Four-way calibration applied to the simultaneous determination of folic acid and methotrexate in urine samples

First-, second- and third-order calibration methods were investigated for the simultaneous determination of folic acid and methotrexate. The interest in the determination of these compounds is related to the fact that methotrexate inhibits the body’s absorption of folic acid and prolonged treatment with methotrexate may lead to folic acid deficiency, and to the use of folic acid to cope with toxic side effects of methotrexate. Both analytes were converted into highly fluorescent compounds by oxidation with potassium permanganate, and the kinetics of the reaction was continuously monitored by recording the kinetics curves of fluorescence emission, the evolution with time of the emission spectra and the excitation–emission matrices (EEMs) of the samples at different reaction times. Direct determination of mixtures of both drugs in urine was accomplished on the basis of the evolution of the kinetics of EEMs by fluorescence measurements and four-way parallel-factor analysis (PARAFAC) or multiway partial least squares (N-PLS) chemometric calibration. The core consistency diagnostic (CORCONDIA) was employed to determine the correct number of factors in PARAFAC and the procedure converged to a choice of three factors, attributed to folic acid, methotrexate and to the sum of fluorescent species present in the urine.      

Simultaneous determination of tyrosine and dopamine in urine samples using excitation-emission matrix fluorescence coupled with second-order calibration

A "green" and quick analytical method for complex compounds was developed for simultaneous determination of tyrosine (Tyr) and dopamine (DA) in urine samples in this paper. The three-way responsive data recorded by excitation-emission matrix fluorescence (EEM) spectrometer was analyzed using second-order calibration methods based on both parallel factor analysis (PARAFAC) and selfweighted alternating trilinear decomposition (SWATLD) algorithms. The EEM spectra of the analytes were overlapped with the background in urine samples. However the second-order advantage of both PARAFAC and SWATLD methods was exploited, even in the presence of unknown interferences and the satisfactory results can be obtained. Furthermore, the linear ranges of Tyr and DA were determined to be 0.042-6.42 μg/mL and 0.18-4.43 μmg/mL, respectively, and the accuracies of both methods were validated by the analytical figures of merit (FOM).     

Standardization of a multivariate calibration model applied to the determination of chromium in tanning sewage

A multivariate standardization procedure was used to extend the lifetime of a multivariate partial least squares (PLS) calibration model for determining chromium in tanning sewage. The Kennard/Stone algorithm was used to select the transfer samples and the F-test was used to decide whether slope/bias correction (SBC) or piecewise direct standardization (PDS) had to be applied. Special attention was paid to the transfer samples since the process can be invalidated if samples are selected which behave anomalously. The results of the F-test were extremely sensitive to heterogeneity in the transfer set. In these cases, it should be taken as an interpretation tool.     

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.     

Adsorptive stripping square wave voltammetry (Ad-SSWV) accomplished with second-order multivariate calibration determination of fenitrothion and its metabolites in river water samples

A method, using stripping square wave voltammetry (Ad-SSWV), for the simultaneous determination of fenitrothion (FEN) and its metabolites: fenitrooxon (OXON) and 3-methyl-4-nitrophenol (3-MET) in environmental samples is reported. All three compounds produce, at mercury electrode (HMDE), an electrochemical signal due to an adsorptive-reductive process. The electrochemical approach shows a very high overlap degree for FEN and OXON voltammograms, however the adsorption kinetic profile could be used as an additional differential variable between both analytes. Second-order multivariate calibration has been tested to solve the mixture of the three compounds. The second-order assayed methods were parallel factor analysis (PARAFAC), unfolded partial least squares (U-PLS), multidimensional partial least squares (N-PLS) and the latest ones were used in combination with the residual bilinearization procedure RBL. U-PLS/RBL model was stated as the best second-order algorithm for the simultaneous determination of these three compounds up to 50 ng mL⁻¹ for each analyte. The detection limits and recovery values were 1.6 ng mL⁻¹ and 92 ± 7% for FEN; 3.7 ng mL⁻¹ and 101 ± 9% for OXON and 0.6 ng mL⁻¹ and 97 ± 8% for 3-MET.     

Determination of anticarcinogenic and rescue therapy drugs in urine by photoinduced spectrofluorimetry using multivariate calibration: comparison of several second-order methods

This paper shows the potential of excitation–emission fluorescence spectroscopy and several second-order methods, such as parallel factor analysis (PARAFAC), multiway partial least-squares (N-PLS) or bilinear least-squares (BLLS), as a multicalibration technique for the analysis of leucovorin (LV) and irinotecan (CPT-11). Although CPT-11 presents native fluorescence, leucovorin has little native fluorescence; however, under irradiation with short-wavelength UV light in the presence of traces of hydrogen peroxide, leucovorin was converted into a highly fluorescent compound. This reaction has been used for the sensitive and selective determination of both compounds. The convenience of analysing the total luminescence spectrum information when using multivariate calibration methods on fluorescence data is demonstrated. Direct determination of mixtures of both drugs in urine was accomplished on the basis of excitation–emission matrices (EEMs) and the three-way multivariate methods.     

