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Inspiratory muscle training (IMT) is known to promote physiological benefits and improve physical performance in endurance sports activities. However, the metabolic adaptations promoted by different IMT prescribing strategies remain unclear. In this work, a longitudinal, randomized, double-blind, sham-controlled, parallel trial was performed to investigate the effects of 11 weeks (3 days·week-1) of IMT at different exercise intensities on the serum metabolomics profile and its main regulated metabolic pathways. Twenty-eight healthy male recreational cyclists (30.4 ± 6.5 years) were randomized into three groups: sham (6 cm·H2O of inspiratory pressure, n = 7), moderate-intensity (MI group, 60% maximal inspiratory pressure (MIP), n = 11) and high-intensity (HI group, 85-90% MIP, n = 10). Blood serum samples were collected before and after 11 weeks of IMT and analyzed by 1H NMR and UHPLC-HRMS/MS. Data were analyzed using linear mixed models and metabolite set enrichment analysis. The 1H NMR and UHPLC-HRMS/MS techniques resulted in 46 and 200 compounds, respectively. These results showed that ketone body metabolism, fatty acid biosynthesis, and aminoacyl-tRNA biosynthesis were upregulated after IMT, while alpha linolenic acid and linoleic acid metabolism as well as biosynthesis of unsaturated fatty acids were downregulated. The MI group presented higher MIP, Tryptophan, and Valine levels but decreased 2-Hydroxybutyrate levels when compared to the other two studied groups. These results suggest an increase in the oxidative metabolic processes after IMT at different intensities with additional evidence for the upregulation of essential amino acid metabolism in the MI group accompanied by greater improvement in respiratory muscle strength.
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Ejercicios Respiratorios , Suero , Humanos , Masculino , Ejercicios Respiratorios/métodos , Cromatografía Líquida de Alta Presión , Fuerza Muscular/fisiología , Espectroscopía de Protones por Resonancia Magnética , Músculos Respiratorios , Estudios LongitudinalesRESUMEN
Individuals with sickle cell disease (SCD) often experience numerous vaso-occlusive crisis events throughout their lives, which can progress to severe damage of several organs, including avascular necrosis, also known as osteonecrosis (ON). Osteonecrosis is one of the most devastating musculoskeletal clinical manifestations of sickle cell disease, afflicting up to 50% of the SCD patients. Herein, a NMR-based untargeted metabolomics approach was used to assess the metabolome alterations of blood plasma and bone marrow interstitial fluid (BMIF) samples of SCD patients with osteonecrosis. Furthermore, biochemical signatures associated with different osteonecrosis stages were assessed by analysing the metabolome of blood plasma and bone marrow interstitial fluid samples of SCD patients with different stages of the disease based on the Fiat and Arlet classification (FAC). Multivariate statistical analysis allowed a clear discrimination between the studied groups and it provided important insights into the different osteonecrosis stages. Citrate was pointed out as a possible biomarker to differentiate SCD patients with and without osteonecrosis. Acetate, creatinine, histidine, tyrosine, glucose, and NI5 seems to be key metabolites associated to different stages of the disease. Although this is a pioneer exploratory study, we acknowledge that fact that it is limited by the group sizes and absence of a validation cohort. Nevertheless, multivariate statistical analyses indicated that the metabolome of blood plasma and BMIF samples encompasses a complex metabolic regulation system for osteonecrosis.
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Anemia de Células Falciformes , Osteonecrosis , Humanos , Líquido Extracelular , Médula Ósea , Metabolómica , PlasmaRESUMEN
Aging process is characterized by a progressive decline of several organic, physiological, and metabolic functions whose precise mechanism remains unclear. Metabolomics allows the identification of several metabolites and may contribute to clarifying the aging-regulated metabolic pathways. We aimed to investigate aging-related serum metabolic changes using a metabolomics approach. Fasting blood serum samples from 138 apparently healthy individuals (20−70 years old, 56% men) were analyzed by Proton Nuclear Magnetic Resonance spectroscopy (1H NMR) and Liquid Chromatography-High-Resolution Mass Spectrometry (LC-HRMS), and for clinical markers. Associations of the metabolic profile with age were explored via Correlations (r); Metabolite Set Enrichment Analysis; Multiple Linear Regression; and Aging Metabolism Breakpoint. The age increase was positively correlated (0.212 ≤ r ≤ 0.370, p < 0.05) with the clinical markers (total cholesterol, HDL, LDL, VLDL, triacylglyceride, and glucose levels); negatively correlated (−0.285 ≤ r ≤ −0.214, p < 0.05) with tryptophan, 3-hydroxyisobutyrate, asparagine, isoleucine, leucine, and valine levels, but positively (0.237 ≤ r ≤ 0.269, p < 0.05) with aspartate and ornithine levels. These metabolites resulted in three enriched pathways: valine, leucine, and isoleucine degradation, urea cycle, and ammonia recycling. Additionally, serum metabolic levels of 3-hydroxyisobutyrate, isoleucine, aspartate, and ornithine explained 27.3% of the age variation, with the aging metabolism breakpoint occurring after the third decade of life. These results indicate that the aging process is potentially associated with reduced serum branched-chain amino acid levels (especially after the third decade of life) and progressively increased levels of serum metabolites indicative of the urea cycle.
