Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 12 de 12
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Bioanalysis ; 4(12): 1429-43, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22793028

RESUMO

BACKGROUND: In support of a pilot clinical trial using acetaminophen as the model compound to assess dried blood spot (DBS) sampling as the method for clinical pharmacokinetic sample collection, a novel sensitive LC-MS/MS method was developed and validated for the simultaneous determination of acetaminophen and its major metabolites, acetaminophen glucuronide and sulfate, in human DBS samples collected by subjects via fingerprick. RESULTS: The validated assay dynamic range was from 50.0 to 5000 ng/ml for each compound using a 1/8´´ (3-mm) disc punched from a DBS sample. Baseline separation of the three analytes was achieved to eliminate the possible impact of insource fragmentation of the conjugated metabolites on the analysis of the parent. The overall extraction efficiency was from 61.3 to 78.8% for the three analytes by direct extraction using methanol. CONCLUSION: The validated method was successfully implemented in the pilot clinical study with the obtained pharmacokinetic parameters in agreement with the values reported in literature.


Assuntos
Acetaminofen/análogos & derivados , Analgésicos não Narcóticos/sangue , Cromatografia de Fase Reversa/métodos , Teste em Amostras de Sangue Seco/métodos , Espectrometria de Massas em Tandem/métodos , Acetaminofen/sangue , Acetaminofen/farmacocinética , Analgésicos não Narcóticos/farmacocinética , Teste em Amostras de Sangue Seco/instrumentação , Humanos , Limite de Detecção , Metanol/química , Projetos Piloto , Padrões de Referência , Extração em Fase Sólida/métodos
2.
Bioanalysis ; 3(17): 1975-86, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21899506

RESUMO

BACKGROUND: Although it is well known that automation can provide significant improvement in the efficiency of biological sample preparation in quantitative LC-MS/MS analysis, it has not been widely implemented in bioanalytical laboratories throughout the industry. This can be attributed to the lack of a sound strategy and practical procedures in working with robotic liquid-handling systems. RESULTS: Several comprehensive automation assisted procedures for biological sample preparation and method validation were developed and qualified using two types of Hamilton Microlab liquid-handling robots. The procedures developed were generic, user-friendly and covered the majority of steps involved in routine sample preparation and method validation. CONCLUSION: Generic automation procedures were established as a practical approach to widely implement automation into the routine bioanalysis of samples in support of drug-development programs.


Assuntos
Automação/métodos , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida/instrumentação , Descoberta de Drogas , Humanos , Imidazóis/análise , Estrutura Molecular , Plasma/química , Piridinas/análise , Padrões de Referência , Reprodutibilidade dos Testes , Robótica , Espectrometria de Massas em Tandem/instrumentação
3.
J Chromatogr B Analyt Technol Biomed Life Sci ; 879(24): 2376-82, 2011 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-21783438

RESUMO

A high-performance liquid chromatography-tandem mass spectrometric (LC-MS/MS) method has been developed and validated for the quantitative analysis of NIM811, a cyclophilin inhibitor, in human dried blood spot (DBS) samples, which were produced by spotting 20 µl whole blood onto FTA cards. A 3mm disc was cut from the DBS samples and extracted using methanol, followed by liquid-liquid extraction with MTBE. The reconstituted extracts were chromatographed using a Halo C(18) column and gradient elution for MS/MS detection. The possible impact of hematocrit, blood sample volume and punching location on DBS sampling was investigated. The results showed that blood sample volume or punching location has no impact on assay performance, but the presence of a high hematocrit resulted in significantly increased analyte concentrations measured from the high QC samples. The current method was fully validated over the range of 10.0-5000 ng/ml with correlation coefficients (r(2)) for three validation batches equal to or better than 0.991. The accuracy and precision (CV) at the LLOQ were -0.7 to 6.0% bias of the nominal value (10.0 ng/ml) and 10.2-2.3%, respectively. For the balance of QC samples (20.0, 50.0, 750, 1500 and 3750 ng/ml), the precision (CV) ranged from 3.2 to 11.7% and from 5.6 to 10.2%, respectively, for the intra-day and inter-day evaluations. The accuracy ranged from -6.8 to 8.5% and -0.2% to 2.7% bias, respectively, for the intra-day and inter-day batches. NIM811 is stable in the DBS samples for at least 24h at room temperature and 4h at 60°C. Interestingly, the long term stability (LTS) assessment showed that the stability of the analyte is better when the DBS samples were stored at a lower storage temperature (e.g. ≤ -60°C) compared to storage at room temperature. This is probably due to the interaction of the additives and/or other materials (e.g. cellulose, etc) on the DBS card with NIM811, a cyclic peptide. The current methodology has been applied to determine the NIM811 levels in DBS samples prepared from a clinical study.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Ciclofilinas/antagonistas & inibidores , Ciclosporina/sangue , Inibidores Enzimáticos/sangue , Espectrometria de Massas em Tandem/métodos , Análise Química do Sangue , Humanos
4.
J Chromatogr B Analyt Technol Biomed Life Sci ; 879(21): 1927-33, 2011 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-21680264

