RESUMO
There is a long-standing concern for the lack of reproducibility of the untargeted metabolomic approaches used in pharmaceutical research. Two types of human plasma samples were split into two batches and analyzed in two individual labs for untargeted GC-MS metabolomic profiling. The two labs used the same silylation sample preparation protocols but different instrumentation, data processing software, and database. There were 55 metabolites annotated reproducibly, independent of the labs. The median coefficient variations (CV%) of absolute spectra ion intensities in both labs were less than 30%. However, the comparison of normalized ion intensity among biological groups, were inconsistent across labs. Predicted power based on annotated metabolites was evaluated post various normalization, data transformation and scaling. For the first time our study reveals the numerical details about the variations in metabolomic annotation and relative quantification using plain inter-laboratory GC-MS untargeted metabolomic approaches. Especially we compare several commonly used post-acquisition strategies and found normalization could not strengthen the annotation accuracy or relative quantification precision of untargeted approach, instead it will impact future experimental design. Standardization of untargeted metabolomics protocols, including sample preparation, instrumentation, data processing, etc., is critical for comparison of untargeted data across labs.
Assuntos
Análise Química do Sangue/métodos , Cromatografia Gasosa-Espectrometria de Massas/métodos , Metabolômica/métodos , Plasma/química , Análise Química do Sangue/instrumentação , Feminino , Cromatografia Gasosa-Espectrometria de Massas/instrumentação , Humanos , Ensaio de Proficiência Laboratorial , Masculino , Metabolômica/instrumentação , Reprodutibilidade dos Testes , SoftwareAssuntos
Metabolômica , Neoplasias/tratamento farmacológico , Injúria Renal Aguda/tratamento farmacológico , Injúria Renal Aguda/metabolismo , Animais , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colite/metabolismo , Sulfato de Dextrana , Descoberta de Drogas , Humanos , Neoplasias/metabolismo , Medicina de PrecisãoRESUMO
Untargeted metabolomics is a promising approach for reducing the significant attrition rate for discovering and developing drugs in the pharmaceutical industry. This review aims to highlight the practical decision-making value of untargeted metabolomics for the advancement of drug candidates in drug discovery/development including potentially identifying and validating novel therapeutic targets, creating alternative screening paradigms, facilitating the selection of specific and translational metabolite biomarkers, identifying metabolite signatures for the drug efficacy mechanism of action, and understanding potential drug-induced toxicity. The review provides an overview of the pharmaceutical process workflow to discover and develop new small molecule drugs followed by the metabolomics process workflow that is involved in conducting metabolomics studies. The pros and cons of the major components of the pharmaceutical and metabolomics workflows are reviewed and discussed. Finally, selected untargeted metabolomics literature examples, from primarily 2010 to 2016, are used to illustrate why, how, and where untargeted metabolomics can be integrated into the drug discovery/preclinical drug development process.
Assuntos
Descoberta de Drogas , Metabolômica , HumanosRESUMO
We have developed a targeted metabolomics screen which consists of using two isotopically labeled glucose compounds to conduct a dual oral glucose tolerance test in rats. This dual isotopic oral glucose tolerance test (DIS-OGTT) can be used to select drug candidates that have "on"- target or have "off"-target effects on oral glucose absorption, hepatic glucose production or glucose disposal. The DIS-OGTT assay utilized intravenously administered [6-13C1-6, 6'-2H2]-glucose and orally administered [U-13C6] glucose to monitor glucose homeostasis. In the experiment, a constant intravenous dose of [6-13C1-6, 6'-2H2] glucose was converted in vivo to a series of [M+1] glucose isotopomers and unlabeled [M] glucose via gluconeogenesis while the orally administered [U-13C6] glucose was converted to a series of [M+3] and [M+2] glucose isotopomers via gluconeogenesis. The detection platform of the assay was based on a negative mode electrospray ionization liquid chromatography tandem mass spectrometry method where the deprotonated glucose anion and its various isotopomers were quantitated in rat plasma using multiple reaction monitoring techniques. The in vivo rat DIS-OGTT assay was a sensitive method for understanding drug candidates underlying postprandial effects on glucose absorption, hepatic glucose production, and insulin controlled glucose disposal. Since glucose derivatization was not required for this assay, a higher sample throughput DIS-OGTT was achieved.
