Novel and conventional techniques for detecting clonazepam in Lucilia cuprina (Diptera: Calliphoridae).

Benzodiazepine drugs, particularly clonazepam, are used as adjunctive therapy to manage epilepsy and control seizures. However, the misuse of these drugs can be fatal. One requirement in forensic investigations is to find evidence of drug residue within a deceased individual's body. In such investigations, entomology can assist by conducting specific pathological and toxicological tests, even when the body is highly decomposed. This study evaluated the effectiveness of two new methods, namely the measurement of protein carbonyls and the use of α,α-diphenyl-β-picrylhydrazyl (DPPH), in detecting clonazepam in Lucilia cuprina (Wiedemann, 1830). The insect’s larvae were fed on the muscle tissues of clonazepam-injected rabbits. These methods were compared to the conventional analytical technique of "High-performance liquid chromatography-mass spectrometry" (HPLC-MS). Additionally, the impact of the drug on L. cuprina development was assessed by measuring morphometric data at the larval, prepupal, and pupal stages. The results demonstrated a significant increase in morphological characteristics (weight, length, and width) in all the groups treated with the drug, when compared to negative and/or positive control groups. Furthermore, the drug caused a noticeable rise in protein carbonyl levels, indicating oxidative damage in the treated groups compared to the control groups. Clonazepam also substantially increased the DPPH inhibition percentage when compared to the negative control. HPLC-MS successfully detected a significant elevation in clonazepam concentration in all the treated L. cuprina specimens. Furthermore, the empty pupariam of L. cuprina has proven its validity to detect the drug.