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

Abstract

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 200mg/kg, i.p.; n=5, respectively) or repeated (REP1 or REP2-3 doses of 25 or 50mg/kg, i.p., 30min 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.