Determination of up to twenty carboxylic acid containing compounds in clinically relevant matrices by o-benzylhydroxylamine derivatization and liquid chromatography-tandem mass spectrometry.

Carboxylic acid containing compounds (R-COOH) are involved in a large number of biological processes and they are relevant for several pathological processes such as neurodegeneration or cancer. Comprehensive methodologies for the quantitative determination of R-COOH in biological samples are required. In this study we have developed a LC-MS/MS method for the quantification of 20 endogenous R-COOH belonging to different pathways such as kynurenine metabolism, serotoninergic pathway, glycolysis, tricarboxylic acid cycle, dopaminergic pathway, short chain fatty acids and glycine metabolism. The approach included derivatization with o-benzylhydroxylamine (reaction time 1 h), liquid-liquid extraction with ethyl acetate and LC-MS/MS detection (run time 10 min). The method was optimized and validated in 5 different matrices (urine, plasma, saliva, brain and liver) following two different approaches: (i) using surrogate matrices and (ii) using actual human samples by standard additions. A suitable linearity was obtained in the endogenous range of the analytes. Adequate intra and inter-assay accuracies (80-120%) and intra- and inter-assay precisions (<20%) were achieved for almost all analytes in all studied matrices. The method was applied in several scenarios to confirm (i) human urinary changes produced in glycolysis after exercise, (ii) metabolic changes produced in rat brain and plasma by methamphetamine administration and (iii) metabolic alterations in human plasma caused by vitamin B6 deficiency. Additionally, the application of the method allowed for establishing previously unreported alterations in R-COOH metabolites under these conditions. Due to the comprehensive analyte and matrix coverage and the wide applicability of the developed methodology, it can be considered as a suitable tool for the study of R-COOH status in health and disease by targeted metabolomics.

Bioavailability of Epigallocatechin Gallate Administered With Different Nutritional Strategies in Healthy Volunteers.

The flavanol epigallocatechin gallate (EGCG) is being tested for the treatment of several diseases in humans. However, its bioavailability and pharmacokinetic profile needs a better understanding to enable its use in clinical trials. There is no consensus on the most appropriate concentration of EGCG in the body to obtain the maximum therapeutic effects. Therefore, the aim of this study is to analyze the bioavailability of EGCG orally administered alone or with different food supplements after overnight fasting in order to determine its optimal conditions (high concentrations in blood and the lowest interindividual variations) to be used as a pharmacological tool in human trials. Ten healthy volunteers (5 men and 5 women) aged 25 to 35 years were recruited prospectively. Three series of clinical experiments with a washout period of seven days among each were performed: 1) Teavigo® (EGCG extract) alone, 2) Teavigo® with a standard breakfast, and 3) FontUp® (Teavigo® commercially prepared with fats, carbohydrates, proteins, vitamins, and minerals). Blood samples were collected at 0, 30, 60, 90, 120, 180, 240, and 360 min after EGCG intake. Free EGCG in plasma was measured using a liquid chromatography and mass spectrometry UPLC-ESI-MS/MS analytical method. The pharmacokinetic variables analyzed statistically were area under the curve (AUC0-360), Cmax, Cav, Cmin, T1/2, and Tmax,. EGCG (Teavigo®) alone was the group with higher AUC0-360, Cmax, and Cav both in men (3.86 ± 4.11 µg/mL/kg/6 h; 5.95 ng/mL/kg; 2.96 ng/mL/kg) and women (3.33 ± 1.08 µg/mL/kg/6 h; 6.66 ng/mL/kg; 3.66 ng/mL). Moreover, FontUp® was the group with the highest value of T1/2 both in men (192 ± 66 min) and women (133 ± 28 min). Teavigo® intake after fasting overnight revealed the highest concentration of EGCG in plasma according to its pharmacokinetic profile, indicating that this is an excellent alternative of administration if the experimental design requires good absorption in the gastrointestinal tract. Moreover, EGCG taken along with food supplements (FontUp®) improved the stability of the molecule in the body, being the best choice if the experimental design wants to reduce interindividual variation.

