RESUMO
Surface-enhanced Raman scattering (SERS) enables the detection of a large number of different adsorbates at extraordinarily low levels. This plasmonics-based technology has undergone a number of remarkable advances since its discovery over 40 years ago, and has emerged from being an investigative tool confined largely to the research laboratory into a much more usable tool across a broad range of investigative studies, both within the laboratory and beyond. The purpose of this Concluding remarks manuscript is to capture, at least in part, the developments in this area since the first Faraday discussion of SERS over a decade ago. It begins with a brief contextual overview and then moves into describing a few of the many highlights from the meeting. Along the way, we have added a few comments and perspectives as a means to more fully stage where the different areas of research with SERS stand today. An addendum is included that collects a few of the recent perspectives on the original work and activities in this area.
Assuntos
Análise Espectral Raman/métodos , Metais/química , Padrões de Referência , Reprodutibilidade dos Testes , Análise Espectral Raman/normasRESUMO
Surface-enhanced Raman scattering (SERS) has enabled the detection of pathogens and disease markers at extremely low levels. This review examines the potential impact of SERS in addressing unmet needs in pathogen diagnostics both in a traditional clinical setting and in the point of care (POC) arena. It begins by describing the strengths and weaknesses of today's diagnostics technologies in order to set a contextual stage for an overview which highlights a few of the many recent developments using SERS in biodefense, human and animal health, and monitoring food and water safety. These sections are followed by discussions of the challenges for the translation of these developments to POC settings, including the performance attributes and metrics for quantification of analytical and clinical figures of merit (e.g., limit of detection and clinical accuracy), and the pathways for large-scale test validation and the build-out of instrumentation and tests kits for POC deployment.
Assuntos
Bactérias/isolamento & purificação , Biomarcadores/análise , Fungos/isolamento & purificação , Sistemas Automatizados de Assistência Junto ao Leito , Análise Espectral Raman , Vírus/isolamento & purificação , Animais , Bactérias/química , Fungos/química , Humanos , Propriedades de Superfície , Vírus/químicaRESUMO
The ability to detect tuberculosis (TB) continues to be a global health care priority. This paper describes the development and preliminary assessment of the clinical accuracy of a heterogeneous immunoassay that integrates a serum pretreatment process with readout by surface-enhanced Raman scattering (SERS) for the low-level detection of mannose-capped lipoarabinomannan (ManLAM). ManLAM is a major virulence factor in the infectious pathology of Mycobacterium tuberculosis (Mtb) that has been found in the serum and other body fluids of infected patients. The effectiveness of ManLAM as a TB diagnostic marker, however, remains unproven for reasons not yet well understood. As reported herein, we have found that (1) ManLAM complexes with proteins and possibly other components in serum; (2) these complexes have a strongly detrimental impact on the ability to detect ManLAM using an immunoassay; (3) a simple pretreatment step can disrupt this complexation; and (4) disruption by pretreatment improves detection by 250×. We also describe the results from a preliminary assessment on the utility of serum pretreatment by running immunoassays on archived specimens from 24 TB-positive patients and 10 healthy controls. ManLAM was measurable in 21 of the 24 TB-positive specimens, but not in any of the 10 control specimens. These findings, albeit for a very small specimen set, translate to a clinical sensitivity of 87.5% and a clinical specificity of 100%. Together, these results both provide much needed evidence for the clinical utility of ManLAM as a TB marker, and demonstrate the potential utility of our overall approach to serve as a new strategy for the development of diagnostic tests for this disease.
