RESUMO
Human purine nucleoside phosphorylase (HsPNP) catalyzes reversible phosphorolysis of nucleosides and deoxynucleosides in the purine cascade. HsPNP has been a target on behalf of the development of new leads for the treatment of a variety of T-cell mediated disorders. Several studies on the HsPNP are focused on the identification of effective, safe, and selective inhibitors. Therefore, this study describes the development of direct, simple, reliable, and inexpensive enzymatic assays to screen HsPNP inhibitors. Initially, HsPNP was covalently immobilized on the surface of magnetic particles (MPs). Due to the versatility of the MPs as solid support for enzyme immobilization, two different methods to monitor the enzyme activity are presented. Firstly, the activity of HsPNP-MPs was assessed offline by HPLC-DAD quantifying the formed hypoxanthine. Then, HsPNP-MPs were trapped in a peek tube, furnishing a microreactor which was inserted on-flow in an HPLC-DAD system to monitor the enzyme activity by the hypoxanthine quantification. Kinetic assays provided KMapp values for the inosine substrate of 488.2 ± 49.1 and 1084 ± 111 µM for the offline and on-flow assays, respectively. For the first time, kinetic studies for Pi as substrate using the HsPNP-MPs exhibits a Michaelis-Menten kinetic, yielding KMapp values for offline and on-flow of 521.2 ± 62.9 µM and 601 ± 66.5 µM, respectively. Inhibition studies conducted with a fourth generation immucillin derivative (DI4G) were employed as proof of concept to validate the use of the HsPNP-MPs assays for screening purposes. Additionally, a small library containing 11 compounds was used to assess the selectivity of the developed assays. The results showed that both presented assays can be applied to selectively recognizing and characterizing HsPNP inhibitors. Particularly, the on-flow method exhibited a high throughput and performance because of its automation and represents an easy and practical approach to reuse the HsPNP-MPs. Besides, this novel enzyme activity assay model can be further applied to other biological targets.
Assuntos
Fenômenos Magnéticos , Purina-Núcleosídeo Fosforilase , Inibidores Enzimáticos/farmacologia , Humanos , Cinética , Nucleosídeos de Purina , Purina-Núcleosídeo Fosforilase/metabolismoRESUMO
The use of affinity-based protein assay produced by covalently linking acetylcholinesterase to magnetic beads, followed by chemical characterization of the selective binders using Liquid Chromatography with tandem High-Resolution Mass Spectrometry (LC-HRMS) is herein described for profiling crude aqueous natural product extracts. The fishing assay was first modulated using galanthamine as a reference ligand and then, the assay condition was adjusted for the aqueous leaves extracts obtained from Lippia gracilis Schauer (genotype 201) that was used as the natural combinatory library. From the experiments, a selective binder has been undisclosed with an accurate mass of 449.1131â¯m/z and identified as eriodictyol 2'-O-glucoside or eriodictyol 3'-O-glucoside. The selectivity of the binding assay was demonstrated, as much as, that erydictiol 7-O-glucoside was not fished, although it was present in the crude aqueous extract. The binding assay platform exhibited high specificity and did not require any sample pretreatment, making it appropriate for profiling binders at natural libraries.
Assuntos
Acetilcolinesterase/química , Bioensaio/métodos , Lippia/química , Extratos Vegetais/química , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas/métodos , Folhas de Planta/químicaRESUMO
This work reports the use of a liquid chromatography ion trap tandem mass spectrometry (LC-IT-MS/MS) system for quantification in human milk samples of both carbamazepine (CBZ) and its active metabolite, carbamazepine 10,11-epoxide (CBZE). An octadecyl restricted-access media bovine serum albumin column (RAM-BSA C(18)) was used in single-column mode. Selectivity, extraction efficiency, accuracy and precision were achieved employing 100 µL of the sample, without preparation, with detection limits of 20.0 ng/mL for CBZ and 40.0 ng/mL for CBZE. The matrix effect was investigated for the compounds by post-column infusion (qualitative) and by on-line extraction (quantitative). It was observed suppression effect for CBZ and CBZE by post-column infusion, ion suppression of 0.80 for CBZ, and enhancement of 1.28 for CBZE by on-line extraction. The developed method was validated and applied to analyze breast milk samples from one nursing mother. CBZ and CBZE were quantified in the concentrations of 2.26 µg/mL and 1.54 µg/mL, respectively. To our knowledge, this is the first report on the simultaneous determination of CBZ and its active metabolite by direct injection of human milk serum.
