Detection of Morphine and Opioids in Fingernails: Immunohistochemical Analysis and Confirmation with Ultra-High-Performance Liquid Chromatography Coupled with High-Resolution Mass Spectrometry

Abstract

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.

Keywords: fingernails; forensic toxicology; high-resolution mass spectrometry; immunohistochemistry; morphine; opioids; ultra-high-performance liquid chromatography; unconventional matrices.

Figure 1

Immunoperoxidase staining for Sheep Anti-Morphine in the fingernail of Subject A. FigPicture (a) shows the 4× magnification, while picture (b) shows the 10× magnification. The figure shows the cytoplasmic staining of nail matrix cells of Subject A, indicating morphine positivity, shown in particular by a black arrow on the 10× picture.

Figure 2

Immunoperoxidase staining for Sheep Anti-Morphine in the fingernail of Subject B, 10× magnification; the picture shows the peroxidase reaction across the nail matrix cells of Subject B, indicating morphine positivity, shown in particular by a black arrow.

Figure 3

Immunoperoxidase staining for Sheep Anti-Morphine in the fingernail of Subject A, 10× magnification. The immunohistochemistry protocol has been modified to observe the presence of any non-specific reaction; in this figure the primary antibody has been used, while the secondary antibody conjugated to the peroxidase enzyme has not been used.

Figure 4

Immunoperoxidase staining for Sheep Anti-Morphine in the fingernail of Subject A, 10× magnification. The immunohistochemistry protocol has been modified to observe the presence of any non-specific reaction; in this figure the primary antibody has not been used, while the secondary antibody conjugated to the peroxidase enzyme has been used.

Figure 5

Immunoperoxidase staining for Sheep Anti-Morphine in the fingernail of Subject C, 10× magnification. The pictures show an absence of positivity for morphine in the nail matrix cells. Counterstaining with hematoxylin. Primary Ab: Polyclonal anti-Morphine Antibody produced in Sheep, dil. 1:100. Secondary Ab: Monoclonal Anti-Goat/Sheep IgG-Peroxidase Antibody produced in Mouse, dil. 1:100, original magnification: 10×.

Figure 6

Immunoperoxidase staining for Sheep Anti-Morphine in the fingernail of Subject C, 10× magnification. The pictures show an absence of positivity for morphine in the nail matrix cells. Counterstaining with hematoxylin. Primary Ab: Polyclonal anti-Morphine Antibody produced in Sheep, dil. 1:100. Secondary Ab: Monoclonal Anti-Goat/Sheep IgG-Peroxidase Antibody produced in Mouse, dil. 1:100, original magnification: 10×.