Determination of colchicine and O-demethylated metabolites in decomposed skeletal tissues by microwave assisted extraction, microplate solid phase extraction and ultra-high performance liquid chromatography (MAE-MPSPE-UHPLC).

Abstract

Microwave assisted extraction (MAE) followed by microplate solid phase extraction (MPSPE) coupled with ultra high performance liquid chromatography (UHPLC) for the semi-quantitative determination of colchicine, 3-demethyl colchicine and 2-demethyl colchicine in postmortem rat bone is described. Rats (n=4) received 50mg/kg colchicine (i.p), and euthanized by CO2 asphyxiation. Remains decomposed to skeleton outdoors and vertebral bones were collected cleaned, and ground to a fine powder. Powdered bone underwent MAE using methanol in a closed microwave system, followed by MPSPE and analysis using UHPLC-PDA. MAE analyte stability was assessed and found to be stable for at least 60 min irradiation time. The majority (>95%) of each analyte was recovered after 15 min. The MPSPE-UHPLC method was linear between 10 and 2,000 ng/mL, with coefficients of variation <20% in triplicate analysis, with a limit of detection of 10 ng/mL for each of the three analytes. Following MAE for 30 min (80°C, 1200W), MPSPE-UHPLC analysis of vertebral bone of colchicine-exposed rats detected colchicine (1.8-4.1 μg/g), 3-demethyl colchicine (0.77-1.8 μg/g) and 2-demethyl colchicine (0.43-0.80 μg/g) in all samples assayed.