Search

Pteridinic derivatives are important biomolecules considered as biomarkers for several diseases, especially in cancer and infectious pathologies. A new fluorimetric-HPLC method for the analysis of nine pteridines in human serum has been reported. Two analytical columns composed by C18 porous and fused core particles were assayed and the results compared. Fused core particle column allows us adequate separation, in only one run and in 15 min. Acid precipitation step of the proteins and clean-up process with an Isolute ENV+ (hydroxylated polystyrene-divinylbenzene copolymer) cartridge of the serum samples have been optimized. Analytes were determined by fluorimetric detection, exciting at 272 nm and measuring the fluorescence emission at 410 nm for isoxanthopterin, at 465 nm for xanthopterin, and at 445 nm for the analysis of the other pteridines. Detection limits between 0.07 and 0.61 ng mL(-1) were calculated according to Clayton criterium. Intraday precision varied from 1.2 to 5.3 and interday precision between 1.2 and 7.4, both expressed as RSD (%). External standard and standard addition calibrations were compared in the analysis of serum samples. The pteridine amounts in serum (expressed as ng mL(-1) ± confidence interval) were 3.69 ± 1.78; 1.35 ± 0.24; 0.46 ± 0.14; 0.54 ± 0.24; 0.84 ± 0.55; 2.10 ± 0.51 and 0.23 ± 0.11 for XAN, NEO, MON, ISO, BIO and 6HMPT, respectively, using the external standard method. Comparable results were obtained by the standard addition method. It is noticeable that 7BIO was not detected in the healthy serum samples analyzed.

Pteridines are important biomarkers metabolites related to several biochemical pathways such as activation of the cell-mediated immune system, biosynthesis of neurotransmitters, etc. The level of pteridinic compounds in urine is considered as an important clinic criterion. In this work, a new liquid chromatography-mass spectrometry (LC-MS) method is proposed to determine several pteridinic biomarkers in urine samples using 6-methylpterin as internal standard (I.S.). Matrix effect was evaluated and several dilutions of urine were tested in order to study the evolution of signal suppression. Sample preparation was limited to 10-fold dilution of the filtered urine followed by injection onto a reversed-phase column. The signal was recorded in selected ion monitoring mode. The lowest limit of detection was found for pterin (values ranged from 1.70 to 3.88 ng mL(-1)) whereas the highest limit was for xanthopterin (values ranged from 10.5 to 49.9 ng mL(-1)) for healthy volunteers between 17 and 51 years old.

The polarographic DPP and DC techniques have been utilized to study the behaviour of 2-pyrilideniminobenzohydroxamic acid (PIBHA). This reagent gives rise to two cathodic waves, the Ep of the first wave is-1.07 V vs Ag/AgCl at pH 3.38 and the Ep of the second wave appeared at-1.45 V at the same pH. The principal characteristics of these waves have been studied and the corresponding possible mechanisms of the electrode process for both waves have been proposed.

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.

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).

A fluorimetric study on the spectral characteristics of two antileukotrienes, cinalukast and montelukast, has been performed. Ionization constants of both of them have been photometrically calculated. Cinalukast p K a in ethanol:water 50:50 (v/v) medium resulted to be 2.2 ± 0.1. Because the spectral characteristics of montelukast are widely affected by the solvent nature, p K a was estimated in two different ethanol:water media, 70:30 (v/v) and 10:90 (v/v) and the values calculated were p K a = 2.9 ± 0.1, and p K a1 = 2.0 ± 0.1 and p K a2 = 6.5 ± 0.1, respectively. It has been proven that the fluorescence of both, cinalukast and montelukast, is significantly intensified in the presence of cyclodextrins (CyDs). The host–guest complexation processes between cinalukast and α-CyD or heptakis-(2,6-di- O -methyl)-β-cyclodextrin (DIMEB) and between montelukast and DIMEB have been investigated by fluorescence spectroscopy. A 1:1 stoichiometric ratio was established for the three studied inclusion complexes. The changes produced on the fluorescence of cinalukast or montelukast, when they are included on the hydrophobic CyD cavity are used to calculate their association constants by a non-linear regression method. Semiempirical MO calculations using AM1 method were performed in order to characterize the studied inclusion complexes. A new method for cinalukast determination in human serum, based on the fluorescence of the complex cinalukast–DIMEB exhibiting limit of detection of 7.95 ng mL −1 has been proposed with satisfactory results. Adequate recovery values between 95 and 103% were calculated at five different concentration levels.