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Ácido Aspártico , Isoleucina , Masculino , Humanos , Adulto Joven , Adulto , Persona de Mediana Edad , Anciano , Femenino , Leucina , Metabolómica/métodos , Metaboloma/fisiología , Envejecimiento , Biomarcadores , Valina , Ornitina , UreaRESUMEN
The extensive use of medicinal herbs to traditionally treat disease persists for generations, and scientific evidence on plant-derived extracts has indicated their numerous biological activities. The Bauhinia, popular known as cow's paw ("pata de vaca"), with more than 60 native species, are extensively used in Brazilian popular medicine for the control of diabetes. Therefore, in 2009, B. forficata, B. variegata and/or B. affinis were included in the Brazilian National List of Medicinal Plants of Interest to SUS (RENISUS - Brazil). In this context, this work reports the results of the chemical differentiation of B. forficata, B. variegata, B. longifolia, and B. affinis using liquid chromatography coupled to high-resolution mass spectrometry and unsupervised chemometric tools. Chromatographic conditions were optimized by using the design of experiments (DoE) and chromatographic knowledge. Furthermore, the chemical profile of the studied species was analyzed by principal component analysis (PCA) and hierarchical cluster analysis that differentiated the four species of Bauhinia, and 55 compounds were also inferred by MS2 experiments, some of them for the first time in B. affinis. In this manner, this work provides important information that could be used in quality control, development of new pharmaceuticals, and food products based on Bauhinia leaves, as well as to explain ethnomedicinal properties, pharmacological and toxicological actions.
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Metabolomics is a discipline that offers a comprehensive analysis of metabolites in biological samples. In the last decades, the notable evolution in liquid chromatography and mass spectrometry technologies has driven an exponential progress in LC-MS-based metabolomics. Targeted and untargeted metabolomics strategies are important tools in health and medical science, especially in the study of disease-related biomarkers, drug discovery and development, toxicology, diet, physical exercise, and precision medicine. Clinical and biological problems can now be understood in terms of metabolic phenotyping. This overview highlights the current approaches to LC-MS-based metabolomics analysis and its applications in the clinical research.
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Medicina , Metabolómica , Cromatografía Liquida , Espectrometría de Masas , MetabolomaRESUMEN
Metabolomics and lipidomics have demonstrated increasing importance in underlying biochemical mechanisms involved in the pathogenesis of diseases to identify novel drug targets and/or biomarkers for establishing therapeutic approaches for human health. Particularly, bioactive metabolites and lipids have biological activity and have been implicated in various biological processes in physiological conditions. Thus, comprehensive metabolites, and lipids profiling are required to obtain further advances in understanding pathophysiological changes that occur in cells and tissues. Chirality is one of the most important phenomena in living organisms and has attracted long-term interest in medical and natural science. Enantioselective separation plays a pivotal role in understanding the distribution and physiological function of a diversity of chiral bioactive molecules. In this context, it has been the goal of method development for targeted and untargeted metabolomics and lipidomic assays. Herein we will highlight the benefits and challenges involved in these stereoselective analyses for clinical samples.