RESUMO

Ultrafiltration of human plasma in combination with LC-MS/MS has been increasingly used in the quantitative analysis of the free fraction of drug candidates for PK/efficacy assessment. In addition to controlling the pre-incubation and centrifugation temperatures, some important factors that must be investigated and addressed include: (1) possible nonspecific binding, (2) possible impact of freeze/thaw cycles of plasma samples and extended storage of plasma samples at room temperature on the analyte recovery prior to ultrafiltration, and (3) identification of the appropriate assay dynamic range to avoid unnecessary dilutions. These factors were explored in the development and validation of a robust LC-MS/MS assay for the quantitative analysis of unbound vadimezan (ASA404) in human plasma. First, to mimic human physiological conditions, all plasma samples were incubated at ~37°C for a minimum of 30 min after thawing and prior to centrifugation to obtain the ultrafiltrate. Second, by passing the calibration standards and QC samples in plasma ultrafiltrate through the ultrafiltration membrane, the observed non-specific binding of the analyte due to the membrane was corrected. Third, the effects of multiple freeze/thaw cycles and/or storage at room temperature for various periods (4, 8, 16 and 24h) were evaluated to determine the impact on analyte concentrations in the ultrafiltrate from the plasma QC samples. Fourth, the appropriate dynamic range was established to accommodate the expected incurred sample free analyte concentrations. The validated assay has a dynamic range of 30.0-30,000 ng/ml for ASA404 in human plasma ultrafiltrate using a sample volume of 30 µl. Quality control pools containing the analyte were prepared at concentrations of 30.0-22,500 ng/ml to cover the assay calibration range. The intra-assay and inter-assay precision and accuracy were ≤ 15% (CV) and within ± 15% (bias) of the nominal values, respectively, for all measured QC concentrations, including the LLOQ. Freeze/thaw for up to three cycles of the plasma samples and/or the extended human plasma sample exposure to room temperature for up to 24h were confirmed to have no impact on the assay results for the free analyte. The validated method was successfully implemented to support clinical studies for the compound.


Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Ultrafiltração/métodos , Xantonas/sangue , Estabilidade de Medicamentos , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Xantonas/química , Xantonas/farmacocinética
5.
Bioanalysis ; 3(9): 967-72, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21545344

RESUMO

BACKGROUND: In this case study, urine samples were collected and transferred before the presence of a small degree of nonspecific binding was identified for the analyte of interest in human urine. The approach taken to address the issue was to use standards and quality controls to mimic the study samples and use Tween-80 (0.5%) to retrieve the adsorbed analyte. The method was validated, however, the incurred sample reanalysis (ISR) failed. RESULTS: Investigation into the ISR failure unveiled ineffective mixing of the study samples, with almost no headspace left inside the sample tubes after the addition of the surfactant using a regular vortex mixer, as the cause of the ISR failure. All samples were reanalyzed using a modified sample mixing method, which resulted in two successful ISR runs. CONCLUSIONS: Thorough sample mixing after the addition of surfactant is one of the important steps in ensuring accurate and reproducible analyses of urine samples with a small degree of analyte nonspecific binding.


