Implications of matrix effects in ultra‐fast gradient or fast isocratic liquid chromatography with mass spectrometry in drug discovery

Abstract

In the analysis of biological samples it is important to reduce the risk of interferences from the matrix itself, other analytes, the dosing vehicle (commonly PEG), and from the MS/MS transitions used for the analysis. Rapid analysis is essential for drug discovery, and even though the requirements for separation may be minimized for speed, the integrity of the analysis is still dependent on the separation. This paper focuses on the potential for interferences from various endogenous and exogenous matrix components commonly encountered in quantitation of analytes and their metabolites from biological matrices. We demonstrate that neither high organic isocratic nor ballistic gradient ultra-fast HPLC show a clearly defined advantage in regards to complex biological matrices. The critical factor in the resolution of matrix interferences still remains in sample preparation.

Development of a two‐step screening ESI‐TOF‐MS method for rapid determination of significant stress‐induced metabolome modifications in plant leaf extracts: The wound response in Arabidopsis thaliana as a case study

“…This step (metabolite fingerprinting) allowed the identification of discriminant ions and the selection of homogeneous plant material. For the metabolite-fingerprinting step, with the aim to detect minute variations in the stressed plants, the fast chromatographic gradient was selected to minimise ion suppression and instrument contamination [23]. The second step consisted of an UPLC-ESI-TOF-MS method with high-resolution profiling of the representative pool, allowing the confirmation and localisation of putative wound biomarkers for further identification.…”

Fast LC/MS in the analysis of small molecules

“…When developing such rapid methods it is important to consider instrument parameters, such as the system volume of the HPLC instrument as well as the acquisition speed of the mass spectrometer. Romanyshyn and Tiller [12,35,36,37,38,39] report that they have optimized the separation power using 20 mm×2 mm 5-µm and 3-µm HPLC columns at flow rates of 1.5-2 mL min -1 with a full linear gradient over as little as 1 min [36]. In a related study [37] the authors conclude that a fast gradient offers advantages over fast isocratic separations in particular for resolving analytes from matrix interferences that commonly elute with the solvent front.…”

“…[9]. Romanyshyn et al [11], Tiller and Romanyshyn [12], and Chi et al [13] all discuss the importance of adequate resolution from the solvent front to avoid ion suppression from endogenous matrix components.…”

“…The specificity of the mass spectrometer has often led to a rather cavalier disregard for the separation process [5], which in turn results in suppression of the MS signal caused by co-eluting interferences from complex matrices [4,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22]. Both ESI and APCI are subject to ion suppression effects, ESI more so than APCI.…”

Identifying and Overcoming Matrix Effects in Drug Discovery and Development

“…Gradient elution gave better separation of the analyte from the poorly retained polar components, and a more complete elution of highly retained contaminants from the column, thereby reducing the potential matrix effects during sample analysis. Moreover, the early and late chromatographic regions can be diverted to waste, thus reducing matrix components that can foul the MS source (Tiller and Romanyshyn, 2002). Ultra Performance Liquid Chromatography (UPLC) provides advantages over traditional HPLC in speed, sensitivity, and resolution of analytes (Novakova et al, 2006;Wren and Tchelitcheff, 2006).…”