Evaluation of benzodiazepines and zolpidem in nails and their stability after prolonged exposure to chlorinated water

Matteo Moretti, Luisa Andrello, Silvia Visonà, Claudia Vignali, Angelo Groppi, Francesca Freni, Antonio Osculati, Luca Tajana, Luca Morini

Research output: Contribution to journalArticlepeer-review


The study aims the development and validation of a LC-MS/MS method for the identification and quantification of benzodiazepines and zolpidem in nails as alternative keratinized matrix to hair in long-term monitoring of anxiolytic and hypnotic drugs. Both fingernail and toenail samples (1-2 mm) were collected by clipping the excess overhang of the nail from volunteers and from postmortem cases. They were washed twice with organic solvents, dried under nitrogen stream, pulverized, immersed in a methanol solution (internal standard: diazepam-D5) and sonicated up to two hours. The solution was then direct injected in the LC-MS/MS system. Mass spectrometry was set in MRM mode, selecting two transitions for each substance. 32 analytes among benzodiazepines, metabolites and hypnotics were included in the list. The method fulfilled the internationally required criteria for validation. Limits of detection ranged from 0.03 pg/mg (zolpidem) to 13.1 pg/mg (bromazepam). 9 subjects under therapy were positive at 7 different benzodiazepines and/or metabolites (lorazepam, desalkylflurazepam, bromazepam, diazepam, alprazolam, lormetazepam and prazepam), while 5 molecules were measured in 4 postmortem cases (diazepam, desmethyldiazepam, delorazepam, 7-aminoclonazepam and zolpidem). In vitro experiments on eight authentic samples suggested that benzodiazepines in nails are influenced by the prolonged exposure to chlorinated water.

Original languageEnglish
Pages (from-to)137-142
Number of pages6
JournalJournal of Pharmaceutical and Biomedical Analysis
Publication statusPublished - Apr 15 2018


  • Benzodiazepines/chemistry
  • Bromazepam/chemistry
  • Chlorine/adverse effects
  • Chromatography, Liquid/methods
  • Diazepam/chemistry
  • Female
  • Forensic Toxicology/methods
  • Humans
  • Limit of Detection
  • Male
  • Nails/chemistry
  • Pyridines/chemistry
  • Tandem Mass Spectrometry/methods
  • Water/adverse effects
  • Zolpidem


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