Abstract

A method for directly coupling ionic HPLC with electrospray ionization mass spectrometry (ESI-MS) utilizing a microdialysis junction interface is described. HPLC eluent was split postcolumn to allow a ∼5–40 μl/min flow into the microdialysis assembly, which consists of a microbore microdialysis fiber and a larger concentric sheath tubing for the introduction of a counter-current dialysis buffer. The salt-containing sample was desalted on-line and transferred directly to the ESI source of a LCQ ion trap mass spectrometer. A sample flow-rate (inside the microdialysis fiber) of 10–20 μl/min was found optimal for both retaining HPLC resolution and achieving efficient on-line desalting. When performed at 50°C, the microdialysis system could provide complete on-line desalting for LC buffers containing up to 50 m M Tris–HCl and 1 M NaCl. The introduction of an organic sheath liquid with 2% acetic acid enhanced the ESI-MS detection sensitivity by as much as tenfold and a sensitivity in the low pmol range (initial LC injection before flow splitting) was achieved for insulin chain A. Successful on-line desalting was also achieved for a protein mixture consisting of ubiquitin, cytochrome c and lysozyme separated by cation-exchange chromatography and a good quality spectrum was obtained for each component. Limitations of the current system include the working pH range (4–11), the temperature tolerance (<60°C) and the molecular mass cut-off (∼1000) imposed by the microdialysis fiber and the low sensitivity of the LCQ mass spectrometer for high molecular mass species.

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