The effect of ionization modifiers added to the mobile phase of nanoflow liquid chromatography-tandem mass spectrometry (nLC-ESI-MS 3) on the simultaneous analysis of all phospholipid (PL) classes in negative ion mode has been investigated. While MS analysis of most PL classes is carried out in negative ion mode, analysis of neutral polar (polar but electrically neutral) lipids like phosphatidylcholine (PC) and sphingomyelin (SM) is highly efficient in positive ion mode. Therefore, analysis of PL mixture samples often requires two separate runs in both positive and negative ion mode. In order to establish run conditions to carry out a single nLC-ESI-MS-MS for all PLs, the ionization efficiency of 13 different types of PL molecules in nLC-ESI-MS has been evaluated in negative ion mode by varying the modifiers and their concentrations. Experiments demonstrated that a mixture of 0.05% ammonium hydroxide and 1mM ammonium formate added to the mobile phase provided effective ionization for all classes of PLs. The optimized conditions were applied to the analysis of a phospholipid mixture extracted from a human urine sample, yielding the identification of a total of 85 PL species. Analysis of the same sample with dual nLC-ESI-MS 2 runs in both positive and negative ion mode confirmed that nLC-ESI-MS 3 with the mixed modifier run only in negative ion mode gave comparable results.
Bibliographical noteFunding Information:
This study was supported by the National Research Foundation (NRF) of Korea grant (No. 2011-0016438 ) funded by the Korean government (MEST) and in part by a grant (No. 2011-0001125 ).
All Science Journal Classification (ASJC) codes
- Analytical Chemistry
- Organic Chemistry