Assuntos
Carbamazepina/análise , Cromatografia Líquida/métodos , Leite Humano/química , Espectrometria de Massas em Tandem/métodos , Animais , Carbamazepina/metabolismo , Bovinos , Cromatografia Líquida/instrumentação , Resíduos de Drogas , Feminino , Humanos , Limite de Detecção , Modelos Lineares , Leite Humano/metabolismo , Reprodutibilidade dos Testes , Soroalbumina Bovina/químicaRESUMO
This work reports the application of restricted access media (RAM) column, in a multidimensional configuration, for simultaneous analysis of sulfamethoxazole (SMX) and trimethoprim (TMP) in whole eggs with ultraviolet detection. The proteins were partially precipitated by adding 0.5 mL of acetonitrile into 1.0 mL of blended egg followed by centrifugation. The supernatant was injected (250 microL) directly into the multidimensional system. At the first dimension, a restricted access medium (RAM) bovine serum albumin (BSA) octadecyl column (100 mm x 46 mm I.D., Luna silica, 10 microm particle size and 100 A pore size), was used for extraction and concentration of the analytes and at second dimension, an octadecyl column (150 mm x 46 mm I.D., Luna silica, 10 microm particle size and 100 A pore size), for analysis. The developed method showed good selectivity, accuracy and precision for quantification of these different compounds in eggs, and the limits of quantification were 80 ng/mL, for both compounds. The validated method is reliable and sensitive for monitoring residues in whole eggs samples and thus, to determine withdraw period for laying hens using veterinary medicine having SMX-TMP combination.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Ovos/análise , Sulfametoxazol/análise , Trimetoprima/análise , Animais , Galinhas , Estrutura Molecular , Reprodutibilidade dos Testes , Sulfametoxazol/química , Trimetoprima/químicaRESUMO
A quick overview of published methods for analyzing compounds in complex biological samples reveals that the most difficult step is the clean-up or extraction of a required compound from the matrix. The strategy required to analyze exogenous compounds in biological fluids depends greatly upon the nature of the compound and upon the biomatrix. Coupled-column separation using restricted-access media as the first dimension in order to exclude macromolecules and retain micromolecules has been successfully used for a number of biological fluids. This paper presents the history of the development of restricted-access media supports and of their application to the direct injection of biological fluid samples in high-performance liquid chromatography.
Assuntos
Técnicas de Química Analítica/instrumentação , Técnicas de Química Analítica/métodos , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Análise Química do Sangue/métodos , Análise Química do Sangue/normas , Líquidos Corporais , Técnicas de Química Analítica/normas , Química Clínica/métodos , Química Clínica/normas , Cromatografia/métodos , Cromatografia Líquida de Alta Pressão/normas , Técnicas de Laboratório Clínico/normas , Humanos , Urinálise/métodosRESUMO
The separation of a series of 23 asymmetric sulfoxides, including the three proton pump inhibitors (PPI) omeprazole, lansoprazole and pantoprazole was investigated by HPLC, under reversed-phase elution with amylose tris(3,5-dimethylphenylcarbamate), amylose tris[(S)-1-phenylethylcarbamate] and amylose tris(3,5-dimethoxyphenylcarbamate) chiral stationary phases, CSP1-3, respectively. The whole set of sulfoxides showed better enantioselectivity and enantioresolution on CSP1. However, the three PPI were enantioseparated only when using CSP1 and CSP3. It was observed an improved enantioselectivity and enantioresolution on CSP3. The mechanisms of retention were evaluated by molecular interaction fields (MIF) generated via GRID force field, which yielded the geometric reasons leading to the scenario outlined. The enantioselective and nonselective interactions are discussed in terms of the reported selectivity. The steric structural outline of the CSP nonselective interaction sites is of major importance to deliver the sulfoxides to the chiral selective sites where the enantioselective interactions take place.
Assuntos
Sulfóxidos/isolamento & purificação , Modelos Moleculares , Estrutura Molecular , EstereoisomerismoRESUMO
Two high-performance liquid chromatographic methods for determination of residual monomer in dental acrylic resins are described. Monomers were detected by their UV absorbance at 230 nm, on a Nucleosil C18 (5 microm particle size, 100 A pore size, 15 x 0.46 cm i.d.) column. The separation was performed using acetonitrile-water (55:45 v/v) containing 0.01% triethylamine (TEA) for methyl methacrylate and butyl methacrylate, and acetonitrile-water (60:40 v/v) containing 0.01% TEA for isobutyl methacrylate and 1,6-hexanediol dimethacrylate as mobile phases, at a flow rate of 0.8 mL/min. Good linear relationships were obtained in the concentration range 5.0-80.0 microg/mL for methyl methacrylate, 10.0-160.0 microg/mL for butyl methacrylate, 50.0-500.0 microg/mL for isobutyl methacrylate and 2.5-180.0 microg/mL for 1,6-hexanediol dimethacrylate. Adequate assay for intra- and inter-day precision and accuracy was observed during the validation process. An extraction procedure to remove residual monomer from the acrylic resins was also established. Residual monomer was obtained from broken specimens of acrylic disks using methanol as extraction solvent for 2 h in an ice-bath. The developed methods and the extraction procedure were applied to dental acrylic resins, tested with or without post-polymerization treatments, and proved to be accurate and precise for the determination of residual monomer content of the materials evaluated.