Assuntos
Avaliação Pré-Clínica de Medicamentos , Glucose/química , Glucose/metabolismo , Homeostase , Metabolômica , Animais , Isótopos de Carbono/análise , Isótopos de Carbono/metabolismo , Teste de Tolerância a Glucose , Masculino , Ratos , Ratos Sprague-Dawley , Urânio/análise , Urânio/metabolismoRESUMO
Monoacylglycerol acyltransferase enzymes (MGAT1, MGAT2, and MGAT3) convert monoacylglycerol to diacylglycerol (DAG). MGAT1 and MGAT2 are both implicated in obesity-related metabolic diseases. Conventional MGAT enzyme assays use radioactive substrates, wherein the product of the MGAT-catalyzed reaction is usually resolved by time-consuming thin layer chromatography (TLC) analysis. Furthermore, microsomal membrane preparations typically contain endogenous diacylglycerol acyltransferase (DGAT) from the host cells, and these DGAT activities can further acylate DAG to form triglyceride (TG). Our mass spectrometry (liquid chromatography-tandem mass spectrometry, or LC/MS/MS) MGAT2 assay measures human recombinant MGAT2-catalyzed formation of didecanoyl-glycerol from 1-decanoyl-rac-glycerol and decanoyl-CoA, to produce predominantly 1,3-didecanoyl-glycerol. Unlike 1,2-DAG, 1,3-didecanoyl-glycerol is proved to be not susceptible to further acylation to TG. 1,3-Didecanoyl-glycerol product can be readily solubilized and directly subjected to high-throughput mass spectrometry (HTMS) without further extraction in a 384-well format. We also have established the LC/MS/MS MGAT activity assay in the intestinal microsomes from various species. Our assay is proved to be highly sensitive, and thus it allows measurement of endogenous MGAT activity in cell lysates and tissue preparations. The implementation of the HTMS MGAT activity assay has facilitated the robust screening and evaluation of MGAT inhibitors for the treatment of metabolic diseases.
Assuntos
Diglicerídeos/análise , Inibidores Enzimáticos/análise , Ensaios de Triagem em Larga Escala , N-Acetilglucosaminiltransferases/antagonistas & inibidores , Acil Coenzima A/metabolismo , Animais , Chlorocebus aethiops , Cromatografia Líquida/métodos , Diglicerídeos/antagonistas & inibidores , Diglicerídeos/biossíntese , Cães , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Haplorrinos , Humanos , Intestinos/efeitos dos fármacos , Intestinos/enzimologia , Cinética , Camundongos , Microssomos/efeitos dos fármacos , Microssomos/enzimologia , N-Acetilglucosaminiltransferases/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/metabolismo , Espectrometria de Massas em Tandem/métodosRESUMO
Monoacylglycerol acyltransferase 2 (MGAT2) catalyzes the synthesis of diacylglycerol (DAG) from free fatty acids (FFA) and sn-monoacylglycerol (MG), the two major hydrolysis products of dietary fat. To demonstrate MGAT2-mediated cellular activity of triglyceride (TG) synthesis, we utilized 1-oleoyl-glycerol-d5 as a substrate to trace MGAT2-driven 1-oleoyl-glycerol-d5 incorporation into TG in HEK293 cells stably expressing human MGAT2. The oleoyl-glycerol-d5 incorporated major TG species were then quantified by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) in a 96-well format. Conventional MGAT2 target-engagement in vivo assays measure the elevation of total plasma TG by orally dosing a bolus of TG oil. We developed a novel LC/ESI/MS/MS-based fat absorption assay to assess the ability of MGAT2 inhibitors to inhibit fat absorption in CD1 mice by a meal tolerance test consisting of a mixture of liquid Boost plus® and 0.59 g/kg U13C-TG oil. The newly resynthesized plasma heavy TGs containing three 13C in the glycerol backbone and two U13C-acyl-chains, which represented the digested, absorbed and resynthesized TGs, were then quantitated by LC/ESI/MS/MS. With this assay, we identified a potent MGAT2 inhibitor that blocked MGAT2-mediated activity in vitro and in vivo. The use of 1-oleoyl-glycerol-d5 and U13C-TG oil followed by LC/ESI/MS/MS detection of stable-isotopic labeled DAG, TG, or glycerol provides a wide range of applications to study pathophysiological regulation of the monoacylglycerol pathway and MGAT2 activity.