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Lipidómica/métodos , Metabolómica/métodos , Aminoácidos/química , Aminoácidos/metabolismo , Animales , Biomarcadores/química , Biomarcadores/metabolismo , Cromatografía Liquida , Humanos , Espectrometría de Masas , EstereoisomerismoRESUMEN
In recent decades, fungi-derived naturally occurring quinazolines have emerged as potential drug candidates. Nevertheless, most studies are conducted for bioactivity assays, and little is known about their absorption, distribution, metabolism, and elimination (ADME) properties. To perform metabolic studies, the synthesis of the naturally occurring quinazolinone, fiscalin B (1), and its chloro derivative, 4-((1H-indol-3-yl)methyl)-8,10-dichloro-1-isobutyl-1,2-dihydro-6H-pyrazino[2,1-b]quinazoline-3,6(4H)-dione (2), disclosed as an antibacterial agent, was performed in a gram scale using a microwave-assisted polycondensation reaction with 22% and 17% yields, respectively. The structure of the non-natural (+)-fiscalin B was established, for the first time, by X-ray crystallography as (1R,4S)-1, and the absolute configuration of the naturally occurring fiscalin B (-)-1 was confirmed by comparison of its calculated and experimental electronic circular dichroism (ECD) spectra as (1S,4R)-1. in vitro metabolic studies were monitored for this class of natural products for the first time by ultra-high-performance liquid chromatography (UHPLC) coupled with high-resolution mass spectrometry (HRMS). The metabolic characteristics of 1 and 2 in human liver microsomes indicated hydration and hydroxylation mass changes introduced to the parent drugs.
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Antibacterianos/metabolismo , Productos Biológicos/metabolismo , Metaboloma/genética , Pirazinas/metabolismo , Antibacterianos/síntesis química , Antibacterianos/química , Productos Biológicos/síntesis química , Productos Biológicos/química , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Cristalografía por Rayos X , Hongos/efectos de los fármacos , Humanos , Indoles/síntesis química , Indoles/química , Indoles/metabolismo , Espectrometría de Masas , Estructura Molecular , Pirazinas/síntesis química , Pirazinas/química , Quinazolinas/síntesis química , Quinazolinas/química , Quinazolinas/metabolismo , EstereoisomerismoRESUMEN
Sickle cell anaemia (SCA) is a debilitating genetic haemoglobinopathy predominantly affecting the disenfranchised strata of society in Africa and the Americas. The most common pharmacological treatment for this disease is the administration of hydroxycarbamide (HC) for which questions remain regarding its mechanism of action, efficacy and long-term toxicity specifically in paediatric individuals. A multiplatform metabolomics approach was used to assess the metabolome of plasma samples from a population of children and adolescents with SCA with and without HC treatment along with non-SCA individuals. Fifty-three metabolites were identified by ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) and 1 H nuclear magnetic resonance (NMR) with a predominance of membrane lipids, amino acids and organic acids. The partial least-squares discriminant analysis (PLS-DA) analysis allowed a clear discrimination between the different studied groups, revealing clear effects of the HC treatment in the patients' metabolome including rescue of specific metabolites to control levels. Increased creatine/creatinine levels under HC treatment suggests a possible increase in the arginine pool and increased NO synthesis, supporting existing models for HC action in SCA. The metabolomics results extend the current knowledge on the models for SCA pathophysiology including impairment of Lands' cycle and increased synthesis of sphingosine 1-phosphate. Putative novel biomarkers are suggested.
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Anemia de Células Falciformes/sangre , Antidrepanocíticos/uso terapéutico , Hidroxiurea/uso terapéutico , Metabolómica , Ácidos/sangre , Síndrome Torácico Agudo/etiología , Adolescente , Aminoácidos/sangre , Anemia de Células Falciformes/complicaciones , Anemia de Células Falciformes/tratamiento farmacológico , Antidrepanocíticos/farmacología , Arteriopatías Oclusivas/etiología , Biomarcadores , Butiratos/sangre , Niño , Cromatografía Líquida de Alta Presión , Creatina/sangre , Creatinina/sangre , Femenino , Humanos , Hidroxiurea/farmacología , Lisofosfolípidos/sangre , Masculino , Espectrometría de Masas , Lípidos de la Membrana/sangre , Modelos Biológicos , Resonancia Magnética Nuclear Biomolecular , Esfingosina/análogos & derivados , Esfingosina/sangreRESUMEN
A potent synthetic α2-adrenergic agonist called PT-31, (3-(2-chloro-6-fluorobenzyl)-imidazolidine-2,4-dione), was recently detected as a potential drug to be used as an adjuvant drug to treat chronic pain. The excellent pharmacological property of PT-31 highlights the importance in elucidating its metabolism, which could provide valuable information about its metabolite profile for further pharmacokinetics studies and additionally to estimate the impact of its metabolites on the efficacy, safety and elimination of PT-31. In this work, the study of the in vitro metabolism of PT-31 was initially carried out by using a liquid chromatography coupled to ion trap multiple-stage mass spectrometer (LC-IT-MSn) and a hybrid triple quadrupole/linear ion trap mass spectrometer (LC-QTrap). The production of at least three unknown oxidative metabolites was observed. Structural identification of the unknown metabolites was carried out by combination of LC-MS experiments, including selected reaction monitoring (SRM) and multi-stage full scan experiments. Further analysis of 1H-NMR led to the structural confirmation of the major metabolite. The results indicated that PT-31 was metabolized by a hydroxylation reaction in the imidazolidine-2,4-dione ring in rat and human liver microsomes, producing the metabolite 3-(2-chloro-6-fluorobenzyl)-5-hydroxyimidazolidine-2,4-dione in rat liver microsomes. A carbon hydroxylation onto the benzyl ring, produced two other minor metabolites of the PT-31 in rat liver microsomes.
