An LCMS-based untargeted metabolomics protocol for cochlear perilymph: highlighting metabolic effects of hydrogen gas on the inner ear of noise exposed Guinea pigs

Introduction Noise-induced hearing loss (NIHL) is an increasing problem in society and accounts for a third of all cases of acquired hearing loss. NIHL is caused by formation of reactive oxygen species (ROS) in the cochlea causing oxidative stress. Hydrogen gas (H 2 ) can alleviate the damage caused by oxidative stress and can be easily administered through inhalation. Objectives To present a protocol for untargeted metabolomics of guinea pig perilymph and investigate the effect of H 2 administration on the perilymph metabolome of noise exposed guinea pigs. Methods The left ear of guinea pigs were exposed to hazardous impulse noise only ( Noise , n = 10), noise and H 2 ( Noise + H2 , n = 10), only H 2 ( H2 , n = 4), or untreated ( Control , n = 2). Scala tympani perilymph was sampled from the cochlea of both ears. The polar component of the perilymph metabolome was analyzed using a HILIC-UHPLC-Q-TOF–MS-based untargeted metabolomics protocol. Multivariate data analysis (MVDA) was performed separately for the exposed- and unexposed ear. Results MVDA allowed separation of groups Noise and Noise + H2 in both the exposed and unexposed ear and yielded 15 metabolites with differentiating relative abundances. Seven were found in both exposed and unexposed ear data and included two osmoprotectants. Eight metabolites were unique to the unexposed ear and included a number of short-chain acylcarnitines. Conclusions A HILIC-UHPLC-Q-TOF–MS-based protocol for untargeted metabolomics of perilymph is presented and shown to be fit-for-purpose. We found a clear difference in the perilymph metabolome of noise exposed guinea pigs with and without H 2 treatment.