Role of ammonium in the ionization of phosphatidylcholines during electrospray mass spectrometry.

RATIONALE: Electrospray mass spectrometry methods for the analysis of phosphatidylcholines (PCs) routinely include ammonium acetate or ammonium formate in the mobile phase. In an effort to justify and optimize the use of these additives, we investigated possible functions of ammonium compounds in the ionization of PCs.
METHODS: Because PCs contain a quaternary amine, the role of ammonium in neutralizing the negatively charged phosphate group was investigated by using deuterated ammonium acetate, adjusting the pH, varying the organic solvent composition, and by comparing the additives ammonium acetate, ammonium formate and ammonium bicarbonate. Seven PC standards were measured ranging from lyso 1-palmitoyl-sn-glycero-3-phosphocholine to 1,2-dieicosapentaenoyl-sn-glycero-3-phosphocholine as well as a mixture of PCs in a krill oil dietary supplement.
RESULTS: Under all conditions tested, aqueous acetonitrile provided more abundant formation of protonated PCs than did aqueous methanol. Regardless of the mobile phase composition and electrospray ion source parameters, no [M + NH4 ]+ ions were detected. Adding deuterated ammonium acetate to the mobile phase failed to form deuterated PCs, indicating that ammonium is not the source of the proton that neutralizes the phosphate negative charge. Instead, water was the source of the proton as deuterated water resulted in the formation of [M + D]+ ions. Addition of organic acids, ammonium formate, ammonium acetate, or ammonium bicarbonate to the mobile phase did not enhance and in most cases suppressed PC ionization.
CONCLUSIONS: Ammonium compounds and organic acids can suppress ionization of PCs when using an aqueous acetonitrile mobile phase during electrospray.