A sensitive LC-MS/MS method for quantifying phosphatidic acid species using basic mobile phases

Phosphatidic acid (PA) is a crucial precursor for various phospholipids in living organisms and also functions as an important intracellular signaling molecule. Due to its characteristic cone-shaped molecular structure, it also plays a role in modulating the curvature of biological membranes. PA exists in various molecular forms due to the combination of its two acyl groups. Therefore, to elucidate the diverse roles of PA in living organisms, it is necessary to measure the abundance of each PA molecular species quantitatively and with high sensitivity. However, reports of analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) are limited due to the strong chelation of the phosphate group in PA with the metal surface of HPLC columns. This study aimed to develop a method for highly sensitive and straightforward phosphatidic acid analysis using LC-MS/MS. We achieved this by using a metal-free column and a basic mobile phase to quantify PA species with high sensitivity and accuracy. Using this method, we analyzed PA species as well as other phospholipid species in HEK293 cells. The fatty acids constituting PA were primarily C16:0 and C18:1, whereas various poly unsaturated fatty acids were detected in the fatty acids constituting other phospholipid classes such as phosphatidylcholine. As another example to illustrate the principle, the amount of PA species were significantly increased in THP-1 cells stimulated with phorbol 12-myristate 13-acetate. These results suggest that our present method is useful to analyze the metabolism and functions of PA in living organisms.