Stability of mephedrone and five of its phase I metabolites in human whole blood.

Mephedrone is a new psychoactive substance known to be unstable in biological matrices stored at room temperature or refrigerated. While the instability of mephedrone has been investigated before, there is currently no data regarding the stability of mephedrone metabolites. In this study, a liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of mephedrone and five of its phase I metabolites (dihydro-mephedrone, nor-mephedrone, hydroxytolyl-mephedrone, 4-carboxy-mephedrone and dihydro-nor-mephedrone) in human whole blood has been developed and validated. Samples were extracted by a mixed mode solid-phase extraction and analyzed on a pentafluorophenylpropyl column. The method was successfully validated for selectivity, linearity (0.2-2 to 10-100 ng/mL), limits of detection (50-500 pg/mL) and quantification (200-2000 pg/mL), precision (0.924-8.27%), accuracy (86.6-115%), carryover, recovery (32.5-88.3%), and matrix effects (71.0-108%). Analyte stability in human whole blood preserved with sodium fluoride/potassium oxalate was assessed at +4°C and -20°C after 24 hours, 48 hours, 4 days, and 10 days of storage. Instability was observed in samples stored at +4°C: nor-mephedrone and 4-carboxy-mephedrone lost 40.2 ± 6.7% and 48.1 ± 4.8%, respectively, of their initial concentration at low concentration level and 33.8 ± 4.2% and 44.6 ± 6.5%, respectively, at high concentration level after 10 days. All analytes were more stable at -20°C where the highest loss of 22.6 ± 6.9% was observed for 4-carboxy-mephedrone after 10 days. This is the first time stability of mephedrone metabolites in human whole blood has been assessed, indicating -20°C to be the recommended storage condition for all analytes in clinical settings.