Assuntos
Artefatos , Manejo de Espécimes/normas , Urina/química , Adsorção , Calibragem , Cromatografia Líquida , Guias como Assunto , Humanos , Polissorbatos , Controle de Qualidade , Padrões de Referência , Reprodutibilidade dos Testes , Projetos de Pesquisa , Manejo de Espécimes/efeitos adversos , Espectrometria de Massas por Ionização por Electrospray , Tensoativos
6.
J Chromatogr B Analyt Technol Biomed Life Sci ; 878(5-6): 583-9, 2010 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-20097141

RESUMO

Analyte loss due to non-specific binding, especially container surface adsorption, is not uncommon in the quantitative analysis of urine samples. In developing a sensitive LC-MS/MS method for the determination of a drug candidate, BAF312, in human urine, a simple procedure was outlined for identification, confirmation and prevention of analyte non-specific binding to a container surface and to recover the 'non-specific loss' of an analyte, if no transfer has occurred to the original urine samples. Non-specific binding or container surface adsorption can be quickly identified by using freshly spiked urine calibration standards and pre-pooled QC samples during a LC-MS/MS feasibility run. The resulting low recovery of an analyte in urine samples can be prevented through the use of additives, such as the non-ionic surfactant Tween-80, CHAPS and others, to the container prior to urine sample collection. If the urine samples have not been transferred from the bulk container, the 'non-specific binding' of an analyte to the container surface can be reversed by the addition of a specified amount of CHAPS, Tween-80 or bovine serum albumin, followed by appropriate mixing. Among the above agents, Tween-80 is the most cost-effective. beta-cyclodextrin may be suitable in stabilizing the analyte of interest in urine via pre-treating the matrix with the agent. However, post-addition of beta-cyclodextrin to untreated urine samples does not recover the 'lost' analyte due to non-specific binding or container surface adsorption. In the case of BAF312, a dynamic range of 0.0200-20.0 ng/ml in human urine was validated with an overall accuracy and precision for QC sample results ranging from -3.2 to 5.1% (bias) and 3.9 to 10.2% (CV), respectively. Pre- and post-addition of 0.5% (v/v) Tween-80 to the container provided excellent overall analyte recovery and minimal MS signal suppression when a liquid-liquid extraction in combination with an isocratic LC separation was employed. The compound was stable in 0.5% Tween-80 treated human urine QC samples for at least 24 h at room temperature, after three freeze/thaw cycles with storage at < or =-60 degrees C and for at least 3 months when stored at < or =-60 degrees C. The current work could serve as a simple example in trouble shooting non-specific binding or container surface adsorption in quantitative analysis of urine samples.


Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Adsorção , Cromatografia Líquida/economia , Humanos , Receptores de Lisoesfingolipídeo/metabolismo , Sensibilidade e Especificidade , Espectrometria de Massas em Tandem/economia
7.
Biomed Chromatogr ; 21(8): 841-51, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17441219

RESUMO

A sensitive and simple liquid chromatography-tandem mass spectrometry method for the determination of midazolam and 1'-hydroxymidazolam in human plasma has been developed and validated with a dynamic range of 0.1-250 ng/mL. The analysis was based on semi-automated liquid-liquid extraction followed by evaporation of the extraction solvent, reconstitution and chromatography on a reversed-phase C(18) column. The mobile phase consists of 5 mm ammonium acetate and methanol and runs in gradient at a flow rate of 0.25 mL/min with column temperature of approximately 20 degrees C. The entire column effluent was transferred into the LC-MS/MS interface operated in positive electrospray ionization mode. The chromatographic run time was 4.3 min per injection, with retention times for midazolam, 1'-hydroxymidazolaml and the internal standard, triazolam, of 2.5, 2.3 and 2.1 min, respectively. The intra-day and inter-day precision (RSD %) and accuracy (bias %) of the quality control samples were <15.0% and within +/-13%, respectively. The current method has been applied to a clinical drug-drug interaction study in human.


