Analytical toxicology: guidelines for sample collection postmortem

The reliability and relevance of any analytical toxicology result is determined in the first instance by the nature and integrity of the specimen(s) submitted for analysis. This article provides guidelines for sample collection, labelling, transport and storage, especially regarding specimens obtained during a postmortem examination. Blood (5 mL) should be taken from two distinct peripheral sites, preferably left and right femoral veins, taking care not to draw blood from more central vessels. Urine (if available), vitreous humour (separate samples from each eye), a representative portion of stomach contents, and liver (10-20 g, right lobe) are amongst other important specimens. A preservative (sodium fluoride, 0.5-2% weight by volume (w/v) should be added to a portion of the blood sample/the sample from one vein, and to urine. Leave a small (10-20% headspace) in tubes containing liquids if they are likely to be frozen. Precautions to minimise the possibility of cross-contamination of biological specimens must be taken, especially if volatile poison(s) may be involved. If death occurred in hospital, any residual antemortem samples should be sought as a matter of urgency. Hair/nail collection should be considered if chronic exposure is suspected, for example, in deaths possibly related to drug abuse. A lock of hair the width of a pen tied at the root end is required for a comprehensive drug screen. The value of providing as full a clinical/occupational/circumstantial history as possible together with a copy of the postmortem report (when available) and of implementing chain-of-custody procedures when submitting samples for analysis cannot be over-emphasised.     

Interpretation of analytical toxicology results in life and at postmortem

Interpretation of analytical toxicology results from live patients is sometimes difficult. Possible factors may be related to: (i) the nature of the poison(s) present; (ii) sample collection, transport and storage; (iii) the analytical methodology used; (iv) the circumstances of exposure; (v) mechanical factors such as trauma or inhalation of stomach contents; and (vi) pharmacological factors such as tolerance or synergy. In some circumstances, detection of a drug or other poison may suffice to prove exposure. At the other extreme, the interpretation of individual measurements may be simplified by regulation. Examples here include whole blood alcohol (ethanol) in regard to driving a motor vehicle and blood lead assays performed to assess occupational exposure. With pharmaceuticals, the plasma or serum concentrations of drugs and metabolites attained during treatment often provide a basis for the interpretation of quantitative measurements. With illicit drugs, comparative information from casework may be all that is available. Postmortem toxicology is an especially complex area since changes in the composition of fluids such as blood depending on the site of collection from the body and the time elapsed since death, amongst other factors, may influence the result obtained. This review presents information to assist in the interpretation of analytical results, especially regarding postmortem toxicology. Collection and analysis of not only peripheral blood, but also other fluids/tissues is usually important in postmortem work. Alcohol, for example, can be either lost from, or produced in, blood especially if there has been significant trauma, hence measurements in urine or vitreous humour are needed to confirm the reliability of a blood result. Measurement of metabolites may also be valuable in individual cases.     

Microbial degradation products of lurasidone and their significance in postmortem toxicology

Recent research reported that lurasidone degrades in unpreserved ante-mortem human whole blood inoculated with microorganisms known to dominate postmortem blood specimens. In vitro degradation occurred at a similar rate to risperidone, known to degrade in authentic postmortem specimens until below analytical detection limits. To identify the lurasidone degradation products formed, an Agilent 6520 liquid chromatograph quadrupole-time-of-flight mass spectrometer (LC-QTOF-MS) operating in auto-MS/MS mode was used. Numerous degradation products not previously reported in prior in vitro or in vivo pharmacokinetic studies or forced degradation studies were detected. Accurate mass data, mass fragmentation data, acetylation experiments, and a proposed mechanism of degradation analogous to risperidone supports initial identification of the major degradation product as N-debenzisothiazole-lurasidone (calculated m/z [M + H]⁺ = 360.2646). A standard was unavailable to conclusively confirm this identification. Retrospective data analysis of postmortem cases involving lurasidone identified the presence of the major degradation product in four of six cases where lurasidone was also detected. This finding is significant for toxicology laboratories screening for this drug in postmortem casework. The major postmortem lurasidone degradation product has consequently been added to the LC-QTOF-MS drug screen at Forensic Science SA (FSSA) to indicate postmortem lurasidone degradation in authentic postmortem blood specimens and as a marker of lurasidone administration in the event lurasidone is degraded to concentrations below detection limits.     

