N-Alkylpyridinium isotope quaternization for matrix-assisted laser desorption/ionization Fourier transform mass spectrometric analysis of cholesterol and fatty alcohols in human hair

Isotope-coded reagents have been developed for labeling of amino acids, phenols and fatty acids, but not for alcohols. In this work, a simple reaction based on direct N-alkylpyridinium isotope quaternization (NAPIQ) was developed for mild derivatization of cholesterol and fatty alcohols. Different from the conventional quaternary reagents with cations on themselves, two simple and charge-neutral reagents: pyridine and d₅-pyridine directly attached N-cationic tag onto the target compounds in the presence of trifluoromethanesulfonic anhydride (Tf₂O) without tedious sample preparation. The derivatization completed in 5 min and achieved charge labeling of the target compounds, which significantly improved the detection limits of analytes by 10³-folds in further analysis by matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI–FTMS). The use of commercially available d₀/d₅-pyridine pairs facilitated isotope-coded chemical derivatization and avoided the use of isotope-labeled internal standards; the excess pyridine did not affect the signals of analytes. Utility of the NAPIQ method was examined in the identification of cholesterol and fatty alcohols in small amount of human hair sample (<0.5 mg). The fluctuation of total cholesterol in human body was profiled during time by quantitatively comparing the different segments of a single strand of hair. This study combines the direct pyridinium quaternization with MALDI–FTMS, which offers a perspective and an alternative tool for the identification and quantification of substances in biological matrix by comparing d₀/d₅ pairs, especially when isotope-labeled internal standards are unavailable.