Principal component analysis (PCA), and soft independent modelling class analogy (SIMCA), were applied to data of content of the various triglycerides, sterols, or both data, to explore their capacity for the typification of a variety of olive oil, belonging to a Spanish origin denomination. This study has demonstrated that it is possible to characterize the oils obtained from a specific type of olives (“Manzanilla Cacerena” of North of Caceres (Extremadura––Spain)) according to their chemical composition. Best results were obtained with the content of triglycerides. The plots of PCs showed that the PC1 is related with the category variable “variety” and the PC2 is related with “maturity”. SIMCA was employed to assign unknown samples into one of two groups or classes, depending on the “variety” of olives, for those which independent PCA models were made. Comman's plot showed that different olive oils are clustered in different groups and each group could be distinguished clearly.

The voltammetric behavior of Vitamin E in the presence of olive oil is studied at the glassy carbon electrode, in a hexane–ethanol medium, with diverse techniques: sampled DC, differential pulse, and square-wave voltammetry. The influence of such variables as the hexane–ethanol proportion, sulfuric acid concentration, and instrumental parameters is studied. Separate voltammetric peaks are obtained for α-tocopherol and δ-tocopherol, but the peaks for β-tocopherol and γ-tocopherol overlap. For the simultaneous determination of α-, β+γ-, and δ-tocopherols in vegetable oils by the PLS-1 multivariate calibration method, the results using sampled DC and DPV voltammograms are compared. The DPV voltammograms are found to be the best data set. The proposed method is applied to the determination of the tocopherols in different vegetable oil samples. The olive oil samples needed a prior cleaning stage by solid-phase extraction on silica cartridges. The results are very acceptable.

Binary mixtures of methotrexate (MTX) and leucovorin (LV) have been resolved by application of first-derivative spectrophotometry and partial least squares calibration (PLS-1). By measuring the first-derivative signals of MTX and LV at 354 and 300 nm, respectively, simultaneous determination was possible. The mean recoveries for urine samples were 91 and 96% for MTX and LV, respectively. Partial least squares (PLS-1) multivariate calibration has been applied to the determination of these compounds in serum and in urine without pretreatment of the samples. The absorption spectra of serum or urine samples spiked with methotrexate and/or leucovorin, were used to optimize the calibration matrixes by the PLS-1 method. The sensitivity and selectivity of the proposed procedures were calculated. Mean recoveries were 101 and 97% for MTX and LV, respectively, for serum samples, and 101 and 98% for MTX and LV, respectively, for urine samples.

A kinetic study of the oxidation of methotrexate (MTX) in acidic medium and in the presence of potassium permanganate has been made on the basis of the fluorescence–time curves. A kinetic method for the determination of MTX was developed with a range of application between 0.22 and 3.30 μM. The proposed kinetic method permits us to determine MTX in human serum and to avoid the natural fluorescence of the serum. A detection limit of 0.18 μM was calculated in the presence of ascorbic acid as activator. Only 100 s per sample is necessary for the analysis. The interference of pteridin derivatives and the rescue agent folinic acid (leucovorin) was tested.

The resolution of binary mixtures of triamterene (TAT) and leucovorin (LV) by application of first-derivative spectrophotometry and by application of Partial Least Squares calibration (PLS-1) was performed. Triamterene is determined in presence of leucovorin directly in the absorption spectra at 358 nm, and leucovorin is determined in the first-derivative spectra at 305.6 nm, zero-crossing of the triamterene. The mean recovery values in urine samples were 102 and 97% for TAT and LV, respectively. Partial Least Squares calibration (PLS-1) multivariate calibration of spectrophotometric data, have been applied to the determination of these compounds in serum and in urine without pretreatment of the samples. The absorption spectra of samples of serum or urine, spiked with triamterene and/or leucovorin, were used to perform the optimization of the calibration matrices by PLS-1 method. Mean recovery values were of 107 and 108% for TAT and LV in serum samples, and 98 and 91% for TAT and LV in urine samples.

