Sheathless capillary electrophoresis-mass spectrometry (CE-MS), using a porous tip sprayer, is proposed for the first time for highly sensitive metabolic profiling.A representative metabolite mixture and human urine were used for evaluation of the sheathless CE-MS platform.For test compounds, RSDs for migration times and peak areas were below 2% and 11%, respectively, and an injection volume of only ~8 nL resulted in detection limits between 11 and 115 nanomolar.Approximately 1200 molecular features were detected in human urine by sheathless CE-MS whereas about 400 molecular features were found with classical sheath-liquid CE-MS.This difference can be attributed to an improved ionization efficiency and increased sensitivity at low flow-rate conditions.The integration of transient-isotachophoresis (t-ITP) as an in capillary preconcentration procedure in sheathless CE-MS further resulted in subnanomolar limits of detection for compounds of the metabolite mixture and more than 1600 molecular features were observed in urine.Compared to the classical CE-MS approaches, the integration of t-ITP combined with the use of a sheathless interface provides a two orders of magnitude sensitivity improvement.Hence, sheathless CE-MS can be used for in-depth metabolic profiling of biological samples, and we anticipate that this approach will yield unique information in the field of metabolomics.