Evaluation of an onsite alcohol testing device for use in postmortem forensic toxicology

The disposable QED saliva alcohol test provides a very simple, fast, and reliable means for quantitative onsite alcohol detection. The purpose of this study was to determine if the QED test would be a useful tool for the determination of postmortem ethanol levels in cases where a rapid result was needed. QED results were compared with ethanol levels determined by headspace GC analysis. Both saliva and vitreous humor specimens were used for the evaluation. QED tests were initially attempted using the oral fluid from 50 individuals. Of these cases, 17 of the tests were valid with 8 positive results. For 23 cases the oral fluid was not attainable, and for 10 cases, the sample was contaminated with blood making the tests invalid. The correlation between the oral fluid results and the blood headspace GC analysis was poor (r = 0.8345) over the range of 0.01-0.29 g/dL. Vitreous specimens were found to be the matrix of choice for analyzing postmortem cases using the QED. Only 6 of 171 specimens were found to be unsuitable. The QED results correlated well with the headspace GC analysis (r = 0.9931, n = 165). When using ethanol levels > 0.02 g/dL (n = 126), an average vitreous (GC)/blood ratio of 1.16 correlated well with the average QED/blood ratio of 1.22. Although the QED saliva alcohol test does not appear to be useful in determining postmortem saliva ethanol levels, it does provide accurate results when using postmortem vitreous humor as the testing matrix.     

Cannabinoids

The British Pharmacological Society conference commenced with the GC Clark symposium on cannabinoids (CBs), an area that was often represented during the general pharmacology sessions of the conference. Endocannabinoids were identified as playing important physiological roles and the CB receptors appear to offer novel therapeutic targets in a range of clinical conditions. This report concentrates on the CB aspects of the meeting.     

Cannabinoids

Since the discovery of an endogenous cannabinoid system, research into the pharmacology and therapeutic potential of cannabinoids has steadily increased. Two subtypes of G-protein coupled cannabinoid receptors, CB(1) and CB(1), have been cloned and several putative endogenous ligands (endocannabinoids) have been detected during the past 15 years. The main endocannabinoids are arachidonoyl ethanolamide (anandamide) and 2-arachidonoyl glycerol (2-AG), derivatives of arachidonic acid, that are produced "on demand" by cleavage of membrane lipid precursors. Besides phytocannabinoids of the cannabis plant, modulators of the cannabinoid system comprise synthetic agonists and antagonists at the CB receptors and inhibitors of endocannabinoid degradation. Cannabinoid receptors are distributed in the central nervous system and many peripheral tissues, including immune system, reproductive and gastrointestinal tracts, sympathetic ganglia, endocrine glands, arteries, lung and heart. There is evidence for some non-receptor dependent mechanisms of cannabinoids and for endocannabinoid effects mediated by vanilloid receptors. Properties of CB receptor agonists that are of therapeutic interest include analgesia, muscle relaxation, immunosuppression, anti-inflammation, antiallergic effects, improvement of mood, stimulation of appetite, antiemesis, lowering of intraocular pressure, bronchodilation, neuroprotection and antineoplastic effects. The current main focus of clinical research is their efficacy in chronic pain and neurological disorders. CB receptor antagonists are under investigation for medical use in obesity and nicotine addiction. Additional potential was proposed for the treatment of alcohol and heroine dependency, schizophrenia, conditions with lowered blood pressure, Parkinson's disease and memory impairment in Alzheimer's disease.     