Quinolonic and cinolonic derivatives are mainly used as antibacterials in fish-farms. In this paper we describe a careful revision of the treatment procedures of samples and a prodedure for the determination of residues of these compounds. Because of the complexity and duration of these procedures, several studies have been carried out and these have lead to a simpler and shorter method. Three approaches have been examined: lyophilization followed by extraction with chloroform, solid-liquid extraction with chloroform and solid-liquid extraction with sodium hydroxide solution, followed by liquid-liquid partition in chloroform. Some previous studies into the partition equilibrium are also included. As a result of our studies we propose a procedure with a lower number of steps than those previously described in the literature. This method has been applied to the analysis of nalidixic, 7-hydroxymethylnalidixic and oxolinic acids and cinoxacin in trout muscle. These analysis have been carried out using an HPLC system equipped with a C18 column and fluorimetric detection. The mobile phase was acetonitrile:oxalic acid. The recoveries obtained were: 70–97% for 7-hydroxymethylnalidixic acid, 75–78% for nalidixic acid, 71–95% for oxolinic acid and 72–85% for cinoxacin.

An HPLC method with ultraviolet and fluorimetric detection has been established for the separation and determination of six quinolonic and cinolonic antibiotics. A Nova-Pak C18 column (150 x 3.9 mm) and a Waters 486 UV and a Waters 470 fluorescence detector have been used. The influence of variables such as mobile-phase composition and flow-rate, has been studied. An acetonitrile-aqueous solution of oxalic acid 4x10(-4) M (28:72, v/v) has been selected as optimum. The wavelength for the photometric detection of the six antibiotics was 265 nm. For the fluorimetric detection two pairs of excitation/emission wavelengths, 260/360 or 270/440 nm, were selected for the determination of nalidixic acid, 7-hydroxymethylnalidixic acid and oxolinic acid, and for the determination of pipemidic acid and cinoxacin, respectively. The analytical parameters and detection and quantification limits of the method have been determined. The proposed method has been applied for the determination of the six compounds in urine, applying different procedures depending on their concentration, the results being very acceptable.

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 utility of first-derivative fluorimetry for the determination of doxycycline and oxytetracycline through the formation of their chelates with aluminium, is discussed. Mixtures of these compounds were determined by first-derivative fluorimetry with linear calibration graphs established between 0.2 and 1.4 μgml−1 for doxycycline and between 0.05 and 0.6 μgml−1 for oxytetracycline. The application of the method to the analysis of these compounds in pharmaceutical preparations is described and a statistical evaluation of the experimental results is reported.

A flow-injection chemiluminescence (CL) method is described for the determination of ciprofloxacin (CIP), norfloxacin (NOR) and ofloxacin (OFL), commonly used antibiotics of the fluoroquinolones family. The method is based on the CL reaction of the fluoroquinolones with tris(2,2′-bipyridyl) ruthenium(II) and Ce (IV), in sulfuric acid medium. The maximum CL emission, given at 0.45 min for CIP, at 0.35 min for NOR and at 0.04 min for OFL, respectively, were measured, allowing the simple application of the proposed method to the routine analysis of the antibiotics. The methods were applied to the determination of CIP, NOR and OFL, in several pharmaceutical preparations, with very satisfactory results, and validated by a previously reported HPLC method. The time-resolved equipment allowed the measurement of the kinetic evolution of the chemiluminescence signals. In base to the differences in the kinetic behaviour of ofloxacin with respect to ciprofloxacin and norfloxacin, binary mixtures of the drugs were resolved by using the time-resolved chemiluminescence signals, in combination with first-order partial least-squares (PLS) multivariate calibration.

Basic studies on the photochemical behaviour of trans-resveratrol and its photoproduct are reported. Photometrically and fluorimetrically calculated acidity constants of the former were determined. The usefulness of the determination of resveratrol by photochemically induced fluorescence and second-derivative photochemically induced fluorescence was also examined. The very weakly fluorescent trans-resveratrol is converted into a highly fluorescent photoproduct by irradiating hydroethanolic solutions of trans-resveratrol containing 40% v/v of ethanol for 60 s with intense UV radiation. The photoproduct presents excitation and emission maxima centred at 260 nm, and 364 and 382 nm, respectively. Under these conditions, a linear relationship between fluorescence intensity and trans-resveratrol concentration was found between 6.6 and 66 ng mL−1. Optimum conditions for the extraction of trans-resveratrol from an aqueous phase at pH 5.0 with diethylether were a phase ratio (aqueous/organic) of 2, a shaking time of 60 s and a buffer concentration of 0.15 mol L−1. An extraction recovery of 100% was reached under these conditions. The optimized extraction procedure was applied to the analysis of resveratrol in wine samples, employing the amplitude between 356 and 364 nm of the second-derivative photoinduced emission spectrum as analytical signal. It was found that there is not matrix effect and recoveries around 100% were obtained at different fortification levels.

