RESUMEN
BACKGROUND: Human cerebrospinal fluid (CSF) is often acquired in Phase I clinical trials to assess the CNS penetration of new pharmacological agents and to search for biomarkers associated with PD effects. Robust methods for neurotransmitter metabolites in CSF have proven elusive, in part due to inadequate reversed phase LC retention. RESULTS: Benzoyl chloride derivatization was used to promote retention for LC-MS/MS for a panel of neurotransmitter metabolites while delivering a concise method for sample preparation. CONCLUSION: A validated assay in human CSF was obtained for 3,4-dihydroxyphenylacetic acid, homovanillic acid, 3,4-dihydroxyphenylglycol and 5-hydroxyindoleacetic acid. This method is differentiated from other LC-MS/MS methods by delivering results in line with full regulatory expectations.
Asunto(s)
Benzoatos/química , Pruebas de Química Clínica/métodos , Neurotransmisores/líquido cefalorraquídeo , Espectrometría de Masas en Tándem , Ácido 3,4-Dihidroxifenilacético/líquido cefalorraquídeo , Ácido 3,4-Dihidroxifenilacético/normas , Animales , Cromatografía Líquida de Alta Presión/normas , Semivida , Ácido Homovanílico/líquido cefalorraquídeo , Ácido Homovanílico/normas , Humanos , Ácido Hidroxiindolacético/líquido cefalorraquídeo , Ácido Hidroxiindolacético/normas , Masculino , Metoxihidroxifenilglicol/análogos & derivados , Metoxihidroxifenilglicol/líquido cefalorraquídeo , Metoxihidroxifenilglicol/normas , Neurotransmisores/química , Neurotransmisores/metabolismo , Control de Calidad , Ratas , Ratas Sprague-Dawley , Espectrometría de Masas en Tándem/normasRESUMEN
A new method utilizing high-performance liquid chromatography with electrochemical detection for the determination of unconjugated 3-methoxy-4-hydroxyphenylglycol, a major metabolite of norepinephrine, in human plasma is described. A novel internal standard, 3-ethoxy-4-hydroxyphenylglycol, was used to correct for variations in extraction efficiency and detector sensitivity. Separation of sample components was achieved isocratically in 20 min per sample in a reversed-phase system. Column temperature had to be carefully regulated. The sensitivity of the assay is suitable for clinical studies, with a limit of detection of 2 pmol/ml. Intra- and interassay precision was good, with observed coefficients of variation of 7.5 and 8.8%, respectively. Accuracy was tested by comparing the results of the present HPLC method to those found with a specific gas chromatographic-mass spectrometric assay. A satisfactory correlation of 0.96 was obtained, with no significant bias between the two methods.