An optimized methodology for the determination of multiclass organic ultraviolet sunscreens and metabolites in human milk through chromatographic and chemometric resolution.

Organic UV filters (UVFS) are used to mitigate the dermal effects associated with health risks from UV radiation, making them essential in personal care products. UVFS are frequently identified in environmental samples due to their high lipophilicity and persistence, underscoring the urgency of comprehensive assessments and regulatory measures aimed at safeguarding ecosystems and human health. The present study reports a multiclass analytical method for determining 16 UV sunscreens and metabolites in breast milk based on an ultrasound-assisted-dispersive liquid-liquid micro-extraction (UA-DLLME) with further chromatographic and chemometric resolution. The experimental conditions of the UA-DLLME were optimized through the implementation of the Design of Experiment tools. To model the responses, least-squares and artificial neural network methodologies were implemented. The optimal conditions were found by employing the desirability function. The samples were analyzed through reverse-phase liquid chromatographic separation, UV diode array, and fast-scanning fluorescence detection. The chromatographic analysis enabled the resolution of 16 analytes in a total time of 13.0 min. Multivariate curve resolution-alternating least-square (MCR-ALS) modelling was implemented to resolve analytes that were not fully resolved and to determine analytes that coeluted with endogenous components of the breast milk samples. An enrichment factor of 5-fold concentration was obtained with this methodology, reaching recoveries between 65 % and 105 % for 13 multiclass UV sunscreens and metabolites in breast milk samples with RSD % and REP % lower than 12 %.

Fluorescence Monitoring Oxidation of Extra Virgin Olive Oil Packed in Different Containers.

'Picual' olive oil was stored in different types of containers for 10 months and monitored via quality parameters. In combination with the mentioned analysis, non-destructive fluorescence spectroscopy was performed combined with multivariate analysis to monitor and quantify oil quality levels. Excitation emission matrices (EMMs) were analyzed using parallel factor analysis (PARAFAC). According to the quality parameters, it was observed that Transparent Crystal (TC) and Opaque Crystal (OC) samples were the ones that deteriorated faster due to their higher exposure to light in comparison with Plastic (P) and Canned (C) samples. In a fast and non-destructive manner, the fluorescence spectroscopy-based prototype successfully monitored the oxidation changes in the EVOOs. Unfolded partial least squares (U-PLS) was used to generate a regression model to quantify quality parameters. Good correlation coefficients were found for the peroxide index, K232 and the oxidative stability index (r2 between 0.90 and 0.94 for cross-validation and validation). For all of that, the results obtained confirmed the ability of fluorescence spectroscopy to monitor the quality of olive oil and EEMs combined with U-PLS can be used to analyze these parameters, eluding the classical methods.

Phenylalanine Photoinduced Fluorescence and Characterization of the Photoproducts by LC-MS.

Phenylalanine (Phe) is a direct precursor of tyrosine and several neurotransmitters. The accumulation of Phe in the brain generates serious and not recoverable pathologies in children. Early detection in newborns is fundamental to apply the appropriate therapy and avoid irreversible health problems. Although fluorescence is a sensitive and selective technique for the determination of amino acids, the fluorescent analysis of Phe is limited since it exhibits a very low fluorescence quantum yield; however, the fluorescence of Phe increases drastically under UV irradiation when a peroxide medium is used. The aim of this research was to analyze the effect of the UV-radiation on Phe aqueous-peroxide solutions and to study the influence of the chemical environment on the photoinducted fluorescence process. The nature and characteristics of the fluorescent photoproducts generated under off-line UV irradiation in hydrogen peroxide medium were achieved by high performance liquid chromatography (HPLC) using a spectrophotometer detector (DAD) coupled in series with a mass spectrometer (MS) or with a fast scan spectrofluorimetric detector (FSFD). Environmental characteristics such as pH, initial concentration of Phe, hydrogen peroxide amount and irradiation time were studied in order to establish their influence on the formation of each one of the photoproducts. As the formation of several highly fluorescent photoproducts has been confirmed, the possibility of designing a chromatographic system with a post-column on-line photoreactor is open. The measure of the total fluorescence signal generated from Phe at the optimized irradiation time, could be used for the determination, with high sensitivity, of the initial amount of Phe in aqueous media, such as human serum or environmental samples. These aspects are being studied at present. .

