Lipid extraction and LC-MS/MS measurements of LPA

Seventeen LPA species were measured using techniques adapted from previously published methods (33). Briefly, either 50 μl of whole plasma or 250 μl of each plasma fraction were extracted using acidified organic solvents; the organic phases were evaporated to dryness and reconstituted for analysis. Fifty picomoles of 17:0 LPA (Avanti Polar Lipids Inc., Alabaster, AL) were included in each sample as an internal standard. LPA was measured using a Shimadzu HPLC coupled with an AB Sciex 6500-QTRAP hybrid linear ion trap triple quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) mode. Samples were separated on a ACQUITY UPLC BEH C8, 1.7 um, 2.1 × 100 mm column with an ACQUITY UPLC BEH C8 VanGuard Precolumn, 1.7 um, 2.1 × 5 mm. Precursor-product ion pairs used for quantitation were as follows: 381.2/152.9 for 14:0, 395.2/153.0 for 15:0, 409.2/153.0 for 16:0, 407.0/153.0 for 16:1, 423.2/153.0 for 17:0, 437.2/152.8 for 18:0, 435.2/153.0 for 18:1, 433.0/153.0 for 18:2, 431.0/153.0 for 18:3, 451.3/152.9 for 19:0, 463.03/153.0 for 20:0, 461.01/153.0 for 20:2, 459.1/153.0 for 20:3, 457.1/152.8 for 20:4, 455.1/153.0 for 20:5, 485.1/153.0 for 22:2, 483.1/153.0 for 22:4, and 481.1/153.0 for 22:6. Data were analyzed using ABSciex Multiquant software, and LPA concentrations (in picomoles) were determined using the internal standard as a reference.