Searching trans-resveratrol in fruits and vegetables: a preliminary screening.

Resveratrol is a phytoalexin with potent anti-inflammatory, anti-oxidant and anti-carcinogenic effects. The object of this work was to determine whether this promising compound was present in the typical fruits and vegetables used in the Mediterranean diet. Our results indicated the presence of trans-resveratrol in concentrations ranging from 0.2 µg/g in tomatoes and 3 lg/g. in strawberries.

Towards the potential use of (1)H NMR spectroscopy in urine samples for prostate cancer detection.

A simple method based in multivariate analysis of (1)H NMR spectra profiles of urine samples can be used to detect patients with prostate cancer.

In vitro/in vivo screening of oxidative homeostasis and damage to DNA, protein, and lipids using UPLC/MS-MS.

Multiple analytical methods are required to comprehensively assess oxidative homeostasis and specific damage to macromolecules. Our aim was to develop a straightforward strategy for the fast assessment of global oxidative status and specific damage to DNA, proteins, and lipids. To this end, an analytical method, based on ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS/MS), was developed and validated for the quantification of 16 oxidative stress (OS) biomarkers. Some of these markers were unstable; thus, an easy sample treatment procedure, including fractionation and derivatization, was set up. The method was validated according to Food and Drug Administration (FDA) guidelines, and it provided good results in terms of intra- and inter-day precision (≤17.2 and 16 %, respectively), accuracy (relative error measurement between -16.6 and 19.8 %), and linearity (R (2) > 0.994). The approach was applied to determine the oxidative insult provoked to cultured rat hepatocytes by cumene hydroperoxide and to analyze the liver and serum samples from patients diagnosed with nonalcoholic steatohepatitis. In both studies, significant differences were found if compared to the corresponding control groups; interestingly, ophthalmic acid was shown as an OS biomarker in both models for the first time. A key advantage of the novel approach in comparison with former multi-method approaches is that now a single method is applied to assess the 16 OS biomarkers. Its comprehensive capacity to profile oxidative homeostasis and damage in both in vitro and clinical samples has been illustrated, which indicates that the proposed approach is a good choice to evaluate whether OS is involved in physiological signals, diseases, or toxic events and to what extent.

Mammalian cell metabolomics: experimental design and sample preparation.

Metabolomics represents the global assessment of metabolites in a biological sample and reports the closest information to the phenotype of the biological system under study. Mammalian cell metabolomics has emerged as a promising tool with potential applications in many biotechnology and research areas. Metabolomics workflow includes experimental design, sampling, sample processing, metabolite analysis, and data processing. Given their influence on metabolite content and biological interpretation of data, a good experimental design and the appropriate choice of a sample processing method are prerequisites for success in any metabolomic study. The use of mammalian cells in the metabolomics field involves harder sample processing methods, including metabolism quenching and metabolite extraction, as compared to the use of body fluids, although such critical issues are frequently overlooked. This review aims to overview the common experimental procedures used in mammalian cell metabolomics based on mass spectrometry, by placing special emphasis on discussing sample preparation approaches, although other aspects, such as cell metabolomics applications, culture systems, cellular models, analytical platforms, and data analysis, are also briefly covered. This review intends to be a helpful tool to assist researchers in addressing decisions when planning a metabolomics study involving the use of mammalian cells.

Solid-phase extraction liquid chromatography-tandem mass spectrometry analytical method for the determination of 2-hydroxy-4-methoxybenzophenone and its metabolites in both human urine and semen.

2-Hydroxy-4-methoxybenzophenone (HMB), which is one of the most commonly used UV filters in sunscreen cosmetics to protect skin from the deleterious effects of the sun, can be percutaneously absorbed, further metabolized, and finally excreted or bioaccumulated. An analytical method for the sensitive determination of HMB and its three metabolites in both human urine and semen is developed. The presented analytical method is based on a solid-phase extraction (SPE) procedure to clean-up and preconcentrate the target analytes from the urine and semen samples followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection. The methodology was fully validated and the standard addition calibration method was used to quantify the target analytes in order to correct the matrix effects observed. Considering this approach, the accuracy of the method was evaluated and the recoveries ranged from 98% to 115% and from 86% to 111% in urine and semen samples, respectively, depending on the analyte. For urine samples, the limits of detection ranged between 0.027 and 0.103 ng mL(-1) and the repeatability of the method, expressed as relative standard deviation, was in the range of 7.2-9.2%, depending on the analyte. In the case of semen samples, the limits of detection ranged between 1 and 3 ng mL(-1) whereas the repeatability was in the range of 2.2-6.4%, depending on the analyte. The described SPE-LC-MS/MS method was satisfactorily applied to both urine and semen samples from a male volunteer who applied a sunscreen cosmetic product containing HMB. HMB and its metabolites were found and quantified in the low ng mL(-1) range in both urine and semen samples, although at a different extent.

