Pancuronium bromide is a steroid and diquaternary ammonium salt. Previous investigators focused on the identification of pancuronium to assist in attempted murder investigations; to confirm the drug in a suicide investigation; and in other cases with access to postmortem blood, serum, and urine analyses. In those reports, tissue samples were not significantly aged, decayed, or exposed to environmental, fungal, or bacterial influences. In the current study, a significantly larger number of samples were successfully assayed for pancuronium after exposure to various external factors that produced widely variable states of decomposition and biological and chemical exposures. The use of solid-phase extraction was integral to routine sample preparation and concentration of the target quaternary ammonium drug from complex biological matrices. The protocols developed can be applied to small-scale or large-scale tissue analysis requirements. Injection-port pyrolysis and gas chromatography-mass spectroscopy analysis of questioned samples facilitated rapid prescreening of quaternary ammonium-type drugs. Contrary to the conclusions of a previous study, m/z 286 is the doubly charged molecular ion of pancuronium, and it forms definitive singly charged product ions larger than the m/z 286 ion. These unique data assure confirmation of pancuronium in questioned samples; this method is particularly important for the unambiguous identification in aged autopsy samples. 7 figures and 16 references