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

ABSTRACT: 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 ?g/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 ?g/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.

SUBMITTER: Godfrey AR

PROVIDER: S-EPMC5366175 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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

Godfrey A Ruth AR Jones Lewis L Davies Mairead M Townsend Rachel R

Analytical and bioanalytical chemistry 20170204 11

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 cha ...[more]

PMID: 28161751

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Project description:BACKGROUND: The number of patients with endometrial carcinoma (EmCa) with advanced stage or high histological grade is increasing and prognosis has not improved for over the last decade. There is an urgent need for the discovery of novel molecular targets for diagnosis, prognosis and treatment of EmCa, which will have the potential to improve the clinical strategy and outcome of this disease. METHODOLOGY AND RESULTS: We used a "drill-down" proteomics approach to facilitate the identification of novel molecular targets for diagnosis, prognosis and/or therapeutic intervention for EmCa. Based on peptide ions identified and their retention times in the first LC-MS/MS analysis, an exclusion list was generated for subsequent iterations. A total of 1529 proteins have been identified below the Proteinpilot® 5% error threshold from the seven sets of iTRAQ experiments performed. On average, the second iteration added 78% new peptides to those identified after the first run, while the third iteration added 36% additional peptides. Of the 1529 proteins identified, only 40 satisfied our criteria for significant differential expression in EmCa in comparison to normal proliferative tissues. These proteins included metabolic enzymes (pyruvate kinase M2 and lactate dehydrogenase A); calcium binding proteins (S100A6, calcyphosine and calumenin), and proteins involved in regulating inflammation, proliferation and invasion (annexin A1, interleukin enhancer-binding factor 3, alpha-1-antitrypsin, macrophage capping protein and cathepsin B). Network analyses revealed regulation of these molecular targets by c-myc, Her2/neu and TNF alpha, suggesting intervention with these pathways may be a promising strategy for the development of novel molecular targeted therapies for EmCa. CONCLUSIONS: Our analyses revealed the significance of drill-down proteomics approach in combination with iTRAQ to overcome some of the limitations of current proteomics strategies. This study led to the identification of a number of novel molecular targets having therapeutic potential for targeted molecular therapies for endometrial carcinoma.

Dataset Information

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

Publications

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

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