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.

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.

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.

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).

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.

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.

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.

Determination of the chemotherapeutic quinolonic and cinolonic derivatives in urine by high-performance liquid chromatography with ultraviolet and fluorescence detection in series.

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.

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.

Host-guest stabilized room temperature phosphorescence in beta-cyclodextrin/ bromoalcohol solutions from 2-naphthyl-oxy-acetic acid and 1-naphthyl-acetic acid.

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

Simultaneous fluorometric determination of nalidixic acid and 7-hydroxymethylnalidixic acid by partial least squares calibration.

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

Synchronous fluorimetric determination of salicylic acid and diflunisal in human serum using partial least-squares calibration.

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

Simultaneous determination of pteridines in multicomponent mixtures using derivative spectrophotometry and partial least-squares calibration.

Simple binary mixtures composed of very similar pteridines (neopterin and pterin) have been resolved by derivative spectrophotometry. Detection limits of 0.30 microgram ml-1 and 0.12 microgram ml-1 for pterin and neopterin, respectively, have been calculated. Also, different mixtures of pteridines considered as disease markers, such as pterin, neopterin, xanthopterin and isoxanthopterin, have been determined by using a partial least-squares (PLS-2) model. Calibration set containing 0-7 micrograms ml-1 for each component was used. The resolution of several mixtures and single determination was tested in artificial 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.

Simultaneous determination of flavor enhancers inosine 5'-monophosphate and guanosine 5'-monophosphate in food preparations by derivative spectrophotometry.

A derivative spectrophotometric method was developed for the quantitative determination of 2 flavor enhancers, inosine 5'-monophosphate (IMP) and guanosine 5'-monophosphate (GMP), in the presence of monosodium glutamate (MSG). Procedures for determining IMP and GMP singly and in binary mixtures are described. Overlapping absorption spectra of both compounds were resolved by using first-derivative spectrophotometry. By measuring the first-derivative signals of IMP and GMP at 253 and 248 nm, respectively, simultaneous determination was possible for IMP and GMP at 5-40 micrograms/mL, in the presence of up to 5000 micrograms/mL MSG. The method was satisfactorily used to determine IMP and GMP in several food preparations.

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.

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.

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.