Assuntos
Antígenos de Bactérias/sangue , Antígenos de Bactérias/metabolismo , Lipopolissacarídeos/sangue , Lipopolissacarídeos/metabolismo , Manose/metabolismo , Mycobacterium tuberculosis/imunologia , Análise Espectral Raman/métodos , Métodos Analíticos de Preparação de Amostras , Biomarcadores/sangue , Biomarcadores/metabolismo , Humanos , Análise Espectral Raman/instrumentaçãoRESUMO
Patient care and prevention of disease outbreaks rely heavily on the performance of diagnostic tests. These tests are typically carried out in serum, urine, and other complex sample matrices, but are often plagued by a number of matrix effects such as nonspecific adsorption and complexation with circulating proteins. This paper demonstrates the importance of sample pretreatment to overcome matrix effects, enabling the low-level detection of a disease marker for tuberculosis (TB). The impact of pretreatment is illustrated by detecting a cell wall component unique to mycobacteria, lipoarabinomannan (LAM). LAM is a major virulence factor in the infectious pathology of Mycobacterium tuberculosis (Mtb) and has been successfully detected in the body fluids of TB-infected individuals; however, its clinical sensitivity - identifying patients with active infection - remains problematic. This and the companion paper show that the detection of LAM in an immunoassay is plagued by its complexation with proteins and other components in serum. Herein, we present the procedures and results from an investigation of several different pretreatment schemes designed to disrupt complexation and thereby improve detection. These sample pretreatment studies, aimed at determining the optimal conditions for complex disruption, were carried out by using a LAM simulant derived from the nonpathogenic M. smegmatis, a mycobacterium often used as a model for Mtb. We have found that a perchloric acid-based pretreatment step improves the ability to detect this simulant by â¼1500× with respect to that in untreated serum. This paper describes the approach to pretreatment, how pretreatment improves the detection of the LAM simulant in human serum, and the results from a preliminary investigation to identify possible contributors to complexation by fractionating serum according to molecular weight. The companion paper applies this pretreatment approach to assays of TB patient samples.
Assuntos
Métodos Analíticos de Preparação de Amostras/métodos , Limite de Detecção , Lipopolissacarídeos/sangue , Lipopolissacarídeos/química , Mycobacterium smegmatis/química , Soluções Tampão , Parede Celular/química , Humanos , Concentração de Íons de Hidrogênio , Peso Molecular , Mycobacterium smegmatis/citologiaRESUMO
N-Hydroxysuccinimide (NHS) ester terminal groups are commonly used to covalently couple amine-containing biomolecules (e.g., proteins and peptides) to surfaces via amide linkages. This one-step aminolysis is often performed in buffered aqueous solutions near physiological pH (pH 6 to pH 9). Under these conditions, the hydrolysis of the ester group competes with the amidization process, potentially degrading the efficiency of the coupling chemistry. The work herein examines the efficiency of covalent protein immobilization in borate buffer (50 mM, pH 8.50) using the thiolate monolayer formed by the chemisorption of dithiobis (succinimidyl propionate) (DSP) on gold films. The structure and reactivity of these adlayers are assessed via infrared spectroscopy (IR), X-ray photoelectron spectroscopy (XPS), electrochemical reductive desorption, and contact angle measurements. The hydrolysis of the DSP-based monolayer is proposed to follow a reaction mechanism with an initial nucleation step, in contrast to a simple pseudo first-order reaction rate law for the entire reaction, indicating a strong dependence of the interfacial reaction on the packing and presence of defects in the adlayer. This interpretation is used in the subsequent analysis of IR-ERS kinetic plots which give a heterogeneous aminolysis rate constant, ka, that is over 3 orders of magnitude lower than that of the heterogeneous hydrolysis rate constant, kh. More importantly, a projection of these heterogeneous kinetic rates to protein immobilization suggests that under coupling conditions in which low protein concentrations and buffers of near physiological pH are used, proteins are more likely physically adsorbed rather than covalently linked. This result is paramount for biosensors that use NHS chemistry for protein immobilization due to effects that may arise from noncovalently linked proteins.
