Therese Koal (Presenter)
BIOCRATES Life Sciences AG
Authorship: Hai Tuan Pham1, Doreen Kirchberg1, Martin Buratti1, Simon Schafferer1, Barbara Wolf1, Glenn Damkroeger2, Madalina Oppermann2, Anastasia Kalli2, Reiko Kiyonami2, David Peake2, Andreas Huhmer2, Therese Koal1
1 Biocrates Life Sciences AG, Innsbruck, AUSTRIA, 2 Thermo Fisher Scientific, San Jose, CA, USA
Standardized, quantitative assays have increasingly been desired in metabolomics, especially in targeted studies. We developed the AbsoluteIDQ® p400 HR Kit to facilitate the quantitative analysis on Thermo Scientific™ Q ExactiveTM Orbitrap™ HRAM MS platform for the first time, bridging the gap between targeted quantitative metabolomics and profiling. The kit quantifies up to 408 metabolites of 11 classes: amino acids, biogenic amines, acylcarnitines, phosphatidylcholines, lysophosphatidylcholines, sphingomyelins, ceramides, cholesteryl esters, diglycerides, triglycerides, and hexoses. A beta-test has been carried out across 3 laboratories on different Q ExactiveTM platforms showing high inter-laboratory comparability, which is mandatory for robust, routine applications in targeted metabolomics.
Multiplexed analysis by targeted metabolomics provides a quantitative snapshot of the metabolic phenotype. Targeted and standardized assays are used in research to improve biological insights, monitor disease biomarker signatures, and enable efforts in personalized medicine. A standardized, quantitative, rapid targeted metabolomics approach covering the metabolites necessary for the assessment of the central metabolic pathways based on amino acids, acylcarnitines, hexoses, biogenic amines, and lipids in plasma, urine, and tissues was developed, combining LC-MS with FIA-MS analysis. Traditionally, quantitative analyses have been carried out on triple quadrupole MS instruments using MRM technology. The high resolution, accurate mass (HRAM) technology on Orbitrap instruments, on the other hand, has predominantly been used in profiling studies. Here, we investigate the combination of these two worlds i.e. application of the quantitative assay on an Orbitrap MS platform for the very first time to demonstrate the power of multiplexed quantitative metabolome analysis, the impact of improved resolution, and increased metabolite coverage.
The newly developed AbsoluteIDQ® p400 HR Kit was based on the well-established AbsoluteIDQ® p180 Kit. For sample preparation, only 10 µL of biofluid samples were required, which were pipetted on a patented 96-well plate, preloaded with internal standards. After a short derivatization the target metabolites were extracted. The extracts were subsequently analyzed with LC-MS (amino acids and biogenic amines) and FIA-MS (acylcarnitines, lipids, and hexoses), carried out on a VanquishTM UHPLC – Q Exactive Focus system. A maximum of 82 samples per kit may be analyzed within 38 hours. MetIDQ™ software was used during the entire automated kit workflow, from sample to result: sample registration and plate layout, sample analysis, data processing for quantification, quality control, technical validation, and data reporting.
We developed the AbsoluteIDQ® p400 HR Kit to facilitate the quantitative analysis on the Thermo Scientific Q Exactive Focus instrument. The kit quantifies up to 408 metabolites of 11 compound classes. These measurements are carried out by LC-MS and FIA-MS injections in UHPLC – Q Exactive instruments.
The LC-MS method provided quantitative analysis of 42 targeted metabolites comprising 21 amino acids and 21 biogenic amines. The measured concentrations are fully comparable to those generated by the triple quadrupole MS-based analyses. Moreover, their performance in terms of LOD, LOQ, accuracy, and precision are very similar. The accuracy in intra-day and inter-day validation experiments was 104.5 ± 8.9 % and 98.5 ± 6.1 %, respectively. The precision was 8.2 ± 4.4 % and 5.4 ± 3.2 %, respectively.
The FIA-MS analysis covers 366 metabolites comprising 55 acylcarnitines, 172 phosphatidylcholines, 24 lysophosphatidylcholines, 31 sphingomyelins, 9 ceramides, 14 cholesteryl esters, 18 diglycerides, 42 triglycerides, and hexoses. The accuracy in intra-day and inter-day validation experiments was 98.2 ± 16.6 % and 95.8 ± 14.6 %, respectively. The precision was 8.4 ± 24.1 % and 10.4 ± 13.0 %, respectively.
The newly adapted standardized and quantitative targeted metabolomics assay has been validated on the Q Exactive Focus and tested on other platforms of the Q Exactive family across three independent laboratories. The results showed excellent lab-to-lab robustness and data comparability. This is amandatory basis for routine applications in quantitative metabolomics profiling.
Conclusions & Discussion
The AbsoluteIDQ® p400 HR Kit has been developed and validated to address the ever increasing need of a standardized and quantitative assay in targeted metabolic phenotyping. It utilizes for the first time the strengths of a multiplex quantitative assay on the Orbitrap-based high resolution MS technology.
References & Acknowledgements:
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IP Royalty: no
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