Development of a novel non-destructive method based on spectral fingerprint for determination of abused drug in insects: An alternative entomotoxicology approach Joyce S. Oliveira a , Tainá C. Baia b , Renata A. Gama b , Kássio M.G. Lima a, ⁎ a Universidade Federal do Rio Grande do Norte, Instituto de Química, Programa de Pós-Graduação em Química, Grupo de Pesquisa em Química Biológica e Quimiometria, CEP 59072-970 Natal, RN, Brazil b Universidade Federal do Rio Grande do Norte, Departamento de Microbiologia e Parasitologia, CEP 59072-970 Natal, RN, Brazil abstract article info Article history: Received 30 January 2014 Accepted 10 February 2014 Available online 16 February 2014 Keywords: Near infrared spectroscopy Wavelength selection Flunitrazepam Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae) Near-infrared spectroscopy (NIRS) is emerging as the tool of choice for non-destructive analysis and the detection of different compounds in biological systems. We evaluate the potential of a novel non-destructive method for the identification of flunitrazepam in Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae) larvae, puparia and adult. Necrophagous insects in particular the larvae found in cadavers have been applied as an alternative at the time of death and can also contribute to the qualitative identification of abused drugs present in the corpse. Using this strategy, we have combined a portable NIR spectrometer with variable selection techniques, such as, genetic algorithm–linear discriminant analysis (GA–LDA) and successive projection algorithm (SPA–LDA) for the identification of this drug in the insects based on the unique spectral “fingerprints” of their biochemical composition. Larva, puparium and adult calibrators containing flunitrazepam at concentra- tions ranging from 0, 4, 8, 16 to 32 pg g -1 were prepared and analyzed. The resulting GA–LDA model successfully classified adult female with respect to their concentration using only 9 wavenumbers. This alternative approach for entomotoxicology requires further testing, but the obtained results suggest that NIR spectra could be used for abused drug identification in insects. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Investigations about the use of arthropods (specifically, arachnids, mites, ticks, scorpions, and spiders) as an alternative toxicological ma- trix and the effects of many compounds (drugs, metals and pesticides) on insect physiology are associated on forensic entomotoxicology. Investigations on the use of carrion-feeding arthropods as alternative toxicological specimens, and on the impact the tissue toxins and drugs have on the development of immature insects feeding on these substances, currently comprise the major avenues of exploration in the emerging field of entomotoxicology [1]. The major interest of entomotoxicology is the determination of these compounds just before death, mainly in skeletonized remains where no tissue or fluids are left [2]. Alcohol [3], drug antidepressants [4–7], barbiturates [8,9], benzodiazepines [10–12], opioids [13–15], metals [16,17], and pesticides [18,19] are commonly involved in cases where entomotoxicology is investigated. Basically these studies have detailed the detection of toxic substances in different developmental stages of insects, a comparison of sample preparation and analytical procedures for each toxic substance. In these reports, the recovered ar- thropods have also been generally homogenized and subsequently processed in a manner similar to that for other, more traditional tissues and fluids, or subjected to extraction techniques developed for the analysis of rigorous tissues, such as hair and nails. The determination of abused drugs in insects is usually performed by gas chromatography–mass spectrometry (GC/MS) [3,12] and liquid chromatography–mass spectrometry (LC/MS) [10,14], coupled to classic extraction techniques such as protein precipitation, liquid–liquid extraction (LLE) or solid phase extraction (SPE). The choice of analytical drug detection/quantification procedures for the analysis of insect tissues depends on the physicochemical properties of the drugs of inter- est and the required selectivity and sensibility. Although the SPE- chromatography methods present high sensitivity, they present several inherent drawbacks for the analysis of insect tissues, such as invasive and destructive technique, besides the use of bulky instrumentation that impairs in-field monitoring. Alternatively, the near-infrared spectroscopy (NIRS) can be utilized to determine the insect metabolic fingerprint (lipids, proteins, cellular processes) [20], emerging as an interesting alternative for a rapid and cost-effective identification of living specimens [21–25]. NIRS is also characterized by a minimum of sample handling. It requires no extrac- tions and is non-destructive. The NIR absorption is affected by the Microchemical Journal 115 (2014) 39–46 ⁎ Corresponding author. Tel.: +55 84 3342 2323; fax: +55 83 3211 9224. E-mail address: kassio@ufrnet.br (K.M.G. Lima). http://dx.doi.org/10.1016/j.microc.2014.02.009 0026-265X/© 2014 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Microchemical Journal journal homepage: www.elsevier.com/locate/microc