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Agonistas de Receptores Adrenérgicos alfa 2/metabolismo , Analgésicos/metabolismo , Microsomas Hepáticos/metabolismo , Agonistas de Receptores Adrenérgicos alfa 2/farmacocinética , Agonistas de Receptores Adrenérgicos alfa 2/uso terapéutico , Analgésicos/farmacocinética , Analgésicos/uso terapéutico , Animales , Dolor Crónico/tratamiento farmacológico , Evaluación Preclínica de Medicamentos , Humanos , Imidazolidinas/metabolismo , Imidazolidinas/farmacocinética , Imidazolidinas/uso terapéutico , Espectroscopía de Resonancia Magnética , Oxidación-Reducción , Ratas , Espectrometría de Masas en TándemRESUMEN
This paper reports an online SPE-LC-MS/MS method for the simultaneous quantification of prostaglandins (PGE2 and PGF2α) in menstrual fluid samples. To meet this goal human peripheral serum was used as surrogate matrix. The analytes were trapped on an OASIS HLB cartridge for 3â¯min, for sample cleanup and enrichment, and then transferred during only 42â¯s to an HSS T3 C18 analytical column, for separation and analysis. Prostaglandins (PGs) were detected by selected reaction monitoring in negative ion mode, PGE2 (m/z 351â¯ââ¯315) and PGF2α (m/z 353â¯ââ¯193) using isotope-labeled internal standard (PGE2-d4, m/z 355â¯ââ¯319). The concentration linear range was of 10.34-1.034â¯ngâ¯mL-1 and the lower limit of quantification (LLOQ) was 10.34â¯ngâ¯mL-1 for both PGs. Validation parameters were successfully assessed according to the European Medicines Agency guideline (EMA), also comprising the FDA normative. The method showed no matrix effect and process efficiency around 100%, in addition to only 15â¯min of analysis time with lower solvent consumption. The method application was carried out using two menstrual fluid sample groups: control (nâ¯=â¯15) and treatment group (nâ¯=â¯7; samples from women that used Tahiti lemon juice). The PGF2α levels were found to be higher in treated group than in control group (pâ¯≤â¯0.05), denoting an effect of the intake of Tahiti lemon juice on the menstrual inflammatory process. The on-line method herein reported could be useful for the analysis of PGs from large research studies.
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Cromatografía Liquida/métodos , Dinoprost/sangre , Dinoprostona/sangre , Menstruación/sangre , Extracción en Fase Sólida/métodos , Espectrometría de Masas en Tándem/métodos , Adolescente , Adulto , Dinoprost/aislamiento & purificación , Dinoprostona/aislamiento & purificación , Femenino , Humanos , Límite de Detección , Modelos Lineales , Reproducibilidad de los Resultados , Adulto JovenRESUMEN
Azo dyes are known as a group of substances with DNA damage potential that depend on the nature and number of azo groups connected to aromatic rings (benzene and naphthalene), chemical properties, e.g. solubility and reactive functional groups, which significantly affect their toxicological and ecological risks. In this paper, we used in vitro models to evaluate the metabolism of selected textile dyes: Disperse Red 73 (DR 73), Disperse Red 78 (DR 78) and Disperse Red 167 (DR 167). To evaluate the mutagenic potential of the textile dyes, the Salmonella mutagenicity assay (Ames test) with strains TA 98 and TA 100 in the presence and absence of the exogenous metabolic system (S9) was used. DR73 was considered the most mutagenic compound, inducing both replacement base pairs (TA 100) and also changing frameshift (TA 98) mutations that are reduced in the presence of the S9 mixture. Furthermore, we used rat liver microsomes in the same experimental conditions of the S9 mixture to metabolize the dyes and the resultant solutions were analyzed using a liquid chromatography coupled to a quadrupole linear ion trap mass spectrometry (LC-MS/MS) to investigate the metabolites formed by the in vitro biotransformation. Based on this experiment, we detected and identified two biotransformation products for each textile dye substrate analyzed. Furthermore, to evaluate the interaction and reactivity of these compounds with DNA, theoretical calculations were also carried out. The results showed that the chemical reaction occurred preferentially at the azo group and the nitro group, indicating that there was a reduction in these groups by the CYP P450 enzymes presented in the rat microsomal medium. Our results clearly demonstrated that the reduction of these dyes by biological systems is a great environmental concern due to increased genotoxicity for the body of living beings, especially for humans.