Detection of exogenous GHB in blood by gas chromatography-combustion-isotope ratio mass spectrometry: implications in postmortem toxicology

Because GHB (gamma-hydroxybutyrate) is present in both blood and urine of the general population, toxicologists must be able to discriminate between endogenous levels and a concentration resulting from exposure. In this paper, we propose a procedure for the detection of exogenous GHB in blood by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Following liquid-liquid and solid-phase extractions, GHB is derivatized to GHB di-TMS before analysis by GC-C-IRMS. Significant differences in the carbon isotopic ratio (delta(13)C-values > 13.5 per thousand) were found between endogenous and synthetic GHB. Indeed, for postmortem blood samples with different GHB concentrations (range: 13.8-86.3 mg/L), we have obtained GHB delta(13)C-values ranging from -20.6 to -24.7 per thousand, whereas delta(13)C-values for the GHB from police seizure were in the range -38.2 to -50.2 per thousand. In contrast to the use of cut-off concentrations for positive postmortem blood GHB concentrations, this method should provide an unambiguous indication of the drug origin.     

Cannabinoids in clinical practice

Cannabis has a potential for clinical use often obscured by unreliable and purely anecdotal reports. The most important natural cannabinoid is the psychoactive tetrahydrocannabinol (delta9-THC); others include cannabidiol (CBD) and cannabigerol (CBG). Not all the observed effects can be ascribed to THC, and the other constituents may also modulate its action; for example CBD reduces anxiety induced by THC. A standardised extract of the herb may be therefore be more beneficial in practice and clinical trial protocols have been drawn up to assess this. The mechanism of action is still not fully understood, although cannabinoid receptors have been cloned and natural ligands identified. Cannabis is frequently used by patients with multiple sclerosis (MS) for muscle spasm and pain, and in an experimental model of MS low doses of cannabinoids alleviated tremor. Most of the controlled studies have been carried out with THC rather than cannabis herb and so do not mimic the usual clincal situation. Small clinical studies have confirmed the usefulness of THC as an analgesic; CBD and CBG also have analgesic and antiinflammatory effects, indicating that there is scope for developing drugs which do not have the psychoactive properties of THC. Patients taking the synthetic derivative nabilone for neurogenic pain actually preferred cannabis herb and reported that it relieved not only pain but the associated depression and anxiety. Cannabinoids are effective in chemotherapy-induced emesis and nabilone has been licensed for this use for several years. Currently, the synthetic cannabinoid HU211 is undergoing trials as a protective agent after brain trauma. Anecdotal reports of cannabis use include case studies in migraine and Tourette's syndrome, and as a treatment for asthma and glaucoma. Apart from the smoking aspect, the safety profile of cannabis is fairly good. However, adverse reactions include panic or anxiety attacks, which are worse in the elderly and in women, and less likely in children. Although psychosis has been cited as a consequence of cannabis use, an examination of psychiatric hospital admissions found no evidence of this, however, it may exacerbate existing symptoms. The relatively slow elimination from the body of the cannabinoids has safety implications for cognitive tasks, especially driving and operating machinery; although driving impairment with cannabis is only moderate, there is a significant interaction with alcohol. Natural materials are highly variable and multiple components need to be standardised to ensure reproducible effects. Pure natural and synthetic compounds do not have these disadvantages but may not have the overall therapeutic effect of the herb.     

Cannabinoids and pain

Recent advances have dramatically increased our understanding of cannabinoid pharmacology: the psychoactive constituents of Cannabis sativa have been isolated, synthetic cannabinoids described and an endocannabinoid system identified, together with its component receptors, ligands and their biochemistry. Strong laboratory evidence now underwrites anecdotal claims of cannabinoid analgesia in inflammatory and neuropathic pain. Sites of analgesic action have been identified in brain, spinal cord and the periphery, with the latter two presenting attractive targets for divorcing the analgesic and psychotrophic effects of cannabinoids. Clinical trials are now required, but are hindered by a paucity of cannabinoids of suitable bioavailability and therapeutic ratio.     

