Quantitative analysis of intracellular <i>N</i>τ-methylhistidine concentration in C2C12 myotubes by liquid chromatography–tandem mass spectrometry

The release of Nτ-methylhistidine from muscle cells into the culture medium is considered an index of muscle protein breakdown. The aim of this study was to establish a quantitative method for measuring the intercellular concentrations of Nτ-methylhistidine and its isomer Nπ-methylhistidine in C2C12 myotubes via liquid chromatography-tandem mass spectrometry with stable isotope dilution analysis. The acceptable linear ranges of detection were both 0.5-250 pmol/mL; r2 = 1.000 (Nτ-methylhistidine), r2 = 0.999 (Nπ-methylhistidine). Culture media and cell extracts from C2C12 myotubes grown in a 6-well plate were considered acceptable samples for detecting the concentration of Nτ-methylhistidine. However, Nπ-methylhistidine was detected in neither the culture medium nor cell extracts. The proposed method can be used to confirm the effects of amino acid depletion on Nτ-methylhistidine levels in C2C12 myotubes. In C2C12 myotubes, culture in an amino acid-depleted medium for 48 h increased intracellular Nτ-methylhistidine levels while decreasing its extracellular level.