Use of the Randox Evidence Investigator immunoassay system for near-body drug screening during post-mortem examination in 261 forensic cases.

BACKGROUND: This paper describes the performance of four Randox drug arrays, designed for whole blood, for the near-body analysis of drugs in a range of post-mortem body specimens. METHODS: Liver, psoas muscle, femoral blood, vitreous humor and urine from 261 post-mortem cases were screened in the mortuary and results were obtained within the time taken to complete a post-mortem. Specimens were screened for the presence of amfetamine, barbiturates, benzodiazepines, benzoylecgonine, buprenorphine, cannabinoids, dextropropoxyphene, fentanyl, ketamine, lysergide, methadone, metamfetamine, methaqualone, 3,4-methylenedioxymetamfetamine, opioids, paracetamol, phencyclidine, salicylate, salicylic acid, zaleplon, zopiclone and zolpidem using the DOA I, DOA I+, DOA II and Custom arrays. RESULTS: Liver and muscle specimens were obtained from each of the 261 post-mortem cases; femoral blood, vitreous humor and urine were available in 98%, 92% and 72% of the cases, respectively. As such, the equivalent of 12,978 individual drug-specific, or drug-group, immunoassay tests were undertaken. Overall >98% of the 12,978 screening tests undertaken agreed with laboratory confirmatory tests performed on femoral blood. CONCLUSIONS: There is growing interest in the development of non-invasive procedures for determining the cause of death using MRI and CT scanning however these procedures are, in most cases, unable to determine whether death may have been associated with drug use. The Randox arrays can provide qualitative and semi-quantitative results in a mortuary environment enabling pathologists to decide whether to remove specimens from the body and submit them for laboratory analysis. Analysis can be undertaken on a range of autopsy specimens which is particularly useful when conventional specimens such as blood are unavailable.

Four fatalities involving 5-IT.

The new designer drug 5-(2-aminopropyl)indole (5-IT) is an indole derivative with stimulant properties. Its synthesis was first described by Albert Hofmann and Franz Troxler in 1962. We report four deaths associated with 5-IT and a validated high-performance liquid chromatography method for quantitation of the drug. In all four deaths, an autopsy was performed, and femoral venous blood, heart blood, urine and vitreous humor were submitted for toxicological analysis. The blood specimens were subjected to comprehensive testing that included alcohol analysis by headspace gas chromatography (GC-FID), acidic/neutral, basic drug and opiates screening by liquid chromatography-mass spectrometry (LC-MS-MS), and acidic/neutral, basic and acidic drugs screening by HPLC. In Case 1, a 25-year-old male, 3,4-methylenedioxymethamphetamine (MDMA; <0.08 mg/L) and 5-IT (preserved femoral blood 1.2 mg/L; unpreserved femoral blood 0.8 mg/L; cardiac blood 1.2 mg/L; vitreous 0.8 mg/L and urine >10 mg/L) were detected, and death was attributed to the toxic effects of 5-IT. In Case 2, a 25-year-old female, 3,4-methylenedioxy-N-methylcathinone (methylone, not quantitated), 6-(2-aminopropyl)benzofuran (6-APB; femoral blood <0.08 mg/L) and 5-IT (preserved femoral blood 1.0 mg/L; unpreserved femoral blood 0.9 mg/L; cardiac blood 2.6 mg/L; vitreous 1.4 mg/L and urine >10 mg/L) were detected, and death was attributed to the toxic 'cocktail effects' of the drugs. In Case 3, a 22-year-old male with a history of epilepsy, 5-IT (0.5 mg/L femoral blood) and 6-APB (0.2 mg/L femoral blood) were detected, and death was attributed to the toxic effects of the drugs, with the role of epilepsy being indeterminate. In Case 4, a 25-year-old female, 5-IT (0.4 mg/L femoral blood), amphetamine (0.4 mg/L femoral blood), MDMA (1.5 mg/L femoral blood), 4-methyl-N-ethylcathione, 3,4-methylenedioxyamphetamine HCl (MDA), benzylpiperazine and 6-APB were detected, and death was attributed to the 'cocktail effect' of the drugs.

