This study aimed to investigate the detection of morphine in fingernails from forensic autopsies using immunohistochemistry (IHC), with confirmation by ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS). A primary antibody specific to morphine and a secondary antibody conjugated to horseradish peroxidase (HRP) was used. IHC on specimens of Subjects A and B (both drug addicts) resulted in the detection of morphine on a cell layer of the nail plate matrix. UHPLC-HRMS and GC-MS analysis showed that Subject A had a morphine concentration of 0.35 ng/mg in the fingernail and 472 ng/mL in the blood, while Subject B reached 1.23 ng/mg in the fingernail and 360 ng/ml in the blood. Most of those matrices were positive for codeine, methadone, EDDP, and 6-MAM. The use of IHC in Subject C (a former addict) showed no positivity for morphine in the fingernail, while the UHPLC-HRMS analysis confirmed its absence in the fingernail and blood. Additionally, an analysis of the scalp or pubic hair of the subjects was carried out using UHPLC-HRMS. The results suggest that IHC can be used to establish the site of accumulation of morphine in the nail matrix; for postmortem diagnosis; and that basic substances can be detected by UHPLC-HRMS. There are no previous studies on the use of IHC as a technique for forensic purposes in unconventional matrices, such as nails.

Polydrug use is a frequent pattern of consumption in Europe. This behavior has mainly been analyzed within restricted groups; more rarely in large populations. Current polydrug use is less studied than simultaneous use. This study focused on the concurrent assumption of polydrug among drivers using hair matrix. Hair matrix, for its biological characteristics, allows to identify illicit drug use more often than other matrices, i.e., urine, and it provides information on the long-term use of them. Hair samples of subjects positive for opiates, cocaine and delta-9-tetrahydrocannabinol (Δ9-THC) collected by the forensic toxicology laboratory of the University of Macerata in the period 2010–2020, were analyzed using a gas chromatography-mass spectrometry method. Our results evidenced that a significant part of the examined population (12.15%) used polydrug. A strong predominance of males over females was evident. Polydrug users were more frequently young people. The abuse of two substances was predominant. Cocaine and Δ9-THC was the most common combination, followed by cocaine and morphine, and morphine and Δ9-THC. The timeframe of polydrug use was also analyzed. Our study shows that polydrug use is a very frequent behavior, and that hair analysis may be a powerful tool to obtain objective biological information of this complex phenomenon.

Keratinized matrices, including nails, are among the most resistant matrices that can be analyzed in cases where remains are deeply decomposed and relatively non -invasive for living people. In order to exploit these new matrices in the search for exogenous substances, it is necessary to develop analytical technologies capable of achieving high levels of sensitivity. In this technical note, an easy method is presented for the simultaneous extraction and quantification of three narcotic substances (morphine, codeine, methadone), two benzodiazepines (BDZ) (clonazepam and alprazolam) and an antipsychotic (quetiapine) from nail matrix by analysis in Ultra High-Performance Liquid Chromatography at High Resolution Mass Spectrometry (HPLC/HR-MS). The method has been validated following the Standard Practices for Method Validation in Forensic Toxicology of the Scientific Working Group for Forensic Toxicology. Nail specimens from eight authentic postmortem cases and thirteen living donor samples were extracted and analyzed. Of the eight postmortem samples, five resulted positive for at least one of the three substances searched. Ten of the thirteen living donor specimens were positive for at least one of the targeted benzodiazepines or quetiapine.

Insects on corpses could be a useful tool for the detection of exogenous substances such as drugs of abuse. The identification of exogenous substances in carrion insects is critical for proper estimation of the postmortem interval. It also provides information about the deceased person that may prove useful for forensic purposes. High-performance liquid chromatography coupled with Fourier transform mass spectrometry is a highly sensitive analytical technique that can identify substances even at very low concentrations, such as in the case of searching for exogenous substances in larvae. In this paper, a method is proposed for the identification of morphine, codeine, methadone, 6-monoacetylmorphine (6-MAM) and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in the larvae of Lucilia sericata, a common carrion fly widely distributed in temperate areas of the world. The larvae, which were reared on a pig meat substrate, were killed once they reached their third stage by immersion in hot water at 80 °C and aliquoted into 400 mg samples. The samples were fortified with 5 ng of morphine, methadone and codeine. After solid-phase extraction, the samples were processed with a liquid chromatograph coupled to a Fourier transform mass spectrometer. This qualitative method has been validated and tested on larvae from a real case. The results lead to the correct identification of morphine, codeine, methadone and their metabolites. This method could prove useful in cases where toxicological analysis must be conducted on highly decomposed human remains, where biological matrices are very limited. Furthermore, it could help the forensic pathologist to better estimate the time of death, as the growth cycle of carrion insects can undergo changes if exogenous substances are taken.