Α 22-year-old obese man was found dead in his room in early autumn. His body was discovered on the floor beside his desk, in a prone position, with blood exiting his nose and dried bloodstains present on his left wrist. Many empty packets of medications were scattered across the floor, on the desk and in a bin, close to the location of the body (Fig. 1). These packets were identified as his prescribed medications, and included paroxetine, quetiapine and alprazolam. The deceased had a history of major depression and panic disorder and was undergoing treatment with these medications. No prior suicide attempts were reported, and no suicidal note was found at the scene or among his belongings. On the outer part of his desk there was a razor blade with a small amount of blood on it. During the previous month, he had been hospitalized for a panic crisis and was discharged one week prior to his body discovery. He was last seen alive by his parents two days prior to his discovery. At the scene, postmortem lividity was observed as confluent on the front side of his face and body, with multiple petechiae in the lividity-dependent areas. Rigor mortis was present throughout the body and the time of death was estimated 18–24 h prior to discovery.

Empty packets of medications scattered on the desk in decedent’s room. A razor was also present at the lower edge of the desk

The body was transferred to the mortuary of the Department of Forensic Medicine and Toxicology, Faculty of Medicine, University of Ioannina, Greece, eight hours after discovery and was preserved in the mortuary refrigeration chamber at 2–40 ºC until autopsy, which was performed 12 h post-transfer. During the postmortem examination, as could be seen in Fig. 2, multiple (more than ten) fresh, linear, superficial incisions, of 10 cm maximum length, were observed on the inner surface of both his lower forearms and wrists (more on his left wrist– of note, the decedent was right-handed according to the police information), running obliquely and towards different directions, suggesting hesitation marks caused by a razor blade. No other injuries or lesions were noted.

(a, b): Cutmarks/superficial incisions on the inner surface of left (a) and right (b) forearms and wrists, respectively (more on the left wrist– right-handed person), running towards different directions, recognized as hesitation marks, corresponding to sharp force injury most likely caused by the razor in Fig. 1

On internal examination, cerebral edema, lung congestion and pulmonary edema were noted. His heart weighed 450 g and presented no anatomical abnormalities, signs of atherosclerosis or myocardial fibrosis. His stomach contained 250 mL of partially digested food mixed with semi-liquid fluids, and gastric mucosa showed early putrefactive changes. The urinary bladder was markedly distended, containing 600 mL clear urine. The prostate gland was normal both in size and composition. All organs showed early putrefactive changes, with no other macroscopic pathological lesions present.

Histological examination was not performed, as the evident putrefactive processes, would yield only vague findings on histology. Blood samples (peripheral blood from the left external iliac vein), along with urine and gastric contents samples were proceeded for toxicological analysis.

The autopsy blood specimen was collected in a 2 mL tube, BD Vacutainer® (Becton Dickinson) with presence of anticoagulant and preservative (sodium fluoride 5 mg and potassium oxalate 4 mg). The urine and stomach contents specimens were stored in 120 mL plastic sterile containers (Medimel P.P.C). All specimens were maintained at 4 °C up to two weeks after sample collection when all analyses were completed and then were stored at −20 °C up to six months (in case that re-analyses were requested).

Ethanol and volatiles analysis were performed in whole blood by head space gas chromatography flame ionization (HS-GC-FID) detection [19]. Screening for opiates, benzodiazepines, amphetamines, cannabinoids and cocaine metabolites, in urine and blood, was performed by immunoassays (SYVA, Abbott Park, IL). Toxicological screening for the presence of common drugs and poisons was carried out by a routine gas chromatography–mass spectrometry (GC–MS) technique in full-scan mode on whole blood and urine extracts, following solid phase extraction (Chem Elut cartridges, Agilent Technologies, Lake Forest, CA, USA). The mass spectral libraries used for identification of compounds were the NIST27 and PMW-tox3. GC–MS analyses were performed using a Shimadzu GC17A-QP5050 GC–MS instrument equipped with an Equity5 column (30 m × 25 mm id), 95% dimethyl-5% diphenylpolysiloxane, film thickness 0.25 μm, purchased from Supelco (Bellefonte, PA). Helium was employed as the carrier gas with a constant flow rate at 1.5 mL/min. For the screening analysis the GC oven temperature was programmed to rise from 60° to 280 °C using a step-temperature program and the total run time was 36.17 min. The GC injector and transfer line were maintained at 260 °C and the injector was operated in the split less mode. Full scan spectra were acquired in the interval 40–550 amu operating in the EI mode at 70 eV. Paroxetine levels in blood were determined by a liquid chromatography–mass spectrometry (LC-MS/MS) method [20], properly modified to apply for urine and gastric contents analysis. Aliquots of 200 µL of whole blood or urine, 200 µL of carbonate buffer (1 M, pH 9.5), and 20 µL of deuterated drugs mixture were extracted with of 1.0 mL methyl tert-butyl ether (MTBE)/0.1 M HCL (37%), stirred, centrifuged and the supernatant was evaporated to dryness with a current of N2, at a temperature of 40 °C. Reconstitution was performed in 50 µL of solvent A: B (88:12%), and samples were transferred to autosampler vials for LC-MS/MS analysis. Analyses were performed in triplicate. Gastric contents of about 1 mL were diluted with 2 mL double distilled water, 2 drops of NaOH, 5 M were added and vortex followed. The mixture was extracted with 3 mL of diethyl ether, vortexed and centrifuged (20 min, 4000 rpm). The supernatant was collected, evaporated and re-dissolved in 50 µL A: B (88:12%), stirred for 30 s and injected into the LC-MS/MS system.

LC-MS/MS instrumentation was a Dionex UltiMate 3000 UHPLC system (Thermo Scientific, Waltham, MA, USA) comprised of a binary pump, coupled to a Q-Trap 5500+™ mass spectrometer (Sciex, Darmstadt, Germany), operated in multiple reaction monitoring (MRM) mode, and equipped with an electrospray ionization (ESI) Turbo V Source in positive mode. The applied ESI inlet conditions were as follows: gas 1, nitrogen (55 psi); gas 2, nitrogen (55 psi); ion-spray voltage of 5500.0 V, positive mode; ion-source temperature, of 550 °C; nitrogen as the curtain gas at 20 psi. Separation was performed on an Accupore C18 column (50 mm × 3 mm, 2.6 μm particle size) equipped with a precolumn cartridge (2.1 mm × 0.2 μm) (Thermo Scientific, Waltham, MA, USA), both operated at 30 °C. Mobile phases were: 10 mM of aqueous ammonium acetate adjusted to pH 3.5 with 0.1% formic acid (eluent A) and acetonitrile UHPLC-MS grade with 0.1% formic acid (eluent B), degassed by Elmasonic S ultrasonic, Germany. The autosampler temperature was 5 °C.