Stability of 3,4-methylenedioxymethampetamine (MDMA), 4-methylmethcathinone (mephedrone) and 3-trifluromethylphenylpiperazine (3-TFMPP) in formalin solution.

Occasionally, the only postmortem samples available for analysis are contaminated with formaldehyde, either due to embalming prior to sampling or because analysis is carried out only when formalin-fixed tissues retained for histological study are available. Formaldehyde reacts with several drugs of forensic interest that contain either a primary or a secondary amine group to form their N-methyl derivatives. We investigated the stability of 3,4-methylenedioxymethampetamine (MDMA), 4-methylmethcathinone (mephedrone) and 3-trifluromethylphenylpiperazine (3-TFMPP) in formalin solutions using three different formaldehyde concentrations (5, 10 and 20%) and three different pHs (3.0, 7.0 and 9.5). Analysis was performed using high-performance liquid chromatography with diode array detection to determine the percentage degradation of each drug over time, up to 60 days. MDMA, mephedrone and 3-TFMPP are unstable in formalin solutions, with the degradation rate increasing with increasing pH. After 28 days in 20% formalin, pH 9.5, there remained 57% of the initial 3-TFMPP concentration, 11% of the initial MDMA concentration and 4% of the initial mephedrone concentration. Forensic toxicologists should be aware that, when analyzing for these drugs in an embalmed body or in tissues stored in formalin solutions, the methylated form of the secondary amine-containing drug could be a more useful analyte than the parent drug.

From the street to the laboratory: analytical profiles of methoxetamine, 3-methoxyeticyclidine and 3-methoxyphencyclidine and their determination in three biological matrices.

Three psychoactive arylcyclohexylamines, advertised as "research chemicals," were obtained from an online retailer and characterized by gas chromatography ion trap electron and chemical ionization mass spectrometry, nuclear magnetic resonance spectroscopy and diode array detection. The three phencyclidines were identified as 2-(ethylamino)-2-(3-methoxyphenyl)cyclohexanone (methoxetamine), N-ethyl-1-(3-methoxyphenyl)cyclohexanamine and 1-[1-(3-methoxyphenyl)cyclohexyl]piperidine. A qualitative/quantitative method of analysis was developed and validated using liquid chromatography (HPLC) electrospray tandem mass spectrometry and ultraviolet (UV) detection for the determination of these compounds in blood, urine and vitreous humor. HPLC-UV proved to be a robust, accurate and precise method for the qualitative and quantitative analysis of these substances in biological fluids (0.16-5.0 mg/L), whereas the mass spectrometer was useful as a confirmatory tool.

Distribution of opiates in femoral blood and vitreous humour in heroin/morphine-related deaths.

The distribution of free morphine (FM), codeine and 6-acetylmorphine (6AM) in vitreous humour (VH) and femoral blood (FB) was measured in 70 cases involving heroin/morphine. The relationship between tissue drug concentrations was assessed with respect to case circumstances. Total morphine (TM) concentrations in FB are also reported. The relative concentrations of FM in VH and FB were influenced by survival time. In rapid deaths (<3h after drug intake; n=34) the median FM concentration in VH (0.13 mg/L) was significantly lower than the corresponding result for FB (0.25mg/L; p<.01). In delayed deaths (>3h; n=12) the VH concentration (median 0.15 mg/L) was higher than in FB (0.092 mg/L; p>.05). Free morphine VH/FB ratios were significantly higher in delayed (median 1.3) compared to rapid deaths (0.64). Although these findings indicate a lag in the distribution of morphine into the VH, overlaps were observed in the VH/FB ratio in rapid and delayed death groups which limits the interpretive use of VH/FB ratios. Codeine and 6AM appeared to distribute more rapidly into the VH. Despite the observation that all opiate analytes were correlated between FB and VH (r ≥ 61; p<.01), our results indicate that in the absence of a blood sample, blood concentrations cannot be reliably inferred from that measured in the VH. In the absence of additional toxicological evidence, the use of FM to TM ratios in blood as an indicator of survival time is not advised.