A review of multivariate calibration methods applied to biomedical analysis

The determination of the contents of therapeutic drugs, metabolites and other important biomedical analytes in biological samples is usually performed by using high-performance liquid chromatography (HPLC). Modern multivariate calibration methods constitute an attractive alternative, even when they are applied to intrinsically unselective spectroscopic or electrochemical signals. First-order (i.e., vectorized) data are conveniently analyzed with classical chemometric tools such as partial least-squares (PLS). Certain analytical problems require more sophisticated models, such as artificial neural networks (ANNs), which are especially able to cope with non-linearities in the data structure. Finally, models based on the acquisition and processing of second- or higher-order data (i.e., matrices or higher dimensional data arrays) present the phenomenon known as “second-order advantage”, which permits quantitation of calibrated analytes in the presence of interferents. The latter models show immense potentialities in the field of biomedical analysis. Pertinent literature examples are reviewed.     

HPLC-DAD data coupled with second-order calibration method applied to food analysis: Simultaneous determination of six benzoylurea insecticides in various fruit samples by selecting time region of chromatogram

This work presents a novel application of second-order calibration based on self-weighted alternating trilinear decomposition(SWATLD)algorithm for analyzing the HPLC-DAD data.The proposed method makes it possible to simultaneously determine teflubenzuron,hexaflumuron,flufenoxuron,chlorfluazuron,diflubenzuron and benzoylurea in different fruit samples,i.e.pear,apple and banana,in the selected time region of chromatogram.The concentration,elution time and spectral information of these benzoylurea insecticides are selectively extracted from complex matrices even in the presence of unknown interferences.The root-mean-square error of prediction(RMSEP)and figures of merit,including sensitivity(SEN),selectivity(SEL)and limit of detection(LOD)are employed to access the performance of the method.The LODs obtained for these insecticides are within the range 0.017–0.26 ppm in pears,0.039–0.33 ppm in apples,0.041–0.44 ppm in bananas,respectively.Such a chemometrics-based protocol holds great potential to be extended as a promising alternative for more practical applications in food safety and quality monitoring.     

Spectrofluorimetry in organized media coupled to second-order multivariate calibration for the determination of galantamine in the presence of uncalibrated interferences

The present article describes the spectrofluorimetric determination of galantamine, a widely used acetylcholinesterase inhibitor, through excitation-emission fluorescence matrices and second-order calibration. With the purpose of enhancing the fluorescence intensity of this substance, the effect of different organized assemblies was evaluated. Although the interaction of galantamine with different cyclodextrins is weak, it was corroborated that the fluorescence intensity of this pharmaceutical in the presence of α-cyclodextrin is increased by a twofold factor. Among the studied micellar media, the anionic surfactant sodium dodecyl sulfate produced the largest signals for the compound of interest (sixfold enhancement), and was selected as auxiliary reagent for the subsequent determinations. The developed approach enabled the determination of galantamine at the ng mL⁻¹ level without the necessity of applying separation steps, and in the presence of uncalibrated interferences. The applied second-order chemometric tools were parallel factor analysis (PARAFAC), unfolded partial least-squares coupled to residual bilinearization (U-PLS/RBL), and multidimensional partial least-squares coupled to residual bilinearization (N-PLS/RBL). The ability of U-PLS/RBL to successfully overcome spectral interference problems is demonstrated. The quality of the proposed method was established with the determination of galantamine in both artificial and natural water samples.     

Simultaneous fluorimetric determination of acetylsalicylic acid metabolites in urine by partial least squares multivariate calibration

A method is described for the simultaneous determination of the main urinary acetylsalicylic acid (aspirin) metabolites, salicyclic, salicyluric and gentisic acids, based on their native fluorescence. The urine was extracted into diethyl ether in acid medium, and back-extracted with glycine/sodium hydroxide buffer solution at pH 9.4. A comparative study of the results found using the excitation, the emission and the combination of the excitation plus the emission spectral data, as analytical signals, was performed. The data set, composed of the excitation plus the emission spectra, was selected as the analytical signal. The optimum wavelengths to record the excitation (_em=444 nm) and the emission spectra (_ex=323 nm) were selected to maximize the contribution from gentisic acid, which is the minor urinary metabolite. Partial least squares (PLS-1) multivariate calibration was then applied for the determination. Recovery values from urine samples spiked with salicyclic, salicyluric and gentisic acids varied from 90.1 to 97.6% (mean 93.6%), from 90.0 to 110% (mean 97.9%) and from 89.9 to 104.7% (mean 98.5%), respectively.     

