Analysis of tramadol and its metabolites in rat skeletal tissues following acute and repeated dose patterns using high performance liquid chromatography tandem mass spectrometry
Date
2020-10-15
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Abstract
The use of skeletal elements for the viable analysis of drugs of abuse has seen increased
prevalence in the past 10 years. Advancements in the analytical methods used, including solid
phase extraction and mass spectrometry, have allowed for increased sensitivity and selectivity.
Previous studies have focused on the influence of dose-death interval, microclimate, differential
patterns of exposure, and the influence of body position. In this work, the opioid analgesic tramadol
was investigated for its pharmacological behaviour when administered as part of three dosage
patterns to male Sprague Dawley rats. The three exposure patterns consisted of an acute low (n =
4, 1 doses, 30 mg/kg) group, a repeated high survived group (n = 5, 3 doses, 30 mg/kg) and a
repeated high overdosed group (n = 11, 3 doses, 30 mg/kg). Drug free rats (n = 4) served as
negative controls. Following euthanasia by CO2 asphyxiation, animals were decomposed to
skeleton outdoors over the summer of 2019 in Sudbury, Ontario. Bones were sorted by animal and
skeletal element (skull, vertebrae, ribs, pelvis, femur, tibia/fibula), then washed and ground to
powder before undergoing dynamic methanolic extraction. Semi-quantitative analysis of tramadol
and four of its metabolites – O-desmethyltramadol, N-desmethyltramadol, N,Odidesmethyltramadol and tramadol N-oxide – was conducted using high-performance liquid
chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) in positive ion mode.
Analyte levels were expressed as a mass-normalized response ratio (RR/m) in order to account for
the exact mass of bone used. Method validation for the analysis of tramadol and its metabolites
was investigated in accordance with the Scientific Working Group of Toxicologists (SWGTOX)
standards of practice, with all criteria except for dilution integrity successfully met at a limit of
detection and limit of quantitation of 1 ng/mL. The effect of exposure pattern on analyte level and
analyte level ratio was assessed using the Kruskal-Wallis test for significant differences (P < 0.05).
A total of 315 pairwise comparisons were performed to assess significant differences, with the
ratio of tramadol to N-desmethyltramadol determined to be the metric most commonly able to
identify these differences in 91% of tests. Additionally, the effect of skeletal element on analyte
level and analyte level ratio was also assessed, with a total of 675 pairwise comparisons. Skeletal
element was determined to be a significant factor in all cases. This data suggests that both skeletal
element and dose pattern are important measures to evaluate with respect to the analysis of drugs
of abuse in bone tissues. Furthermore, different metrics, including analyte level and analyte level
ratios, may be useful for discriminating between these different dosing patterns.
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Keywords
Tramadol, tandem mass spectrometry, skeletal tissues, forensic toxicology