Assuntos
Glicerídeos/metabolismo , Glicerol/metabolismo , Metabolismo dos Lipídeos , N-Acetilglucosaminiltransferases/metabolismo , Espectrometria de Massas em Tandem/métodos , Animais , Cromatografia Líquida/métodos , Inibidores Enzimáticos/farmacologia , Células HEK293 , Humanos , CamundongosRESUMO
INTRODUCTION: The target-based drug discovery process, including target selection, screening, hit-to-lead (H2L) and lead optimization stage gates, is the most common approach used in drug development. The full integration of in vitro and/or in vivo data with in silico tools across the entire process would be beneficial to R&D productivity by developing effective selection criteria and drug-design optimization strategies. AREAS COVERED: This review focuses on understanding the impact and extent in the past 5 years of in silico tools on the various stage gates of the target-based drug discovery approach. EXPERT OPINION: There are a large number of in silico tools available for establishing selection criteria and drug-design optimization strategies in the target-based approach. However, the inconsistent use of in vitro and/or in vivo data integrated with predictive in silico multiparameter models throughout the process is contributing to R&D productivity issues. In particular, the lack of reliable in silico tools at the H2L stage gate is contributing to the suboptimal selection of viable lead compounds. It is suggested that further development of in silico multiparameter models and organizing biologists, medicinal and computational chemists into one team with a single accountable objective to expand the utilization of in silico tools in all phases of drug discovery would improve R&D productivity.
Assuntos
Simulação por Computador , Desenho de Fármacos , Terapia de Alvo Molecular , Animais , Descoberta de Drogas/métodos , HumanosRESUMO
Metabolism catalyzed by the cytochrome P450 enzymes (CYPs) represents the most important pathway for drug metabolism and elimination in humans. Identification of the CYPs responsible for metabolism of existing and novel drugs is critical for the prediction of adverse reactions caused by drug-drug interactions or individual genetic polymorphism. An integrated approach is described for CYP-mediated metabolic reaction phenotyping using both recombinant enzymes and human liver microsomes in combination of selective inhibitors or inhibitory antibodies. The in vitro method described includes screening of recombinant CYPs for metabolic activity, chemical inhibition or antibody neutralization, and correlation analysis with isoform-selective marker activities. The primary focus is on identification of the most common enzymes including CYP1A2, 2C9, 2C19, 2D6, and 3A4, although the same strategy could potentially be used for identification of other isoforms.
Assuntos
Hidrocarboneto de Aril Hidroxilases/genética , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP2C19 , Citocromo P-450 CYP2C9 , Humanos , Hidroxilação , Técnicas In Vitro , Inativação Metabólica/genética , Microssomos Hepáticos/enzimologia , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Determination of drug distribution in brain and other tissues is important in pharmaceutical research. Tissue drug levels need to be determined routinely as they are usually diagnostic for both efficacy and toxicity. Determination of tissue levels in small organ subregions is frequently performed due to important functional considerations. These measurements have traditionally been very tedious requiring extensive dissection and specimen pooling to achieve detection of analytes of interest. Direct and indirect methods utilizing mass spectrometry have been reported for detection of analytes in tissue specimens. Typically, these require very specialized MS or sampling equipment and are only partially successful due to analyte response. We have developed a novel approach for quantitation of tissue sections called Functional Tissue Microanalysis (FTM) in which small circular samples are removed from subregions of interest, extracted and analyzed by conventional LC/MS/MS utilizing electrospray ionization. This allows direct measurement of regional concentrations without dissection and homogenization of tissue specimens as many subregions can be sampled from a single mounted section. Utilization of the FTM approach for analysis of both sagittal and coronal rat brain sections is shown for quantitation of raclopride and rimonabant. Reproducibility of this approach and comparison to conventional methods is reported.
Assuntos
Encéfalo/metabolismo , Cromatografia Líquida/métodos , Preparações Farmacêuticas/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Fígado/metabolismo , Ratos , Distribuição TecidualRESUMO
A major strategy used in drug design is the inhibition of enzyme activity. The ability to accurately measure the concentration of the inhibitor which is required to inhibit a given biological or biochemical function by half is extremely important in ranking compounds. Since the concept of the half maximal inhibitory concentration (IC(50)) is used extensively for studying reversible inhibition enzymatic reactions, it is important to clearly understand the experimental design and the mathematical modeling techniques used to generate IC(50) values. The most important part of the experimental design is to measure the rate of production of [P] during the linear phase of the time course of the reaction and to prove that the enzyme-catalyzed reaction is reversible. The most important part of the mathematical modeling is to select the correct model and to have a firm understanding on how to handle outliers in the data. These topics are discussed in greater detail along with a discussion on how much quantitative and mechanistic information can be reasonably deduced from an experiment.
