title: Quantitative analysis of benzodiazepines in vitreous humor by high-performance liquid chromatography.
abstract: OBJECTIVE
  Benzodiazepines are frequently screened drugs in emergency toxicology, drugs of abuse testing, and in forensic cases. As the variations of benzodiazepines concentrations in biological samples during bleeding, postmortem changes, and redistribution could be biasing forensic medicine examinations, hence selecting a suitable sample and a validated accurate method is essential for the quantitative analysis of these main drug categories. The aim of this study was to develop a valid method for the determination of four benzodiazepines (flurazepam, lorazepam, alprazolam, and diazepam) in vitreous humor using liquid-liquid extraction and high-performance liquid chromatography.
  
  
  METHODS
  Sample preparation was carried out using liquid-liquid extraction with n-hexane: ethyl acetate and subsequent detection by high-performance liquid chromatography method coupled to diode array detector. This method was applied to quantify benzodiazepines in 21 authentic vitreous humor samples. Linear curve for each drug was obtained within the range of 30-3000 ng/mL with coefficient of correlation higher than 0.99.
  
  
  RESULTS
  The limit of detection and quantitation were 30 and 100 ng/mL respectively for four drugs. The method showed an appropriate intra- and inter-day precision (coefficient of variation < 10%). Benzodiazepines recoveries were estimated to be over 80%. The method showed high selectivity; no additional peak due to interfering substances in samples was observed.
  
  
  CONCLUSIONS
  The present method was selective, sensitive, accurate, and precise for the quantitative analysis of benzodiazepines in vitreous humor samples in forensic toxicology laboratory.
authors.affiliation: Legal Medicine Research Center, Legal Medicine Organization, Forensic Toxicology Department, Tehran, Iran.
authors.forename: Elham
authors.initials: E
authors.lastname: Bazmi
authors.affiliation: Department of Forensic Medicine, School of Medicine, Tehran University of Medical Sciences (TUMS), Tehran, Iran.
authors.forename: Behnam
authors.initials: B
authors.lastname: Behnoush
authors.affiliation: Legal Medicine Research Center, Legal Medicine Organization, Forensic Toxicology Department, Tehran, Iran.
authors.forename: Maryam
authors.initials: M
authors.lastname: Akhgari
authors.affiliation: Legal Medicine Research Center, Legal Medicine Organization, Forensic Toxicology Department, Tehran, Iran.
authors.forename: Leila
authors.initials: L
authors.lastname: Bahmanabadi
keywords: Benzodiazepines; forensic toxicology; high-performance liquid chromatography; liquid–liquid extraction; method validation; vitreous humor