Forensic intoxication with clobazam: HPLC/DAD/MSD analysis

Abstract

Clobazam (Castillium 1 , Urbanil 1 ), a benzodiazepine often used as an anxiolytic and in the treatment of epilepsy, is considered a relatively safe drug. The authors present a fatal case with a 49-year-old female, found dead at home. She had been undergoing psychiatric treatment and was a chronic alcoholic. The autopsy findings were unremarkable, except for multivisceral congestion, steatosis and a small piece of a plastic blister pack in the stomach. Bronchopneumonia, bronchitis and bronchiolitis were also diagnosed. Anhigh-performance liquid chromatography (HPLC)/diode array detector (DAD)/mass spectrometry detection (MSD) with electrospray method was developed in order to detect, confirm and quantify clobazam in the post-mortem samples. In the chromatographic separation, a reversed-phase column C 18 (2:1 Â 150 mm, 3.5 mm) was used with a mobile phase of methanol and water, at a 0.25 ml/min flow rate. Carbonate buffer (pH 10.5) and 20 ml of prazepam (100 mg/ml) as internal standard were added to the samples. A simple and reliable liquid-liquid extraction method for the determination of clobazam in post-mortem samples was described. Calibration curves for clobazam were performed in blood, achieving linearity between 0.01 and 10 mg/ml and a detection limit of 1.0 ng/ml. The clobazam concentration found in post-mortem blood was 3.9 mg/ml, higher than the reported therapeutic concentration (0.1-0.4 mg/ml). The simultaneous acquisition by photodiode array detection and mass spectrometry detection results allowed benzodiazepines to be identified with sufficient certainty.An examination of all the available information suggested that death resulted from respiratory depression due to clobazam toxicity.

Efficacy of Clobazam as Add-on Therapy for Refractory Epilepsy

“…We found only 1 report of severe intoxication with CLB that could have led to respiratory depression. 23 The serum level of CLB in this case was 3.9 2g/mL, much higher than the reported therapeutic concentration (0.1-0.4 2g/mL). Larger prospective studies should be performed to confirm our findings and detect uncommon or rare side effects.…”

Clobazam Therapeutic Drug Monitoring

“…Four of these cases 41,42,84,85 had N-desmethylclobazam concentrations >3000 ng/mL; 3 cases 41,42,85 had concentrations > 4 times 3000 ng/mL, including 1 case 85 >10 times 3000 ng/mL. N-desmethylclobazam concentrations were not measured in 2 cases, 83,86 but their clobazam concentrations were >3000 ng/mL or 10 times higher than the upper recommended limit of 300 ng/mL.…”

“…This patient appears also to be case report 2 in another article. 22 f According to Proença et al 86 there is no tissue redistribution after death; thus postmortem blood concentrations reflect well the concentrations before death. g A 70-year-old French women was found dead. The contribution of other drugs was eliminated by the toxicology report.…”

“…Table 2 describes 6 reports of clobazam intoxication, 41,42,83–86 including 2 deaths 85,86 that occurred at elevated serum concentrations. Four of these cases 41,42,84,85 had N-desmethylclobazam concentrations >3000 ng/mL; 3 cases 41,42,85 had concentrations > 4 times 3000 ng/mL, including 1 case 85 >10 times 3000 ng/mL.…”

Development and validation of a liquid chromatographic/electrospray ionization mass spectrometric method for the quantitation of prazepam and its main metabolites in human plasma

“…15 An LC/MS method with an electrospray ionization (ESI) interface has been reported for the determination of clobazam in blood using prazepam as the internal standard. 16 Although the above-described methods include several successful approaches, there has been no report of the determination of prazepam and its major metabolites in human plasma by LC/MS with ESI.…”