Single-step, chloroform-free extraction of lysophosphatidylcholine from plasma for cancer biomarker analysis

Abstract

Lysophosphatidylcholine (LPC) has been the subject of research for many years, but its role in lipid turnover is still not fully understood, neither its role in cancer development and progression. A crucial aspect in LPC research is its efficient and fast extraction from plasma and tissues to use LPC as a biomarker in clinical settings. The extraction methods commonly in use like Bligh & Dyer require the use of toxic halogenated solvents and are time consuming due to multiple extraction steps and subsequent solvent evaporation. In this study, a new, salt-assisted one-step extraction protocol, which avoids halogenated solvents, is presented. Saturated ammonium acetate solution was used as a salt component and acetonitrile:isopropanol (2.5:1 v/v) as eluent. The new extraction is characterized by its simplicity, robustness and short total process time of 18 minutes. For validation according to the ICH M10 guideline for bioanalytical method validation, LPC species extracted from plasma were quantified by LC-MS/MS, using LPC 19:0 as internal standard. In addition to these advantages, the new extraction procedure showed a slightly but statistically significant better recovery compared to Bligh & Dyer (93.2 % vs. 87.5 %; p < 0.05; n = 5), as shown by a t-test. The applicability of the new method was demonstrated in a pilot study, in which the plasma of 15 healthy volunteers was analyzed for its content of various LPC species.