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.

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.

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.

A rapid high-performance liquid chromatographic method has been developed to determine piromidic acid in trout muscle tissue and in urine, in the presence of nalidixic, 7-hydroxymethylnalidixic, oxolinic and pipemidic acids and cinoxacin. A Nova-Pak C18 column was used with acetonitrile-4x10(-4) M oxalic acid (40:60, v/v) as the mobile phase. A post-column change of pH was made with NaOH. Fluorimetric detection at 456 nm (lambda ex 275 nm) was used. The instrumental detection limit was 5.91 ng/ml, based on height of peak. Pretreatment of the urine samples was not necessary and fish samples were extracted with sodium hydroxide solutions and cleaned by means of an extraction with chloroform. Detection limit was 147 ng/ml for urine and 5.91 ng/g for trout muscle. Good separation without interference from any other components was obtained. Recovery was better than 87% in urine and better than 72% in trout muscle tissue.

The solid complexes that 2-benzylideniminobenzohydroxamic, 2-pyrilideniminobenzo-hydroxamic and 2-salicylideniminobenzohydroxamic acids form with Fe(III) and Cu(II) ions have been isolated. The six complexes have been characterized by chemical analysis and IR spectroscopy. Their thermal behaviour has also been studied.

2-Pyrilideniminobenzohydroxamic acid (PIBHA) has been synthesized by the reaction ofo-aminobenzohydroxamic acid and pyridine-2-carbaldehyde. Its solubility in several solvents, spectral characteristics andpKa values are reported. Its reactivity with inorganic ions, in aqueous media and by extraction with chloroform, isoamyl alcohol, toluene, and a toluenic solution of trioctylmethylammonium chloride (Adogen 464) has been investigated. ThepK values corresponding to the formation constants of the complexes formed between PIBHA and Cd(II), Mn(II), Co(II), Ni(II) and Cu(II) in ethanol-water medium are also reported.

The reaction of Ti(IV) with 2-pyrilideniminobenzohydroxamic acid (2-PIBHA) was studied in aqueous medium and by using an extraction in isoamyl alcohol. The stoichiometries of the complexes formed are 1:1 and 1:3 (Ti(IV):reagent) in both cases. The extraction and spectrophotometric determination of titanium with 2-PIBHA can be performed in a concentration range from 0.1 to 1.4 μg/ml of Ti(IV). The apparent molar absorptivity in isoamyl alcohol is 39600 1.mol−1.cm−1 at 375 nm. The relative error of the method is ± 0.64% for 25 μg of Ti(IV) in the aqueous phase (50 ml) and the extraction constant is 1.45×1013.

Although pteridines are excreted in the urine of normal healthy persons, levels of some of these compounds seem to be modified by several diseases. Creatinine is considered to be the best reference for study of the excretion of these markers in urine. A fast, very sensitive, and precise HPLC method with ultraviolet and fluorimetric detection has been established for the simultaneous determination of pterin-6-carboxylic (CAR), neopterin (NEO), xanthopterin (XAN), isoxanthopterin (ISO), biopterin (BIO), and creatinine (CREA) in urine. The influence of conditions such as mobile-phase composition and flow rate, have been studied. 0.015m Tris-HCl-10−3m NaCl buffer solution, pH 6.8, was selected as mobile phase. These five pteridine derivatives are well resolved in less than six minutes. CREA is determined by photometric detection at 230 nm and pteridines by fluorimetric detection at 444 nm, with excitation at 280 nm. The detection limits are below 25 pg for all pteridines and 0.30 ng for creatinine. The method has been applied to the determination of these compounds in urine. A non-clean-up concentration step is necessary before the separation. The urine sample is oxidized before HPLC injection. Values for the pteridine-to-creatinine ratios found for two groups of healthy persons (younger and older than 30 years) are reported.