Quantitative Analysis of Lysophosphatidic Acid in Human Plasma by Tandem Mass Spectrometry

Analysis of lysophosphatidic acids (LPAs) is of clinical importance as they can serve a potential marker for ovarian and other gynecological cancers and obesity. It is critically important to develop a highly sensitive and specific method for the early detection of gynecological cancers to improve the overall outcome of this disease. We have established a novel quantification method of LPAs in human plasma by negative ionization tandem mass spectrometry (MS-MS) using multiple reaction monitoring (MRM) mode without the conventional TLC step. Protein-bound lipids, LPAs in plasma were extracted with methanol : chloroform (2:1) containing LPA C14:0 as an internal standard under acidic condition. Following back extraction with chloroform and water, the centrifuged lower phase was evaporated and reconstituted in methanol. The reconstituted solution was directly injected into electrospray source of MS/MS. For MRM mode, Q1 ions selected were m/z 409, 433, 435, 437 and 457 which corresponds to molecular mass [M-H]- of C16:0, C18:2, C18:1, C18:0 and C20:4 LPA, respectively. Q2 ions selected for MRM were m/z 79, phosphoryl product. Using MS/MS with MRM mode, all the species of LPAs were completely separated from plasma matrix without severe interferences. This method allowed simultaneous detection and quantification of different species of LPAs in a plasma over a linear dynamic range of 0.01-25 ㎛olL-1 . The detection limit of the method was 0.3 pmol/mL, with a correlation coefficient of 0.9983 in most LPAs analyzed. When applied to the plasmas of normal and gynecological cancer patients, this new method differentiated two different groups by way of total LPA level.