Simultaneous fluorimetric determination of acetylsalicylic acid metabolites in urine by partial least squares multivariate calibration

A method is described for the simultaneous determination of the main urinary acetylsalicylic acid (aspirin) metabolites, salicyclic, salicyluric and gentisic acids, based on their native fluorescence. The urine was extracted into diethyl ether in acid medium, and back-extracted with glycine/sodium hydroxide buffer solution at pH 9.4. A comparative study of the results found using the excitation, the emission and the combination of the excitation plus the emission spectral data, as analytical signals, was performed. The data set, composed of the excitation plus the emission spectra, was selected as the analytical signal. The optimum wavelengths to record the excitation (lambda(em)=444 nm) and the emission spectra (lambda(ex)=323 nm) were selected to maximize the contribution from gentisic acid, which is the minor urinary metabolite. Partial least squares (PLS-1) multivariate calibration was then applied for the determination. Recovery values from urine samples spiked with salicyclic, salicyluric and gentisic acids varied from 90.1 to 97.6% (mean 93.6%), from 90.0 to 110% (mean 97.9%) and from 89.9 to 104.7% (mean 98.5%), respectively.     

Potentiometric sensor array for the determination of lysine in feed samples using multivariate calibration methods

A potentiometric sensor array has been developed for the determination of lysine in feed samples. The sensor array consists of a lysine biosensor and seven ion-selective electrodes for NH4+, K+, Na+, Ca2+, Mg2+, Li+, and H+, all based on all-solid-state technology. The potentiometric lysine biosensor comprises a lysine oxidase membrane assembled on an NH4+ electrode. Because the selectivity of the lysine biosensor towards other cation species is not sufficient, there is severe interference with the potentiometric response. This poor selectivity can be circumvented mathematically by analysis of the richer information contained in the multi-sensor data. The sensor array takes advantage of the cross-selectivity of lysine for each electrode, which differs from the other species and quantification of lysine in complex feed sample extracts is accomplished with multivariate calibration methods, such as partial least-squares regression. The results obtained are in a reasonable agreement with those given by the standard method for amino acid analysis.     

Multivariate calibration applied to synchronous fluorescence spectrometry. Simultaneous determination of polycyclic aromatic hydrocarbons in water samples

Synchronous fluorescence spectra of mixtures containing ten polycyclic aromatic hydrocarbons (anthracene, benz[a]anthracene, benzo[a]pyrene, chrysene, fluoranthene, fluorene, naphthalene, perylene, phenanthrene and pyrene) have been used for the determination of these compounds by Partial Least Squares Regression (PLSR), using both PLS-1 and PLS-2. Different procedures have been used for the pretreatment of the data in order to obtain better models, and the size of the calibration matrix has also been studied. The best models have been used for the determination of the above mentioned PAHs in spiked natural water samples at concentration levels between 4 and 20 ng ml⁻¹. Recoveries ranged from 80 to 120% in most cases, although fluorene gave significantly lower results.     

Second-order multivariate calibration procedures applied to high-performance liquid chromatography coupled to fast-scanning fluorescence detection for the determination of fluoroquinolones

Different second-order multivariate calibration algorithms, namely parallel factor analysis (PARAFAC), N-dimensional partial least-squares (N-PLS) and multivariate curve resolution-alternating least-squares (MCR-ALS) have been compared for the analysis of four fluoroquinolones in aqueous solutions, including some human urine samples (additional four fluoroquinolones were simultaneously determined by univariate calibration). Data were measured in a short time with a chromatographic system operating in the isocratic mode. The detection system consisted of a fast-scanning spectrofluorimeter, which allows one to obtain second-order data matrices containing the fluorescence intensity as a function of retention time and emission wavelength. The developed approach enabled us to determine eight analytes, some of them with overlapped profiles, without the necessity of applying an elution gradient, and thus significantly reducing both the experimental time and complexity. The study was employed for the discussion of the scopes of the applied second-order chemometric tools. The quality of the proposed technique coupled to each of the evaluated algorithms was assessed on the basis of the figures of merit for the determination of fluoroquinolones in the analyzed water and urine samples. Univariate calibration of four analytes led to limits of detection in the range 20–40 ng mL⁻¹ and root mean square errors for the validation samples in the range 30–60 ng mL⁻¹ (corresponding to relative prediction errors of 3–8%). The ranges for second-order multivariate calibration (using PARAFAC and N-PLS) of the remaining four analytes were: limit of detection, 2–8 ng mL⁻¹, root mean square errors, 3–50 ng mL⁻¹ and relative prediction errors, 1–5%.     

Photoinduced spectrofluorimetric determination of fluoroquinolones in human urine by using three- and two-way spectroscopic data and multivariate calibration

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.     