Combination of fluorescence excitation emission matrices in polar and non-polar solvents to obtain three- and four- way arrays for classification of Tempranillo grapes according to maturation stage and hydric status.

The applicability of front-face excitation-emission fluorescence spectroscopy to compare grape water extracts of two consecutive sampling dates, corresponding with two maturation stages, and subjected to full irrigation and non-irrigation, was carried out. The decomposition of the obtained three way grape samples was initially analyzed by means of parallel factor analysis (PARAFAC). A tentative identification of fluorophores was done by matching PARAFAC score values with HPLC measurements. It was found that the first PARAFAC component was highly correlated with the sum of concentrations of catechin and epicatechin. The decomposition of the three way fluorescence data by linear discriminant analysis (LDA)-PARAFAC and LDA-unfolding partial least squares (U-PLS) allowed to discriminate between the first and the second maturation stage. The incorporation of an additional mode to the data, achieved by a diethyl ether extraction, giving rise to a four-way excitation-emission-solvent-samples data set, allowed to differentiate between irrigated and non-irrigated samples with the same assayed algorithms. As far as we know, the use of four-way data arrays for classification issues has been reported for the first time.

Front-face fluorescence excitation-emission matrices in combination with three-way chemometrics for the discrimination and prediction of phenolic response to vineyard agronomic practices.

Phenolic extracts from cv Tempranillo grapes subjected to water stress and irrigation treatment, both of them with high and low crop load, were analyzed by front-face fluorescence. Excitation-emission matrices (EEMs) were analyzed by means of unsupervised parallel factor analysis (PARAFAC), PARAFAC supervised by linear discriminant analysis, and discriminant unfolded partial least-squares. All algorithms allowed to differentiate between water stress and irrigation grape samples when the fluorescence maxima region of catechin and epicatechin, and resveratrol was considered. A central composite design was employed for the calibration of catechin, epicatechin and resveratrol. Resveratrol was quantified by U-PLS in both, irrigated and water stressed samples, and levels between 3.46 ±â€¯0.22 and 4.67 ±â€¯0.08 µg mL-1 and 2.43 ±â€¯0.60 and 3.03 ±â€¯0.10 µg mL-1, respectively, were found. PARAFAC only allowed the determination of the sum of catechin plus epicatechin (R2 = 0.9397). The determination of total catechin plus epicatechin by means of PARAFAC was successfully validated by liquid chromatography.

Phenanthrene metabolites determination in human breast and cow milk by combining elution time-emission fluorescence data with multiway calibration.

Phenanthrene is the most released polycyclic aromatic hydrocarbon into the environment by anthropogenic action. Because of the absorption and biotransformation pathways, this compound is metabolized and the most abundant metabolites are hydroxylated derivatives, such as 1-, 2-, 3-, 4- and 9-hydroxyphenanthrene, which are excreted through biological fluids, included mammals milk. For the resolution and quantitation of co-eluted analytes, elution time-emission fluorescence matrices were analysed with different second-order calibration algorithms: n-way and unfolded partial least squares, both coupled with residual bilinearization (N-PLS/RBL and U-PLS/RBL), and multivariate curve resolution-alternative least squares (MCR-ALS). Once optimized the chromatographic parameters, in isocratic mode, the elution time was of 5.5 min. The second-order data were obtained exciting at 250 nm, with an emission range from 330 to 430 nm, each 1 nm, and elution time from 0 to 5.5 min each 5.4 s. The ranges for the second-order multivariate methods in validation samples were from 1.0 to 9.0 ng mL-1 for 1-, 2-, 3- and 4-hydroxyphenanthrene, and from 5.0 to 45.0 ng mL-1 for 9-hydroxyphenanthrene. Root mean square errors of prediction between 0.45 and 1.82 ng mL-1 (relative errors of prediction 7-22%) were obtained. The optimized procedures were applied in the analysis of human breast milk and in whole and semi-skimmed commercial cow milk. N-PLS/RBL and U-PLS/RBL algorithms show satisfactory results for the five metabolites with recoveries ranging between 82% and 115%.

