Determination of Methyl Group Positions in Long-Chain Aliphatic Methyl Ethers and Alcohols by Gas Chromatography/Orbitrap Mass Spectrometry

Methylated long-chain aliphatic compounds such as terminal methyl ethers are a common compound type found on the epicuticular layer of arthropods, e.g., spiders. Because complex mixtures are encountered in small amounts when analyzing these mixtures, GC/MS is the method of choice for characterizing the individual constituents. However, the methyl branch location cannot be deduced from the original spectra due to the easy loss of methanol, resulting in nonspecific spectra, and a complex derivatization scheme has been employed to address this issue. We noted that although mass spectra obtained by EI-quadrupol and EI-Orbitrap ionization are superficially quite similar, a +2.0 V C-trap offset of the latter leads to reduced fragmentation. The high-resolution Orbitrap spectra contain enough information to allow for methyl group localization in the chain. However, the spectra of the methyl ethers contain many ions, making individual analysis quite time-consuming. Therefore, scripts using Excel and R were developed with the help of ChatGPT 4.0, resulting in ion series spectra (ISS) that contained only ions of a specific ion series. The analysis of 11 synthetic methyl ethers showed that especially the ion series C_<i>n</i>H_2<i>n</i>+1O (ISS45) and C_<i>n</i>H_2<i>n</i>-2 (ISS40) are of high diagnostic value, together with some methoxy group-induced fragmentation. The approach was successfully tested with lipids from the spider <i>Tetragnatha versicolor</i>, which had been previously analyzed by derivatization, and with web extracts of <i>Erigone atra</i>, revealing 1-methoxy-2,16-dimethylhenicosane as a male-specific component─the first spider methyl ether in a volatility range that would allow detection via the gas phase. This approach can also be applied to structurally related primary alcohols, although the diagnostic ions are of lower intensity.