A novel analytical methodology for the determination of hydroxy polycyclic aromatic hydrocarbons in breast and cow milk samples.

Hydroxy polycyclic aromatic hydrocarbons (OHPAHs) in biological fluids, such as milk, are considered as biomarkers of exposure to polycyclic aromatic hydrocarbons (PAHs) in organism. The presence of OHPAHs in milk samples indicates a potential contamination on human organisms and milk producing animals. In this way, infants can be contaminated by lactation through the consumption of milk of both, human and animal origins. In this paper, eight OHPAHs have been analyzed in commercial cow milks and in human breast milk using HPLC and fast scanning fluorimetric detection (FSFD). Extraction and cleaning procedures of OHPAHs from milk samples have been investigated, and the experimental results using two bibliographic protocols and a new proposed protocol have been compared. The new protocol using enzymatic hydrolysis, proteins precipitation and, solvent extraction using acetonitrile, was proposed as the most adequate for the determination of 2-hydroxyfluorene, 1-/9-, 2-/3- and 4-hydroxyphenanthrenes, 1-hydroxypyrene and 3-hydroxybenzo[a]pyrene. The method recoveries ranged from 80-102% and 75-91% for fresh cow milk and for human breast milk, respectively, for all components except for 3-OHBz[a] Py. Low recovery values were calculated for 3-hydroxybenzo[a]pyrene in all cases. No statistical difference in the method performance was observed between fresh cow milk and human breast milk.

Optimization and validation of a rapid liquid chromatography method for determination of the main polyphenolic compounds in table olives and in olive paste.

A high performance liquid chromatography method, coupled to diode-array and fluorescence detectors, with a previous solid-liquid extraction, has been developed for the simultaneous detection and quantification of polyphenolic compounds in table olives and in olive paste. The effects of extraction variables have been studied by response surface methodology. The best conditions were extraction with 100% methanol (2mM NaF) during 30min for table olives, and 91% methanol (2mM NaF) during 40min for olive paste. Chromatographic separation of 26 polyphenols from different families was optimized. This method provides high linearity, in all cases higher than 98.65%, and high sensitivity whose detection limits ranged between 0.08 and 1.11µg/mL. The validated method has been applied for the determination of polyphenols in table olive and olive paste samples. The intra-day and inter-day assay repeatability, in the analysis of real samples was less than 7.6 and 11%, respectively.

HPLC determination of serum pteridine pattern as biomarkers.

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.

Rapid and sensitive on-line solid phase extraction-ultra high performance liquid chromatography-electrospray-tandem mass spectrometry analysis of pesticides in surface waters.

A simple, fast and sensitive method has been developed for the determination of 37 LC-amenable pesticides in surface water samples. On-line solid phase extraction (SPE) coupled to ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS. was employed for pre-concentration and analysis of all compounds in 16min. SPE parameters were evaluated in order to increase sample throughput and detectability. Thus, injected sample volume, sample loading flow, carryover effects and reusability of the cartridges employed were studied, observing that 70 extractions can be performed with the same cartridge. Validation parameters were performed and good linearity (R(2)>0.99 in all cases) and precision (interday relative standard deviation values were lower than 14%) were obtained. Limits of detection (LOD) and limits of quantification (LOQ) were lower than 6.0 and 18.0ngL(-1) applying an injection sample volume of 1.5mL, respectively, with exception of thifensulfuron methyl, whose limits were 10.0 and 33.0ngL(-1), respectively. On-line SPE recoveries were evaluated for three concentration levels (0.01, 0.03, 0.10 and 0.20µgL(-1)) and acceptable values were found. The on-line SPE method was also compared with off-line SPE. Matrix effects were observed for majority of compounds and standard addition method was selected for analysis of real water samples. Finally, surface water samples were analyzed and, in all cases, the pesticide concentrations were below than the allowable limit in water for human consumption.

A simple HPLC-ESI-MS method for the direct determination of ten pteridinic biomarkers in human urine.

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.

Development of a non-aqueous capillary electrophoresis method with UV-visible and fluorescence detection for phenolics compounds in olive oil.

Response surface methodology has been applied to the optimization of a simple and rapid non-aqueous capillary electrophoresis method for the separation and determination of several phenolic compounds belonging to the different families present in olive oil. A Box-Behnken design was employed and a total of 27 experiments were performed using olive oil samples spiked with the phenols and injected directly in the capillary after dilution 1:1 with 1-propanol. Finally, the background electrolyte (BGE) was constituted of 25 mM boric acid and 18 mM KOH in a mixture of 74:26 (v/v) 1-propanol/methanol. The hydrophobicity of the BGE allows its miscibility with the olive oil and, as a consequence, the possibility of characterizing and determining these kinds of compounds in this sample without any pretreatment. A hydrodynamic injection (6 s, -30 mbar) was applied and the separation was carried out using 35 °C and +20 kV of separation temperature and voltage, respectively. A capillary with two detection windows for serial online UV and fluorescence detection was satisfactorily employed. The validation of the method was carried out by setting the calibration curves, and the figures of merit were finally obtained. A lineal relationship between the corrected peak area and concentration and limits of detection in the order of micrograms per milliliter were found.

Comparison of the predictive ability of several second-order multivariate methods in the simultaneous determination of two therapeutic drugs in human urine.

