Background:　Droplet　microfluidics　with　push-pull　and　microdialysis　sampling　from　brain　slices,　cultured　cells　and　engineered　tissues　produce　low　volume　mass　limited　samples　containing　analytes　sampled　from　the　extracellular　space.　This　sampling　approach　coupled　to　mass　spectrometry　(MS)　detection　allows　evaluation　of　time-　dependent　chemical　changes.　Our　goal　is　an　approach　for　continuous　sampling　and　segregation　of　extracellular　samples　into　picoliter　droplets　followed　by　the　characterization　of　the　droplets　using　nanoelectrospray　ionization　(nESI)　MS.　The　main　focus　here　is　the　optimization　of　the　carrier　oil　for　the　microfluidic　device　that　neither　affects　the　stability　of　picoliter　droplets　nor　compatibility　with　MS　detection　of　a　range　of　analytes.　Results:　We　developed　and　characterized　a　1-octanol-assisted　ultra-small　volume　droplet　microfluidic　nESI　MS　system　for　the　analysis　of　neurotransmitters　in　distinct　samples　including　cerebrospinal　fluid　(CSF).　The　use　of　a　1-octanol　oil　phase　was　effective　for　generation　of　aqueous　droplets　as　small　as　65　pL　and　enabled　detection　of　acetylcholine　(ACh)　and　gamma-aminobutyric　acid　(GABA)　in　water　and　artificial　CSF.　Continuous　MS　analysis　of　droplets　for　extended　periods　up　to　220　min　validated　the　long-term　stability　of　droplet　generation　and　analyte　detection　by　nESI-MS.　As　an　example,　ACh　response　demonstrated　a　linear　working　range　(R2　2　=　0.99)　between　0.4　mu　M　and　25　mu　M　with　a　limit　of　detection　of　370　nM　(24　amol),　enabling　its　quantitation　in　rodent　CSF.　Significance:　The　established　droplet　microfluidics　-　nESI　MS　approach　allows　the　analysis　of　microenvironments　at　high　spatiotemporal　resolution.　The　approach　may　allow　microsampling　and　monitoring　of　spatiotemporal　dynamics　of　neurochemicals　and　drugs　in　the　brain　and　spinal　cord　of　live　animals.