Ultrasonically assisted electrospray ionization for LC/MS determination of nucleosides from a transfer RNA digest

Anal Chem. 1994 Feb 1;66(3):406-14. doi: 10.1021/ac00075a015.


The use of an ultrasonic nebulizer to assist electrospray ionization mass spectrometry (ESI-MS) has been described and demonstrated with the analysis of a transfer RNA digest by microcolumn LC. The restricted range of mobile-phase compositions amenable to the electrospray process has traditionally placed a severe limitation on the types of LC applications that can be used with ESI-MS. For this reason, an ultrasonic nebulizer configured for LC has been developed that can generate the fine dispersion of liquid required for ESI-MS from any type of mobile phase. In the case presented here, a transfer RNA was enzymatically digested into its substituent nucleosides, which were then analyzed by microcolumn LC. The required mobile-phase gradient (beginning at 5% methanol) falls outside the solvent range that can be used with conventional electrospray. The ultrasonic nebulizer, however, resolves this problem. The fundamental behavior of the four most common nucleosides (cytidine, adenosine, guanosine, uridine) was studied, and conclusions concerning the effects of solution chemistry were drawn. Specifically, signal from the H+ adducts of these species seems to be strongly dependent on the pKa value. Also, effects from several source operating variables were examined. These included capillary exit voltage, drying and focusing gases, and nebulizer frequency. Performance was found to be consistent over a wide range (0-100% methanol) of mobile-phase compositions. The limit of detection for adenosine injected onto a microcolumn was found to be 100 amol. Finally, nucleosides from as little as 150 fmol of RNA (amount prior to digestion) could be detected.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Chromatography, Liquid*
  • Mass Spectrometry*
  • Nucleosides / analysis*
  • RNA, Transfer / chemistry*
  • Reproducibility of Results
  • Ultrasonics


  • Nucleosides
  • RNA, Transfer