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.

Liquid chromatography mass spectrometry for quantifying plasma lysophospholipids: potential biomarkers for cancer diagnosis.

Cancer is a complex disease with many genetic and epigenetic aberrations that result in development of tumorigenic phenotypes. While many factors contribute to the etiology of cancer, emerging data implicate lysophospholipids acting through specific cell-surface, and potentially intracellular, receptors in acquiring the transformed phenotype propagated during disease. Lysophospholipids bind to and activate specific cell-surface G protein-coupled receptors (GPCRs) that initiate cell growth, proliferation, and survival pathways, and show altered expression in cancer cells. In addition, a number of enzymes that increase lysophospholipid production are elevated in particular cell lineages and cancer patients' cells, whereas in a subset of patients, the enzymes degrading lysophospholipids are decreased. Thus, ideal conditions are established to increase lysophospholipids in the tumor microenvironment. Indeed, ascites from ovarian cancer patients, which reflects both the tumor environment and a tumor-conditioned media, exhibits markedly elevated levels of specific lysophospholipids as well as one of the enzymes involved in production of lysophospholipids: autotaxin (ATX). The potential sources of lysophospholipids in the tumor microenvironment include tumor cells and stroma, such as mesothelial cells, as well as inflammatory cells and platelets activated by the proinflammatory tumor environment. If lysophospholipids diffuse from the tumor microenvironment into the bloodstream and persist, they have the potential to serve as early diagnostic markers as well as potential monitors of tumor response to therapy. Many scientific and technical challenges need to be resolved to determine whether lysophospholipids or the enzymes producing lysophospholipids alone or in combination with other markers have the potential to contribute to early diagnosis. Breast cancer is the most frequently diagnosed cancer among women. Mammography is associated with morbidity and has a high false positive and false negative rate. Thus, there is a critical need for biomarkers that can contribute to reduced false positive and false negative diagnoses, and to identify, stage, and/or predict prognosis of this disease to improve patient management. Here we describe a technical approach that can be applied to human blood plasma to measure the concentration of growth factor-like lysophospholipids contained in circulation. Using liquid chromatography mass spectrometry (LC/MS/MS), we quantified the amount of lysophosphatidic acid (16:0, 18:0, 18:1, 18:2, and 20:4), lysophosphatidylinositol (18:0), lysophosphatidylserine (18:1), lysophosphatidylcholine (16:0, 18:0, 18:1, 18:2, and 20:4), sphingosine-1-phosphate, and sphingosylphosphorylcholine species from human female plasma samples with malignant, benign, or no breast tumor present. Other methods described here include handling patient blood samples, lipid extraction, and factors that affect lysophospholipid production and loss during sample handling.