Mass spectrometric analysis of the amiodarone-induced alteration of phosphatidylcholines in living single cells.

Amiodarone is one of the cationic amphiphilic drugs which cause the drug-induced phospholipidosis (DIPL). With the accumulation of phospholipids, the multiple-membrane structures called lamellar bodies emerge in the cell. Since the pathogenesis mechanism has not been clarified yet, DIPL is still concerned in the clinical use of amiodarone and the new drug development processes. In this study, we have focused on phosphatidylcholines (PCs) which are the representative phospholipids and have developed a single-cell analytical method for the determination of PCs accumulated in the lamellar bodies. By a nanoelectrospray ionization-tandem mass spectrometry (nanoESI-MS/MS), PCs were sensitively and selectively detected at a single-cell level with their product ions of m/z 184. The amounts of PCs increased in the amiodarone-treated cells, and some of them were found higher in the lamellar bodies than in the cytoplasm. The exact mass analysis by a high-resolution mass spectrometry and the identification of the side chains of PCs revealed that the two PCs of PC34:1 (16:0_18:1) and PC32:0 (16:0_16:0) may accumulate in the lamellar bodies. The developed method can be applied to the regioselective analyses of other phospholipids and metabolites at a single-cell level and is promising to clarify the mechanism of DIPL.