INCORPORATION OF A RAM CELL IN A MICROCOLUMN FOR ON-COLUMN DETECTION OF RADIOLABELED MOLECULES FOR LC-RAM-ESI-MS

The objective of this work was to design a nondestructive solid scinti llation radioactivity monitor (RAM) cell for the detection of radiolab eled molecules for use with packed liquid chromatography (LC) microcol umns coupled to electrospray ionization-mass spectrometry (ESI-MS). To take advantage of the increased mass sensitivity offered by microcolu mns coupled to ESI-MS, the contributions to band broadening due to the solid scintillant was minimized by incorporation of the cell within t he microcolumn, resulting in on-column detection of radiolabeled molec ules. Our work shows that the contributions to band broadening and RAM sensitivity depend on the particle size of the solid scintillant, cel l volume, and cell diameter. Our results show that a solid scintillati on cell packed with silanated lithium glass with a mean particle size of 9.6 +/- 2.6 mu m gives a 24% increase in peak area compared to a ce ll packed with a mean particle size of 21.0 +/- 2.5 mu m. Our results show that a. 508-mu m-internal-diameter (i.d.) cell provided a 15% inc rease in peak area compared to a 762-mu m-i.d. cell. This work demonst rates the feasibility of detection of radiolabeled molecules using mic rocolumns. We have developed a 1.2-mu L volume on-column RAM cell for use with 250-mu m-i.d. packed microcolumns which can detect less than 200 radioactive disintegrations per minute (dpm) (1 dpm = 60 Bq) for a 1-min-wide chromatographic peak. Further development of solid scintil lants with a mean particle size of 3-5 mu m and spherical shape will p rovide additional sensitivity and a decrease in the band broadening co ntributed by the RAM cell. Additionally, such particles may be slurrie d with stationary phase and pack:ed into microcolumns for characteriza tion of on-column dispersion processes. The development of an on-colum n RAM solid scintillation cell for microcolumns combined with ESI-MS w ill have applications including the characterization of radiolabeled m olecules and their metabolites and the determination of ligand-protein binding sites through the use of radiolabeled ligands combined with o n-line protein digestion using microcolumn LC-RAM-ESI-MS. (C) 1998 Joh n Wiley & Sons, Inc.