Analysis of skeletal muscle has potential value in the assessment of cocaine-related deaths.

This study assesses the interpretive value of cocaine, benzoylecgonine (BZE) and cocaethylene (COET) in skeletal muscle (rectus femoris) in cocaine-using decedents. The distribution of these analytes in cardiac muscle (CM), vitreous humour (VH), femoral blood (FB) and cardiac blood (CB) is also reported. In rectus femoris muscle, the spatial distribution of the analytes was examined across the whole rectus femoris muscle collected from seven fatalities in which cocaine was detected. In six of these cases, death was attributed to trauma and in one case the cause of death was undetermined but suspected to be drug related. In two additional cases analytes were detected in the blood and/or VH but not in the muscle. The muscle was sectioned into 12-15 approximately equal segments, each of which was analysed after homogenisation. Tissue and bio-fluid samples were extracted by solid phase extraction with confirmation and quantification by GC-ion trap-MS/MS. No significant variation was observed in the concentration of any analyte throughout the muscle in the 7 cases analysed. The results reported here are in contrast to a previous study in which great variation in the concentration of some basic drugs (mainly tricyclic antidepressants and benzodiazepines) was observed throughout the thigh muscle bulk (Williams and Pounder, 1997). Analyte concentrations in skeletal muscle (SM) correlated well with those in FB (p<0.01). In general, the concentration of cocaine and COET followed the order VH > CM > SM > FB ≥ CB. Cocaine concentrations measured in VH were significantly higher than in blood and muscle. Inter-matrix variations in the concentrations of BZE and COET were less marked. The concentration of BZE exceeded that of cocaine in all matrices and in all cases except one where the time between death and drug intake was suspected to be short. In this case, the cocaine to BZE ratio measured in SM (2.66), CM (2.91) and VH (2.19) was higher than that measured in FB (0.97). Given that the concentrations of cocaine and its metabolites were uniformly distributed throughout the muscle and considering the good correlation observed between muscle and blood, muscle could be of interpretive value in cocaine related deaths. Further, since cocaine is known to have greater post-mortem stability in muscle than blood, concentrations measured in muscle may reflect more closely those at the time of death and might be of particular value in cases with an extended period between death and tissue sampling.

Phenazepam: the drug that came in from the cold.

In the past few years there has been concern in Western Europe and in the US about the rise in abuse of phenazepam, a benzodiazepine that was originally developed in the USSR in the mid- to late 1970s.(1-4) Although phenazepam is one of the most widely prescribed benzodiazepines in Russia and other commonwealth of independent state (CIS) countries, it has not been licensed elsewhere in the world. Due to very limited licensed geographical distribution, there is very little peer-reviewed literature that is not written in Russian. In this article, we review the current state of what is currently known about phenazepam. This information on phenazepam and how it can be detected in biological specimens should assist the forensic community in identifying phenazepam in routine toxicology screening and interpreting any phenazepam concentrations that are obtained.

Meptazinol and ethanol: a fatal intoxication.

Meptazinol (Meptid(®)) is an analgesic drug that is used to treat mild to moderate pain including postoperative pain, obstetrical pain, and the pain of renal colic. This case reports a death due to the combined effects of meptazinol and alcohol in a man with significant heart disease and alcoholic liver disease. A 57-year-old male was found unresponsive in his bed at home with empty blister packets of meptazinol around him. A general drug screen detected the presence of meptazinol, and caffeine and metabolites, in cardiac blood. Analysis, both quantitative (HPLC-DAD) and qualitative (HPLC-DAD, LC-MS), of meptazinol was carried out. Meptazinol was found at the following concentrations: 15.5 mg/L in unpreserved femoral blood; 18.6 mg/L in preserved (fluoride-oxalate) femoral blood; 52.1 mg/L in unpreserved cardiac blood; 16.8 mg/L in preserved vitreous; 61.7 mg/L in unpreserved urine; and 9.8 g/L in stomach contents. Ethanol, analyzed by headspace GC-FID, was present in preserved (fluoride-oxalate) femoral venous blood, urine, and vitreous at concentrations of 232 mg/100 mL, 297 mg/100 mL, and 192 mg/100 mL, respectively. Death was attributed to meptazinol and ethanol toxicity, with atherosclerotic coronary artery disease as a contributing factor.

