Institute Hermes Pardini

Vespasiano, Brazil

Institute Hermes Pardini

Vespasiano, Brazil
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Diniz M.E.R.,Institute Hermes Pardini | Dias N.L.,Institute Hermes Pardini | Paulo B.P.,Institute Hermes Pardini | Andrade F.V.,Institute Hermes Pardini | And 2 more authors.
Brazilian Journal of Analytical Chemistry | Year: 2017

Clonazepam and clobazam are potent benzodiazepines derivatives that have been used primarily as anticonvulsants. Methods based on liquid chromatography tandem mass spectrometry (LC-MS/MS) are considered the gold standard in therapeutic monitoring. In this work a simple, rapid and sensitive LC-MS/MS method for the determination of clonazepam, clobazam and N-desmethylclobazam in serum was developed and validated. The method consisted in a simple liquid-liquid extraction, in which 200 μL of serum containing the internal standard (temazepam or stable isotopes of clonazepam and clobazam) were treated with ethyl acetate and subjected to LC-MS/MS analysis using positive electrospray ionization. Chromatographic separation was performed on a C18 column and isocratic mobile phase containing methanol:water:acetonitrile (50:30:20, v/v/v) with 0.05% of formic acid at 400 μL min-1. The linear analytical range of the procedure was between 10.0 and 160.0 ng mL-1 for clonazepam, 25.0 and 525.0 ng mL-1 for clobazam and 100.0 and 5,000.0 ng mL-1 for N-desmethylclobazam. The sample dilution of 1:2 and 1:10 for clobazam/N-desmethylclobazam and 1:2 for clonazepam was validated for the samples that exceed the method linearity. Relative standard deviations for intra and inter-day precision were lower than 4.3% for clonazepam, 7.5% for N-desmethylclobazam and 9.7% for clobazam. The recovery range was between 95 and 109% for all analytes. The LC-MS/MS method has been developed and validated successfully for the quantitative analysis and therapeutic monitoring of these benzodiazepines. The method has been applied successfully in the clinical laboratory routine. © 2017, DKK Comunicacao. All rights reserved.

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