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Compuestos Azo/metabolismo , Colorantes/metabolismo , ADN/química , Pruebas de Mutagenicidad , Animales , Biotransformación , Cromatografía Liquida , Microsomas Hepáticos/metabolismo , Modelos Teóricos , Mutágenos , Ratas , Salmonella , Salmonella typhimurium , Espectrometría de Masas en TándemRESUMEN
Perturbation of organic anion transporter (OAT) 1- and OAT3-mediated transport can alter the exposure, efficacy, and safety of drugs. Although there have been reports of the endogenous biomarkers for OAT1/3, none of these have all of the characteristics required for a clinical useful biomarker. Cynomolgus monkeys were treated with intravenous probenecid (PROB) at a dose of 40 mg/kg in this study. As expected, PROB increased the area under the plasma concentration-time curve (AUC) of coadministered furosemide, a known substrate of OAT1 and OAT3, by 4.1-fold, consistent with the values reported in humans (3.1- to 3.7-fold). Of the 233 plasma metabolites analyzed using a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolomics method, 29 metabolites, including pyridoxic acid (PDA) and homovanillic acid (HVA), were significantly increased after either 1 or 3 hours in plasma from the monkeys pretreated with PROB compared with the treated animals. The plasma of animals was then subjected to targeted LC-MS/MS analysis, which confirmed that the PDA and HVA AUCs increased by approximately 2- to 3-fold by PROB pretreatments. PROB also increased the plasma concentrations of hexadecanedioic acid (HDA) and tetradecanedioic acid (TDA), although the increases were not statistically significant. Moreover, transporter profiling assessed using stable cell lines constitutively expressing transporters demonstrated that PDA and HVA are substrates for human OAT1, OAT3, OAT2 (HVA), and OAT4 (PDA), but not OCT2, MATE1, MATE2K, OATP1B1, OATP1B3, and sodium taurocholate cotransporting polypeptide. Collectively, these findings suggest that PDA and HVA might serve as blood-based endogenous probes of cynomolgus monkey OAT1 and OAT3, and investigation of PDA and HVA as circulating endogenous biomarkers of human OAT1 and OAT3 function is warranted.
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Biomarcadores/sangre , Ácido Homovanílico/sangre , Proteína 1 de Transporte de Anión Orgánico/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Ácido Piridóxico/sangre , Animales , Transporte Biológico/fisiología , Línea Celular , Células HEK293 , Humanos , Macaca fascicularis , Metabolómica/métodos , Probenecid/metabolismoRESUMEN
This work presents an in vitro investigation of the effect of protein restriction on the metabolism of albendazole (ABZ). This study was conducted using liver microsomal fractions obtained from Wistar rats. For the quantitative analysis, a multidimensional High Performance Liquid Chromatography (2D HPLC) method was fully validated for the determination of the ABZ metabolites: albendazole sulfoxide, albendazole sulfone and albendazole 2-aminesulfone. The target compounds were directly extracted using a C8-RAM-BSA column (5.0x0.46 cm i.d.) and analyzed on a chromatographic chiral column containing amylose tris(3,5-dimethylphenylcarbamate) (150x4.6 mm i.d.). The in vitro biotransformation results showed that the protein restriction influenced the oxidative metabolism of ABZ. The production of R-(+)-ABZ-SO (1309 nmol/L) and S-(-)-ABZ-SO (1456 nmol/L) was higher in the control animals than in the animals fed with a diet containing 6% protein, which produced 778.7 nmol/L and 709.5 nmol/L for R-(+) and S-(-)-ABZ-SO enantiomers, respectively. These results were statistically inspected by Student´s t test and the results showed a significant difference between the two means (p<0.05). Moreover, the production of ABZ-SO enantiomers was enantioselective where the S-(-)-ABZ-SO was formed in greater amounts than the R-(+)-ABZ-SO in control animals (p=0.0231). However, the enantioselectivity was not observed when the in vitro biotransformation of ABZ was conducted using the microsomal fractions obtained from protein restriction animals (p>0.05). Furthermore, animal nutritional condition could affect the pattern of ABZ sulphoxidation indicating that the protein nutrition affect primarily the formation of R-(+)-ABZSO and S-(-)-ABZ-SO enantiomers.