Detection and quantification of extra virgin olive oil adulteration by means of autofluorescence excitation-emission profiles combined with multi-way classification.

Within olive oils, extra virgin olive oil is the highest quality and, in consequence, the most expensive one. Because of that, it is common that some merchants attempt to take economic advantage by mixing it up with other less expensive oils, like olive oil or olive pomace oil. In consequence, the characterization and authentication of extra virgin olive oils is a subject of great interest, both for industry and consumers. This paper reports the potential of front-face total fluorescence spectroscopy combined with second-order chemometric methods for the detection of extra virgin olive oils adulteration with other olive oils. Excitation-emission matrices (EEMs) of extra virgin olive oils and extra virgin olive oils adulterated with olive oils or with olive pomace oils were recorded using front-face fluorescence spectroscopy. The full information content in these fluorescence images was analyzed with the aid of unsupervised parallel factor analysis (PARAFAC), PARAFAC supervised by linear discriminant analysis (LDA-PARAFAC), and discriminant unfolded partial least-squares (DA-UPLS). The discriminant ability of LDA-PARAFAC was studied through the tridimensional plots of the canonical vectors, defining a surface separating the established categories. For DA-UPLS, the discriminant ability was established through the bidimensional plots of predicted values of calibration and validation samples, in order to assign each sample to a given class. The models demonstrated the possibility of detecting adulterations of extra virgin olive oils with percentages of around 15% and 3% of olive and olive pomace oils, respectively. Also, UPLS regression was used to quantify the adulteration level of extra virgin olive oils with olive oils or with olive pomace oils.

HPLC-fast scanning fluorimetric detection determination of risk exposure to polycyclic aromatics hydrocarbons biomarkers in human urine.

AIM: An HPLC method for the determination of 2-hydroxyfluorene (2-OHF), various hydroxyphenanthrene metabolites (1-, 2-, 3-, 4- and 9-hydroxyphenanthrene, OHPhs), 1-hydroxypyrene (1-OHPy) and 3-hydroxybenzo[a]pyrene (3-OHB[a]Py) in human urine, has been developed using fast scanning fluorimetric detection and gradient elution mode. MATERIALS & METHODS: All reagents were of analytical grade. Standard solutions were prepared separately, by exact weighing or dilution with ultrapure acetonitrile, and were stored at 4 ºC in darkness. The standard addition method was used for the analysis of urine samples. RESULTS: In the optimized conditions, 2- and 3-hydroxyphenanthrene, and 1- and 9-hydroxyphenanthrene metabolites eluted at the same retention time; however, all other hydroxy-polycyclic aromatic hydrocarbons were well resolved. Multi-emission detection allows us to monitor each metabolite at its most sensitivity emission wavelength. Detection limits ranged between 0.9 and 4.26 ng ml-1. CONCLUSION: Fortified urine samples of nonexposure and nonsmoker volunteers, previous precipitation step with acetonitrile, were used to test the proposed method. The obtained results confirm the goodness of the method.

Front-face fluorescence spectroscopy combined with second-order multivariate algorithms for the quantification of polyphenols in red wine samples.

