RESUMEN
Aim: To develop a high sensitivity and specific analytical method to measure endogenous levels of leukotriene B4 (LTB4) in human plasma. Methodology: LC-MS/MS and ELISA. Results: An LC-MS/MS method was developed with a sensitivity of 1.0 pg/ml, and within and between batch precision of <16% and <13% RSD, respectively. Conclusion: We have developed a sensitive LC-MS/MS method that can detect endogenous LTB4 in human plasma. The LC-MS/MS method displayed correlation with a commercial LTB4 ELISA when analyzing in ex vivo ionophore-stimulated blood samples. For untreated plasma this correlation was lost. Endogenous LTB4 was shown to be unstable in plasma during storage at -20°C and subject to stereoisomer formation. Neither of the assays could quantify endogenous plasma LTB4 in samples stored for long term.
Asunto(s)
Leucotrieno B4/sangre , Cromatografía Liquida , Ensayos Clínicos Fase I como Asunto , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Leucotrieno B4/antagonistas & inhibidores , Leucotrieno B4/química , Masculino , Estructura Molecular , Espectrometría de Masas en TándemRESUMEN
We describe a simple and robust tube-based ex vivo whole blood stimulation procedure suitable for use in clinical laboratories by multiple operators on repeated occasions to study cytokine production in phase 1 human trials of investigational medicines, developed by the authors specifically to study IL-17A expression in man. The stimulation procedure is further proposed as a useful tool for biomarker assay development and validation, for example to prepare quality control samples without reliance on recombinant materials for spiking.