Assuntos
Cromatografia Líquida/métodos , Midazolam/análogos & derivados , Midazolam/sangue , Espectrometria de Massas em Tandem/métodos , Calibragem , Humanos , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
8.
J Chromatogr B Analyt Technol Biomed Life Sci ; 846(1-2): 57-68, 2007 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-16962398

RESUMO

For the first time, a liquid chromatographic method with tandem mass spectrometric detection (LC-MS/MS) for the simultaneous determination of ribavirin and rabavirin base was developed and validated over the concentration range of 10-5,000 ng/ml, respectively, using a 0.025 ml monkey plasma sample. Ribavirin, ribavirin base, and the internal standards were extracted from monkey plasma via protein precipitation. After evaporation of the supernatant, the extract was reconstituted with 5% methanol (containing 0.1% formic acid) and injected onto the LC-MS/MS system. Optimum chromatographic separation was achieved on a Waters Atlantis dc18 (150 mm x 2.1mm, 5 microm) column with mobile phase run in gradient with 100% water containing 0.5% formic acid (A) and 90% acetonitrile (containing 0.5% formic acid (B). The flow rate was 0.4-0.6 ml/min with total cycle time of approximately 7.0 min. Post-column addition of acetonitrile (containing 0.1% formic acid) at 0.3 ml/min was used to increase the ionization efficiency in the MS source. The method was validated for sensitivity, linearity, reproducibility, stability and recovery. Lack of adverse matrix effect and carry-over was also demonstrated. The intra-day and inter-day precision and accuracy of the quality control (QC) samples were <9.0% relative standard deviation (R.S.D.) and 10.8% bias for ribavirin, and 10.3% R.S.D. and 11.3% bias for ribavirin base. The current specific, accurate and precise assay is useful in support of the toxicokinetic and pharmacokinetic studies of these compounds.


Assuntos
Antivirais/sangue , Cromatografia Líquida de Alta Pressão/métodos , Ribavirina/sangue , Espectrometria de Massas em Tandem/métodos , Animais , Haplorrinos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
9.
Toxicology ; 213(1-2): 1-12, 2005 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-15970369

RESUMO

Transplantation in nonhuman primates, in particular using solid organs from porcine donors, requires an efficacious induction immunosuppression. Besides biologicals, the low molecular weight drugs used include cyclophosphamide (CyP) and methotrexate (MTX). As these compounds generally have a narrow therapeutic window, we performed tolerability studies in baboons and cynomolgus monkeys, with/without maintenance immunosuppressants such as cyclosporine A, everolimus, mycophenolate sodium and FTY720. In both species, a four-dose CyP regimen of 40, 20, 30 and 30 mg/kg i.v. on days 1, 2, 4 and 6 is not tolerated, but the regimen is tolerated upon individual adjustment of the third and fourth dose to 18-25 and 8-20mg/kg, respectively, based on white blood cell count. In cynomolgus monkeys, a 5-day course of MTX i.v. at 0.5 mg/(kg d) is well tolerated, but not MTX at 1.0 mg/(kg d); in combination with maintenance immunosuppression, the 0.5mg/(kg d) dose can cause adverse effects. Combinations of CyP and MTX are tolerated using the 5-day course of MTX at 0.25 mg/(kg d) and a four-dose regimen of CyP at 10, 2.5, 7.5 and 7.5 mg/kg. These regimens are tolerated in combination with maintenance immunosuppressants. The data provide base values for investigators using nonhuman primates in experimental studies, particularly in xenotransplantation requiring effective induction immunosuppression that is close to maximum tolerated dose levels.


Assuntos
Ciclofosfamida/toxicidade , Imunossupressores/toxicidade , Metotrexato/toxicidade , Animais , Ciclofosfamida/farmacologia , Ciclosporina/farmacologia , Sinergismo Farmacológico , Everolimo , Feminino , Cloridrato de Fingolimode , Imunossupressores/farmacologia , Contagem de Leucócitos , Macaca fascicularis , Masculino , Metotrexato/farmacologia , Ácido Micofenólico/farmacologia , Transplante de Órgãos/veterinária , Papio , Propilenoglicóis/farmacologia , Sirolimo/análogos & derivados , Sirolimo/farmacologia , Esfingosina/análogos & derivados
10.
Xenotransplantation ; 12(1): 72-5, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15598276

RESUMO

BACKGROUND: In vivo xenotransplantation modeling in large animal species is often performed in nonhuman primates, including baboons. For proper data interpretation, reference values for clinical chemistry and hematology are required. METHODS: These values are available from baseline levels in animals subjected to tolerability/pharmacokinetic studies. For each individual study two tests for clinical chemistry and hematology were performed before the start of treatment. RESULTS AND CONCLUSION: Baseline levels were determined in 27 males and 15 females, in total 106 determinations (53 in males, and 53 in females). For a number of parameters levels in cynomolgus monkeys show clear differences from those in baboons. These data are reported here to provide baseline values for veterinarians and investigators using nonhuman primates in experimental studies.


