An Experimental Rib Pig Model to Verify Postmortem Xenobiotics Recovery and Bone Conditions After Controlled Aging Experimental Rib Pig Model to Verify Postmortem Xenobiotics Recovery and Bone Conditions After Controlled

In forensic practice the unavailability of traditional biological samples for toxico logical examinations, such as in decomposed body, is not rare. The continuous scientific research of possible alternative matrices has identified the bone tissue as a substrate usable for the toxicological purpose. Particularly, rib bone marrow (BM) seems to be an optimal specimen for xenobiotics recovery. The aim of the present study was a suit ability assessment of rib BM to detect acute and previous xenobiotics consumption. Rib segments of about 5 g were spiked with antidepressant (Amitriptyline, Citalopram, Venlafaxine and Sertraline) and antipsychotic (Haloperidol, Chlorpromazine, Clotiapine, Methotrimeprazine, Promazine) at the concentration levels of 200 and 500 ng/g. Pig ribs were examined at time 0 and after 15, 30, 60 and 90 days to verify the persistency of the substances. Histological study to verify any structural alterations in pig rib morphol ogy after extraction procedure were carried out both fresh and following incubation at 25°C for 3 months. All the substances were still measurable after three months aging. Only haloperidol was no longer detectable at T 90 . No significant recovery differences were noted between ribs fortified with 200 or 500 ng/g, both for antidepressants and antipsychotics, at the other sampling times. No structural alterations in pig rib morphology neither after leaching nor after the aging process (25°C for 3 months) were found. There is limited scientific evidence to show that bone/BM drug concentrations reflect those of blood at time of death, yet. However, bone/BM drug detection is helpful in forensic investigation to give information on xenobiotics intake.

The aim of the present study was a suitability assessment of rib BM to detect acute and previous xenobiotics consumption.
To this end, we performed analyses on pig ribs fortified with antidepressant and antipsychotic mix concentrations. The pig ribs were examined by leaching both while fresh and following incubation at 25°C (to mimic a postmortem corruption). Specifically, pig ribs were examined at time 0 and after 15, 30, 60 and 90 days of aging to verify the recovery of the substances. The pig ribs, both fresh and following incubation at 25°C for 3 months, underwent histological study to evaluate their conditions after leaching and aging. the promising results suggest that rib bone tissue could be a usable substrate for toxicological detection of xenobiotics when conventional matrices are not available.

Samples
The study was performed on pig ribs fortified with antidepressant (Amitriptyline, Citalopram, Venlafaxine and Sertraline) and antipsychotic (Haloperidol, Chlorpromazine, Clotiapine, Methotrimeprazine, Promazine). Two different mix concentrations (200 and 500 ng/g) were used. Groups of seven pig ribs for each experimental concentration were examined both fresh and following incubation at 25°C for 3 months (to mimic a post mortem corruption).

Extraction Procedure
The extraction was carried out on rib segments of about 5 g.
Each rib was cleaned of any remains of muscles and connective tissues by sharp dissection. The ribs were then washed with deionized water to remove external contaminations and left to dry in the air. Each sample was spiked with 500 ng/g of bupivacaine as internal standard. The extraction was performed by leaching process using absolute ethanol + 1% ammonia solution. The eluate was acidified with HCl (37%) to pH 5-6 and then evaporated to dryness by rotavapor. The residue was reconstituted in 8 mL of chlorobutane, and sodium borate buffer (pH 11.5) until pH 9 was achieved. After centrifugation at 3000 rpm for 5 min, the organic phase was transferred in a vial, while the aqueous phase was

Histological Evaluation
Collected bone fragments, both fresh and following incubation at 25°C for 3 months, were fixed in 4% formalin and then decalcified in EDTA (ethylene-diamin-tetra-acetate) for 36 hours.
After decalcification samples were processed for routine paraffin inclusion. Transverse sections, 6μm thick, were cut and stained with haematoxylin-eosin staining.

Histological Evaluation
The results of the histological evaluation are illustrated in Figures  bacteria, animals and plants [2]. , However, the marrow also has an extensive blood supply and its great vascularization and lipid-rich matrix allow the xenobiotics to enter it.
The nutritive artery provides the supply of blood, and its capillaries make a system of sinusoids inside the medullary cavity  [1,34]. According with other authors, our data show that ribs BM is an excellent analytical sample for xenobiotics recovery [2,13,14,25,26,30,31].
In fact, a good recovery of all drugs has been obtained, although with small individual differences. A higher recovery rate was observed in the ribs treated with 500 ng/g, as showed in Tables 1 &   2, soon after fortification (T0). No significant recovery differences were noted between ribs fortified with 200 or 500 ng/g, both for antidepressants and antipsychotics, at the other sampling times.
Among antidepressants, the best recovery was observed for Venlaxfaxine, followed by Citalopram and Amitriptyline; Sertraline showed the lowest recovery.
Among antipsychotics, the best recovery was observed for Clotiapine and Haloperidol; for Chlorpromazine, Methotrimeprazine and Promazine a lower recovery was achieved. All the substances were still measurable after three months aging. Only haloperidol was no longer detectable at T90. The extraction method used in the study reported here did not damage bone tissues as demonstrated by histology. In fact, no structural alterations in pig rib morphology neither after leaching nor after the aging process (25°C for 3 months) were found.

Conclusion
At the current state of knowledge, there is limited scientific evidence to show that bone/BM drug concentrations reflect those of blood at time of death. However, bone/BM drug detection is helpful in forensic investigation because it may reveal intake before death. Our study has been able to quantify, in ribs, nine substances of toxicological interest three months after aging. Further experimental data and validated analytical assays are necessary to make qualitative and quantitative determination in human BM samples.