The potential of front-face fluorescence spectroscopy combined with second-order chemometric methods was investigated for the quantification of the main polyphenols present in wine samples. Parallel factor analysis (PARAFAC) and unfolded-partial least squares coupled to residual bilinearization (U-PLS/RBL) were assessed for the quantification of catechin, epicatechin, quercetin, resveratrol, caffeic acid, gallic acid, p-coumaric acid, and vanillic acid in red wines. Excitation-emission matrices of different red wine samples, without pretreatment, were obtained in front-face mode, recording emission between 290 and 450 nm, exciting between 240 and 290 nm, for the analysis of epicatechin, catechin, caffeic acid, gallic acid, and vanillic acid; and excitation and emission between 300-360 and 330-400nm, respectively, for the analysis of resveratrol. U-PLS/RBL algorithm provided the best results and this methodology was validated by an optimized liquid chromatographic coupled to diode array and fluorimetric detectors procedure, obtaining a very good correlation for vanillic acid, caffeic acid, epicatechin and resveratrol.

Fluorescence properties of flavonoid compounds. Quantification in paprika samples using spectrofluorimetry coupled to second order chemometric tools.

The influence of pH on the fluorescence of flavonoid compounds was investigated and the highest fluorescence emission was obtained in basic medium. Selected conditions to improve this signal were: pH 9.5, 0.14 M Britton Robinson buffer and methanol between 5% and 10%. The excitation-emission fluorescence matrices of a set of 36 samples of Spanish paprika were analyzed by means of parallel factor analysis (PARAFAC). Thus, the profiles of possible fluorescence components (PARAFAC loadings) were obtained. One of these profiles was identified by matching PARAFAC scores with LC analysis on the same samples. Two clusters of samples were obtained when score values were plotted against each other. Spectrofluorimetry coupled to second order multivariate calibration methods, as unfolded-partial least squares with residual bilinearization (U-PLS/RBL) and multidimensional-partial least-squares with residual bilinearization (N-PLS/RBL), was investigated to quantify quercetin and kaempferol in those samples. Good results were obtained for quercetin by this approach.

Influence of the presence of natural monosaccharides in the quantification of α-dicarbonyl compounds in high content sugar samples. A comparative study by ultra-high performance liquid chromatography-single quadrupole mass spectrometry using different derivatization reactions.

The goal of this work is to evaluate the formation of side α-dicarbonyl compounds in high content sugar samples. These compounds may be originated from fructose and glucose, during different derivatization reactions. The formation of D-glucosone, 3-deoxyglucosone, glyoxal, and methylglyoxal, using three derivatization agents (5,6-diamino-2,4-hydroxypyrimidine, 2,4,5-triamine-6-hydroxypyrimidine, and o-phenylenediamine), and ultra-high pressure liquid chromatography in combination with MS detection, has been assessed in the presence of different levels of monosaccharides. 2,4,5-triamine-6-hydroxy-pyrimidine appears to be the most suitable for the analysis of α-dicarbonyl compounds, in this kind of food samples, and was selected as optimum reagent for the quantification of these compounds. The validation of the method was performed through the establishment of external standard calibration curves and analytical figures of merit, and it showed good linearity over a wide concentration range (r(2)>0.99), and limits of detection and quantification lower than 42µgL(-1) and 142µgL(-1), respectively. The validated method has been successfully applied to the determination of the target compounds in honey samples. The intraday and interday assay variability in the analysis of real samples was below 2.3 and 5.7%, respectively, for all analytes.

Determination of chemotherapeutic drugs in human urine by capillary electrophoresis with UV and fluorimetric detection using solid-supported liquid-liquid extraction for sample clean-up.