Cannabinoids and pain

Convincing evidence from preclinical studies demonstrates that cannabinoids can reduce pain responses in a range of inflammatory and neuropathic pain models. The anatomical and functional data reveal cannabinoid receptor-mediated analgesic actions operating at sites concerned with the transmission and processing of nociceptive signals in brain, spinal cord and the periphery. The precise signalling mechanisms by which cannabinoids produce analgesic effects at these sites remain unclear; however, significant clues point to cannabinoid modulation of the functions of neurone and immune cells that mediate nociceptive and inflammatory responses. Intracellular signalling mechanisms engaged by cannabinoid receptors-like the inhibition of calcium transients and adenylate cyclase, and pre-synaptic modulation of transmitter release-have been demonstrated in some of these cell types and are predicted to play a role in the analgesic effects of cannabinoids. In contrast, the clinical effectiveness of cannabinoids as analgesics is less clear. Progress in this area requires the development of cannabinoids with a more favourable therapeutic index than those currently available for human use, and the testing of their efficacy and side-effects in high-quality clinical trials.     

Antemortem and postmortem rodenticide analysis in forensic toxicology as a part of an LC-MS/MS-based multi-target screening strategy

Since rodenticides represent a substance group relevant in toxicological analyses, the aim of this work was the development of a complex multi-target screening strategy for the identification with liquid chromatography–tandem mass spectrometry. A simple protein precipitation was used as the sample preparation strategy. Further, a Luna 5 μm C18 (2) 100 Å, 150 × 2 mm analytical column was applied for the separation of relevant analytes with a Shimadzu HPLC. Signal detection was performed with a SCIEX API 5500 QTrap MS/MS system. The rodenticides investigated (α-chloralose, brodifacoum, bromadiolone, coumatetralyl, difenacoum, and warfarin) could be incorporated effectively into a multi-target screening strategy covering about 250 substances representing different groups with a limit of detection appropriate for substance identification. The strategy can easily be modified to perform semi-quantitative measurements for this substance group and could be supplemented by quantification based on standard addition.     

Cannabinoids and cancer

Marijuana has been used in medicine for millennia, but it was not until 1964 that delta9-tetrahydrocannabinol (delta9-THC), its major psychoactive component, was isolated in pure form and its structure was elucidated. Shortly thereafter it was synthesized and became readily available. However, it took another decade until the first report on its antineoplastic activity appeared. In 1975, Munson discovered that cannabinoids suppress Lewis lung carcinoma cell growth. The mechanism of this action was shown to be inhibition of DNA synthesis. Antiproliferative action on some other cancer cells was also found. In spite of the promising results from these early studies, further investigations in this area were not reported until a few years ago, when almost simultaneously two groups initiated research on the antiproliferative effects of cannabinoids on cancer cells: Di Marzo's group found that cannabinoids inhibit breast cancer cell proliferation, and Guzman's group found that cannabinoids inhibit the growth of C6 glioma cell. Other groups also started work in this field, and today, a wide array of cancer cell lines that are affected is known, and some mechanisms involved have been elucidated.     

Cannabinoids and neuroinflammation

Growing evidence suggests that a major physiological function of the cannabinoid signaling system is to modulate neuroinflammation. This review discusses the anti-inflammatory properties of cannabinoid compounds at molecular, cellular and whole animal levels, first by examining the evidence for anti-inflammatory effects of cannabinoids obtained using in vivo animal models of clinical neuroinflammatory conditions, specifically rodent models of multiple sclerosis, and second by describing the endogenous cannabinoid (endocannabinoid) system components in immune cells. Our aim is to identify immune functions modulated by cannabinoids that could account for their anti-inflammatory effects in these animal models.     

