Among all phospholipid classes, phosphatidic acid (PA) and phosphatidylserine (PS) tend to elute as broad peaks under the reversed phase LC conditions. In this study, we aimed to detect sharp peaks of phosphorylated compounds, such as phospholipids, by using a metal-free column, and bio-inert HPLC coupled to tandem MS, and adding acetylacetone to mobile phase. PA and PS were eluted as peaks of broad and low peak intensity using a stainless steel column. By contrast, they were eluted as sharp peaks using a metal-free column. Moreover, the peak intensity were improved 3-fold by using the mobile phase including in 1% acetylacetone. We suggested that acetylacetone was suppressed interactions of the metal contacts with PA and PS, and improved the ionization efficiency.