Capillary electrophoresis was used for the rapid determination of three chemotherapeutic drugs employed to treat colorectal cancer: irinotecan, tegafur, and leucovorin, and their main metabolites (7-ethyl-10-hydroxycamptothecin and 5-fluorouracil), in human urine samples. A phosphate buffer (pH 11.34; 20 mM) was selected as the background electrolyte. A hydrodynamic injection (9 s, 30 mbar) was applied and the separation was carried out using a separation temperature and voltage of 25°C and 25 kV, respectively. A capillary with two detection windows for serial online UV and fluorescence detection was satisfactorily employed. A solid-supported liquid-liquid extraction procedure was optimized for the clean-up of the urine samples and the extraction of the analytes. Matrix effects were assessed and signal suppression was observed for three of the analytes, thus, matrix-matched calibration was used for compensating residual matrix effects on these analytes. The proposed method allows the separation and quantification of the chemotherapeutics in less than 6 min. Detection limits range between 0.01 and 0.30 mg/L. The method was satisfactorily applied to the determination of the target compounds in human urine samples, with recoveries of 92.4-107.7%.

Green analytical determination of emerging pollutants in environmental waters using excitation-emission photoinduced fluorescence data and multivariate calibration.

An eco-friendly strategy for the simultaneous quantification of three emerging pharmaceutical contaminants is presented. The proposed analytical method, which involves photochemically induced fluorescence matrix data combined with second-order chemometric analysis, was used for the determination of carbamazepine, ofloxacin and piroxicam in water samples of different complexity without the need of chromatographic separation. Excitation-emission photoinduced fluorescence matrices were obtained after UV irradiation, and processed with second-order algorithms. Only one of the tested algorithms was able to overcome the strong spectral overlapping among the studied pollutants and allowed their successful quantitation in very interferent media. The method sensitivity in superficial and underground water samples was enhanced by a simple solid-phase extraction with C18 membranes, which was successful for the extraction/preconcentration of the pollutants at trace levels. Detection limits in preconcentrated (1:125) real water samples ranged from 0.04 to 0.3 ng mL(-1). Relative prediction errors around 10% were achieved. The proposed strategy is significantly simpler and greener than liquid chromatography-mass spectrometry methods, without compromising the analytical quality of the results.

Evaluation of liquid chromatographic behavior of lumazinic derivatives, from α-dicarbonyl compounds, in different C18 columns: application to wine samples using a fused-core column and fluorescence detection.

Several C18 columns, packed with totally porous particles of different sizes and shell thicknesses, have been compared for simultaneous determination of α-dicarbonyl compounds, previous derivatization to lumazinic derivatives. Chromatographic conditions for the separation have been optimized for each column, and chromatographic parameters have been calculated and exhaustively compared. A core-shell C18 column provided the best results, and a HPLC method with fluorimetric detection has been proposed. The developed method has been validated in terms of linearity, precision, and sensitivity. Detection and quantification limits obtained were comprised between 0.02 and 0.30 and 0.07 and 1.0 ng mL(-1), respectively, while RSD values obtained were lower than 6% and 5% in intraday and interday repeatability studies, respectively. The method has been applied to analysis of the α-dicarbonyl compounds in different types of wines. The higher levels of the total α-dicarbonyl compounds were found in sweet wines and the lower levels in white wines.

Development of a method for the determination of advanced glycation end products precursors by liquid chromatography and its application in human urine samples.

A liquid chromatographic method with fluorimetric detection has been developed to determine the most abundant α-dicarbonyl compounds, generated as intermediates in the Maillard's reaction, previous derivatization to high fluorescent pteridinic derivatives. Hence, the biomarkers D-glucosone, 3-deoxyglucosone, glyoxal, methylglyoxal, diacetyl, 2,3-pentanedione, and phenylglyoxal were quantified using a gradient elution mode. The experimental conditions of the derivatization reaction and mobile phase composition were optimized. Linearity ranges (peak area versus α-dicarbonyl compound concentration) from 1.0 to 100.0 ng mL(-1) were obtained. Detection limits were comprised between 0.3 and 11.0 ng mL(-1). The high sensitivity of the method allows the determination of α-dicarbonyl compounds present in human urine, such as D-glucosone, 3-deoxyglucosone, glyoxal, and methylglyoxal, that are used as biomarkers, in order to investigate their roles in several diseases, with special emphasis in diabetes mellitus. With the aim of avoiding the interferences due to pteridinic compounds present in urine, a cleanup step with an ISOLUTE ENV+ cartridge was carried out. The concentrations of these urinary biomarkers have been reported as a normalized ratio to urinary creatinine, and determined in healthy and in diabetic volunteers, of different ages and sex. In all urine samples, standard addition and external calibration procedures were applied and compared.

