Nano-LC-MS/MS for the monitoring of angiotensin IV in rat brain microdialysates: limitations and possibilities

To broaden our knowledge about the central role of the angiotensin IV (Ang IV) peptide, we aimed to monitor its extracellular concentration in the brain using in vivo microdialysis. Ang IV was measured in the dialysates using a previously developed nano-LC-MS/MS assay with an LOD of 50 pM. Using this assay, baseline levels of Ang IV in dialysates from different brain structures were undetectable. However, immediately after microdialysis probe insertion, Ang IV could be detected in a concentration that varied between 120 and 187 pM. Using the zero-net-flux method, the extracellular levels of Ang IV in the striatum were estimated at 46 pM. These data may indicate that Ang IV is mainly present intracellularly. In addition, Ang IV was clearly measurable after striatal perfusion of Ang II. On the other hand, our nano-LC-MS/MS method was successful for the detection of Met-enkephalin and neurotensin in dialysates from the rat. In conclusion, the nano-LC-MS/MS method coupled with microdialysis is well suited to monitor the biologically significant conversion between Ang II and Ang IV in vivo, but physiological extracellular levels of Ang IV appear too low to be detected.