The Laboratory of Metabolomics at the Institute of Physiology of the Czech Academy of Sciences (IPHYS CAS) provides fee-based services for liquid chromatography–mass spectrometry-based (LC−MS) analysis of polar metabolites, complex lipids, and exposome compounds (including drugs) in biological materials such as plasma, serum, tissues, and cells. These compounds are usually in the range of 50 to 2000 Da and their concentrations may span several orders of magnitude.
For the analysis of investigational medicinal products (IMP) and pharmaceuticals, the laboratory is OECD GLP certified and regularly inspected by the National Authority (State Institute for Drug Control).
LIMeX (LIpids, Metabolites, and eXposome compounds) LC−MS workflow is used for the untargeted analysis of complex lipids, polar metabolites, and exposome compounds (such as drugs).
Bi-phase extraction method using methanol, methyl-terc butyl ether (MTBE), and water provides two phases: one containing polar metabolites (aka “metabolomics”) and different exposome compounds such as drugs (aka “exposomics”), and the other with complex lipids (aka “lipidomics”). During untargeted analyses, all detectable compounds are acquired using validated LC–MS methods.
Since no single LC–MS method can cover the full metabolome/lipidome/exposome, we use different chromatography modes (reversed-phase liquid chromatography, RPLC and hydrophilic interaction chromatography, HILIC) and electrospray ionization modes to comprehensively characterize these isolated fractions.
A high-resolution mass spectrometer Thermo Q Exactive Plus coupled with a liquid chromatograph (Thermo Vanquish) is routinely used for LIMeX workflow. The mass spectrometer collects full scan MS1 data as well as data-dependent MS/MS spectra for all samples. While MS1 data is used for quantification, MS/MS spectra are used for compound annotation using MS/MS library search or for compound confirmation. The acquired data sets are processed using MS-DIAL 4 software, including annotation of complex lipids, polar metabolites, and other detected compounds using in-house, open-source as well as commercial MS/MS spectral libraries (NIST20, MassBank, MoNA).
LIMeX workflow for untargeted and targeted analysis of complex lipids, polar metabolites, and exposome compounds.
For human cohort studies, we usually report over 500 complex lipids, 100 polar metabolites, and tens of exposome compounds (mainly food components and drugs) using LIMeX−4D (i.e., 4 LC−MS platforms).
All compounds are quantified as peak heights and reported in “arbitrary units”. These values are used as inputs for one-dimensional or multidimensional statistical methods (e.g., t-test, fold-change, heatmap, principal component analysis, partial least squares-discriminant analysis).
The LIMeX workflow uses over 60 different internal standards, which can be used for estimations of absolute concentrations for various metabolites detected in plasma/serum and tissues.
Targeted quantitative LC−MS/MS analysis is available for specific low-abundant lipid mediators (eicosanoids, endocannabinoids, and fatty acid esters of hydroxy fatty acids (FAHFA)) and steroids.
The sample preparation for these low-abundant compounds is more demanding and solid-phase extraction (SPE) is involved in the workflow to remove high-abundant compounds (e.g., phospholipids).
A quadrupole/linear ion trap mass spectrometer (SCIEX QTRAP 5500) operated in multiple reaction monitoring (MRM) coupled with a liquid chromatograph (Dionex/Thermo Ultimate 3000 RSLC) is used for analysis.
Targeted LC−MS methods are used to measure metabolites labeled with stable isotopes using 13C/2H glucose and 2H2O tracers. LIMeX protocol is used for sample extraction followed by LC−MS analysis in which case the mass spectrometer (Q Exactive Plus) is operated in ultra-high-resolution mode (140,000 FWHM at m/z 200).
Theoretical isotopologues are calculated for annotated metabolites in Python and instrumental files are processed through MRMPROBS software. Peak heights are adjusted for the natural abundance of elements and tracer purity using IsoCor.
Targeted LC−MS methods are used for Good Laboratory Practice (GLP) and non-GLP analysis of investigational medicinal products (IMP) and pharmaceutical compounds, including also potential drug candidates within absorption, distribution, metabolism and excretion (ADME) studies.
The lab is GLP certified for analytical and clinical chemistry testing.
For GLP studies, the following three steps are essential:
A high-resolution mass spectrometer Thermo Q Exactive Plus coupled with a liquid chromatograph (Thermo Vanquish) is routinely used for the analysis of pharmaceutical compounds.
To submit your samples, please use the following submission form. All platforms have a minimum sample requirement of 10 samples. The form also contains information on a minimum amount or volume requirement per sample for each platform.
Send this form to Dr. Tomáš Čajka and deliver your samples to:
After receiving your samples, a unique project ID is generated and is used within all platforms, for the final report, and data archiving.
doc. Ing. Tomáš Čajka, Ph.D. Head of the Laboratory
Ing. Jiří Hricko Research Assistant LC–MS, GLP Mgr. Michaela Paučová Research Assistant LC–MS, GLP Mgr. Tatyana Kobets, Ph.D. Research Assistant statistics, bioinformatics Ing. Michaela Nováková Research Assistant statistics, bioinformatics Mgr. Petra Zedníková (maternity leave) Research Assistant
The laboratory has provided metabolomics and lipidomics data for several projects worldwide. Below is the list of papers published in peer-reviewed journals.