Assuntos
Aminas/química , Proteínas Imobilizadas/química , Succinimidas/química , Boratos/química , Soluções Tampão , Ésteres , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Propriedades de SuperfícieRESUMO
Proteomic analyses of readily obtained human fluids (e.g., serum, urine, and saliva) indicate that the diagnosis of complex diseases will be enhanced by the simultaneous measurement of multiple biomarkers from such samples. This paper describes the development of a nanoparticle-based multiplexed platform that has the potential for simultaneous read-out of large numbers of biomolecules. For this purpose, we have chosen pancreatic adenocarcinoma (PA) as a test bed for diagnosis and prognosis. PA is a devastating form of cancer in which an estimated 86% of diagnoses resulted in death in the United States in 2010. The high mortality rate is due, in part, to the asymptomatic development of the disease and the dearth of sensitive diagnostics available for early detection. One promising route lies in the development of a serum biomarker panel that can generate a signature unique to early stage PA. We describe the design and development of a proof-of-concept PA biomarker immunoassay array coupled with surface-enhanced Raman scattering (SERS) as a sensitive readout method.
Assuntos
Adenocarcinoma/diagnóstico , Antígeno CA-19-9/sangue , Metaloproteinase 7 da Matriz/sangue , Neoplasias Pancreáticas/diagnóstico , Análise Espectral Raman , Biomarcadores Tumorais/sangue , Ouro , Humanos , Imunoensaio , Nanopartículas Metálicas , Prognóstico , ProteômicaRESUMO
Botulinum neurotoxins (BoNTs) are classified at a highest degree of threat in biodefense, due largely to their high lethality. With the growing risk of biowarfare, the shortcomings of the gold standard test for these neurotoxins, the mouse bioassay, have underscored the need to develop alternative diagnostic testing strategies. This paper reports on the detection of inactivated Clostridium botulinum neurotoxin serotype A (BoNT-A) and serotype B (BoNT-B), the two most important markers of botulism infection, by using a sandwich immunoassay, gold nanoparticle labels, and surface-enhanced Raman scattering (SERS) within the context of two threat scenarios. The first scenario mimics part of the analysis needed in response to a "white powder" threat by measuring both neurotoxins in phosphate-buffered saline (PBS), a biocompatible solvent often used to recover markers dispersed in a powdered matrix. The second scenario detects the two neurotoxins in spiked human serum to assess the clinical potential of the platform. The overall goal is to develop a test applicable to both scenarios in terms of projections of required levels of detection. We demonstrate the ability to measure BoNT-A and BoNT-B in PBS at a limit of detection (LoD) of 700â¯pg/mL (5 pM) and 84â¯pg/mL (0.6 pM), respectively, and in human serum at 1200â¯pg/mL (8 pM) and 91â¯pg/mL (0.6 pM), respectively, with a time to result under 24â¯h. The steps required to transform this platform into an onsite biodefense screening tool that can simultaneously and rapidly detect (<1â¯h) these and other agents are briefly discussed.
RESUMO
BACKGROUND: The population served by Federally Qualified Health Centers (FQHCs) has lower levels of cancer screening compared with the general population and suffers a disproportionate cancer burden. To address these disparities, 3 federal agencies and a primary care association established and tested the feasibility of a Regional Cancer Collaborative (RCC) in 2005. METHODS: RCC faculty implemented a learning model to improve cancer screening across 4 FQHCs that met explicit organizational readiness criteria. Regional faculty trained "care process leaders," who worked with primary care teams to plan and implement practice changes. FQHCs monitored progress across the following measures of screening implementation: self-management goal-setting; number and percent screened for breast, cervical, and colorectal cancer; percent timely results notification; and percent abnormal screens evaluated within 90 days. Progress and plans were reviewed in regular teleconferences. FQHCs were encouraged to create local communities of practice (LCOP) involving community resources to support cancer screening and to participate in a monthly teleconference that linked the LCOPs into a regional community of practice. Summary reports and administrative data facilitated a process evaluation of the RCC. chi test and test of trends compared baseline and follow-up screening rates. RESULTS: The RCC taught the collaborative process using process leader training, teleconferences, 2 regional meetings, and local process improvement efforts. All organizations created clinical tracking capabilities and 3 of the 4 established LCOPs, which met monthly in an regional community of practice. Screening documentation increased for all 3 cancers from 2005 to 2007. Colorectal cancer screening increased from 8.6% to 21.2%. CONCLUSIONS: A regional plan to enable collaborative learning for cancer screening implementation is feasible, and improvements in screening rates can occur among carefully selected organizations.
