Analysis of Dextromethorphan and Three Metabolites in Decomposed Skeletal Tissues by UPLC–QToF–MS: Comparison of Acute and Repeated Drug Exposures

Ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC–QToF–MS) analysis of dextromethorphan (DXM) and its metabolites—dextrorphan, 3-methoxymorphinan (3-MEM) and 3-hydroxymorphinan—in skeletal remains of rats exposed to DXM under different dosing patterns is described. Rats (n = 20) received DXM in one of four dosing patterns: acute (ACU1 or ACU2—100 or 200 mg/kg, i.p.; n = 5, respectively) or repeated (REP1 or REP2—3 doses of 25 or 50 mg/kg, i.p., 30 min apart; n = 5, respectively). Drug-free animals (n = 5) served as negative controls. Following euthanasia, the animals decomposed to skeleton outdoors. Bones were sorted by animal and skeletal element (vertebra, femur, pelvis, tibia, rib and skull), washed, air-dried and pulverized prior to dynamic methanolic drug extraction, filtration/pass-through extraction and analysis by UPLC–QToF–MS in positive electrospray ionization mode. Analyte levels (expressed as mass-normalized response ratios, RR/m) differed significantly between ACU1 and ACU2 (Mann–Whitney (MW), P < 0.05) in all skeletal elements for all analytes investigated, and between REP1 and REP2 in most skeletal elements for 3-MEM and 3-HOM, but in all skeletal elements for DXM. Between ACU1 and ACU2, and between REP1 and REP2, analyte level ratios (RRi/RRj) differed significantly (MW, P < 0.05) in 3/6 to 6/6 skeletal elements, depending on the ratios concerned, with no analyte level ratio differing significantly between both ACU1 vs ACU2 and REP1 vs REP2. Kruskal–Wallis (KW) analysis showed skeletal element to be a main effect for all analyte levels and analyte level ratios in all ACU and REP groups examined (P < 0.05). For data pooled only according to exposure pattern, KW analysis showed dose pattern to be a main effect for both analyte levels and analyte level ratios (P < 0.05). These data illustrate a dependence of these measures on dose, dose pattern and skeletal element, suggesting that some exposure patterns may be distinguished by toxicological analysis of bone.