Soy Isoflavone Extract Does Not Increase the Intoxicating Effects of Acute Alcohol Ingestion in Human Volunteers.

Soy beans contain isoflavones, including daidzein and genistein, with biological activities related to therapeutic effects in reducing osteoporosis, decreasing adverse menopausal manifestations, providing protection from cardiovascular diseases, and reducing hormone-dependent cancers and age-related cognitive-decline. Daidzein has been described as inhibiting the aldehyde-dehydrogenase-2 enzyme (ALDH2), and reducing alcohol use in clinical pilot studies. Our aim was to evaluate the possible interactions between a soy extract product and alcohol in a crossover, single blind, randomized study. Ten healthy male volunteers participated in two experimental sessions: one with a single dose of alcohol (0.5 g/kg, Vodka Absolut, Sweden), and the other with four capsules of a soy extract product (Super-Absorbable Soy Isoflavones, Life-Extension, United States) and, 2 h later, the same dose of alcohol. Results showed no differences in vital signs except a slightly higher significative reduction in diastolic blood pressure at 2, 3, 4, and 8 h after administration with alcohol alone in comparison with soy extract+alcohol. Ethanol-induced subjective and adverse effects were similar for both conditions with the exception of headache (higher at 8 h after alcohol alone). Our results demonstrate that a single dose of a soy isoflavone extract did not influence alcohol pharmacokinetics and pharmacological effects and did not induce any disulfiram-reaction symptoms. Soy extract and alcohol did not interact and can be administered safely.

Dose-dependent metabolic disposition of hydroxytyrosol and formation of mercapturates in rats.

Hydroxytyrosol (HT), one of the major polyphenols present in olive oil, is known to possess a high antioxidant capacity. The aim of the present study was to investigate dose dependent (0, 1, 10 and 100 mg/kg) alterations in the metabolism of HT in rats since it has been reported that metabolites may contribute to biological effects. Special attention was paid to the activation of the semiquinone-quinone oxidative cycle and the formation of adducts with potential deleterious effects. Thus, we developed a novel analytical methodology to monitor the in vivo formation of the HT mercapturate, N-acetyl-5-S-cysteinyl-hydroxytyrosol in urine samples. Biomarkers of hepatic and renal toxicity were evaluated within the dose range tested. Following HT administration, dose-dependent effects were observed for the recovery of all the metabolites studied. At the lowest dose of 1 mg/kg, the glucuronidation pathway was the most relevant (25-30%), with lower recoveries for sulfation (14%), while at the highest dose of 100 mg/kg, sulfation was the most prevalent (75%). In addition, we report for the first time the formation of the mercapturate conjugate of HT in a dose-dependent manner. The biochemical data did not reveal significant toxic effects of HT at any of the doses studied. An increase in the GSH/GSSG ratio at the highest dose was observed indicating that the products of HT autoxidation are counteracted by glutathione, resulting in their detoxification. These results indicate that the metabolic disposition of HT is highly dependent on the dose ingested.

Serotonergic neurotoxic thioether metabolites of 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy"): synthesis, isolation, and characterization of diastereoisomers.

3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) is a synthetic recreational drug of abuse that produces long-term toxicity associated with the degeneration of serotonergic nerve terminals. In various animal models, direct administration of MDMA into the brain fails to reproduce the serotonergic neurotoxicity, implying a requirement for the systemic metabolism and bioactivation of MDMA. Catechol-thioether metabolites of MDMA, formed via oxidation of 3,4-dihydroxymethamphetamine and 3,4-dihydroxyamphetamine (HHMA and HHA) and subsequent conjugation with glutathione (GSH), are selective serotonergic neurotoxicants when administered directly into brain. Moreover, following systemic administration of MDMA, the thioether adducts are present in rat brain dialysate. MDMA contains a stereogenic center and is consumed as a racemate. Interestingly, different pharmacological properties have been attributed to the two enantiomers, (S)-MDMA being the most active in the central nervous system and responsible for the entactogenic effects, and most likely also for the neurodegeneration. The present study focused on the synthesis and stereochemical analysis of the neurotoxic MDMA thioether metabolites, 5-(glutathion-S-yl)-HHMA, 5-(N-acetylcystein-S-yl)-HHMA, 2,5-bis-(glutathion-S-yl)-HHMA, and 2,5-bis-(N-acetylcystein-S-yl)-HHMA. Both enzymatic and electrochemical syntheses were explored, and methodologies for analytical and semipreparative diastereoisomeric separation of MDMA thioether conjugates by HPLC-CEAS and HPLC-UV, respectively, were developed. Synthesis, diastereoisomeric separation, and unequivocal identification of the thioether conjugates of MDMA provide the chemical tools necessary for appropriate toxicological and metabolic studies on MDMA metabolites contributing to its neurotoxicity.

