Characterizing Cancer Tumours Using HILIC–MS


The Column

ColumnThe Column-03-09-2016
Volume 4
Issue 12

Characterizing Cancer Tumours using HILIC–MS

Photo Credit: SCIEPRO/Getty Images

Early screening procedures are key in the fight against cancer. Michal Holcapek from the University of Pardubice, in Pardubice, the Czech Republic, spoke to Kate Mosford of The Column about his work using hydrophilic interaction liquid chromatography coupled to mass spectrometry (HILIC–MS) in characterizing kidney cancer tumours.


Q. In 2015, your group published a study characterizing the differences between polar lipid classes in cancerous tumours and in the surrounding normal tissue using hydrophilic interaction liquid chromatography (HILIC) and electrospray ionization mass spectrometry (ESI–MS). What led your group to begin this study?



We previously developed various cutting-edge separations of lipid isomers, but without strong biological applications. Later, I decided that I wanted to start up something with clear and easily explainable “benefits for humanity”, so we started with the bioanalysis of human body fluids. We are now working on the large-scale ERC CZ project with the goal of finding lipid cancer biomarkers. In the framework of this project, we are analyzing a large number of clinical samples of tumour and healthy tissues obtained from oncological surgery, plasma, and urine of both cancer patients and healthy volunteers. The goal of this project is to find statistically relevant differences in the lipidomic composition of healthy individuals and those suffering from selected types of cancer, and then using this data to develop early diagnosis screening procedures.   

Q. What were your key findings?



It is a well-known fact that tumour cells and normal cells are not identical. We already know some of the general differences between their lipidomes, such as the downregulation of phospholipids containing arachidonic acid with the expected upregulation of eicosanoids formed from arachidonic acid, which is currently being researched. Another general difference is the upregulation of some phospholipids containing combinations of saturated and monounsaturated fatty acyls, because those fatty acids can be synthesized

de novo

during the growth of tumour cells. Significant changes are also observed in the area of sphingolipids and glycosphingolipids. We are optimizing ultrahigh-performance liquid chromatography–mass spectrometry (UHPLC–MS) and ultrahigh-performance supercritical fluid chromatography coupled to MS (UHPSFC–MS) methods for their accurate quantitation.  

Q. What were the main challenges you encountered and how did you overcome them?



By far the most serious problem in this type of research is biological variability. Proving statistically significant differences in cell composition are the result of the disease and not the result of biological variability is a major prerequisite in biomarker discovery. It is impossible to exclude or diminish this issue, because biological variability can be caused by many unavoidable variances, such as different living style, nutrition habits, physical activity, body-mass-index, age, sex, etc. This issue is the main reason why many previous biomarker studies failed in part or completely. We can still guarantee the quality of our analytical workflow using well optimized and validated quantitative methods, removing any additional uncertainty caused by natural sample-to-sample variations in the concentrations of individual metabolites.   Q. What other analytical methods have been used in the analysis of lipids in tumours and how does your group’s method compare?   A: In recent years, MS-based approaches are the method of choice for a comprehensive quantitative analysis of lipids based on the following requirements: Internal standards per each lipid class to compensate for different extraction and ionization efficiencies, internal standards and analytes are ionized at the same time to avoid matrix effects and ion suppression, and finally the full validation of the whole analytical method. The combination of these factors sets the highest standards for lipidomic quantitation.  

Q. Kidney cancer is one of most prevalent cancers in both men and women. Could this analytical method be used to analyze polar lipids in other cancerous tumours?



Yes, our method could be applied to study various cancer types and other diseases with altered lipid metabolism. As well as kidney cancer, we can also analyze samples of breast, pancreas, prostate, and lung cancers. We focus mainly on cancer types with a high mortality, morbidity, with available treatment options, but a lack of reliable screening procedures.  

Q. What is your group working on next?



We are still proceeding with this research because it was started less than three years ago. At the moment, we are measuring large sample sets, processing the data, and statistically evaluating them. When we find dysregulated lipids, we try to search for the biological consequences of these changes using metabolic pathways. Unfortunately we are often unable to fully complete this task within our analytical laboratory, so we co-operate with skillful individuals with a strong biological background in lipidomics. The next step will be integrating our research into the context of metabolomics, proteomics focused on enzymes responsible for the lipid biosynthesis, and cancer biology. This type of research is certainly multidisciplinary.  

Q. You are chairing the upcoming HPLC 2017 symposium in Prague, have you started planning yet? Is there anything that the conference will focus on differently to previous years? 



We are already hard at work organizing and planning because we want to attract a large number of scientists from all over the world. The main goal of the symposium is to enable the active participation of many students and young researchers and ensure that they are given adequate space in the conference programme. We are preparing a range of interesting offers for younger scientists, such as low conference fees, cheaper short courses, travel grants, scientific awards, and a programme track devoted solely to short oral presentations by young researchers combined with tutorials given by leading experts during the whole conference. Our organizational efforts and plans for young scientists can be followed on the conference website ( and Facebook (  

Michal Holčapek

is a professor of analytical chemistry and head of the mass spectrometry group at the Department of Analytical Chemistry at the University of Pardubice, Czech Republic, where he obtained his Ph.D. in analytical chemistry in 1999. His research started with the structural analysis of organic and organometallic compounds using mass spectrometry (MS). This was then coupled with liquid chromatography (LC) and focused mainly on the metabolism of various pharmaceutical drugs and natural compounds. His present specialization is in the lipidomic analysis of biological samples using liquid chromatography (LC) or supercritical fluid chromatography (SFC) coupled to MS, shotgun electrospray ionization coupled to MS (ESI-MS), matrix-assisted laser desorption–ionization coupled to MS (MALDI-MS), and MS imaging. The main emphasis is on clinical research with an aim to find biomarkers for cancers and other serious human diseases. He has co-authored over 110 papers in peer-reviewed journals with a h-index of 32 and given over 300 conference presentations including more than 20 plenary or keynote lectures. He is a member of editorial boards on

Analytical Chemistry


Analytical and Bioanalytical Chemistry



; a guest editor of special volumes on Mass spectrometry – innovation and application in

Journal of Chromatography A

(2010, 2012 and 2016), editor of special issues on lipidomics in

Analytical Chemistry

(2014) and

Analytical and Bioanalytical Chemistry

(2015), editor of the book

Extreme Chromatography: Faster, Hotter, Smaller

(American Oil Chemical Society, 2011), head of the Czech Mass Spectrometry Section and national representative in International Mass Spectrometry Foundation (2005–2013), chairman of several international and national conferences. 

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