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Development and validation of a HILIC- MS/MS multi-targeted method for metabolomics applications

Significance Statement

Metabolomics is a newly emerging field of “omics” research concerned with the comprehensive characterization of the small molecule basic polar metabolites in biological systems. The overall goal of metabolomics is to link the metabolite concentration information with clinical or physiology data and to assess the multi-parametric response of a living system to pathophysiological stimuli or genetic modification. Metabolites may describe phenomena which often are not successfully described or predicted by genomics or transcriptomics, such as the effects of xenobiotics or gut microbiome or the positive response or no response to pharmaceutical intervention.

The analysis of polar metabolites represents a challenging task for contemporary science. LC-MS presents a growing popularity as the platform for metabolomic studies due to its high throughput, robustness and wide coverage of metabolites. The success of a LC-MS-based metabolomic study often depends on multiple experimental steps. The comprehensive analysis of complex samples still requires meticulous method development due to lack of analyte free matrix and experience on both LC and MS methods.

To address this challenge we developed a HILIC-MS/MS profiling method for the quantification of ca 100 metabolites of high significance in central biosynthetic routes. The proposed method provides the identification and the generation of definite quantitative data. The method has been optimised and fully validated to accurately quantify polar metabolites in a single injection of 40 min. The use of a single analytical station enhances assay/practitioner control, reporting and inter-laboratory testing.

The developed method was applied first to the analysis of IVF extracellular culture medium of IVF procedure and subsequently to other biological matrices. The results from the analysis provided information on the metabolic activity reflecting from embryos with different growth potential. Differences in the profiles of media deriving from chromosomally normal embryos that resulted in blastocyst and abnormal that stayed in arrested stage could be detected.  Multivariate statistical analysis showed that alanine, glucose, lactic acid and creatine revealed to be the most significant to the differentiation of embryos with different growth potential.

Additional results showed that the developed method can sensitively detect the metabolic content of different matrices and could be used as a tool for detecting differences in metabolic signature and for different purposes in life sciences (diagnostic, nutritional assessment and other). Although the development of such an analytical tool may require time, hard work and dedication it is in our opinion definitely worth the effort.

Development and validation of a HILIC- MS MS multi-targeted method for metabolomics applications. Global Medical Discovery

Journal Reference

Electrophoresis. 2015:Volume 36: issue 18:2215-2225.

Virgiliou C1, Sampsonidis I1, Gika HG2, Raikos N3, Theodoridis GA1.

Show Affiliations
  1. Department of Chemistry, Aristotle University, Thessaloniki, 54124, Greece.
  2. Department of Chemical Engineering, Aristotle University, Thessaloniki, 54124, Greece.
  3. Department of Medicine, Aristotle University, Thessaloniki, 54124, Greece.

Abstract

The paper reports the development of a multi-analyte method and its application in metabolic profiling of biological fluids. The initial aim of the method was the quantification of metabolites existing in cell culture medium used in in-vitro fertilisation (IVF) and in other biological fluids related to embryo growth. Since most of these analytes are polar primary metabolites a hydrophilic interaction liquid chromatography (HILIC) system was selected. The analytical system comprised Ultra (High) performance liquid chromatography (UHPLC) with detection on a triple quadrupole mass spectrometer operating in both positive and negative modes. Mobile phase and gradient elution conditions were studied with the aim to achieve the highest coverage of metabolic space in a single injection namely the largest number of analytes that could be detected and quantified. The developed method provides absolute quantitation of ca. 100 metabolites belonging to key metabolite classes such as sugars, amino acids, nucleotides, organic acids and amines. Following validation, the method was applied for the metabolic profiling of hundreds of samples of spent culture medium originating from human IVF procedures and several hundreds of biological samples such as amniotic fluid, human urine and blood serum from pregnant women. The bioanalytical end-point was to provide assistance in the process of embryo transfer and improving IVF success rates but also to provide insight in complications related to the subsequent embryo growth during pregnancy. This article is protected by copyright. All rights reserved.

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