RESUMO
In this research, we developed and validated a liquid chromatography coupled to mass spectrometry (LC-QToF-MS) method for simultaneous quantification of the anti-tuberculosis drugs ethambutol, isoniazid, pyrazinamide and rifampicin in human plasma. Plasma samples spiked with cimetidine (internal standard) were extracted using protein precipitation with acetonitrile containing 1% formic acid. Separation was performed using a C18 column under flow gradient conditions with water and acetonitrile, both containing 5 mm ammonium formate and 0.1% formic acid. The method was validated according to the ANVISA and US Food and Drug Administration guidelines for bioanalytical method validation. The calibration curve was linear over a concentration range of 0.2-5 µg ml-1 for ethambutol, 0.2-7.5 µg ml-1 for isoniazid, 1-40 µg ml-1 for pyrazinamide and 0.25-2 µg ml-1 for rifampicin, all with adequate precision and accuracy. The method was reproducible, selective and free of carryover and matrix effects. The validated LC-QToF-MS method was successfully applied to real samples and shown to be applicable to future therapeutic and pharmacokinetic monitoring studies.
Assuntos
Antituberculosos/sangue , Cromatografia Líquida de Alta Pressão/métodos , Etambutol/sangue , Isoniazida/sangue , Espectrometria de Massas/métodos , Pirazinamida/sangue , Rifampina/sangue , Humanos , Plasma/químicaRESUMO
With the increased cases of multidrug- or rifampicin-resistant tuberculosis and co-infection with HIV globally, it is difficult to achieve ideal clinical responses because of poor drug absorption and drug-drug interactions. Herein, a bioanalytical UPLC-MS/MS method was developed and validated to quantify five anti-TB agents in human plasma samples for detecting blood drug concentrations to improve therapeutic effects. To overcome the matrix effects, stable isotope labeled analogue of each analyte was used for internal standardization. A simple single-step protein precipitation by acetonitrile was employed for the sample preparation, then the analytes including rifampicin, rifabutin, pyrazinamid, ethambutol, isoniazid and their isotope labeled internal standards (ILISs) were implemented on an HILIC silica column with a gradient mode. The linear range for each analyte was covering the peak drug concentration (Cmax) in the 20 times diluted plasma samples. The coefficient of variation of intra- and inter-day precision was less than 17.0 %, and the accuracy ranged between 91.5 and 110.0 %. The extraction recoveries of all agents were ≥90.2 %, and the matrix effects with internal standard-normalization for all agents were 97.1-110.0 %. The optimal blood sampling time was designed basing on the results of stability validation. This UPLC-MS/MS method with a run time of 3.5â¯min was successfully applied to routine therapeutic monitoring of the five anti-TB agents in patient plasma.
Assuntos
Antituberculosos/sangue , Monitoramento de Medicamentos/métodos , Etambutol/sangue , Isoniazida/sangue , Pirazinamida/sangue , Rifabutina/sangue , Rifampina/sangue , Infecções Oportunistas Relacionadas com a AIDS/sangue , Calibragem , Cromatografia Líquida de Alta Pressão/métodos , Monitoramento de Medicamentos/instrumentação , Humanos , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Massas em Tandem/métodos , Tuberculose/sangueAssuntos
Antituberculosos/sangue , Pirazinamida/sangue , Tuberculose Pulmonar/sangue , Antituberculosos/administração & dosagem , Brasil , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Mycobacterium tuberculosis/efeitos dos fármacos , Pirazinamida/administração & dosagem , Fatores Sexuais , Estatísticas não Paramétricas , Fatores de Tempo , Resultado do Tratamento , Tuberculose Pulmonar/tratamento farmacológicoAssuntos
Humanos , Masculino , Feminino , Pirazinamida/sangue , Tuberculose Pulmonar/sangue , Antituberculosos/sangue , Pirazinamida/administração & dosagem , Fatores de Tempo , Tuberculose Pulmonar/tratamento farmacológico , Brasil , Fatores Sexuais , Resultado do Tratamento , Estatísticas não Paramétricas , Relação Dose-Resposta a Droga , Mycobacterium tuberculosis/efeitos dos fármacos , Antituberculosos/administração & dosagemRESUMO
Intestinal barrier function and serum concentrations of rifampin, isoniazid and pyrazinamide were studied in healthy controls and patients with active pulmonary tuberculosis. A case-control study of 29 controls and 30 cases attending at the Health Center, July, 2004 to December, 2005 was conducted. The body mass index was significantly reduced in cases compared to controls (p < 0.001). The intestinal paracellular transport of lactulose was significantly (p = 0.019) reduced in cases compared to controls. The transcellular transport of mannitol and the lactulose:mannitol ratio were not significantly (p = 0.0698) reduced in cases compared to controls. Low serum concentrations of rifampin, isoniazid and pyrazinamide were observed in 81 percent (48/59), 92 percent (54/59) and 28 percent (12/59), respectively, in all individuals. The results demonstrated a marked decrease on intestinal paracellular transport in patients with active pulmonary tuberculosis and reduced serum concentrations of rifampin and isoniazid in both groups.
Assuntos
Adulto , Feminino , Humanos , Masculino , Antituberculosos/farmacocinética , Absorção Intestinal/fisiologia , Tuberculose Pulmonar/metabolismo , Antituberculosos/sangue , Antituberculosos/uso terapêutico , Estudos de Casos e Controles , Isoniazida/sangue , Isoniazida/farmacocinética , Isoniazida/uso terapêutico , Pirazinamida/sangue , Pirazinamida/farmacocinética , Pirazinamida/uso terapêutico , Rifampina/sangue , Rifampina/farmacocinética , Rifampina/uso terapêutico , Fatores de Tempo , Tuberculose Pulmonar/sangue , Tuberculose Pulmonar/tratamento farmacológicoRESUMO
Intestinal barrier function and serum concentrations of rifampin, isoniazid and pyrazinamide were studied in healthy controls and patients with active pulmonary tuberculosis. A case-control study of 29 controls and 30 cases attending at the Health Center, July, 2004 to December, 2005 was conducted. The body mass index was significantly reduced in cases compared to controls (p < 0.001). The intestinal paracellular transport of lactulose was significantly (p = 0.019) reduced in cases compared to controls. The transcellular transport of mannitol and the lactulose:mannitol ratio were not significantly (p = 0.0698) reduced in cases compared to controls. Low serum concentrations of rifampin, isoniazid and pyrazinamide were observed in 81% (48/59), 92% (54/59) and 28% (12/59), respectively, in all individuals. The results demonstrated a marked decrease on intestinal paracellular transport in patients with active pulmonary tuberculosis and reduced serum concentrations of rifampin and isoniazid in both groups.