An aliquot of total lipid extract was analyzed with a Micromass Quattro Ultima triple quadrupole mass spectrometer equipped with a nanoelectrospray source. Samples were loaded into thin-wall nanoflow capillary tips (Waters) and analyzed by ESI-MS-MS in both positive (for phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylserine (PS) and phosphatidylethanolamine (PE)), and negative ion mode (for PI, phosphatidylglycerol (PG), phosphatidic acid (PA), PS and PE). Capillary/cone voltages were 0·7 kV/50 V and 0·9 kV/50 V for positive ion and negative ion modes, respectively. Tandem mass spectra (MS-MS) were obtained using argon as the collision gas (~3·0 mTorr) with collision offset energies as follows: 35 V, PC in positive ion mode; 25 V, PE in positive ion mode; 22 V, PS in positive ion mode; 50 V, PE in negative ion mode; 28 V, PS in negative ion mode; 45 V, PI in negative ion mode; and 50 V, all glycerophospholipids detected by precursor scanning for m/z 153 in negative ion mode. MS-MS daughter ion scanning was performed with a collision-offset energy of 35 V. In positive ion mode, ions in the PC, PE, and PS spectra were annotated based on their [M+H-NMe3]+ for PC, and the corresponding fragment ions [M-140] and [PA-H] daughter ions for PE and PS respectively, and compared with that of their theoretical values. In negative ion mode, PL class peaks were assigned according to their [lyso-H]−, [lyso-H20-H]−, [lysoPA-H]−, or [lysoPA-H20-H]− daughter ion derivatives. FAs were assigned based on their [M−H]− values. Saturated and unsaturated FAs were assumed to be esterified to the sn-1 and sn-2 position of PLs, respectively. Each spectrum (600–1000 m/z) encompasses at least 50 repetitive scans, each of 4 s duration. Spectra were normally processed by subtraction of background and smoothed using Micronass processing algorithms unless otherwise indicated. The internal standards were used to ensure efficient ionization and fragmentation and as a control for sample variability.