Identification of the Biotransformation Products of 2-Ethylhexyl 4-(N,N-Dimethylamino)benzoate.

Nowadays, 2-ethylhexyl 4-(N,N-dimethylamino)benzoate (EDP) is one of the most widely used UV filters in sunscreen cosmetics and other cosmetic products. However, undesirable processes such as percutaneous absorption and biological activity have been attributed to this compound. The in vitro metabolism of EDP was elucidated in the present work. First of all, the phase I biotransformation was studied in rat liver microsomes and two metabolites, N,N-dimethyl-p-aminobenzoic acid (DMP) and N-monomethyl-p-aminobenzoic acid (MMP), were identified by GC-MS analysis. Secondly, the phase II metabolism was investigated by means of LC-MS. The investigated reactions were acetylation and glucuronidation working with rat liver cytosol and with both human and rat liver microsomes, respectively. Analogue studies with p-aminobenzoic acid (PABA) were carried out in order to compare the well established metabolic pathway of PABA with the unknown biotransformation of EDP. In addition, a method for the determination of EDP and its two phase I metabolites in human urine was developed. The methodology requires a solid-phase extraction prior to LC-MS analysis. The method is based on standard addition quantification and has been fully validated. The repeatability of the method, expressed as relative standard deviation, was in the range 3.4-7.4% and the limit of detection for all quantified analytes was in the low ng mL(-1) range.

Determination of hydroxylated benzophenone UV filters in sea water samples by dispersive liquid-liquid microextraction followed by gas chromatography-mass spectrometry.

A new analytical method for the determination of four hydroxylated benzophenone UV filters (i.e. 2-hydroxy-4-methoxybenzophenone (HMB), 2,4-dihydroxybenzophenone (DHB), 2,2'-dihydroxy-4-methoxybenzophenone (DHMB) and 2,3,4-trihydroxybenzophenone (THB)) in sea water samples is presented. The method is based on dispersive liquid-liquid microextraction (DLLME) followed by gas chromatography-mass spectrometry (GC-MS) determination. The variables involved in the DLLME process were studied. Under optimized conditions, 1000 microL of acetone (disperser solvent) containing 60 microL of chloroform (extraction solvent) were injected into 5 mL of aqueous sample adjusted to pH 4 and containing 10% NaCl. Before injecting into the GC-MS system, the DLLME extracts were evaporated under an air stream and then reconstituted with N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA), thus allowing the target analytes to be converted into their trimethylsilyl derivatives. The best conditions for the derivatization reaction were 75 degrees C and 30 min. High enrichment factors for all the target analytes (ranging from 58 to 64) and good repeatability (RSD around 6%) were obtained. The limits of detection were in the range of 32-50 ng L(-1), depending on the analyte. The recoveries obtained by using the proposed DLLME-GC-MS method evidenced the presence of matrix effects for some of the target analytes, and thereby the standard addition calibration method was employed. Finally, the validated method was applied to the analysis of sea water samples.

Development of a fully automated sequential injection solid-phase extraction procedure coupled to liquid chromatography to determine free 2-hydroxy-4-methoxybenzophenone and 2-hydroxy-4-methoxybenzophenone-5-sulphonic acid in human urine.

2-Hydroxy-4-methoxybenzophenone and 2-hydroxy-4-methoxybenzophenone-5-sulphonic acid, commonly known as benzophenone-3 (BZ3) and benzophenone-4 (BZ4), respectively, are substances widely used as UV filters in cosmetic products in order to absorb UV radiation and protect human skin from direct exposure to the deleterious wavelengths of sunlight. As with other UV filters, there is evidence of their percutaneous absorption. This work describes an analytical method developed to determine trace levels of free BZ3 and BZ4 in human urine. The methodology is based on a solid-phase extraction (SPE) procedure for clean-up and pre-concentration, followed by the monitoring of the UV filters by liquid chromatography-ultraviolet spectrophotometry detection (LC-UV). In order to improve not only the sensitivity and selectivity, but also the precision of the method, the principle of sequential injection analysis was used to automate the SPE process and to transfer the eluates from the SPE to the LC system. The application of a six-channel valve as an interface for the switching arrangements successfully allowed the on-line connection of SPE sample processing with LC analysis. The SPE process for BZ3 and BZ4 was performed using octadecyl (C18) and diethylaminopropyl (DEA) modified silica microcolumns, respectively, in which the analytes were retained and eluted selectively. Due to the matrix effects, the determination was based on standard addition quantification and was fully validated. The relative standard deviations of the results were 13% and 6% for BZ3 and BZ4, respectively, whereas the limits of detection were 60 and 30 ng mL(-1), respectively. The method was satisfactorily applied to determine BZ3 and BZ4 in urine from volunteers that had applied a sunscreen cosmetic containing both UV filters.