Oskar González (1,2) María Encarnación Blanco (1), Rosa M. Alonso (1), Thomas Hankemeier (2)
(1) Analytical Chemistry Department, Faculty of Science and Technology, University of the Basque Country (UPV/EHU) (2) Division of Analytical Biosciences, Leiden Academic Centre for Drug Research
Continuous post-column infusion of standards is a very useful tool to obtain additional and very valuable information about a LC-MS method. Here we show multiple applications of this technique in method development and routine analysis based on our experience in the laboratory: detection of compounds causing ion suppression, aid in sample treatment choice, understanding of abnormal results, compensation of matrix effect and some others. Considering all these benefits, we encourage to use this technique in clinical analysis to improve the reliability of LC-MS methods and to obtain more accurate results.
Post Column Infusion of standards (PCI) is frequently used in LC-MS method validation to study in a qualitative way the ion suppression/enhancement areas in a chromatogram. But in addition, it can be an extremely powerful tool to detect and solve pitfalls that emerge during LC-MS analysis. Here we show how PCI can help to find out the cause of several problems that may occur during LC-MS analysis by means of real cases from our research laboratory. Thanks to the PCI phospholipid accumulation in the column was detected, differences between anticoagulants were observed, degradation and ion suppression phenomena were distinguished, the robustness and sensitivity decay of the method was controlled and many others.
PCI can be also extremely valuable during method development, for instance during sample treatment optimization. It is well known that the matrix effect of an analyte can be studied by infusing a standard after the chromatographic separation and monitoring it while running blank samples (which is known as matrix effect profile) (1). In this way, matrix effect profiles for different sample treatments can be compared to study their efficiency. Here we show an explanatory example using two different sample treatments for plasma (standard protein precipitation and phospholipid removal cartridges). This approach can also be used to see how a LC-MS method will behave when there is a change in the matrix, for example if we try to transfer a method developed for urine to plasma samples.
Finally, we will briefly introduce an innovative technique to compensate for the matrix effect using PCI (2). This approach solves some of the problems caused by the ion suppression in quantitative analysis and dramatically improves the repeatability, accuracy, robustness and dynamic range of the method.
Summing up, with this contribution we would like to show, using practical examples, that PCI can be an extremely useful tool in clinical analysis for method development and routine analysis. Considering all its benefits, we believe that this technique is underused and we would like to encourage the analytical community to employ it.
(1) H. Stahnke et al. "Compensation of Matrix Effects by Postcolumn Infusion of a Monitor Substance in Multiresidue Analysis with LC−MS/MS" Analytical Chemistry (2009) 81, 2185-92.
(2) O. Gonzalez et al. “Matrix effect compensation in small molecule profiling for a LC-TOF platform using multi-component post-column infusion” Analytical Chemistry (2015) 87(12), 5921-9.
Acknowledgements: The authors thank the Basque Government for support of this research via a post-doctoral grant (Programa Posdoctoral de perfeccionamiento de doctores del Gobierno Vasco) and a pre-doctoral grant.