Assuntos
Neoplasias Colorretais/diagnóstico , Disparidades em Assistência à Saúde , Programas de Rastreamento/organização & administração , Serviços Preventivos de Saúde/organização & administração , Atenção Primária à Saúde/organização & administração , Programas Médicos Regionais/organização & administração , Idoso , Neoplasias Colorretais/epidemiologia , Neoplasias Colorretais/prevenção & controle , Relações Comunidade-Instituição , Feminino , Humanos , Masculino , Programas de Rastreamento/estatística & dados numéricos , Pessoa de Meia-Idade , Padrões de Prática Médica/organização & administração , Serviços Preventivos de Saúde/estatística & dados numéricos , Atenção Primária à Saúde/estatística & dados numéricos , Avaliação de Processos em Cuidados de Saúde , Avaliação de Programas e Projetos de Saúde , Garantia da Qualidade dos Cuidados de Saúde , Estados UnidosRESUMO
This paper describes preliminary results on the surprising impact of human serum as a sample matrix on the detectability of protective antigen (PA) and lethal factor (LF), two antigenic protein markers of Bacillus anthracis, in a heterogeneous immunometric assay. Two sample matrices were examined: human serum and physiological buffer. Human serum is used as a specimen in the diagnostic testing of potentially infected individuals. Physiological buffers are often applied to the recovery of biomarkers dispersed in suspicious white powders and other suspect specimens and as a serum diluent to combat contributions to the measured test response from nonspecific adsorption. The results of these experiments using a sandwich immunoassay read out by surface-enhanced Raman scattering yielded estimates for the limit of detection (LOD) for both markers when using spiked human serum that were remarkably lower than those of spiked physiological buffer (â¼70,000× for PA and â¼25,000× for LF). The difference in LODs is attributed to a degradation in the effectiveness of the capture and/or labeling steps in the immunoassay due to the known propensity for both proteins to denature in buffer. These findings indicate that the use of physiological buffer for serum dilution or recovery from a powdered matrix is counter to the low-level detection of these two antigenic proteins. The potential implications of these results with respect to the ability to detect markers of other pathogenic agents are briefly discussed.
Assuntos
Antígenos de Bactérias/sangue , Toxinas Bacterianas/sangue , Imunoensaio/métodos , Soro/química , Anticorpos/imunologia , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Biomarcadores/sangue , Soluções Tampão , Ouro/química , Humanos , Limite de Detecção , Nanopartículas Metálicas/química , Análise Espectral Raman/métodosRESUMO
The development and use of HPLC-MS for the study of metabonomics is reviewed. To date the technique has been applied to the analysis of urine samples obtained from studies in rodents in investigations of physiological variation (e.g., factors such as strain, gender, diurnal variation, etc.) and toxicity. Examples are provided of the use of conventional HPLC, capillary methods and the recently introduced high-resolution systems based on a combination of high pressure and small particle size ("UPLC"). Comparison is also made of the use of 1H NMR spectroscopy and HPLC-MS for the analysis of biofluid samples and the advantages and limitations of the two approaches are assessed. Likely future developments are considered.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas/métodos , Animais , Líquidos Corporais/química , Feminino , Masculino , Ratos , Distribuição TecidualRESUMO
(1)H NMR and HPLC-MS were used to generate metabolite fingerprints for the metabonomic analysis of urine obtained from both male and female Zucker obese (fa/fa) rats, used as a model of type II diabetes, and normal male Wistar-derived animals. The resulting data were subjected to chemometric analysis (principal components analysis and partial least squares discriminant analysis) to investigate the effects of strain, diurnal variation is strain, diurnal variation and gender and gender on metabolite profiles. In the case of strain, (1)H NMR spectroscopic analysis revealed increased taurine, hippurate and formate and decreased betaine, alpha-ketoglutarate, succinate and acetate in samples from Zucker-obese compared to Wistar-derived rats. HPLC-MS analysis detected increased hippurate and ions at m/z 255.0640 and 285.0770 in positive, and 245.0122 and 261.0065 in negative electrospray ionisation (ESI), respectively, for the Zucker obese samples. Both techniques enable the detection of diurnal variation in the urine of male and female Zucker rats, marked by increases in taurine, creatinine, allantoin and alpha-ketoglutarate by (1)H NMR, and ions at m/z 285.0753, 291.0536 and 297.1492 (positive ESI) and 461.1939 (negative ESI) using HPLC-MS, in the evening samples. Differences between male and female Zucker rats were also observed. Compared to samples from male rats hippurate, succinate, alpha-ketoglutarate and dimethylglycine ((1)H NMR) were elevated in the urine of female animals together with ions at, e.g., m/z 431.1047, 325.0655, 271.0635 and 447.0946 (positive ESI) and m/z 815.5495 and 459.0985 (negative ESI) by HPLC-MS. Both analytical techniques used in this study were able to detect differences between normal and Zucker obese rats, which may provide markers of metabolic disease.
