Scrutinizing different ionization responses of polar lipids in a reversed-phase gradient by implementing a counter-gradient

Lipidomics studies strive for a comprehensive identification and quantification of lipids. While reversed phase (RP) liquid chromatography (LC) coupled to high resolution mass spectrometry (MS) offers unrivalled selectivity and thus is the preferred method for lipid identification, accurate lipid quantification remains challenging. The widely adopted one-point lipid class specific quantification (one internal standard per lipid class) suffers from the fact that ionization of internal standard and target lipid occurs under different solvent composition as a consequence of chromatographic separation. To address this issue, we established a dual flow injection and chromatography setup that allows to control solvent conditions during ionization enabling isocratic ionization while running a RP gradient through the use of a counter-gradient. Using this dual LC pump platform, we investigated the impact of solvent conditions within a RP gradient on ionization response and arising quantification biases. Our results confirmed that changing solvent composition significantly influences ionization response. Quantification of human plasma (SRM 1950) lipids under gradient and isocratic ionization conditions further confirmed these findings as significant differences between the two conditions were found for the majority of lipids. While the quantity of sphingomyelins with >40 C atoms was consistently overestimated under gradient ionization, isocratic ionization improved their recovery compared to consensus values. However, the limitation of consensus values was demonstrated as overall only small changes in z-score were observed because of high uncertainties of the consensus values. Furthermore, we observed a trueness bias between gradient and isocratic ionization when quantifying a panel of lipid species standards which is highly dependent on lipid class and ionization mode. Uncertainty calculations under consideration of the trueness bias as RP gradient uncertainty revealed that especially ceramides with >40 C atoms had a high bias leading to total combined uncertainties of up to 54%. The assumption of isocratic ionization significantly decreases total measurement uncertainty and highlights the importance of studying the trueness bias introduced by a RP gradient to reduce quantification uncertainty.