Luca Morini1, Fulvia Pozzi2, Angelo Groppi2. 1. Department of Public Health, Experimental and Forensic Medicine, University of Pavia, via forlanini 12, 27100, Pavia, Italy. Electronic address: luca.morini@unipv.it. 2. Department of Public Health, Experimental and Forensic Medicine, University of Pavia, via forlanini 12, 27100, Pavia, Italy.
Abstract
INTRODUCTION: An in vitro study on authentic positive samples was carried out, aiming the evaluation of the effect of chlorinated water on benzodiazepines in hair. Two subjects exposed to chlorinated water for several consecutive times were also investigated. METHOD: Seven hair samples collected from autopsy cases, previously tested positive for benzodiazepines, were washed with dichloromethane and methanol. They were longitudinally divided in six aliquots of about 30mg. An aliquot was processed without treatment while other five ones were soaked in chlorinated water (0.1% sodium dichloroisocyanurate and 0,1M sulfuric acid at pH 5.5) for 4, 20, 24 and 30h respectively. Hair samples were then processed following a fully validated and previously published method. Briefly hair samples were sonicated in 600 microliters methanol containing halazepam (IS) up to two hours. Ten microliters were injected in a liquid chromatographic tandem mass spectrometric (LC-MS/MS) system. Analytes were eluted from a C18 reversed-phased column. Two transitions on multiple reaction monitoring and positive ionization mode were monitored for each compound. RESULT AND DISCUSSIONS: Six compounds among benzodiazepines and metabolites were identified and quantified in the seven hair samples: diazepam (575pg/mg), desmethyldiazepam (562pg/mg), chlordesmethyldiazepam (173pg/mg), desalkylflurazepam (320pg/mg), clonazepam (three cases-195, 119 and 111pg/mg respectively), lormetazepam (two cases-182 and 416pg/mg respectively). Traces of 7-aminoclonazepam were identified into 2 samples. Stability of benzodiazepines in water was evaluated by soaking an aliquot of hair for up to 30h in deionized water. No significant degradation was observed. Samples soaked in chlorinated water showed considerable decreasing from the initial concentration even after the 4-h treatment: the fastest degradation was provided by clonazepam that showed a 61% loss. The greatest loss was measured for diazepam (86% loss after 30-h soaking). CONCLUSION: To the best of our knowledge this is the first in vitro study that evaluated benzodiazepines stability in hair after prolonged exposure to chlorinated water. The results showed that the longer the exposure the higher the degradation. Prolonged exposure to chlorinated water and sunlight must be always taken into account as possible causes of false negative results.
INTRODUCTION: An in vitro study on authentic positive samples was carried out, aiming the evaluation of the effect of chlorinated water on benzodiazepines in hair. Two subjects exposed to chlorinated water for several consecutive times were also investigated. METHOD: Seven hair samples collected from autopsy cases, previously tested positive for benzodiazepines, were washed with dichloromethane and methanol. They were longitudinally divided in six aliquots of about 30mg. An aliquot was processed without treatment while other five ones were soaked in chlorinated water (0.1% sodium dichloroisocyanurate and 0,1M sulfuric acid at pH 5.5) for 4, 20, 24 and 30h respectively. Hair samples were then processed following a fully validated and previously published method. Briefly hair samples were sonicated in 600 microliters methanol containing halazepam (IS) up to two hours. Ten microliters were injected in a liquid chromatographic tandem mass spectrometric (LC-MS/MS) system. Analytes were eluted from a C18 reversed-phased column. Two transitions on multiple reaction monitoring and positive ionization mode were monitored for each compound. RESULT AND DISCUSSIONS: Six compounds among benzodiazepines and metabolites were identified and quantified in the seven hair samples: diazepam (575pg/mg), desmethyldiazepam (562pg/mg), chlordesmethyldiazepam (173pg/mg), desalkylflurazepam (320pg/mg), clonazepam (three cases-195, 119 and 111pg/mg respectively), lormetazepam (two cases-182 and 416pg/mg respectively). Traces of 7-aminoclonazepam were identified into 2 samples. Stability of benzodiazepines in water was evaluated by soaking an aliquot of hair for up to 30h in deionized water. No significant degradation was observed. Samples soaked in chlorinated water showed considerable decreasing from the initial concentration even after the 4-h treatment: the fastest degradation was provided by clonazepam that showed a 61% loss. The greatest loss was measured for diazepam (86% loss after 30-h soaking). CONCLUSION: To the best of our knowledge this is the first in vitro study that evaluated benzodiazepines stability in hair after prolonged exposure to chlorinated water. The results showed that the longer the exposure the higher the degradation. Prolonged exposure to chlorinated water and sunlight must be always taken into account as possible causes of false negative results.