Assuntos
Biomarcadores/urina , Cromatografia Líquida de Alta Pressão/métodos , Imageamento por Ressonância Magnética/métodos , Obesidade/urina , Espectrometria de Massas por Ionização por Electrospray/métodos , Acetatos/urina , Animais , Betaína/urina , Ritmo Circadiano , Feminino , Formiatos/urina , Glutaratos/urina , Hipuratos/urina , Masculino , Ratos , Ratos Endogâmicos , Ratos Wistar , Ratos Zucker , Fatores Sexuais , Ácido Succínico/urina , Taurina/urinaRESUMO
Ultra-performance liquid chromatography (UPLC) coupled to orthogonal acceleration time-of-flight mass spectrometry (oa-TOFMS) in positive electrospray ionization mode was used to obtain metabolite profiles for urine obtained from three strains of Zucker rat. These were the Zucker lean, the Zucker (fa/fa) obese and the Zucker lean/(fa) cross. Clear age- and strain-related differences were noted with the leptin-deficient (fa/fa) obese animal showing significant differences from both the other Zucker rat strains with respect to metabolite profiles.
Assuntos
Fatores Etários , Obesidade/genética , Ratos Zucker/genética , Ratos Zucker/metabolismo , Magreza/genética , Animais , Cromatografia Líquida , Cruzamentos Genéticos , Espectrometria de Massas , Ratos , Ratos Zucker/urina , Espectrometria de Massas por Ionização por Electrospray , Fatores de TempoRESUMO
Analysis of biological fluids using ultra-performance liquid chromatography/mass spectrometry (UPLC/MS) (metabonomics) can allow new insights to be gained into disease processes, with advances in chromatographic techniques enabling the detection of thousands of metabolites. In this work metabonomics has been used to investigate the metabolic processes involved in type II diabetes in the Zucker obese rat. Plasma was analyzed from three different strains, the Zucker (fa/fa) obese, Zucker lean and the lean/(fa) obese cross. Using UPLC/MS, ca. 10,000 ions were detected due to the narrow peak widths and excellent peak shapes achieved with this technology. Confidence in the chromatographic performance was demonstrated by the use of quality control standards. The positive and negative ion total ion chromatograms obtained from the three strains were readily distinguishable using multivariate statistical analysis. The greatest difference was observed between the Zucker lean and Zucker lean/(fa) rats compared to the Zucker (fa/fa) obese rats. Positive ions m/z 220 (4.36 min), 282(3.78 min), 359 (5.33 min) and 405 (7.77 min) were elevated in the plasma derived from Zucker lean rats whilst ions m/z 385 (6.80 min) and 646 (4.36 min) were at a lower concentration compared to the plasma from the Zucker (fa/fa) obese animals. Negative ions elevated in the Zucker lean rats included m/z 212 (2.30 min), 514 (2.85 min), 295 (4.39 min), 329 (3.11 min), 343 (2.86 min) and 512 (2.86 min) with ions m/z 538 (4.18 min), 568 (4.18 min), 568 (5.09 min) and 612 (4.30 min) being raised in the samples derived from Zucker (fa/fa) obese animals. The ion m/z 514 (3.85 min) was found to correspond to taurocholate, providing further support for an involvement of taurine metabolism in diabetes.
