Miltefosine: a novel internal standard approach to lysophospholipid quantitation using LC-MS/MS

Understanding and determining levels of lysophospholipids (LPLs) is of increasing interest to the bioanalytical community as they may be targeted for preparative removal as a matrix interference or as a lead substance as a biomarker of disease. Studies monitoring levels of LPLs have used a range of approaches for quantitation whereby those using an internal standard have used either deuterated analogues of the target LPL or alternative LPLs containing an odd number of carbon atoms within its chain, which can be expensive and difficult to distinguish with other LPLs, respectively. A structural analogue, miltefosine, was investigated as a novel internal standard to quantify a selection of lysophosphatidylcholines (LPCs) of clinical interest. A reverse phase C18 LC-MS/MS method was characterised for 16:0-LPC, 18:1-LPC and 18:0-LPC, showing good sensitivity and linearity for all compounds, with limit of detection (LOD) values <1 μg/mL and R ² ≥ 0.97. Quality control (QC) samples were studied to determine accuracy and precision of the method, with values <15% variation for each compound at multiple concentrations. As an example application, we have used this method to detect the amount of LPC breakthrough following solid phase extraction (SPE) of plasma to quantify LPCs as a target species and to remove them as matrix interferences under various conditions typical to clinical work. This study showed that changes in sample pH could adversely affect the capture of the LPCs and their contribution as matrix interferences, with 3.6 μg/mL of 18:1-LPC observed following plasma extraction. Graphical Abstract A novel internal standard approach to lysophospholipid quantitation in extracted plasma using miltefosine, with analysis by LC-MS/MS.