Effects of mobile phase additives, solution pH, ionization constant, and analyte concentration on the sensitivities and electrospray ionization mass spectra of nucleoside antiviral agents

The effects of various mobile-phase additives, solution pH, pK(a), and anal yte concentration on electrospray ionization mass spectra of a series of pu rine and pyrimidine nucleoside antiviral agents were studied in both positi ve and negative ion modes. The use of 1% acetic acid resulted in good HPLC separation and the greatest sensitivity for [M + H](+) ions. In the negativ e ion mode, 50 mM ammonium hydroxide gave the greatest sensitivity for [M - H](-) ions. The sensitivities as [M + H](+) ions were significantly larger than the sensitivities as [M - H](-) ions for purine antiviral agents. Vid arabine monophosphate and pyrimidine antiviral agents, however, showed comp arable or greater sensitivities as [M - H](-) ions. The sensitivity as [M H](+) showed no systematic variation with pH; however, the sensitivity as [M - H](-) did increase with increasing pH, At constant pH, the ion intensi ty of the protonated species increased with increasing pK(a), At higher ana lyte concentrations, dimer (M2H+) and trimer (M3H+) ions were observed. [M + Na](+) adducts were the dominant ions with 0.5 mM sodium salts for these compounds. The spectra of the more basic purine antiviral agents showed no [M + NH4](+) adductions, but [M + NH4](+) ions were the major peaks in the spectra of the less basic pyrimidine antiviral agents with ammonium salts. The ammonium adduct ion was formed preferentially when the proton affinity of the analyte was dose to that of NH3, Abundant [M + OAc](-) ions were obs erved for all of the antiviral agents except vidarabine monophosphate from solutions with added HOAc, NaOAc, and NH4OAc. The utility of mobile phases containing 1% HOAc or 50 mM NH4OH was demonstrated for chromatographic sepa rations.