RESUMEN
BACKGROUND: Dried blood spot-based bioanalysis potentially introduces novel matrix effects that need to be eliminated or controlled. Within nonregulatory drug discovery these can be defined as ≤20% and ≤30% for nominal peak area, respectively. RESULTS: Controlling matrix effects for a panel of compounds by simple 1D-HPLC-MS/MS was not achievable and the optimization of 2D-HPLC-MS/MS is reported here. Simple inclusion of a 'trapping' stage was not sufficient to improve matrix effects and optimization of the reconstitution solvent, reconstitution volume and injection volume was required for a generic system to be developed. CONCLUSION: A generic 2D-LC-MS/MS system has been developed that eliminates paper-based matrix effects and eliminates or controls dried blood spot matrix effects for a panel of compounds extracted from FTA Elute™ with methanol.
Asunto(s)
Artefactos , Recolección de Muestras de Sangre/métodos , Sangre , Cromatografía Líquida de Alta Presión/métodos , Descubrimiento de Drogas , Papel , Espectrometría de Masas en Tándem/métodos , Animales , Guanidinas/química , Inyecciones , Isotiocianatos/química , Metanol/química , Ratas , Solventes/química , Factores de TiempoRESUMEN
BACKGROUND: Targeting the gonadotropin-releasing hormone pathway for the treatment of endometriosis leads to an interest in monitoring for endogenous modulators of this pathway (RFRP3 and kisspeptin) as baseline controls for treatment development. RESULTS: Stabilization of RFRP3 was shown to be extremely difficult in a highly enzymatically active matrix, such as rat blood. Sample denaturing with solvent at collection was necessary due to enzyme inhibition being unsuccessful at stabilization leading to difficulties in sample processing. Monitoring multiple fragments formed in blood can aid in profiling these peptides once in-source conversion is controlled. CONCLUSION: generic high-sensitivity LC-MS/MS assay was developed for RFRP3 and the fragments formed from it in whole blood. Use of 2D chromatography circumvents concentration and retention issues related to small fragments with a normal flow setup, making a more open-access approach feasible.
Asunto(s)
Análisis Químico de la Sangre/métodos , Neuropéptidos/sangre , Animales , Humanos , Neuropéptidos/química , Neuropéptidos/farmacocinética , Ratas , Ratas WistarRESUMEN
BACKGROUND: Dried blood spots (DBS) are rapidly gaining a foothold in the pharmaceutical industry. However, applications in exploratory drug discovery are limited mainly owing to method development time. The development of a generic DBS assay is presented with its application in serial microsampling from mice. RESULTS: A generic 'fit-for-purpose' assay was developed on FTA(®) Elute, which allowed 90% of compounds tested to reach sensitivity levels of 1 ng/ml. A ten-time point serial mouse pharmacokinetic study was conducted using 20-µl microsamples and DBS. Application of generic 'fit-for-purpose' approach did not compromise data delivery or quality. CONCLUSIONS: Serial microsampling and DBS in exploratory mouse pharmacokinetic has been shown to provide superior data quality when compared with traditional plasma-based composite studies.
