Introduction Benzodiazepines and sedative hypnotics, such as zolpidem, zaleplon and zopiclone are some of the most commonly prescribed and abused drugs in the USA (1–4). Approximately 20 benzodiazepines have been approved for use in the USA and are prescribed as anxiolytics, muscle relaxants, anesthetic adjuncts, anticonvulsants, insomnia treatment and treatment for obsessive-compulsive disorders (1, 2). In forensic toxicology, this means that benzodiazepines and sedative hypnotics can been seen in antemortem (AM) cases, such as driving under the influence of drug(s) and drug-facilitated sexual assaults where both blood and urine could be analyzed (1, 5–9). They are also important in postmortem (PM) toxicology casework, where these same drugs can be used in suicides and analyzed in blood and/or tissue samples. For crime laboratories that analyze both AM and PM casework, it is ideal to have an extraction method that can treat all sample matrices: blood, urine, liver, brain and stomach contents, the same for shorter turnaround times. Due to benzodiazepine and sedative hypnotics’ popularity and frequency of abuse, forensic toxicology laboratories need a rapid, sensitive and inexpensive method for determining these drug concentrations in various matrices at the same time (10). Traditionally, benzodiazepine analysis was performed using gas chromatography–mass spectrometry (GC–MS). Unlike GC–MS, ultra performance liquid chromatography triple quadrupole mass spectrometry (LC–MS-MS) can tolerate injections with aqueous content. Therefore, the high aqueous content found in urine, AM blood, PM blood and tissue homogenate samples is not detrimental to benzodiazepine analysis by LC–MS-MS. In addition, only minimal amounts of specimen were needed to achieve the sensitivity required with elimination of matrix interferences (10). Sample preparation methods, therefore, focused on providing clean, non-aqueous extracts for derivatization. While liquid–liquid extractions (LLE) had been historically....

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