Assuntos
Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Inibidores Enzimáticos/farmacologia , Animais , Biocatálise , Relação Dose-Resposta a Droga , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Concentração Inibidora 50RESUMO
Various CYP time-dependent inhibition (TDI) assays have been widely implemented in drug discovery and development which has led to great success in positively identifying compounds with mechanism-base inhibition liability. However, drug-drug interaction (DDI) predictions by various in-silico models utilizing kinetic parameters obtained from TDI assays have met with significant challenges including questionable kinetic data, over-simplified in-vitro models and unreliable mathematic algorithms. Although significant efforts have been made to standardize the TDI assay and refine mathematical models, recent evaluation studies have revealed that the kinetic parameters of TDI, the most important in-vitro data required by all DDI prediction models, are significantly impacted by a variety of experimental variables including microsomal protein concentration, metabolic stability, CYP-specific probes, and post-incubation time. This review attempts to provide medicinal chemists a brief overview on the current status of TDI assays, determination of kinetic parameters and in silico DDI predictions with emphasis on the complexity of the TDI kinetics and limitations of current in-vitro models and DDI prediction methodologies.
Assuntos
Inibidores das Enzimas do Citocromo P-450 , Inibidores Enzimáticos/farmacologia , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Descoberta de Drogas , Interações Medicamentosas , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Cinética , Relação Estrutura-Atividade , Fatores de TempoRESUMO
Increasing pressure on the pharmaceutical industry to reduce cost and focus internal resources on "high value" activities is driving a trend to outsource traditionally "in-house" drug discovery activities. Compound collections are typically viewed as drug discovery's "crown jewels"; however, in late 2007, Johnson & Johnson Pharmaceutical Research & Development (J PRD) took a bold step to move their entire North American compound inventory and processing capability to an external third party vendor. The authors discuss the combination model implemented, that of local compound logistics site support with an outsourced centralized processing center. Some of the lessons learned over the past five years were predictable while others were unexpected. The substantial cost savings, improved local service response and flexible platform to adjust to changing business needs resulted. Continued sustainable success relies heavily upon maintaining internal headcount dedicated to vendor management, an open collaboration approach and a solid information technology infrastructure with complete transparency and visibility.
Assuntos
Descoberta de Drogas/organização & administração , Indústria Farmacêutica/organização & administração , Preparações Farmacêuticas , Descoberta de Drogas/economia , Descoberta de Drogas/normas , Indústria Farmacêutica/economia , Indústria Farmacêutica/normas , Peso Molecular , Preparações Farmacêuticas/economia , Preparações Farmacêuticas/normasRESUMO
Diacylglycerol acyltransferase (DGAT) catalyzes the final step in triglyceride (TG) synthesis. There are two isoforms, DGAT1 and DGAT2, with distinct protein sequences and potentially different physiological functions. To date, the ability to determine clear functional differences between DGAT1 and DGAT2, especially with respect to hepatic TG synthesis, has been elusive. To dissect the roles of these two key enzymes, we pretreated HepG2 hepatoma cells with (13)C(3)-D(5)-glycerol or (13)C(18)-oleic acid, and profiled the major isotope-labeled TG species by liquid chromatography tandem mass spectrometry. Selective DGAT1 and DGAT2 inhibitors demonstrated that (13)C(3)-D(5)-glycerol-incorporated TG synthesis was mediated by DGAT2, not DGAT1. Conversely, (13)C(18)-oleoyl-incorporated TG synthesis was predominantly mediated by DGAT1. To trace hepatic TG synthesis and VLDL triglyceride (VLDL-TG) secretion in vivo, we administered D(5)-glycerol to mice and measured plasma levels of D(5)-glycerol-incorporated TG. Treatment with an antisense oligonucleotide (ASO) to DGAT2 led to a significant reduction in D(5)-glycerol incorporation into VLDL-TG. In contrast, the DGAT2 ASO had no effect on the incorporation of exogenously administered (13)C(18)-oleic acid into VLDL-TG. Thus, our results indicate that DGAT1 and DGAT2 mediate distinct hepatic functions: DGAT2 is primarily responsible for incorporating endogenously synthesized FAs into TG, whereas DGAT1 plays a greater role in esterifying exogenous FAs to glycerol.
