HybridSPE: A novel technique to reduce phospholipid-based matrix effect in LC-ESI-MS Bioanalysis

Abstract

When complex biological materials are analyzed without an adequate sample preparation technique, MS signal and response undergo significant alteration and result in poor quantification and assay. This problem generally takes place due to the presence of several endogenous materials component in samples. One of the major causes of ion suppression in bioanalysis is the presence of phospholipids during LC-MS analysis. The phospholipid-based matrix effect was investigated with a commercially available electro spray ionization (ESI) source coupled with a triple quadrupole mass spectrometer. HybridSPE dramatically reduced the levels of residual phospholipids in biological samples, leading to significant reduction in matrix effects. This new procedure that combines the simplicity of precipitation with the selectivity of SPE allows obtaining much cleaner extracts than with conventional procedures. HybridSPE-precipitation procedure provides significant improvement in bioanalysis and a practical and fast way to ensure the avoidance of phospholipids-based matrix effects. The present review outlines the HybridSPE technique to minimize phospholipids-based matrix effects on LC–ESI-MS bioanalysis.

The impact of phospholipids and phospholipid removal on bioanalytical method performance

“…While this paper does not specifically mention phospholipids, the technique of post-column infusion to identify matrix effects is widely accepted as an essential part of analytical method validation. Post-column infusion involves infusing a standardized solution of the analytes in question during MS analysis, for the purpose of identifying regions of ionization suppression or enhancement caused by the matrix ions, thus giving the chromatographer the opportunity to resolve the analytes from the regions of ion suppression/enhancement to avoid interference in quantification related to matrix ions (Ahmad et al, 2012;Dams et al, 2003). Only components, whether they are endogenous metabolites, phospholipids, proteins or other interfering compounds, that co-elute with the target(s) will affect the signal intensity produced in the MS detection of the analyte (s) (Bonfiglio et al, 1999).…”

“…Absolute matrix effects are defined as a difference in response between a neat solution sample and a spiked post-extraction sample, and this type of interference primarily affects the accuracy of the method. Relative matrix effects are defined as difference in response between various lots of spiked post-extraction samples, and often impact the precision of the method (Ahmad et al, 2012;Ismaiel et al, 2007). Matrix effects associated with co-elution of phospholipid components can manifest as ionization suppression or signal enhancement, an indicated in Fig.…”

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).…”