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Albendazol/farmacocinética , Dieta con Restricción de Proteínas , Hígado/metabolismo , Desnutrición Proteico-Calórica/metabolismo , Albendazol/administración & dosificación , Albendazol/análogos & derivados , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Biotransformación , Modelos Animales de Enfermedad , Masculino , Microsomas Hepáticos/metabolismo , Estado Nutricional , Oxidación-Reducción , Desnutrición Proteico-Calórica/etiología , Desnutrición Proteico-Calórica/fisiopatología , Ratas WistarRESUMEN
RATIONALE: Pharmacokinetic data to support clinical development of pharmaceuticals are routinely obtained from liquid plasma samples. The plasma samples require frozen shipment and storage and are extracted off-line from the liquid chromatography/tandem mass spectrometry (LC/MS/MS) systems. In contrast, the use of dried blood spot (DBS) sampling is an attractive alternative in part due to its benefits in microsampling as well as simpler sample storage and transport. However, from a practical aspect, sample extraction from DBS cards can be challenging as currently performed. The goal of this report was to integrate automated serial extraction of large numbers of DBS cards with on-line liquid chromatography/high-resolution accurate mass spectrometry (LC/HRAMS) bioanalysis. METHODS: An automated system for direct DBS extraction coupled to a LC/HRAMS was employed for the quantification of midazolam (MDZ) and α-hydroxymidazolam (α-OHMDZ) in human blood. The target analytes were directly extracted from the DBS cards onto an on-line chromatographic guard column followed by HRAMS detection. No additional sample treatment was required. The automated DBS LC/HRAMS method was developed and validated, based on the measurement at the accurate mass-to-charge ratio of the target analytes to ensure specificity for the assay. RESULTS: The automated DBS LC/HRAMS method analyzed a DBS sample within 2 min without the need for punching or additional off-line sample treatment. The fully automated analytical method was shown to be sensitive and selective over the concentration range of 5 to 2000 ng/mL. Intra- and inter-day precision and accuracy was less than 15% (less than 20% at the LLOQ). The validated method was successfully applied to measure MDZ and α-OHMDZ in an incurred human sample after a single 7.5 mg dose of MDZ. CONCLUSIONS: The direct DBS LC/HRAMS method demonstrated successful implementation of automated DBS extraction and bioanalysis for MDZ and α-OHMDZ. This approach has the potential to promote workload reduction and sample throughput increase.
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Cromatografía Liquida/métodos , Pruebas con Sangre Seca/métodos , Espectrometría de Masas en Tándem/métodos , Automatización , Humanos , Modelos Lineales , Masculino , Midazolam/análogos & derivados , Midazolam/sangre , Modelos Químicos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Online automated extraction of dried blood spots (DBS) via direct extraction to a solid-phase extraction (SPE) cartridge and bioanalysis by high-resolution accurate mass spectrometry was examined. The methodology was validated and used to investigate the effect of hematocrit on assay bias using partial and whole spot extractions from accurately dispensed blood samples. RESULTS: The completed analysis of a DBS sample was accomplished within 2 to 3 min using the online DBS-SPE platform. Hematocrit related bias was observed (>15%) for the partial DBS extractions, but not when the whole DBS was eluted. CONCLUSION: RESULTS demonstrate successful implementation of automated online DBS-SPE high-resolution accurate mass spectrometry analysis and the remediation of hematocrit bias using a capillary micro dispenser for accurate spotting of blood samples.
