Distinguishing between structural isomers of metabolites typically requires reference standards or comparative data like retention times. A new method uses hydrogen-deuterium exchange (HDX) to differentiate 4- and 5-hydroxy positional isomers of hydroxy-N,N-dimethyltryptamine (psilocin and bufotenine) based on their absolute pKa differences. The indole amine hydrogen exchanges quickly, while the aromatic hydroxy hydrogen exchanges at a rate dependent on ring position. After back-exchange on the LC column, only the 4-hydroxy isomer shows appreciable deuterium retention within hours, enabling clear differentiation via high-resolution mass spectrometry. This approach could characterize unknown structural isomers without relying on external standards or instrument-specific data.
A chemical derivatization technique called trimethylation enhancement using diazomethane (TrEnDi) improves the sensitivity of mass spectrometry for detecting 13 different drugs of misuse. All analytes were successfully permethylated, with 11 showing improved analytical characteristics. Sensitivity enhancements ranged from 1.2-fold to 24.2-fold for psilocybin, and most species showed increased reversed-phase chromatographic retention. Using 13C-isotopically labeled TrEnDi reagents resolved isobaric interference between three pairs of controlled substances. In an unconventional aprotic solvent system for electrospray ionization, TrEnDi-modified amphetamine was easily measured while unmodified amphetamine was not detected. Applied to morphine in real urine, TrEnDi achieved a percent recovery of 103.1% and a 2.4-fold sensitivity enhancement, demonstrating its versatility for trace analysis.