Quantitative analysis of acyl-lysophosphatidic acid in plasma using negative ionization tandem mass spectrometry

Analysis of acyl-lysophosphatidic acids (LPAs) has clinical importance as a potential biomarker for ovarian and other gynecological cancers or obesity from the point of view of prevention. Here we report a simple sample preparation and analytical method with high sensitivity and specificity for the early detection of gynecological cancers to improve the overall outcome of this disease. We established a novel quantification method for acyl-LPAs in plasma by electrospray negative ionization tandem mass spectrometry (MS-MS) using multiple reaction monitoring mode without conventional TLC step. Protein-bound lipids, acyl-LPAs in plasma were extracted with methanol/chloroform (2:1) containing LPA C(14:0) as internal standard under acidic conditions. Following back-extraction with chloroform and water, the centrifuged lower phase was evaporated and reconstituted in methanol and then analyzed. Using ESI-MS-MS with negative ionization MRM mode, all the species of LPAs were completely separated from plasma matrix without severe interference. For MRM mode, Q1 ions selected were m/z 409, 433, 435, 437 and 457 which corresponds to molecular mass [M-H](-) of C(16:0), C(18:2), C(18:1), C(18:0) and C(20:4) LPA, respectively. Q2 ions selected for MRM was m/z 79, phosphoryl product. Using MS-MS with MRM mode, all the species of LPAs were completely separated from plasma matrix without severe interference. This method allowed simultaneous detection and quantification of different species of LPAs in plasma over a linear dynamic range of 0.01-25 micromol/l. The method detection limit was 0.3 pmol/ml with correlation coefficient of 0.9983 in most LPAs analyzed. When applied to plasma from normal and gynecological cancer patients, this new method differentiated two different groups by way of total LPA level.