Analytical characterization and rapid determination of 2-(diphenylmethyl)pyrrolidine in blood and application to an internet product.

The increased availability of new psychoactive substances ("legal highs") from retail shops or internet sources has caught the imagination of consumers, law enforcement and scientific communities. The present study describes the identification of 2-(diphenylmethyl)pyrrolidine (DPMP, desoxy-D2PM) as the key constituent found in an internet product called "A3A New Generation". Analytical characterization of this new generation "legal high" product was based on gas chromatography (EI/CI) ion trap mass spectrometry and liquid chromatography (HPLC) using electrospray ionization tandem mass spectrometry and diode array detection. A quantitative method was also developed and validated for the detection of DPMP in human whole blood using HPLC single wavelength ultraviolet detection (210 nm). Evaluation of standard method validation parameters was found to be satisfactory. The circulation of DPMP on the market is another example of psychoactive substances described decades ago in the patent literature which are beginning to be rediscovered by recreational drug communities. The ability to unambiguously identify and detect this psychoactive compound should therefore be of interest to those who are exposed to the recreational drugs field.

Virtual casting of stab wounds in cartilage using micro-computed tomography.

In homicidal stabbings using a serrated knife, stab wounds involving costal cartilage leave striations on the cut surface of the cartilage from the serration points on the blade edge. Class characteristics of the serrated blade can be determined from the striation marks, and individualizing characteristics may be seen also. The traditional method for recording the striation marks involves the pernickety technique of casting with dental impression material. We assessed the potential utility of micro-computed tomography scanning of the stab track as a technique for nondestructive recording of striation patterns and virtual casting of them. Stab tracks in porcine cartilage, produced with a coarsely serrated blade, were scanned with a bench-top micro-computed tomography scanner. The typical irregularly regular striation pattern could be demonstrated, and the images manipulated, using proprietary software to produce a virtual cast. Whether the technology will have sufficient resolution to image not only class characteristic tool marks but also the much finer individualizing tool marks remains to be evaluated, but the technology shows considerable promise.

An experimental model of tool mark striations in soft tissues produced by serrated blades.

Stab wounds produced by serrated blades are generally indistinguishable from stab wounds produced by non-serrated blades, except when visible tool mark striations are left on severed cartilage. We explored the possibility that similar striations may be produced on the soft tissues of internal organs. Loin of beef, bovine kidney, and pig heart, liver, and aorta were each stabbed 20 times with a coarsely serrated blade. The walls of the stab tracks were exposed and documented by photography, cast with dental impression material, and the casts photographed. Striations were identified in all of the tissues in every stabbing, but their consistency and quality varied between tissues. Striations were most easily seen in liver, heart, and aorta. Tool mark striations in soft tissues other than cartilage have not been described in homicidal stabbings, likely because they have not been sought. We suggest that the walls of stab wound tracks should be exposed, and tissue striations should be sought as a means of identifying the weapon as having a serrated blade.

Tool mark striations in pig skin produced by stabs from a serrated blade.

Stab wounds produced by serrated blades are generally indistinguishable from stab wounds produced by non-serrated blades, except when visible tool mark striations are left on severed cartilage. Using a pig-skin experimental model, we explored the possibility that similar striations may be left in skin. Stabs into pig skin were made using a straight spine coarsely serrated blade (121), a drop point finely serrated blade (20), a clip point irregular coarsely serrated blade (20), a drop point coarsely serrated blade (15), and as controls 2 non-serrated blades (40). Tool mark striations could be seen on the skin wall of the stab canal in all stabs made using serrated blades but in none with non-serrated blades.The striation pattern, reflecting the class characteristics of the serrated blade, was the same as that described in cartilage but less well defined. Fixation of the specimen with Carnoy's solution best preserved visible striations, and fixation with formaldehyde after staining with 5% Neutral Red was also satisfactory. Casting with vinyl polysiloxane dental impression material greatly facilitated photo-documentation. Applying the technique to homicidal stabbings may help identify stab wounds produced with serrated blades.