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Pruebas con Sangre Seca/métodos , Extracción en Fase Sólida/métodos , Espectrometría de Masas en Tándem/métodos , Automatización/métodos , Cromatografía Liquida/métodos , HumanosRESUMEN
An integrated automated approach has been developed for the direct determination of drug concentrations using a SCAP DBS system for online extraction and analysis of dried blood spots (DBS) from DBS paper cards to a multidimensional liquid chromatography system coupled to a high-resolution QTOF mass spectrometry (LC-HRMS). An accurate, precise, selective, and sensitive two-dimensional liquid chromatography-high-resolution mass spectrometry (2D LC-HRMS) assay was developed and validated using small volumes of rat blood (approximately 1.25 µL) from a 2 mm DBS punch. The methodology was validated according to internationally accepted regulated bioanalysis acceptance criteria in order to establish the validity of the combination of online DBS assay and use of HRMS for quantitative bioanalysis. The fully automated procedure exhibited acceptable linearity (r(2) > 0.997) over the concentration range of 5 to 1000 ng/mL. Intra- and interday precision and accuracy runs indicated relative errors less than 20% at the LLOQ level and less than 15% at all other levels. The direct extraction and analysis of DBS samples resulted in a 5-fold improvement in assay sensitivity compared to conventional off-line extraction of punched DBS samples. In addition, the impact of blood hematocrit (Hct) on accurate quantification of the studied drugs also was evaluated, comparing Hct values of 30% and 60% against standards prepared at 45%. Hematocrit experiments show that Hct can influence the accuracy of drugs quantified by DBS and needs to be thoroughly evaluated prior to committing to validating a DBS assay. The online DBS system coupled to the LC-HRMS was then successfully applied to a pharmacokinetic study performed on male Sprague-Dawley rats after administration of a single dose of 5 and 10 mg/kg for midazolam and desipramine, respectively.
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Cromatografía Liquida/normas , Desipramina/sangre , Pruebas con Sangre Seca/normas , Midazolam/sangre , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/normas , Animales , Automatización de Laboratorios , Cromatografía Liquida/métodos , Desipramina/farmacocinética , Masculino , Midazolam/farmacocinética , Ratas , Ratas Sprague-Dawley , Extracción en Fase Sólida , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
The use of DBS is an appealing approach to employing microsampling techniques for the bioanalysis of samples, as has been demonstrated for the past 50 years in the metabolic screening of metabolites and diseases. In addition to its minimally invasive sample collection procedures and its economical merits, DBS microsampling benefits from the very high sensitivity, selectivity and multianalyte capabilities of LC-MS, which has been especially well demonstrated in newborn screening applications. Only a few microliters of a biological fluid are required for analysis, which also translates to significantly reduced demands on clinical samples from patients or from animals. Recently, the pharmaceutical industry and other arenas have begun to explore the utility and practicality of DBS microsampling. This review discusses the basis for why DBS techniques are likely to be part of the future, as well as offering insights into where these benefits may be realized.
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Cromatografía Liquida/normas , Pruebas con Sangre Seca/normas , Espectrometría de Masas en Tándem/normas , Animales , Recolección de Muestras de Sangre/normas , Celulosa/química , Cromatografía Liquida/instrumentación , Doping en los Deportes/métodos , Pruebas con Sangre Seca/instrumentación , Filtración , Ciencias Forenses/métodos , Hematócrito , Humanos , Recién Nacido , Ratones , Porosidad , Estándares de Referencia , Espectrometría de Masas en Tándem/instrumentaciónRESUMEN
In this work, the development of two multidimensional liquid chromatography methods coupled to a fluorescence detector is described for direct analysis of microsomal fractions obtained from rat livers. The chiral multidimensional method was then applied for the optimization of the in vitro metabolism of albendazole by experimental design. Albendazole was selected as a model drug because of its anthelmintics properties and recent potential for cancer treatment. The development of two fully automated achiral-chiral and chiral-chiral high performance liquid chromatography (HPLC) methods for the determination of albendazole (ABZ) and its metabolites albendazole sulphoxide (ABZ-SO), albendazole sulphone (ABZ-SO2) and albendazole 2-aminosulphone (ABZ-SO2NH2) in microsomal fractions are described. These methods involve the use of a phenyl (RAM-phenyl-BSA) or octyl (RAM-C8-BSA) restricted access media bovine serum albumin column for the sample clean-up, followed by an achiral phenyl column (15.0×0.46cmI.D.) or a chiral amylose tris(3,5-dimethylphenylcarbamate) column (15.0×0.46cmI.D.). The chiral 2D HPLC method was applied to the development of a compromise condition for the in vitro metabolism of ABZ by means of experimental design involving multivariate analysis.