Assuntos
Diacilglicerol O-Aciltransferase/metabolismo , Ensaios Enzimáticos/métodos , Glicerol/metabolismo , Fígado/enzimologia , Ácido Oleico/metabolismo , Animais , Diacilglicerol O-Aciltransferase/antagonistas & inibidores , Diacilglicerol O-Aciltransferase/genética , Inibidores Enzimáticos/farmacologia , Esterificação/efeitos dos fármacos , Ácidos Graxos/biossíntese , Ácidos Graxos/metabolismo , Células Hep G2 , Humanos , Marcação por Isótopo , Lipoproteínas VLDL/metabolismo , Masculino , Camundongos , Oligonucleotídeos Antissenso/genética , Triglicerídeos/biossínteseRESUMO
A highly unusual rearrangement in collision-induced dissociation mass spectrometry is reported that involves intramolecular transfer of the fluorobenzyl cation between two remote amidic nitrogen atoms separated by five chemical bonds. The same intramolecular transfer was also observed for two related analogs. It is postulated that the ionic reactions are initiated by protonation of the first amidic nitrogen, resulting in formation of the fluorobenzyl cation and a neutral partner that are maintained together in the gas phase by electrostatic interactions as an intermediate ion-neutral complex. In the ion-neutral complex, the nascent fluorobenzyl cation approaches geometrically to the second amidic nitrogen atom on the neutral partner, and subsequently forms a new C-N bond and an isomeric precursor ion as the charge is retained on the amidic nitrogen. The newly formed isomeric precursor ion eventually undergoes the final fragmentation by amide bond cleavage. Alternatively, the ionic reactions proceed through a direct intramolecular transfer mechanism by which the molecular ion adopts to a ring-like configuration in the gas phase, so that both the donor and recipient nitrogens are geometrically close to each other within a bonding distance to permit a direct transfer between two sites even though they are separated by multiple chemical bonds.
Assuntos
Cátions/química , Fluorbenzenos/química , Gases/química , Nitrogênio/química , Isomerismo , Conformação Molecular , Preparações Farmacêuticas/química , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyzes the terminal step in triglyceride (TG) synthesis using diacylglycerol (DAG) and fatty acyl-CoA as substrates. In the liver, the production of VLDL permits the delivery of hydrophobic TG from the liver to peripheral tissues for energy metabolism. We describe here a novel high-content, high-throughput LC/MS/MS-based cellular assay for determining DGAT activity. We treated endogenous DGAT-expressing cells with stable isotope-labeled [¹³C18]oleic acid. The [¹³C18]oleoyl-incorporated TG and DAG lipid species were profiled. The TG synthesis pathway assay was optimized to a one-step extraction, followed by LC/MS/MS quantification. Further, we report a novel LC/MS/MS method for tracing hepatic TG synthesis and VLDL-TG secretion in vivo by administering [¹³C18]oleic acid to rats. The [¹³C18]oleic acid-incorporated VLDL-TG was detected after one-step extraction without conventional separation of TG and recovery by derivatizing [¹³C18]oleic acid for detection. Using potent and selective DGAT1 inhibitors as pharmacological tools, we measured changes in [¹³C18]oleoyl-incorporated TG and DAG and demonstrated that DGAT1 inhibition significantly reduced [¹³C18]oleoyl-incorporated VLDL-TG. This DGAT1-selective assay will enable researchers to discern differences between the roles of DGAT1 and DGAT2 in TG synthesis in vitro and in vivo.