Stereochemical analysis of 3,4-methylenedioxymethamphetamine and its main metabolites by gas chromatography/mass spectrometry.

3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) is consumed as the racemate but some metabolic steps are enantioselective. In addition, chiral properties are preserved during MDMA biotransformation. A quantitative analytical methodology using gas chromatography/mass spectrometry (GC/MS) to determine enantioselective disposition in the body of MDMA and its main metabolites including 3,4-methylenedioxyamphetamine (MDA), 4-hydroxy-3-methoxymethamphetamine (HMMA), and 4-hydroxy-3-methoxyamphetamine (HMA) was developed. Plasma and urine samples were collected from a male volunteer. The analysis of MDMA, MDA, and 4-hydroxy-3-methoxy metabolites by GC/MS required a two-step derivatization procedure. The first step consisted of derivatization of the amine with enantiomerically pure Mosher's reagent ((R)-MTPCl). Triethylamine was used as a base to neutralize hydrochloric acid formed during the reaction allowing quantitative derivatization, which resulted in a substantial improvement in the sensitivity of the method compared with other previously described techniques. Further treatment with ammonium hydroxide was required since both amine and hydroxyl groups underwent derivatization in the reaction. Ammonium hydroxide breaks bonds formed with hydroxyl groups without affecting amine derivatives. The second derivatization step using hexamethyldisilazane was needed for metabolites containing phenol residues. This derivatization method permitted the stereochemically specific study of MDMA and its main monohydroxylated metabolites by GC/MS. A detailed study of the chemical reactions involved in the derivatization steps was indispensable to develop a straightforward, sensitive, and reproducible method for the analysis of the parent drug compound and its metabolites.

Determination of MDMA and its metabolites in blood and urine by gas chromatography-mass spectrometry and analysis of enantiomers by capillary electrophoresis.

A gas chromatography-mass spectrometry (GC-MS) method was used for the simultaneous quantitation of 3,4-methylenedioxymethamphetamine (MDMA) and the 3,4-methylenedioxyamphetamine (MDA), 4-hydroxy-3-methoxymethamphetamine (HMMA), and 4-hydroxy-3-methoxyamphetamine (HMA) metabolites in plasma and urine samples after the administration of 100 mg MDMA to healthy volunteers. Samples were hydrolyzed prior to a solid-phase extraction with Bond Elut Certify columns. Analytes were eluted with ethyl acetate (2% ammonium hydroxide) and analyzed as their trifluoroacyl derivatives. Linear calibration curves were obtained at plasma and urine concentration ranges of 25-400 ng/mL and 250-2000 ng/mL for MDMA and HMMA, and of 2.5-40 ng/mL and 100-1000 ng/mL for MDA and HMA. Following the same urine preparation procedure but without the derivatization step, a capillary electrophoresis (CE) method for enantiomerical resolution of compounds was developed using (2-hydroxy)propyl-beta-cyclodextrin at two different concentrations (10 and 50mM in 50mM H3PO4, pH 2.5) as chiral selector. Calibration curves for the CE method were prepared with the corresponding racemic mixture and were linear between 125 and 2000 ng/mL, 50 and 1000 ng/mL, and 125 and 1500 ng/mL for each enantiomer of MDMA, MDA, and HMMA, respectively. Stereoselective disposition of MDMA and MDA was confirmed. HMMA disposition seems to be in apparent contradiction with MDMA findings as the enantiomer ratio is close to 1 and constant over the time.