Phosphatidylcholine (PC) species in human plasma are used as biomarkers of disease. PC biomarkers are often limited by the inability to separate isobaric PCs. In this work, we developed a targeted shotgun approach for analysis of isobaric and isomeric PCs. This approach is comprised of two MS methods: a precursor ion scanning (PIS) of mass m/z 184 in positive mode (PIS m/z +184) and MS(3) fragmentation in negative mode, both performed on the same instrument, a hybrid triple quadrupole ion-trap mass spectrometer. The MS(3) experiment identified the FA composition and the relative abundance of isobaric and sn-1, sn-2 positional isomeric PC species, which were subsequently combined with absolute quantitative data obtained by PIS m/z +184 scan. This approach was applied to the analysis of a National Institute of Standards and Technology human blood plasma standard reference material (SRM 1950). We quantified more than 70 PCs and confirmed that a majority are present in isobaric and isomeric mixtures. The FA content determined by this method was comparable to that obtained using GC with flame ionization detection, supporting the quantitative nature of this MS method. This methodology will provide more in-depth biomarker information for clinical and mechanistic studies.

Department of Biomedical Sciences University of North Dakota School of Medicine and Health Sciences University of North Dakota

Grand Forks ND 58201

USDA ARS Grand Forks Human Nutrition Research Center Grand Forks ND 58203

USDA ARS Grand Forks Human Nutrition Research Center Grand Forks ND 58203 Department of Chemistry University of North Dakota

USDA ARS Grand Forks Human Nutrition Research Center Grand Forks ND 58203 Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic 16610 Prague 6 Czech Republic

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