Cannabinoids in pain and inflammation

Cannabinoids exhibit medicinal properties including analgesic, anti-inflammatory and immunosuppressive properties. This paper reviews some of the recent findings in the study of cannabinoids in pain and inflammation. Some of the effects of cannabinoids are receptor mediated and others are receptor independent. Endocannabinoids naturally reduce pain and are cerebroprotective. Natural and synthetic cannabinoids have the potential to reduce nociception, reverse the development of allodynia and hyperalgesia, reduce inflammation and inflammatory pain and protect from secondary tissue damage in traumatic head injury.     

Cannabinoids for therapeutic use

The two main modes of cannabinoid administration, oral ingestion of tetrahydrocannabinol (THC) and smoking of dry cannabis plant material, both have specific advantages and disadvantages. Disadvantages of oral ingestion include slow and erratic absorption, delayed onset of action and low systemic bioavailability, whereas disadvantages of smoking include mucosal damage and short duration of effect. In recent years several new modes of cannabinoid delivery have been tested. Alternative routes of systemic pulmonary administration include inhalation with a vaporizer and the use of cannabinoids in aerosol form. They avoid or reduce the formation of carcinogenic combustion products found in cannabis smoke. Sublingual (buccal) administration of liquid cannabis extracts has been tested in clinical trials in the UK. This mode is easy to administer and might enable easier dose titration than oral capsules. Transdermal delivery achieves a sustained and steadier action than inhalation or oral administration of THC, and is being investigated in preclinical studies by groups in the US and Israel. Use of ethosomal carriers has been shown to enhance skin permeation by the lipophilic cannabinoids. Rectal administration of THC-hemisuccinate suppositories has been tested in some patients; systemic bioavailability is twice as high as with oral administration because of the reduced first-pass effect. Water-soluble agonists to the cannabinoid receptor that allow intravenous administration have been developed. Dexanabinol, a non-psychotropic neuroprotective cannabinoid derivative, was given intravenously in clinical studies to decrease the consequences of severe closed head injuries. Increasing water solubility, for example by the use of cyclodextrin technology, also improved the possibilities of topical cannabinoid administration to the eye for glaucoma treatment. Several of these new approaches to cannabinoid delivery now under preclinical and clinical investigation may find their way into clinical practice within a few years.     

Pharmacogenomics as molecular autopsy for postmortem forensic toxicology: genotyping cytochrome P450 2D6 for oxycodone cases

Pharmacogenomics, the study of the impact of heritable traits on pharmacology and toxicology, may serve as an adjunct for certifying opioid fatalities. Oxycodone, frequently prescribed for the relief of moderate to severe pain, is metabolized by cytochrome P450 (CYP) 2D6, encoded by a polymorphic gene with three mutations (*3, *4, and *5) with a combined 95% allelic frequency and about 10% prevalence. Individuals with variant alleles are more susceptible to oxycodone toxicity. By assessing the prevalence of CYP2D6 polymorphisms and covariables, we hypothesized that oxycodone fatality may be partially due to poor drug metabolism caused by CYP2D6 variant alleles. From the Milwaukee County Medical Examiner's Office (MCMEO), a retrospective analysis of 15 oxycodone cases was followed by genotyping blood samples for the variant alleles by conventional and real-time PCRs. Institutional Review Board approval was obtained. Oxycodone, extracted from blood and/or urine, was quantitated by GC-MS. The results show two homozygous for 2D6*4 and four heterozygous for 2D6*4. The MCMEO was not significantly different from those in the control group (n = 26) (p > 0.05, Fisher's Exact Test). However, genotyping CYP2D6 provided a more definitive interpretation of the oxycodone toxicity in four cases. Therefore, pharmacogenomics may serve as an adjunct in the determination of the cause and manner of death in forensic toxicology and a pharmacogenomic algorithm for genotyping has been proposed.     

Cannabinoids and neuropathic pain

After a brief overview of the endocannabinoid system (CB receptors, and endocannabinoids) and of the cannabinergic ligands, some general issues related to cannabinoids and pain are commented. Finally, the most important findings regarding cannabinoids and neuropathic pain are discussed in detail.