Assuntos
Cromatografia Líquida de Alta Pressão , Diabetes Mellitus Tipo 2/metabolismo , Obesidade/sangue , Preparações Farmacêuticas/sangue , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Fenótipo , Análise de Componente Principal , Ratos , Ratos Endogâmicos , Ratos Zucker/genética , Especificidade da Espécie , Biologia de SistemasRESUMO
The field of drug metabolism has been revolutionized by liquid chromatography/mass spectrometry (LC/MS) applications with new technologies such as triple quadrupoles, ion traps and time-of-flight (ToF) instrumentation. Over the years, these developments have often relied on the improvements to the mass spectrometer hardware and software, which has allowed users to benefit from lower levels of detection and ease-of-use. One area in which the development pace has been slower is in high-performance liquid chromatography (HPLC). In the case of metabolite identification, where there are many challenges due to the complex nature of the biological matrices and the diversity of the metabolites produced, there is a need to obtain the most accurate data possible. Reactive or toxic metabolites need to be detected and identified as early as possible in the drug discovery process, in order to reduce the very costly attrition of compounds in late-phase development. High-resolution, exact mass measurement plays a very important role in metabolite identification because it allows the elimination of false positives and the determination of non-trivial metabolites in a much faster throughput environment than any other standard current methodology available to this field. By improving the chromatographic resolution, increased peak capacity can be achieved with a reduction in the number of co-eluting species leading to superior separations. The overall enhancement in the chromatographic resolution and peak capacity is transferred into a net reduction in ion suppression leading to an improvement in the MS sensitivity. To investigate this, a number of in vitro samples were analyzed using an ultra-performance liquid chromatography (UPLC) system, with columns packed with porous 1.7 mum particles, coupled to a hybrid quadrupole time-of-flight (ToF) mass spectrometer. This technique showed very clear examples for fundamental gains in sensitivity, chromatographic resolution and speed of analysis, which are all important factors for the demands of today's HTS in discovery.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Dextrometorfano/farmacocinética , Microssomos Hepáticos/metabolismo , Proclorperazina/farmacocinética , Espectrometria de Massas por Ionização por Electrospray/métodos , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Taxa de Depuração Metabólica , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
LC/MS is an analytical technique that, due to its high sensitivity, has become increasingly popular for the generation of metabolic signatures in biological samples and for the building of metabolic data bases. However, to be able to create robust and interpretable (transparent) multivariate models for the comparison of many samples, the data must fulfil certain specific criteria: (i) that each sample is characterized by the same number of variables, (ii) that each of these variables is represented across all observations, and (iii) that a variable in one sample has the same biological meaning or represents the same metabolite in all other samples. In addition, the obtained models must have the ability to make predictions of, e.g. related and independent samples characterized accordingly to the model samples. This method involves the construction of a representative data set, including automatic peak detection, alignment, setting of retention time windows, summing in the chromatographic dimension and data compression by means of alternating regression, where the relevant metabolic variation is retained for further modelling using multivariate analysis. This approach has the advantage of allowing the comparison of large numbers of samples based on their LC/MS metabolic profiles, but also of creating a means for the interpretation of the investigated biological system. This includes finding relevant systematic patterns among samples, identifying influential variables, verifying the findings in the raw data, and finally using the models for predictions. The presented strategy was here applied to a population study using urine samples from two cohorts, Shanxi (People's Republic of China) and Honolulu (USA). The results showed that the evaluation of the extracted information data using partial least square discriminant analysis (PLS-DA) provided a robust, predictive and transparent model for the metabolic differences between the two populations. The presented findings suggest that this is a general approach for data handling, analysis, and evaluation of large metabolic LC/MS data sets.
Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Metabolismo/fisiologia , Interpretação Estatística de Dados , Análise Discriminante , Humanos , Urinálise/métodosRESUMO
The use of Ultra Performance Liquid Chromatography (UPLC), with a rapid 1.5 minute reversed-phase gradient separation on a 1.7 microm reversed-phase packing material to provide rapid "high throughput" support for metabonomic screening is demonstrated. The peak capacity and the number of marker ions detected using these fast UPLC separations and oa-TOF MS was found to be similar to that generated by conventional HPLC-MS methods with a 10 minute separation. The UPLC-MS methodology was applied to the analysis of urine samples from rodents, including normal and Zucker obese rats and three strains of mice (of both sexes), and was found to provide rapid discrimination between age, strain, gender and diurnal variation.
Assuntos
Urinálise/métodos , Envelhecimento/urina , Animais , Cromatografia Líquida/métodos , Ritmo Circadiano/fisiologia , Feminino , Masculino , Espectrometria de Massas/métodos , Camundongos , Camundongos Nus , Ratos , Ratos Zucker , Caracteres SexuaisRESUMO
The combination of a new 1.7 mum reversed-phase packing material, and a chromatographic system, operating at ca. 12,000 psi, (so-called ultra performance liquid chromatography, UPLC) has enabled dramatic increases in chromatographic performance to be obtained for complex mixture separation. This increase in performance is manifested in improved peak resolution, together with increased speed and sensitivity. Here, we show that UPLC offers significant advantages over conventional reversed-phase HPLC amounting to a more than doubling of peak capacity, an almost 10-fold increase in speed and a 3- to 5-fold increase in sensitivity compared to that generated with a conventional 3.5 microm stationary phase. The first functional genomic application of UPLC-MS technology is illustrated here with respect to multivariate metabolic profiling of urines from males and females of two groups of phenotypically normal mouse strains (C57BL19J and Alpk:ApfCD) and a "nude mouse" strain. We have also compared this technology to conventional HPLC-MS under similar analytical conditions and show improved phenotypic classification capability of UPLC-MS analysis together with increased ability to probe differential pathway activities between strains as a result of improved analytical sensitivity and resolution.
Assuntos
Cromatografia Líquida/métodos , Genômica , Espectrometria de Massas/métodos , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos NusRESUMO
Ultra-performance liquid chromatography (UPLC) utilizes sub-2 microm particles with high linear solvent velocities to effect dramatic increases in resolution, sensitivity and speed of analysis. The reduction in particle size to below 2 microm requires instrumentation that can operate at pressures in the 6000-15,000 psi range. The typical peak widths generated by the UPLC system are in the order of 1-2 s for a 10-min separation. In the present work this technology has been applied to the study of in vivo drug metabolism, in particular the analysis of drug metabolites in bile. The reduction in peak width significantly increases analytical sensitivity by three- to five-fold, and the reduction in peak width, and concomitant increase in peak capacity, significantly reduces spectral overlap resulting in superior spectral quality in both MS and MS/MS modes. The application of UPLC/MS resulted in the detection of additional drug metabolites, superior separation and improved spectral quality.
Assuntos
Bile/química , Cromatografia Líquida de Alta Pressão/métodos , Microquímica/métodos , Midazolam/análise , Nanotecnologia/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Tamanho da Partícula , Ratos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Integração de SistemasRESUMO
The process of metabolite identification is essential to the drug discovery and development process; this is usually achieved by liquid chromatography/tandem mass spectrometry (LC/MS/MS) or a combination of liquid chromatography/mass spectrometry (LC/MS) and nuclear magnetic resonance (NMR) spectroscopy. Metabolite identification is, however, a time-consuming process requiring an experienced skilled scientist. Multivariate statistical analysis has been used in the field of metabonomics to elucidate differences in endogenous biological profiling due to a toxic effect or a disease state. In this paper we show how a combination of liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) and multivariate statistical analysis can be used to detect drug metabolites in a biological fluid with no prior knowledge of the compound administered.