The aim of this study was the establishment of a UHPLC-QqQ-MS/MS method to determine methotrexate in postmortem biological samples and quantify the postmortem distribution of methotrexate in a case of fatal intoxication of this drug. A volume of 100 μL or 100 mg of postmortem specimens was precipitated with 400 μL of cold methanol and then analyzed using UHPLC-QqQ-MS/MS. The validation parameters of the method were as follows: limit of quantification: 0.1−1.0 ng/mL or ng/g, coefficient of determination: >0.998 (R2), matrix effect, intra- and inter-day accuracies and precisions: not greater than 13.6%, 14.8% and 17.4%, respectively. The recoveries were: 89.0−113.6%. The postmortem distribution studies revealed methotrexate concentrations as follows: blood—7.2 ng/mL, vitreous humor—0.8 ng/mL, liver—43.7 ng/g, kidney—20.6 ng/g, bone marrow—29.9 ng/g, lumbar vertebra—20.0 ng/g. The highest concentrations of methotrexate after poisoning were found in the tissues with the most rapidly dividing cells. The method described is simple, precise and selective. Methotrexate concentrations can be routinely determined in postmortem specimens. Determination of methotrexate in the postmortem biological material is possible after a few days of intensive treatment.
Keywords: LC-MS; distribution; fatal intoxication; methotrexate; poisoning.