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Albendazol/análisis , Antihelmínticos/metabolismo , Antineoplásicos/metabolismo , Cromatografía Líquida de Alta Presión/métodos , Microsomas Hepáticos/metabolismo , Albendazol/metabolismo , Animales , Antihelmínticos/análisis , Antineoplásicos/análisis , Microsomas Hepáticos/química , Análisis Multivariante , RatasRESUMEN
This work evaluated the potential of the isoforms of methamidophos to cause organophosphorus-induced delayed neuropathy (OPIDN) in hens. In addition to inhibition of neuropathy target esterase (NTE) and acetylcholinesterase (AChE), calpain activation, spinal cord lesions and clinical signs were assessed. The isoforms (+)-, (±)- and (-)-methamidophos were administered at 50mg/kg orally; tri-ortho-cresyl phosphate (TOCP) was administered (500mg/kg, po) as positive control for delayed neuropathy. The TOCP hens showed greater than 80% and approximately 20% inhibition of NTE and AChE in hen brain, respectively. Among the isoforms of methamidophos, only the (+)-methamidophos was capable of inhibiting NTE activity (approximately 60%) with statistically significant difference compared to the control group. Calpain activity in brain increased by 40% in TOCP hens compared to the control group when measured 24h after dosing and remained high (18% over control) 21 days after dosing. Hens that received (+)-methamidophos had calpain activity 12% greater than controls. The histopathological findings and clinical signs corroborated the biochemical results that indicated the potential of the (+)-methamidophos to be the isoform responsible for OPIDN induction. Protection against OPIDN was examined using a treatment of 2 doses of nimodipine (1mg/kg, i.m.) and one dose of calcium gluconate (5mg/kg, i.v.). The treatment decreased the effect of OPIDN-inducing TOCP and (+)-methamidophos on calpain activity, spinal cord lesions and clinical signs.
Asunto(s)
Insecticidas/toxicidad , Síndromes de Neurotoxicidad/etiología , Compuestos Organotiofosforados/toxicidad , Tritolilfosfatos/toxicidad , Acetilcolinesterasa/efectos de los fármacos , Acetilcolinesterasa/metabolismo , Administración Oral , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Calcio/metabolismo , Gluconato de Calcio/farmacología , Calpaína/efectos de los fármacos , Calpaína/metabolismo , Hidrolasas de Éster Carboxílico/antagonistas & inhibidores , Hidrolasas de Éster Carboxílico/metabolismo , Pollos , Femenino , Homeostasis , Síndromes de Neurotoxicidad/prevención & control , Nimodipina/farmacología , Médula Espinal/efectos de los fármacos , Médula Espinal/patología , Factores de TiempoRESUMEN
Analytical and semipreparative high performance liquid chromatography methods using polysaccharide-based chiral stationary phases were developed for the enantiomeric resolution of albendazol sulfoxide. The enantioseparation of this compound was evaluated with four chiral stationary phases: cellulose and amylose tris(3,5-dimethylphenylcarbamate), amylose tris[(S)-1-phenylethylcarbamate] and amylose tris(3,5-dimethoxyphenylcarbamate), under three elution conditions: normal, reversed-phase and polar organic mode. The influences of the mobile phase and of the structure of the chiral stationary phase on the enantiomeric separation are discussed. The best chiral performances were achieved on an amylose tris(3,5-dimethylphenylcarbamate) phase under normal (R(s)=4.96) and polar organic mode (R(s)=2.60 and 3.09). A polar organic condition using methanol as mobile phase offered shorter retention factors (k(1)=0.34) and was scaled up to semipreparative HPLC to obtain milligram quantities of both albendazole sulfoxide enantiomers for further in vitro studies. Optical rotation and circular dichroism of both enantiomers of albendazole sulfoxide was determined. The compounds ABZ, ABZ-SO, (R)-(+)-ABZ-SO and (S)-(-)-ABZ-SO were all evaluated regarding their capacity to inhibit the in vitro growth of three human tumor cell lines: MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer) and A375-C5 (melanoma). In addition, the effect of the (R)-(+)-ABZ-SO compound in the cell cycle profile and apoptosis of MCF-7 cells were also studied. Results indicated that compound ABZ was the most potent regarding cell growth inhibition and that the (+)-(R)-ABZ was a more potent inhibitor of cell growth than the (S)-(-)-ABZ-SO, particularly in the MCF-7 cell line. In addition, the (R)-(+)-ABZ-SO significantly increased the levels of apoptosis of the MCF-7 cells.