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

Abstract

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. Copyright © 2016 John Wiley & Sons, Ltd.

Figure 1

Chemical structure of phosphatidylcholine (PC) and hypothetical role of ammonium in protonation during positive ion electrospray. As indicated in the text, no evidence of [M+NH₄]⁺ formation was obtained.

Figure 2

Positive ion electrospray LC-MS/MS analysis of 1 μL of 0.01 mg/mL of krill oil. The detection of lyso palmitoyl PC (SRM of the protonated molecule of m/z 496 forming the abundant fragment ion of m/z 184) is shown to compare effects of methanol/water (80:20; v/v), methanol/water + 5 mM ammonium formate or acetonitrile/water (80:20/v/v) on PC ionization. The two peaks detected in the methanol traces represent separation of the isomeric sn¹ and sn² lyso palmitoyl PCs.

Figure 3

Positive ion electrospray mass spectra of DHA DHA PC in acetonitrile/water (80:20; v/v) containing A) 5 mM ammonium acetate; or B) no ammonium acetate. Protonated PC and adducts with sodium and potassium were detected, but no [M+NH₄]⁺ was observed at m/z 895.6. The addition of ammonium acetate did not eliminate or reduce the relative abundances of [M+Na]⁺ and [M+K]⁺ ions. Similar results were obtained when ammonium formate or ammonium bicarbonate was substituted for ammonium acetate (data not shown).

Figure 4

Positive ion electrospray mass spectra of DHA DHA PC infused in A) 100% acetonitrile containing 5 mM [d7]-ammonium acetate; or B) acetonitrile/D₂O (80:20; v/v) containing 5 mM ammonium acetate. Note that the formation of [M+D]⁺ ions at m/z 879.6 occurred only when using D₂O, indicating that water and not ammonium donates the proton during the ionization of PCs.

Figure 5

Comparison of mobile phase additives for the measurement of EPA EPA PC in krill oil using positive ion electrospray LC-MS/MS with SRM ([M+H]⁺ m/z 827.1 to m/z 184). Note that formic acid, acetic acid, ammonium formate, and ammonium acetate were less effective than ammonium bicarbonate, which was nearly equivalent to neat acetonitrile/water (80:20; v/v) as the mobile phase.