SIGNAL ENHANCEMENT FOR GRADIENT REVERSE-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY ELECTROSPRAY-IONIZATION MASS-SPECTROMETRY ANALYSIS WITH TRIFLUOROACETIC AND OTHER STRONG ACID MODIFIERS BY POSTCOLUMN ADDITION OF PROPIONIC-ACID AND ISOPROPANOL

Trifluoroacetic acid (TFA) and other volatile strong acids, used as mo difiers in reverse-phase high-performance liquid chromatography, cause signal suppression for basic compounds when analyzed by electrospray ionization mass spectrometry (ESI-MS). Evidence is presented that sign al suppression is caused by strong ion pairing between the TFA anion a nd the protonated sample cation of basic sample molecules. The ion-pai ring process ''masks'' the protonated sample cations from the ESI-MS e lectric fields by rendering them ''neutral.'' Weakly basic molecules a re not suppressed by this process. The TFA signal suppression effect i s independent from the well-known spray problem that electrospray has with highly aqueous solutions that contain TFA. This previously report ed spray problem is caused by the high conductivity and surface tensio n of aqueous TFA solutions. A practical method to enhance the signal f or most basic analytes in the presence of signal-suppressing volatile strong acids has been developed. The method employs postcolumn additio n of a solution of 75% propionic acid and 25% isopropanol in a ratio 1 :2 to the column flow. Signal enhancement is typically 10-50 times for peptides and other small basic molecules. Thus, peptide maps that use ESI-MS for detection can be performed at lower levels, with conventio nal columns, without the need to use capillary chromatography or reduc ed mass spectral resolution to achieve satisfactory sensitivity. The m ethod may be used with similar results for heptafluorobutyric acid and hydrochloric acid. A mechanism for TFA signal suppression and signal enhancement by the foregoing method, is proposed.