Assuntos
Diacilglicerol O-Aciltransferase/metabolismo , Ensaios Enzimáticos/métodos , Fígado/enzimologia , Animais , Radioisótopos de Carbono/metabolismo , Células Cultivadas , Cromatografia Líquida , Hepatócitos/citologia , Hepatócitos/enzimologia , Humanos , Insetos/citologia , Insetos/enzimologia , Insetos/virologia , Rim/citologia , Rim/embriologia , Rim/enzimologia , Lipoproteínas VLDL/química , Lipoproteínas VLDL/metabolismo , Masculino , Ácido Oleico/metabolismo , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas em Tandem , Triglicerídeos/química , Triglicerídeos/metabolismoRESUMO
Inhibition curve shift is a commonly used approach for screening of time-dependent CYP inhibitors which requires parallel paired incubations to obtain two inhibition curves for comparison. For the control incubation, a test compound is co-incubated with a probe substrate in human liver microsomes (HLM) fortified with NADPH; for the time-dependent incubation (TDI), the test compound is pre-incubated with NADPH-fortified HLM followed by a secondary incubation with a probe substrate. For both incubations, enzyme activity is measured respectively by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis of the CYP-specific metabolite, and a TDI inhibitor can be readily identified by inhibition curve shifting as a result of CYP inactivation by the test compound during the pre-incubation. In the present study, we describe an alternative approach to facilitate TDI screening in which stable isotope labeled CYP-specific probes are used for the TDI, and non-labeled substrates are included in the control incubation. Because CYP-specific metabolites produced in the TDI are stable isotope labeled, two sets of incubation samples can be combined and then simultaneously analyzed by LC/MS/MS in the same batch run to reduce the run time. This new method has been extensively validated using both a number of known competitive and TDI inhibitors specific to five most common CYPs such as 1A2, 2C9, 2C19, 2D6, and 3A4. The assay is performed in a 96-well format and can be fully automated. Compared to the traditional method, this approach in combination with sample pooling and a short LC/MS/MS gradient significantly enhances the throughput of TDI screening and thus can be easily implemented in drug discovery to evaluate a large number of compounds without adding additional resource.
Assuntos
Cromatografia Líquida/métodos , Inibidores das Enzimas do Citocromo P-450 , Inibidores Enzimáticos/análise , Ensaios de Triagem em Larga Escala/métodos , Espectrometria de Massas em Tandem/métodos , Humanos , Marcação por Isótopo , Microssomos Hepáticos/química , Microssomos Hepáticos/enzimologiaRESUMO
Integrating physicochemical, drug metabolism, pharmacokinetics, ADME, and toxicity assays into drug discovery in order to reduce the attrition rates in clinical development is reviewed. The review is organized around three main decision points used in discovery including hit generation, lead optimization and final candidate selection stages. The preclinical strategies used at each decision point are discussed from a drug discovery perspective. Typically, preclinical data produced at these stages use lower throughput assays, smaller amounts of compounds and operate within a timeframe that is consistent with the iterative cycle of most drug discovery research projects. Understanding the false positive rates of these drug discovery preclinical assays is a must in reducing attrition rates in development.
Assuntos
Descoberta de Drogas , Farmacocinética , Carcinógenos/farmacocinética , Mutagênicos/farmacocinética , Permeabilidade , Espécies Reativas de Oxigênio/farmacocinética , SolubilidadeRESUMO
Constant neutral loss (CNL) and precursor ion (PI) scan have been widely used for the in vitro screening of glutathione conjugates derived from reactive metabolites, but these two methods are only applicable to triple quadrupole or hybrid triple quadrupole mass spectrometers. Additionally, the success of CNL and PI scanning largely depends on structure and CID fragmentation pathways of GSH conjugates. In the present study, a highly efficient methodology has been developed as an alternative approach for high-throughput screening and structural characterization of reactive metabolites using the linear ion trap mass spectrometer. In microsomal incubations, a mixture of glutathione [GSH, gamma-glutamyl-cystein-glycin] and the stable-isotope labeled compound [GSX, gamma-glutamyl-cystein-glycin-(13)C2-(15)N] was used to trap reactive metabolites, resulting in formation of both labeled and unlabeled conjugates at a given isotopic ratio. A mass difference of 3.0 Da between the natural and labeled GSH conjugate (mass tag) at a fixed isotopic ratio constitutes a unique mass pattern that can selectively trigger the data-dependent MS(2) scan of both isotopic partner ions, respectively. In order to eliminate the response bias of GSH adducts in the positive and negative mode, a polarity switch is executed between the mass tag-triggered data dependent MS(2) scan, and thus ESI- and ESI+ MS(2) spectra of both labeled and nonlabeled GSH conjugates are obtained in a single LC-MS run. Unambiguous identification of glutathione adducts was readily achieved with great confidence by MS(2) spectra of both labeled and unlabeled conjugates. Reliability of this method was vigorously validated using several model compounds that are known to form reactive metabolites. This approach is not based on the appearance of a particular product ion such as MH(+) - 129 and anion at m/z 272, whose formation can be structure-dependent and sensitive to the collision energy level; therefore, the present method can be suitable for unbiased screening of any reactive metabolites, regardless of their CID fragmentation pathways. Additionally, this methodology can potentially be applied to triple quadrupole or hybrid triple quadrupole mass spectrometers.