Assuntos
Resinas Acrílicas/química , Cromatografia Líquida de Alta Pressão/métodos , Materiais Dentários/química , Metacrilatos/análise , Metilmetacrilato/análise , Calibragem , Fracionamento Químico/métodos , Reprodutibilidade dos Testes , Espectrofotometria UltravioletaRESUMO
A bidimensional HPLC method for the simultaneous determination of sulfamethoxazole (SMX) and trimethoprim (TMP) in bovine milk has been developed and validated. After centrifugation, aliquots (150 microl) of milk samples were directly injected to a column-switching HPLC system. At the first step a RAM octyl-BSA column was employed to automatically remove proteins that otherwise would interfere with milk analysis. The mobile phase 0.01 M phosphate buffer pH 6.0:acetonitrile (95:5, v/v) was used in the first 5 min for the elution of milk proteins and then 0.01 M phosphate buffer pH 6.0:acetonitrile (83:17, v/v) for transfer SMX and TMP to the analytical column. The separation of SMX and TMP from one another and from other remaining milk components was performed on an octyl column using the mobile phase 0.01 M phosphate buffer pH 5.0:acetonitrile (82:18, v/v), which were detected by UV at 265 nm. The calibration graphs were linear in the concentration ranges of 25-800 ng/ml and 50-400 ng/ml for SMX and TMP, respectively. The intra- and inter-assay coefficients of variation were less than 15% for both drugs. The validated method was applied to the analysis of milk samples of twelve (two groups of six) cows after administration (intramuscular or subcutaneous) of a single recommended therapeutic dose of the SMX-TMP combination.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Leite/química , Sulfametoxazol/análise , Trimetoprima/análise , Animais , Bovinos , Estabilidade de Medicamentos , Feminino , Congelamento , Sistemas On-Line , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Combinação Trimetoprima e Sulfametoxazol/uso terapêuticoRESUMO
A direct injection high-performance liquid chromatographic (HPLC) method, with column-switching, for the determination of omeprazole enantiomers in human plasma is described. A restricted access media (RAM) of bovine serum albumin (BSA) octyl column has been used in the first dimension for separation of the analyte from the biological matrix. The omeprazole enantiomers were eluted from the RAM column onto an amylose tris(3,5-dimethylphenylcarbamate) chiral column by the use of a column-switching valve and the enantioseparation was performed using acetonitrile-water (60:40 v/v) as eluent. The analytes were detected by their UV absorbance at 302 nm. The validated method was applied to the analysis of the plasma samples obtained from 10 Brazilian volunteers who received a 40 mg oral dose of racemic omeprazole and was able to quantify the enantiomers of omeprazole in the clinical samples analyzed. The assay was able to determine the cytochrome P450 2C19 phenotype of the subjects participating in this study.
Assuntos
Antiulcerosos/sangue , Cromatografia Líquida de Alta Pressão/métodos , Inibidores Enzimáticos/sangue , Omeprazol/sangue , Antiulcerosos/farmacocinética , Área Sob a Curva , Inibidores Enzimáticos/farmacocinética , Humanos , Omeprazol/farmacocinética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , EstereoisomerismoRESUMO
This work reports the use of multidimensional HPLC by coupling a restricted access medium (RAM) bovine serum albumin (BSA) octadecyl column (100 x 4.6 mm I.D., 10 microm particle size and 120 A pore size) to an octadecyl Hypersil column (150 x 4.6 mm I.D., 5 microm particle size and 120 A pore size) to the analysis of amoxycillin in human plasma by direct injection. Ion pairing was necessary to extract amoxycillin with good recovery from the plasma proteins. To prepare the spiked samples, aliquots (60 microl) of the appropriated standard solutions were added to each culture tube containing an 180 microl of plasma and a solution of 0.30 mM tetrabuthylammonium phosphate (60 microl). They were vortexed for 15 s and then 290 microl were transferred to autosampler vials. Aliquots (250 microl) of the spiked plasma samples were injected to a column-switching HPLC system. An analysis time of 25 min with no time spent on sample preparation was achieved. The developed method showed good selectivity, sensitivity, accuracy and precision for direct analysis of this polar low wavelength ultraviolet absorption antibiotic using only 180 microl of human plasma. The validated method proved to be reliable and sensitive for the determination of amoxycillin in plasma samples of five healthy volunteers to whom test and reference formulations were administered as an oral dose (500 mg).