Phospholipids in liquid chromatography/mass spectrometry bioanalysis: comparison of three tandem mass spectrometric techniques for monitoring plasma phospholipids, the effect of mobile phase composition on phospholipids elution and the association of phospholipids with matrix effects

Abstract

Because plasma phospholipids may cause matrix effects in bioanalytical liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods, it is important to establish optimal mass spectrometric techniques to monitor the fate of phospholipids during method development and application. We evaluated three MS/MS techniques to monitor phospholipids using positive and negative electrospray ionization (ESI). The first technique is based on using positive precursor ion scan of m/z 184, positive neutral loss scan of 141 Da and negative precursor ion scan of m/z 153. The second technique is based on using class-specific positive and negative selected reaction monitoring (SRM) transitions to monitor class-representative phospholipids. The third technique, previously reported, utilizes in-source collision-induced dissociation (CID)-based positive SRM of m/z 184 --> 184. We recommend the all-inclusive technique 1 for use in qualitative assessment of all classes of phospholipids and technique 2 for use in quantitative assessment of class-representative phospholipids. Secondly, we evaluated the elution behaviors of the plasma phospholipids under different reversed-phase mobile phase conditions. The phospholipid-eluting strength of a mobile phase was mainly dependent on the type and amount (%) of the organic eluent and the strength increased in the order of methanol, acetonitrile and isopropyl alcohol. Under the commonly used gradient and isocratic elution schemes in LC/MS/MS bioanalysis, not all the phospholipids are eluted off the column. Thirdly, we investigated the association between phospholipids and matrix effects in positive and negative ESI using basic, acidic and neutral analytes. While the phospholipids caused matrix effects in both positive and negative ESI, the extent of ionization suppression was analyte-dependent and was inversely related to the retention factor and broadness of the phospholipids peaks. The lysophospholipids which normally elute earlier in reversed-phase chromatography are more likely to cause matrix effects compared to the later-eluting phospholipids in spite of the larger concentrations of the latter in plasma.

Utilizing Internal Standard Responses to Assess Risk on Reporting Bioanalytical Results from Hemolyzed Samples

“…Lipids, such as phospholipids, are present in plasma, and it has been demonstrated that they cause significant matrix effect (29,30). Evaluation of matrix factor and selectivity in hyperlipemic samples has been routinely conducted in bioanalytical laboratories.…”

Reverse-Polynomial Dilution Calibration Methodology Extends Lower Limit of Quantification and Reduces Relative Residual Error in Targeted Peptide Measurements in Blood Plasma

“…Although we did not find that phospholipid-depleted RPD was significantly different from C18 SPE RPD in terms of error and precision in this study, phospholipid depletion did increase ion intensity signals for two peptides and may thus extend the LLOQ for certain analytes. Routine deployment of combination SPE-phospholipid clean-up for all samples in a run may improve the performance of the chromatography column overall, resulting in more accurate measurements; and as these clean-up methods do not induce extra sample-handling, it may be best practice for multiplexed assays of nonphospho-analytes (22)(23)(24)(25). Another aspect for consideration is whether the type of regression equation used for curve fitting has a significant or indeed determining effect on the difference in relative residual error and precision between SID and "Reverse" calibration approaches.…”

“…Several simple methods have independently demonstrated the ability to increase accuracy in various hyphenated-MS assays in complex matrices: "Reverse" curves utilize the stable-isotope analog not as an internal standard but as a surrogate calibration analyte to circumvent interference from the endogenous analyte signal and extend assay Lower Limit(s) of Quantification (LLOQ), and nonlinear calibration techniques have proven to more accurately reflect the concentration-MS detector response at the low and high end of concentration gradients (8, 14, 18 -21). Specifically in the case of biological matrices, phospholipids are particularly deleterious ion suppressing elements because of their easily ionizable, polar, and hydrophobic moieties that can have complex interactions with co-eluting analytes as well as the chromatography stationary and mobile phases required for most other analytes (22)(23)(24)(25). Combination solid-phase extraction (SPE) and phospholipid removal techniques have proved to effectively minimize ion suppression effects in ESI-MS assays (22)(23)(24)(25).…”

“…Specifically in the case of biological matrices, phospholipids are particularly deleterious ion suppressing elements because of their easily ionizable, polar, and hydrophobic moieties that can have complex interactions with co-eluting analytes as well as the chromatography stationary and mobile phases required for most other analytes (22)(23)(24)(25). Combination solid-phase extraction (SPE) and phospholipid removal techniques have proved to effectively minimize ion suppression effects in ESI-MS assays (22)(23)(24)(25).…”

Analytical Methods for Quantification of Drug Metabolites in Biological Samples

“…The main source of the commonly observed ME of plasma samples is believed to be endogenous phospholipids and proteins. The lysophospholipids which normally elute earlier in reversed phase chromatography are more likely to cause matrix effects compared to the later eluting phospholipids in spite of the larger concentrations of the latter in plasma [106]. Phospholipids cause ion suppression in both, positive and negative ESI modes and must be removed or resolved chromatographically.…”