Francesco Paolo Busardò1, Chrystalla Kyriakou2, Roberta Tittarelli2, Giulio Mannocchi2, Flaminia Pantano2, Alessandro Santurro2, Simona Zaami2, Giovanni Baglìo3. 1. Department of Anatomical, Histological, Forensic and Orthopaedic Sciences, Sapienza University of Rome, Viale Regina Elena 336, 00161 Rome, Italy. Electronic address: fra.busardo@libero.it. 2. Department of Anatomical, Histological, Forensic and Orthopaedic Sciences, Sapienza University of Rome, Viale Regina Elena 336, 00161 Rome, Italy. 3. National Institute for Health, Migration and Poverty (NIHMP), Via di S. Gallicano, 25/a, 00153 Roma, Italy.
Abstract
AIMS: The aim of this work is to test the stability of mephedrone added to whole blood collected from alive and dead mephedrone free-users and stored at three different temperatures (-20, +4 and +20°C) with and without preservatives up to 6 months, trying to establish the best storage condition in order to reduce possible analyte loss/degradation during the storage period. MATERIALS AND METHODS: Different sources of blood were obtained as follow: 10 samples of blood came from 10 alive mephedrone free-users (mean age 34±15.8 years old) (Group 1), whereas 10 post mortem blood samples were obtained from 10 cadavers, in which the post mortem interval was between 24 and 36h (Group 2). The cause of death in post mortem cases (mean age 45±14.2 years old) was not drug related. Pools of blood were spiked with mephedrone at the concentration of 1mg/L and 1mL aliquots were transferred in 2mL Eppendorf capped tubes with and without preservatives as follow: with ethylenediaminetetraacetic acid (EDTA) 3%; with sodium fluoride/potassium oxalate (NaF/KOx) 1.67%/0.2%, respectively; without preservatives. All samples were stored at three different temperatures: -20°C, 4°C and 20°C and extracted and analyzed in duplicate by GC-MS according to a previously published method by Dickson et al., every other day during the first month and then weekly up to 6 months. RESULTS AND CONCLUSIONS: our study allow us to affirm that -20°C is the best storage temperature for mephedrone stability in ante-mortem and post-mortem blood samples in comparison to the other two tested temperatures (+4 and +20°C), showing higher values in both groups in samples stored with and without preservatives (p<0.0001). The comparison of Group 1 (samples coming from alive subjects) and Group 2 (post-mortem samples) highlights a better stability of mephedrone in Group 1 (p<0.001) at all tested storage conditions. Finally, the analysis of blood specimens stored with and without preservatives in both groups suggests that specimens stored with NaF/KOx maintain mephedrone stability better than those stored with EDTA (p<0.001) and those stored without preservatives (p<0.0001), therefore, we strongly recommend in order to maintain the highest mephedrone stability in blood, to store specimens at -20°C adding NaF/KOx as preservative.
AIMS: The aim of this work is to test the stability of mephedrone added to whole blood collected from alive and dead mephedrone free-users and stored at three different temperatures (-20, +4 and +20°C) with and without preservatives up to 6 months, trying to establish the best storage condition in order to reduce possible analyte loss/degradation during the storage period. MATERIALS AND METHODS: Different sources of blood were obtained as follow: 10 samples of blood came from 10 alive mephedrone free-users (mean age 34±15.8 years old) (Group 1), whereas 10 post mortem blood samples were obtained from 10 cadavers, in which the post mortem interval was between 24 and 36h (Group 2). The cause of death in post mortem cases (mean age 45±14.2 years old) was not drug related. Pools of blood were spiked with mephedrone at the concentration of 1mg/L and 1mL aliquots were transferred in 2mL Eppendorf capped tubes with and without preservatives as follow: with ethylenediaminetetraacetic acid (EDTA) 3%; with sodium fluoride/potassium oxalate (NaF/KOx) 1.67%/0.2%, respectively; without preservatives. All samples were stored at three different temperatures: -20°C, 4°C and 20°C and extracted and analyzed in duplicate by GC-MS according to a previously published method by Dickson et al., every other day during the first month and then weekly up to 6 months. RESULTS AND CONCLUSIONS: our study allow us to affirm that -20°C is the best storage temperature for mephedrone stability in ante-mortem and post-mortem blood samples in comparison to the other two tested temperatures (+4 and +20°C), showing higher values in both groups in samples stored with and without preservatives (p<0.0001). The comparison of Group 1 (samples coming from alive subjects) and Group 2 (post-mortem samples) highlights a better stability of mephedrone in Group 1 (p<0.001) at all tested storage conditions. Finally, the analysis of blood specimens stored with and without preservatives in both groups suggests that specimens stored with NaF/KOx maintain mephedrone stability better than those stored with EDTA (p<0.001) and those stored without preservatives (p<0.0001), therefore, we strongly recommend in order to maintain the highest mephedrone stability in blood, to store specimens at -20°C adding NaF/KOx as preservative.
Authors: Flaminia Pantano; Roberta Tittarelli; Giulio Mannocchi; Roberta Pacifici; Alessandro di Luca; Francesco Paolo Busardò; Enrico Marinelli Journal: Curr Neuropharmacol Date: 2017 Impact factor: 7.363