DETERMINATION OF CLONAZEPAM IN HAIR AND NAIL USING LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY (LC-MS/MS)

Clonazepam, (5-(2-Chlorophenyl)-7-nitro-1,3-dihydro-1,4-benzodiazepin-2-one), a nitrobenzodiazepine and a scheduled drug, is one of the most highly abused drugs in recent times. A sensitive analytical methodology using Liquid Chromatography tandem Mass Spectrometry (LC-MS/MS) was developed for the determination of clonazepam in human hair and nail samples. The method involves the decontamination of hair and nail using 2mL each of 0.1% SDS (sodium dodecyl sulfate), water, and methanol, dried followed by incubation in phosphate buffer (pH=8), extraction with diethyl ether:chloroform (70:30, v/v), separation, and detection using liquid chromatography-tandem mass spectrometry. The method was totally validated with the limit of detection (LOD) of clonazepam estimated as 0.50pg/mg and the limit of quantification (LOQ), 2.0pg/mg of hair and nail. Linearity was observed in the range 2.0pg/mg to 5000pg/mg with r2>0.9980. The extraction recoveries, intra-day and inter-day precisions, accuracy, and matrix effect were also studied for the developed method. The amount of clonazepam detected in the hair and nail of different subjects was found to be in the range of 0.1156-1.040ng/mg and 0.0713-0.880ng/mg, respectively. The method is highly sensitive, precise, and reliable for the determination of clonazepam in hair and nail, which can be adopted by law enforcement laboratories for routine analysis.