: An Automated Workflow to Account for Isotopologue-Related Interferences in Electrospray Ionization High-Resolution Mass Spectra of Phospholipids.

In the past decade, hydrophilic interaction liquid chromatography (HILIC) has emerged as an efficient alternative to reversed-phase chromatography (RPC) for the analysis of phospholipid (PL) mixtures based on mass spectrometric detection. Since the separation of PL by HILIC is chiefly based on their headgroup, the mass spectrum of each class can be obtained by spectral averaging under the corresponding HILIC band. Using experimental / values resulting from high mass resolution/accuracy instruments, the sum compositions of PL in a specific class can be thus inferred but partial overlapping may occur between signals related to the M + 0 isotopologue of one species and the M + 2/M + 4 isotopologues of species having one/two more C═C bonds in their chemical structures. Here, an automated workflow, named (lipid isotopic pattern interference correction), is proposed to account for such interferences. Starting from the experimentally verified assumption that peaks in isotope patterns are Gaussian, predicts, as a function of / ratio, signal intensities due to M + 2 and M + 4 isotopologues of species with one or two more C = C bonds than the target one and calculates the corrected intensity for the M + 0 isotopologue of the latter. Thanks to an iterative procedure, the suggested algorithm compensates also for slight shifts occurring between experimental and theoretical / ratios related to isotopologue peaks. Examples of applications to simulated and experimental mass spectra of two PL classes, i.e., phosphatidylcholines (PC) and cardiolipins (CL), emphasize the increased extent of correction at the increase of molecular masses of involved species.