The aim of this paper is to study the applicability of second-order multivariate methods in the simultaneous determination of two therapeutic drugs in human urine samples. The studied drugs, irinotecan and thalidomide, are used in the treatment of malignant tumours. Irinotecan (CPT-11) is used to treat colon cancer; recent studies have shown the benefits of using thalidomide in combination with CPT-11 in the treatment of this disease. CPT-11 is highly fluorescent, but the native fluorescence of thalidomide is very weak. The second-order methods assayed were parallel factor analysis (PARAFAC), unfolded partial least-squares (U-PLS) and multidimensional partial least-squares (N-PLS), both combined with the residual bilinearization procedure (RBL). The excitation-emission matrices (EEMs) of the samples were recorded as analytical signal. The accuracy and precision of the algorithms were evaluated through the root mean square error of prediction (RMSEP) and the elliptical joint confidence region test (EJCR), obtaining better results with PARAFAC, which was successfully applied to the determination of thalidomide and CPT-11 in human urine samples, after a previous liquid-liquid extraction with chloroform.

On line photochemically induced excitation-emission-kinetic four-way data: analytical application for the determination of folic acid and its two main metabolites in serum by U-PLS and N-PLS/residual trilinearization (RTL) calibration.

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.

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.

Spectrofluorimetric determination of irinotecan in the presence of oxidant agents and metal ions.

In this work four spectrofluorimetric methods for the determination of irinotecan (CPT-11) in human urine and pharmaceuticals have been developed. Initially, the fluorescent characteristics of irinotecan (CPT-11) have been studied in both acidic and basic media. Later, the fluorescence emission generated by the oxidation of CPT-11 with several agents was studied. A quenching of fluorescence could be observed in the presence of Ce(IV) and I(2)/I(-). Also, the reaction between several divalent and trivalent metal ions with CPT-11 was studied, and one method in presence of Fe(3+) was developed since it was the only metal ion that changes the fluorescence of the analyte. The proposed methods present limit of detection comprises between 0.46 and 2.57ngmL(-1). The spectrofluorimetric methods were applied to human urine. No pre-treatment of the sample was necessary, only a dilution 1:20 with water was made. No interference of the matrix was observed in the conditions used. Recoveries were comprises between 100.0 and 104.3%. Also, pharmaceuticals preparations were analyzed with recoveries between 106.7 and 119.7%. The proposed spectrofluorimetric methods were validated by RP-HPLC, obtaining that the oxidation with iodine is the best method to analyze urine samples, while than, the fluorimetric method developed at acidic pH value is the best for the analysis of pharmaceuticals.

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).

Resolution of ofloxacin-ciprofloxacin and ofloxacin-norfloxacin binary mixtures by flow-injection chemiluminescence in combination with partial least squares multivariate calibration.

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.

Complexation study of cinalukast and montelukast with cyclodextrines.

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 pK(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, pK(a) was estimated in two different ethanol:water media, 70:30 (v/v) and 10:90 (v/v) and the values calculated were pK(a)=2.9+/-0.1, and pK(a1)=2.0+/-0.1 and pK(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 alpha-CyD or heptakis-(2,6-di-O-methyl)-beta-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.

Determination of resveratrol in wine by photochemically induced second-derivative fluorescence coupled with liquid-liquid extraction.

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.

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.

Comparison of different fluorimetric signals for the simultaneous multivariate determination of tocopherols in vegetable oils.

This paper deals with the simultaneous determination of the quaternary mixture of tocopherols (alpha-, beta-, gamma-, and delta-T) performed using fluorimetric techniques and partial least squares (PLS-1) multivariate analysis. In this study, PLS-1 was applied to matrices made up of fluorescence excitation and emission spectra (EEM) and with fluorescence excitation, emission, and synchronous spectra (EESM) of tocopherols dissolved in hexane: diethyl ether (70:30 v/v). A calibration set of 55 samples based in a central composite plus a full factorial plus a fractionated factorial design was constructed. When synthetic samples were analyzed, recoveries around 100% were obtained and detection limits were calculated using EEM and EESM. For the analysis of the oils, the samples, diluted in hexane, were cleaned in silica cartridges and tocopherols were eluted with hexane: diethyl ether (90:10 v/v). The developed method was applied to different edible oils. The results are satisfactory for alpha-, beta-, and gamma-, but they are worse for delta-T.

Comparison of UV derivative-spectrophotometry and partial least-squares (PLS-1) calibration for determination of methotrexate and leucovorin in biological fluids.

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.

Kinetic fluorimetric determination of the antineoplastic methotrexate (MTX) in human serum.

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 microM. 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 microM 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.

Determination of triamterene and leucovorin in biological fluids by UV derivative-spectrophotometry and partial least-squares (PLS-1) calibration.

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.

Kinetic fluorimetric study of the oxidation reaction of folinic acid (leucovorin) with potassium permanganate. Determination in human urine.

In this study a kinetic fluorimetric method for the determination of folinic acid (leucovorin, LV) in human urine has been developed. The fluorescence emission generated by the oxidation reaction between LV and potassium permanganate in alkaline medium has been monitored at 360nm (excitation wavelength 290nm). The effect of instrumental and experimental variables on the reaction was investigated and a 0.17M sodium hydroxide, 3.3x10(-5)M KMnO(4) concentration and a temperature of 70 degrees C were selected for the reaction. The concentration range has been optimized between 10 and 700ngml(-1) of LV. The correlation coefficient was 0.9989. The sensitivity of the proposed method is 9.5ngml(-1) (expressed as limit of detection in accordance with the Clayton criterion). The determination time per sample is smaller than 200s. The proposed kinetic fluorimetric method has been applied to the direct determination of this compound in human urine. Recovery values from urine samples, containing LV, range from 82 to 110% (mean 96%).