Sensitized synchronous fluorimetric determination of trans-resveratrol and trans-piceid in red wine based on their immobilization on nylon membranes.

It has been carried out the determination of trans-resveratrol and trans-piceid in red wine samples by using room temperature synchronous fluorescence, sensitized through their retention on nylon membranes, in front-face mode. These compounds are weakly fluorescent in solution but their retention allows using the native fluorescence of these compounds as analytical signal, due to the increase in the medium rigidity. To determine these compounds in red wine, a previous liquid-liquid extraction is necessary and in the case of trans-resveratrol it is also necessary a previous cleanup stage using C18 cartridges. Diethylether and ethyl acetate are the selected extractant solvents for trans-resvertarol and trans-piceid, respectively. The retention on nylon membranes was carried out by immersion of the membranes in solutions of these compounds. Variables involved in the retention and measurement processes were optimized, and the analytical figures of merit were obtained under optimal conditions. Ethanol:water 10:90 v:v and ethyl acetate were the solvents used for the retention of trans-resveratrol and trans-piceid, respectively and, for each case a immersion time of 300 and 600s was selected. Satisfactory linear relation between fluorescence intensity and concentration was found in the intervals 0.040 and 0.242mgL(-1) of trans-resvertarol and 0.009 and 0.288mgL(-1) of trans-piceid. Concentration of 1.08±0.21mgL(-1) for trans-resveratrol and 1.49±0.36mgL(-1) for trans-piceid were found in a wine sample obtained from a pool of commercial red wines.

Usefulness of fluorescence excitation-emission matrices in combination with PARAFAC, as fingerprints of red wines.

The possibility of using front-face fluorescence spectroscopy to characterize red wines was investigated, and a tentative identification of their main fluorescent components was attempted. Fifty-seven red wine samples from different origins were included in the present study. Their fluorescence excitation-emission matrices (EEMs) were registered directly on 3-mL aliquots of untreated samples. The assayed excitation and emission ranges were 245-340 and 300-500 nm, respectively. The set of 57 EEMs was analyzed by means of parallel factor analysis (PARAFAC). Thus, the spectral excitation and emission profiles of possible "pure" fluorescence components (PARAFAC loadings) and the relative contribution of each component to the individual EEMs (PARAFAC scores) were obtained. The red wine system contained four main fluorescence components, and the excitation and emission loadings had maxima at the wavelength pairs 260/380, 275/323, 330/410, and 280/364 nm, respectively. A tentative identification of fluorophores was done by matching PARAFAC score values with HPLC measurements on the same 57 samples, as well as fluorescence measurements on pure compounds typically present in red wine. It was found that the third component was highly correlated with concentrations of catechin and epicatechin. When the PARAFAC score values were plotted against each other, they did to some extent discriminate the wines according to origin (country) and grape variety.

Post-column on-line photochemical derivatization for the direct isocratic-LC-FLD analysis of resveratrol and piceid isomers in wine.

