuracy in quantification than the PLS-1 method performed by using chromatograms obtained at one selected wavelength. In order to demonstrate the validity and applicability of the method, it has been applied to the resolution of iprodione, procymidone, chlorothalonil, folpet and triazophos pesticide mixtures in ground water and soil.

A kinetic-photometric method for the individual determination of imipramine and desipramine and a differential kinetic method for the resolution of mixtures of these tricyclic antidepresant based on the stopped-flow mixing technique were developed for the first time and applied to the determination of these species in serum. Both methods rely on the oxidative coupling of these compounds with 3-methylbenzothiazolin-2-one hydrazone in the presence of ammonium iron(III) sulphate in hydrochloric acid. On the basis of the initial rate and absorbance amplitude of the kinetic curves obtained for each analyte, the resolution of the mixture was accomplished by using the proportional equation method. The linear range of the calibration graphs was 0.3-7.0 μ ml−1 for impramine and 0.3-5.0 μg ml−1 for desipramine. Mixtures of imipramine and desipramine in ratios between 2:1 and 1:8 were satisfactory resolved. The determination of these analytes in serum called for a prior separation step for matrix removal.

2-Furfuraldehyde, 5-hydroxymethylfurfuraldehyde and malonaldehyde are known to react with 2-thiobarbituric acid when heated in an acidic medium. A study of the influence of several chemical and physico-chemical variables on the reaction was undertaken. The products of reaction show high absorption in the visible spectral region with several degrees of overlapping. The resolution of ternary mixtures of these compounds was accomplished by several chemometric approaches. A comparative study of the results obtained for simultaneous determinations in mixtures by using derivative spectrophotometry and partial least-squares analysis is presented. The multi-wavelength factor analysis-based method is demonstrated to be the method of choice for the multi-component analysis of ternary mixtures of these compounds.

A very simple approach for the determination of fluorescent and absorbing species based on stopped-flow mixing methodology is reported. By using a special procedure to introduce samples and their solvent into the observation cell of a spectrofluorimeter or spectrophotometer with the aid of a stopped-flow module, one can rapidly obtain a kinetic curve, the slope and amplitude of which are proportional to the concentration of the fluorescent or absorbing solute concerned. Various chemical systems, including quinine, imipramine, potassium permanganate and Methylene Blue were examined in order to find a plausible explanation for the kinetic effect observed and whether it applied to any fluorescent or absorbing species. The proposed approach was satisfactorily applied to the determination of imipramine in serum.

The quantitative predictive abilities of derivative spectral analsis by using the zero-crossing point method are compared with the results obtained by the use of a multivariate calibration method. Mixtures of two pesticides (carbaryl and chlorpyrifos) were resolved of derivative spectrophotometry. Partial least squares (PLS), a full spectrum calibration method, was also applied with previous optimization of the calibration matrix by the PLS-2 method. Both approaches were satisfactorily applied to the simultaneous determination of carbaryl and chlorpyrifos in commercial formulations. The simplicity of the derivative method makes it the method of choice for simple binary mixtures.

A liquid chromatographic method for the simultaneous analysis of marker pteridins and biopterin reduced forms, in urine samples is proposed. A Zorbax Eclipse XDB-C18 column was used for the chromatographic separation, using a 98/2 (v/v), citrate buffer (pH 5.5)-acetonitrile mobile phase, in isocratic mode. A post-column photoderivatization was carried out with an on-line photoreactor, located between a diode array detector (DAD) and a fast scanning fluorescence detector (FSFD). Neopterin (NEO), biopterin (BIO), pterin (PT) and dihydrobiopterin (BH2) were determined by measuring native fluorescence, using the photoreactor in OFF-mode, and tetrahydrobiopterin (BH4) was determined by measuring of the induced fluorescence of the generated photoproducts, using the photoreactor in ON-mode. In addition, Creatinine (CREA), as a reference of metabolites excrection in urine, was simultaneously determined using the DAD detector. Detection limits were 0.2, 13.0, 0.3, 0.3 and 3.5 ng mL(-1), for NEO, BH2, BIO, PT and BH4, respectively, and 0.4 microg mL(-1) for CREA. Ratio values for NEO/CREA, PT/CREA, BH4/CREA, BH2/CREA, NEO/BIO and BIO(total)/CREA, in urine samples, of healthy children and adults, phenylketonuric children and infected mononucleosis children, are reported. A comparative study, about the mean values obtained for each of the compounds, by the present procedure and by the classical iodine oxidation method (Fukushimas method), has been performed, in urine samples belonging to healthy volunteers. The values obtained were BH4/CREA: 0.41, BH2/CREA: 0.31 and BIO(total)/CREA: 0.73, by the proposed method, and BH4/CREA: 0.35, BH2/CREA: 0.20 and BIO(total)/CREA: 0.48, by iodine oxidation method.