Endogenous γ-hydroxybutyric acid concentrations in saliva determined by gas chromatography-mass spectrometry.

Endogenous γ-hydroxybutyric acid (GHB) concentrations in blood and urine are well documented, but there are very little data on natural levels in saliva, a biological matrix increasingly used for drug testing. We measured endogenous GHB concentrations in 120 unpaid volunteers who also provided anonymous epidemiological data. Samples were analyzed using a rapid and reliable method, utilizing liquid-liquid extraction, silyl-derivatization, and gas chromatographic-mass spectrometric analysis. One sample, between the lower limit of quantitation (0.2 mg/L) and limit of detection (0.1 mg/L), was split to 0.15 mg/L for statistical purposes. Salivary GHB concentrations ranged from 0.15 to 3.33 mg/L (mean = 1.29; median = 1.13). Statistical analysis using the Mann-Whitney test indicated that endogenous GHB concentrations in saliva were not significantly affected by age, gender, medical conditions, use of medications, and recent food/drink consumption. Interpreting GHB concentrations in biological samples poses difficulties because of its endogenous presence and rapid elimination, and this is true for saliva as well as blood and urine. However, saliva has the merit of being easy to collect by law enforcement personnel.

Mephedrone (4-methylmethcathinone)-related deaths.

Four deaths related to the drug 4-methylmethcathinone (mephedrone) are reported. Qualitative and quantitative analysis of mephedrone was performed by high-performance liquid chromatography-diode-array detection. Of the four deaths, one was attributed to the adverse effects of mephedrone, with cardiac fibrosis and atherosclerotic coronary artery disease as a contributing factor. A 49-year-old female insufflated mephedrone; analysis disclosed mephedrone in femoral venous blood (0.98 mg/L). The second death was attributed solely to mephedrone. A 19-year-old male took mephedrone as well as alcohol and "ecstasy"; analysis disclosed mephedrone (2.24 mg/L femoral venous blood) and 3-trifluoromethylphenylpiperazine (3-TFMPP). In the third fatality, a 55-year-old female was found dead in bed; the death was attributed to the combined effects of mephedrone and methadone. Analysis of femoral venous blood revealed the prescribed drugs diazepam, nordiazepam, olazepine, and chlorpromazine metabolites together with methadone (0.3 mg/L) and mephedrone (0.13 mg/L). In the fourth case, a 17-year-old male car driver was involved in a vehicular collision and died of multiple blunt force injuries. Analysis revealed mephedrone in femoral venous blood (0.24 mg/L).

How can we reduce the number of coroner autopsies? Lessons from Scotland and the Dundee initiative.

Currently the medicolegal dissection rate for England and Wales is 22% (110,000 coronial autopsies for 500,000 deaths per annum), yet there is a general lack of evidence about the utility of and justification for such a high level of activity, which is between double and triple the rate in other jurisdictions. The government is currently consulting on how to reduce the numbers, and the Coroners and Justice Act 2009 is permissive of external examinations as an alternative to dissections. We describe the philosophy and practice of the Scottish system of postmortem external examinations, and the 20-year experience of a local initiative to maximize use of such external examinations. Currently our regional medicolegal dissection rate is 6%, which if applied to England and Wales would reduce the number of dissections from 110,000 to 30,000 per annum, with all of the social, resource and management implications. While the autopsy is an important tool in modern death investigation, an almost automatic recourse to it is inappropriate. In our view external examinations are not only cost-effective but also a necessary element in any death investigative system which wishes to strike an appropriate balance between intrusion by the state and the rights of the bereaved.