An online nano-liquid-chromatography tandem mass spectrometry (HPLC-MS/MS) with ultra low push-pull sampling for determining brain acetylcholine levels

Acetylcholine (ACh) is believed to play a major role in attentional processing in cortex, thereby contributing to memory formation and maintenance. Monitoring ACh level-changes during behavior will help to understand their role in neuronal communication and function. In contrast to other highly sensitive detectors like electrochemistry (EC), which are commonly used in the monitoring of changes of extracellular brain fluid (EBF), mass-spectrometry methods enable direct determination of Ach without additional enzyme reactor. Here, a push and pull sampling method with flow rates in the range of nano-liters/min is used to reduce depletion of the EBF, allowing for lower flow rates compared to conventional microdialysis. To match the requirement to determine amounts of Ach in the attomole amount of acetylcholine from an EBF sample, a nano HPLC-MS/MS with semi online sampling system was set up and tested. The injection assembly included a high pressure injection pump and a series of computer-controlled nanopeak valves. For each injection a 250nl sample obtained in 5-min sampling interval was pumped out from the sampling capillary tubing. Chromatographic separation was performed on a capillary column of 75 µm I.D. Each HPLC run was finished in 7 min with a stepwise elution profile used for fast analysis. After the injection, the sample was trapped by flushing the column with pure water for 2 min, and subsequently with high concentration of acetonitrile favored by electrospray ionization MS. In-vitro test shows that acetylcholine could be detected with a low detection-limit of 30 attomole. By repeating this method in vivo we were able to reliably detect acetylcholine in the awake macaque. Future experiments will apply this methodology in combined neurochemical-electrophysiological experiments in behaving animals