Tandem mass spectrometry with online high‐flow reversed‐phase extraction and normal‐phase chromatography on silica columns with aqueous‐organic mobile phase for quantitation of polar compounds in biological fluids

Abstract

In this work, high-flow online reversed-phase extraction was coupled with normal phase on silica columns with aqueous-organic mobile phase liquid chromatography/tandem mass spectrometry (LC/MS/MS) to quantify drug candidates in biological fluids. The orthogonal separation effect obtained from this configuration considerably reduced matrix effects and increased sensitivity for highly polar compounds as detected by selected reaction monitoring. This approach also significantly improved the robustness and limit of detection of the assays. An evaluation of this system was performed using a mixture of albuterol and bamethan in rat plasma. Assay validation demonstrated acceptable accuracy (< 8% difference) and precision (< 6% CV) for these model compounds. The system has been used for the quantitation of polar ionic compounds in biological fluids in support of drug discovery programs. This assay was used to analyze samples for a BMS proprietary compound (A) in a rat pharmacokinetic study and is shown as an example to demonstrate the precision, accuracy, and sufficient sensitivity of this system.

An overview of matrix effects in liquid chromatography–mass spectrometry

“…The results demonstrated that a reduction in ME was obtained when sufficient orthogonality of the phase systems was achieved. High-flow on-line reverse-phase extraction was coupled with normal phase on silica columns with aqueous-organic mobilephase LC-ESI-MSMS to quantify drug candidates in biological fluids (Deng et al, 2005). The orthogonal separation effect obtained from this configuration considerably reduced ME and increased sensitivity for highly polar compounds.…”

Development and validation of sensitive LC-MS/MS assays for quantification of HP-#x03B2;-CD in human plasma and CSF

“…Coupling of reversed phase and HILIC has been successfully used to reduce matrix effects and increase sensitivity for highly polar compounds ( 20,21 ). A 2D-LC method was developed to separate HP-␤ -CD from the endogenous matrix using two orthogonal chromatographic separation mechanisms.…”

Chromatographic Separation Methods

“…Highly aqueous mobile phases used for conventional online extraction conditions may cause peak distortion and " breakthrough " on HILIC analytical columns because water is a stronger solvent on HILIC. A gradient elution starting from high organic content (e.g., 90% acetonitrile in water) can provide better peak shape and retention (Deng et al, 2005 ).…”