A 1:1 stoichiometric ratio and a formation constant of 130 1 mol −1 were obtained for the binary inclusion complex between acenaphthene and β-cyclodextrin (CD). On addition of 2-bromoethanol, effective quenching of the fluorescence emission and enhancement of the room-temperature phosphorescence (RTP) of acenaphthene is observed. The use of 2-bromoethanol as an external “heavy atom” to obtain room-temperature phosphorescence in fluid solutions is discussed. The RTP emission from acenaphthene included in β-CD was optimized and characterized. The apparent formation constant of the ternary associate was determined to be 880 1 mol −1 by the use of RTP enhancement in the presence of 1% 2-bromoethanol. The data indicate a ternary association between acenaphthene, β-CD and the bromo alcohol. The use of sodium sulfite, for chemical deoxygenation, to obtain RTP emission from a non-heavy atom-containing luminophor is shown to be feasible. The kinetics of the deoxygenation reaction were established and a photochemical catalytic effect on the reaction was demonstrated.

First-derivative synchronous spectrofluorimetry was applied to the simultaneous resolution of propranolol and hydralazine in pharmaceutical preparations. Chemical and instrumental parameters were optimized for the determination of mixtures of these drugs. The analysis is performed by measuring the first-derivative synchronous spectra at certain selected pairs of excitation and emission wavelengths where the responses exhibit no contribution to each other. The optimization of the synchronous scanning path was accomplished by analysis of the three-dimensional excitation-emission matrices.

The determination of folic acid and its two main serum metabolites, 5-methyltetrahydrofolic acid and tetrahydrofolic acid, has been accomplished using four-way data modelled by the third-order multivariate calibration methods unfolded and N-dimensional partial least-squares (U-PLS and N-PLS), in combination with the separate procedure known as residual trilinearization (RTL). The four-way data were acquired by following the photochemical reaction of these compounds by on line irradiation with a UV lamp. The excitation-emission matrices (EEMs) were recorded as a function of the irradiation time, using a fast scanning spectrofluorimeter. The method achieves selectivity from the different rates at which the corresponding photoproducts of the folic acid derivatives are formed and degraded. Several N-dimensional chemometric algorithms were used and the method was applied to the determination of these compounds in serum samples. The best algorithms to perform the multivariate calibration were U-PLS and N-PLS in combination with the separate residual trilinearization procedure, achieving the second-order advantage. The approach allows minimizing or eliminating traditionally time-consuming sample pre-treatments and can facilitate quantifying an analyte in its native environment.

The differentiation of chromatographic bands with respect to time is useful for improving the resolution of overlapping peaks owing to the reduced band widths of derivatives in comparison with the original bands. An IBM XT 286 microcomputer was interfaced to a Perkin-Elmer liquid chromatographic system and software was developped to allow the acquisition and digitization of chromatographic data for subsequent manipulation, storage and graphical representation. Smoothing and differentiation of chromatographic bands with respect to time were done by the Savitzky-Golay method. The method was applied to the determination of a mixture of antioxidants used as additives in nutrition. The overlapping peaks corresponding to propyl gallate and tert-butylhydroquinone were resolved by time-domain differentiation.