)**  
Since first isolated from human urine hydrolysates, 3-methylhistidine (3-MH) has been employed as a muscle protein turnover marker in a wide range of human and animal studies and is widely accepted as a marker for determining periods of increased muscle catabolism. As with all endogenous compounds, quantitative determination of 3-MH requires arduous method development, especially as its isomer, 1-methylhistidine (1-MH), which is a product of anserine breakdown, is widely present in most biological matrices of interest [1]. Traditionally, the individual determination of 1- and 3-MH requires either the use of ion chromatography or the utilisation of a derivatisation technique in combination with high performance liquid chromatography, to avoid co-elutions and determine the two isomers separately, and with gas chromatography, to enhance their volatility and facilitate the analysis. This is mainly due to the fact that the two compounds present almost identical retention behaviour in reverse phase chromatography and poor gas chromatographic behaviour as non-derivatised entities [2; 3; 4]. Here, we examine the possibility of facilitating unique spectral and ion abundance differences during collision induced dissociation between the two isomers along with different retention behaviour in certain hydrophilic interaction liquid chromatography conditions in order to develop a liquid chromatography coupled to tandem mass spectrometry method for the simultaneous separation and analysis of the two isomers without the need for derivatisation. Such a method will enable the analysis of large numbers of samples which are usually associated with muscle catabolism studies in an efficient and economically friendly manner. This work was supported by the project \"Foodomics-GR\" (MIS 5029057), funded by the Operational Programme NSRF 2014-2020 and co-financed by Greece and European Regional Development Fund. ## References  
* [1] Phillips, S.M. ; Tipton, K.D. ; Aarsland, A.; Wolf, S.E. ; Wolfe, R.R. Mixed muscle protein synthesis and breakdown after resistance exercise in humans. _Am. J. Physiol. Endocrinol. Metabol._**1997**, _273_, E99-E107. * [2] Min, S.; Yisheng, Y.; Lu, Y. Determination of urinary 3-methylhistidine by high-performance liquid chromatography with o-phthaldialdehydedebyde precolumn derivatization. _J. Chromatogr. B Biomed. Sci. Appl._**1992**, _581_, 272-276. * [3] Houweling, M.; van der Drift, S.G.A. ; Jorritsma, R.; Tielens, A.G.M. Quantification of plasma 1- and 3-methylhistidine in dairy cows by high-performance liquid chromatography-tandem mass spectrometry. _J. Quiry Sci._**2012**, _95_, 3125-3130. * [4] Wang, H.; Hu, P.; Jiang, J. Measurement of 1-and 3-methylhistidine in human urine by ultra performance liquid chromatography-tandem mass spectrometry. _Clin. Chim. Acta_**2012**, _413_, 131-138. * [5]_16.32. Combination of Library-Free and Library-Based Metabolomics Protocols Form the Characterization of Anti-Cancer Drug Carfilzomib administrated to Mice_
* [6]**I. Barla 1**, **P. Efentakis 2**, **S. Lamprou 2**, **P.-E. Nikolaou 2**, **E. Kastritis 3**, **M. A. Dimopoulos 3**, **E. Terpos 3**, **I. Andreadou 2**, **N. Thomaidis 1** and **E. Gikas 1,*
* [7] Department of Chemistry, National and Kapodistrian University of Athens, 10431 Athens, Greece
* [8] Department of Chemistry, National and Kapodistrian University of Athens, 10431 Athens, Greece
* [9] Department of Medicine, National and Kapodistrian University of Athens, 10431 Athens, Greece
* [10] Correspondence: vgikas@chem.uoa.gr  
Metabolomic profiling studies are powerful weapons for the comprehension of biological systems. Metabolites include all compounds of low-molecular-weight and have a pivotal role, as metabolome is affected by every external or internal perturbation. Due to this sensitive response, the investigation of metabolites is frequently employed to provide \"clues\" in several health issues, e.g., the definition of pathophysiological processes and the discovery of disease biomarkers, including those of drug efficacy and drug toxicity.