Resveratrol is a stilbene produced by plants, e.g. grapes, in response to stress. Resveratrol is extracted during winemaking, being present in wine as 3-O-ß-d-gluocoside (piceid) and as aglycone. Both, resveratrol and piceid exist in two isomeric forms, trans and cis. Resveratrol and piceid are weakly fluorescent in both of their isomeric forms, but highly fluorescent compounds are obtained when the original molecules are UV-irradiated. A chromatographic method with post-column on-line photoderivatization, has been developed for the analysis of resveratrol and piceid isomers. The four analytes are firstly separated in a C18 column (150mm×3.9mm i.d., 4µm) by isocratic elution, at 15°C, with a mobile phase consisting of a mixture acetonitrile:o-phosphoric acid (0.04%), 18:82, v:v, at 0.9mLmin(-1), and secondly they are on-line phototransformed into their fluorescent photoproducts in a 3m PTFE tube coiled around a 4W xenon lamp. The elution conditions have been chemometrically optimized by means of the experimental design and the response surface methodology. Linearity ranges from 0.10 to 1.50 and from 0.10 to 1.00µgmL(-1) and LOD around 0.001 and 0.01µgmL(-1) have been calculated for trans- and cis-isomers, respectively. The method has been satisfactorily applied to red and white wine samples by standard addition and external calibration, respectively.

Isocratic chromatography of resveratrol and piceid after previous generation of fluorescent photoproducts: wine analysis without sample preparation.

Resveratrol and its 3-glucoside (piceid), are stilbene-like molecules produced by plants. Both of them are weakly fluorescent, but highly fluorescent compounds are obtained when their hydroethanolic solutions are UV-irradiated, which implies a substantial improvement in the sensitivity of analytical methods. Experimental design (central composite design) together with the response surface methodology have been used to find optimum conditions for the fast, sensitive, and precise chromatographic analysis (with isocratic elution) of resveratrol and piceid in wine samples. These compounds have been UV-transformed into their respective photoproducts, which have been separated in a C18 column (Novapack C(18) 150x3.9 mm, 4 microm) by isocratic elution, using a mobile phase made up of acetonitrile and 4.1 vol% aqueous acetic acid, 19:81 v/v, at a flow rate of 0.8 mL/min, and fluorimetrically detected at 364 nm (lambda(exc) = 260 nm). Detection limits (S/N = 3) are 0.29 and 0.28 microg/L for resveratrol and piceid, respectively. The method has been applied to the analysis of these compounds in wine samples without a clean-up step. The analysis is completed in only 20 min. The standard addition method has been applied to the analysis of a commercial red wine and average recoveries near 100% were obtained for resveratrol and piceid. Three wine pools were satisfactorily analysed by external calibration.

High-performance liquid chromatographic determination of glyoxal and methylglyoxal in urine by prederivatization to lumazinic rings using in serial fast scan fluorimetric and diode array detectors.

Glyoxal and methylglyoxal are two important markers of oxidative stress and both are involved in the evaluation of several diseases. A new HPLC method for determining glyoxal and methylglyoxal in urine was developed. The method is based on the reaction of alpha-dialdehydes, glyoxal and methylglyoxal, with 5,6-diamino-2,4-hydroxypyrimidine sulfate in basic medium to form highly fluorescent lumazine derivatives. Creatinine was also included in the method even though it does not react with the reagent. The derivatives and creatinine are separated on a C(18) reversed-phase column with a mobile phase consisting of acetonitrile:citrate buffer, pH 6.0 (3:97 v/v). The flow rate was 1.0mLmin(-1) and the effluent was monitored photometrically at 250 nm for determination of creatinine and fluorimetrically at 500 nm (exciting at 330 nm) for determination of glyoxal and methylglyoxal derivatives. Recording time of the separation is less than 10 min. Determination of the analytes is performed in urine after incubation of the sample, with the reagent in alkaline medium, for 30 min at 60 degrees C. Urinary levels of glyoxal and methylglyoxal, expressed as glyoxal/creatinine and methylglyoxal/creatinine ratios, in healthy young women and men were determined. For women, values of 0.80+/-0.37 and 0.60+/-0.22 microg/mg of creatinine were found for glyoxal and methylglyoxal, respectively. For men, values of 0.63+/-0.15 and 0.49+/-0.05 microg/mg of creatinine were found for glyoxal and methylglyoxal, respectively. These results were also related to the body mass index of each individual.