Photoinduced fluorimetric determination of folic acid and 5-methyltetrahydrofolic acid in serum using the kinetic evolution of the emission spectra accomplished with multivariate second-order calibration methods

Second-order multivariate calibration methods in combination with a continuous flow system, which allows for the continuous on-line irradiation of the analytes, have been employed for the determination of folic acid and its main metabolite 5-methyltetrahydrofolic acid in serum samples. An experimental central composite design, together with response surface methodology, has been used to find the optimum instrumental variables to perform the photochemical reaction. The time evolution of the emission spectra of the generated photoproducts, in the range 330-540 nm, after irradiation at 275 nm for 20 min, provided the three-way data set employed. On the basis of the differences on the kinetic rates of the photoreaction of both analytes, direct determination of the compounds in human plasma has been accomplished. The second-order methods assayed were parallel factor analysis (PARAFAC), self-weighted alternating trilinear decomposition (SWATLD), and unfolded partial least-squares (U-PLS), multidimensional partial least-squares (N-PLS), and bilinear least-squares (BLLS), all three in combination with the residual bilinearization procedure (RBL).     

Different strategies for the direct determination of amoxicillin in human urine by second-order multivariate analysis of kinetic-spectrophotometric data

The kinetic evolution of UV-visible absorption spectra of amoxicillin in the presence of copper(II) ions has been processed by the second-order multivariate methods parallel factor analysis (PARAFAC) and also by a novel approach based on partial least-squares with residual bilinearization (PLS/RBL). The latter one is employed for the first time to evaluate kinetic-spectral information. The mechanism of the analyte metal-catalyzed hydrolysis involves a reaction intermediate and a final reaction product, both with spectra which may allow for the determination of amoxicillin in human urine, even in the presence of unsuspected sample components. This is possible thanks to the second-order advantage exploited by the employed chemometric algorithms, among which PARAFAC and PLS/RBL gave the best results. Amoxicillin was determined in a series of spiked and real urine samples, which allowed to perform, respectively, a recovery study and a comparison with the reference high-performance liquid chromatographic technique. The best figures of merit were obtained with PLS/RBL, namely sensitivity, 0.5 AU L mg⁻¹ (AU = absorbance units), analytical sensitivity, 500 L mg⁻¹ and limit of detection, 6 mg L⁻¹. Relative advantages and disadvantages of the employed algorithms are discussed.     

Photostability studies for micellar liquid chromatographic determination of nifedipine in serum and urine samples

Nifedipine is a photosensitive compound that is converted into its 4-(2-nitrophenyl) pyridine and 4-(2-nitrosophenyl) pyridine homologue. In order to obtain the most adequate conditions for handling nifedipine solutions in the analytical laboratory, a number of studies on the decomposition of this compound were performed. A simple micellar liquid chromatographic procedure was described to determine nifedipine in different biological matrices such as serum and urine, and to control its decomposition. To perform the analysis, nifedipine was dissolved in 0.1 m SDS at pH 3 and chromatographed using a mobile phase containing 0.125 m SDS-3% pentanol, pH 3 on a C18 column and UV detection at 235 nm. The chromatographic analysis time was 8 min. The response of the drug for both biological matrices was linear in the 1-100 microg/mL range, with r2>0.997 at all times. Repeatability, intermediate precision (CV, %) and limits of quantification and detection (ng/mL) were 0.19, 4.3, 104 and 31 in serum and 0.81, 2.1, 136 and 41 in urine. The method developed here does not show interferences or matrix effects produced by endogenous compounds. Micellar media and mobile phases have the advantage of stabilising the compounds, thus preventing photodegradation and allowing the direct injection of biological samples.     

Second-order and higher-order multivariate calibration methods applied to non-multilinear data using different algorithms

We discuss and evaluate the current state of second-order and higher-order multivariate calibration methods devoted to the determination of compounds in non-multilinear data systems. We examine possible causes of multilinearity deviations:

(1)

a non-linear relationship between signal and analyte concentration;

(2)

a signal for a given sample that is non-multilinear; and,

(3)

component profiles that are not constant across the different samples.

We discuss the advantages and the limitations of the algorithms available to cope with these different situations.The review covers relevant analytical problems found in samples of environmental and biological interest, highlighting some significant examples, and evaluating the advantages and the limitations of the different algorithms available.     

Gallium determination in biological samples

A sensitive, simple and time-saving method has been developed for the neutron activation analysis of gallium at concentrations around 10⁻⁴ ppm in biological tissues. After a 24-hour irradiation in a thermal neutron flux of 2.8·10¹³ n·cm⁻²·s⁻¹ and a purification by ion-exchange chromatography to eliminate troublesome elements such as sodium, iron and copper, the⁷²Ga activity is measured with enough accuracy for the method to be applicable in animal physiology and clinical toxicology.