Assuntos
Química Clínica/normas , Hematologia/normas , Macaca fascicularis/sangue , Transplante Heterólogo , Animais , Feminino , Masculino , Valores de Referência
11.
Xenotransplantation ; 11(6): 511-6, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15479460

RESUMO

BACKGROUND: In vivo xenotransplantation modeling in large animal species is often performed in nonhuman primates, including baboons. For proper data interpretation, reference values for clinical chemistry and hematology are required. METHODS: These values are available from baseline levels in animals subjected to tolerability/pharmacokinetic studies. For each individual study two tests for clinical chemistry and hematology were performed before the start of treatment. RESULTS AND CONCLUSION: We present such data from 17 male and 16 female baboons, with body weights ranging between 4.4 and 14.0 kg (males) and 4.1 and 15.0 kg (females), respectively. The number of duplicate samples per animal determined before each individual study ranged between one and five. These data are reported here to provide baseline values for veterinarians and investigators using baboons in experimental studies, particularly in xenotransplantation.


Assuntos
Química Clínica/normas , Hematologia/normas , Papio , Transplante Heterólogo , Animais , Análise Química do Sangue/normas , Feminino , Masculino , Padrões de Referência
12.
J Clin Pharmacol ; 43(2): 163-70, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12616669

RESUMO

Treatment of hyperglycemia in patients with diabetes mellitus and renal insufficiency is complicated by altered pharmacokinetics of hypoglycemic agents. This study evaluated the pharmacokinetic profile and safety of nateglinide, an amino acid derivative that improves early phase insulin secretion and reduces mealtime glucose excursions. This open-label, single-dose, two-center study included patients (mean age = 57 +/- 10 years) with type 1 or 2 diabetes with impaired renal function (IRF) (n = 10) or with renal failure undergoing hemodialysis (n = 10). Both groups were compared with age-, sex-, height-, and weight-matched healthy controls (n = 20). All participants received a single 120-mg dose of nateglinide immediately before breakfast. Pharmacokinetic and safety evaluations were undertaken up to 48 hours postdose. All 40 subjects completed the study. Plasma nateglinide concentrations increased rapidly in patients undergoing dialysis and matched healthy subjects (tmax = 0.95 vs. 0.78 h, respectively) and was comparable with patients with IRF and matched healthy subjects (tmax = 0.80 vs. 0.65 h, respectively). There were no statistically significant differences for Cmax or AUC0-t between the groups. Nateglinide was eliminated rapidly in all groups (t1/2 = 1.9-2.8 h). There was no correlation between the level of renal function and systemic exposure. There was a low extent of renal excretion of nateglinide in healthy subjects (11%) and diabetic patients with IRF (3%). Nateglinide was well tolerated. These data suggest that nateglinide is suitable for use in diabetic patients with IRF or with renal failure undergoing dialysis. Given the comparable absorption and elimination profiles of nateglinide in renally impaired and healthy subjects, no dose adjustment appears necessary in the renally impaired.


Assuntos
Cicloexanos/farmacocinética , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Hipoglicemiantes/farmacocinética , Fenilalanina/farmacocinética , Adulto , Idoso , Área Sob a Curva , Glicemia/efeitos dos fármacos , Estudos de Casos e Controles , Cicloexanos/sangue , Cicloexanos/farmacologia , Feminino , Meia-Vida , Humanos , Hipoglicemiantes/sangue , Hipoglicemiantes/farmacologia , Falência Renal Crônica/metabolismo , Masculino , Taxa de Depuração Metabólica , Pessoa de Meia-Idade , Nateglinida , Fenilalanina/análogos & derivados , Fenilalanina/sangue , Fenilalanina/farmacologia , Diálise Renal
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...