Direct coupling of ionic high-performance liquid chromatography with electrospray ionization mass spectrometry utilizing a microdialysis junction interface

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 similar to 5-40 mu l/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 mu l/min was found optimal for both retaining HPLC resolution and achieving efficient on-line desalting. When performed at 50 degrees C, the microdialysis system could provide complete on-line desalting for LC buffers containing up to 50 mM 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 degrees C) and the molecular mass cut-off (similar to 1000) imposed by the microdialysis fiber and the low sensitivity of the LCQ mass spectrometer for high molecular mass species. (C) 1999 Elsevier Science B.V. All rights reserved.