Background::The detection of new designer benzodiazepines in biological fluids and tissues, together with the traditional ones, could represent an important analytical update for laboratories performing clinical
and forensic toxicological analysis.
Objective::A liquid chromatography tandem mass spectrometry method (LC-MS/MS) has been developed, fully
validated, and applied to a cohort of real urine samples collected from patients under withdrawal treatment and
from intoxication cases.
Methods::100 µL urines were added to a buffer solution containing deuterated internal standards; the samples
were then extracted through a liquid/liquid procedure, dried under a nitrogen stream, and reconstituted in mobile phase. The chromatographic separation was performed in reverse phase through a C18 column with gradient elution. Mass spectrometry operated in positive polarization and multiple reaction monitoring mode.
Results::25 molecules were optimized for instrumental analysis: 9 designer benzodiazepines and 16 traditional
compounds (parent drugs and main metabolites). Sensitivity, specificity, linearity, accuracy, imprecision, recovery, matrix effects, and carry-over have been evaluated for all molecules. Only cinazepam did not satisfy all
acceptance criteria for validation. 10 among the 50 analyzed samples tested positive for at least one of the
monitored molecules. In particular, two different samples collected from the same case provided positive results
for flubromazepam, a designer benzodiazepine.
Conclusion::The method was proven to be useful in detecting not only traditional benzodiazepines but also new
designer ones. The identification of a New Psychoactive Substance in real samples confirmed that analytical
procedures should be updated to include as many substances as possible.