Forensic toxicology is a field dealing with applications of accepted scientific methods in investigating drug related cases, analytical results of which can be issued and used in court. As unique aspects of forensic medicine/toxicology, various specimens ranging from blood, urine, body fluids to solid tissues can be dealt with in analysis. In addition, target substances to be subjected can also be varied from medicines, abused drugs, chemicals, to daily used substances. In analysis, various high-sensitive instruments such as gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) are being employed throughout the world.
Thus, it should be taken into consideration that various phenomenon including postmortem distribution/redistribution of drugs in the body and matrix effects on analysis can influence results of analysis, making the interpretation of analytical results very complex and difficult in forensic investigations. In this review, we have presented comprehensive perspectives on toxicological analysis and pharmacological information on 88 psychoactive substances in “Narcotics and Psychotropics Control Act” in Japan, which should be of use in drug related cases. It is desirable for examiners to investigate and consider the cause of death based on comprehensive medical perspective, keeping in mind that the results of toxicological analysis are only one of factors in determining the cause of death. In addition, it is also quite important to record details of the samples and analytical procedures employed, so that the previous results can be verified later. These details on toxicological analysis can provide “chain of custody” of the investigation.
The endocannabinoid system (ECS) is known to be involved in the retrograde suppression of signal transduction at synapse. Although there are several reports investigating the effect of ECS on dopamine (DA) signaling, consistent results have not been reported. Therefore, in this study, PC12 cells differentiated with nerve growth factor were used as model cells for dopaminergic neuron, and the true effects of endocannabinoid on DA release in these cells were accurately evaluated using mass spectrometry. The induction of DA release by hexanal was significantly suppressed by co-treatment with endocannabinoid 2-arachidonylglycerol (2-AG). In addition, it was suggested that the changes in intracellular calcium kinetics mediated by cannabinoid receptors were involved in the suppression of DA release by co-treatment with 2-AG. We have also shown in previous studies that DA enhances the function of the ECS in glial cells. These results suggest that glial ECS may act as a suppression system for DA signaling in situations where DA signaling is abnormally enhanced in the central nervous system.
Chronic kidney disease (CKD) is a common disorder and cause of death in cats. In the classification proposed by the International Renal Interest Society (IRIS), stage 1 and 2 CKD are difficult to diagnose accurately using markers, in comparison with normal controls. We recently described a simple and highly reproducible tandem mass tag labelling method for identifying potential disease-marker candidates among low-abundance urine proteins. In the current study, urine samples were obtained from 90 normal control cats as the control group and from 50 cats with CKD (stage 1). To identify new urine biomarkers for CKD, two pool urine samples (normal controls and CKD stage 1 cats) were differentially labelled with TMT, subjected to analysis using SDS-PAGE, digested with trypsin and subjected to analysis using LC-MS/MS. Kidney injury molecule-1 (KIM-1) was identified as a protein with higher levels in cats with CKD (stage 1). An ELISA of urine KIM-1 showed within-run (3.2–4.5%) and between-day (3.4–4.8%) reproducibility. Urine KIM-1 levels measured with this assay were significantly greater in CKD (stage 1) cats than in normal cats (63.7±10.7 vs. 35.7±9.7 μg/g Cre, p<0.001). These results indicate that KIM-1 may be useful as a complementary marker with p-Cre and BUN for detection of CKD (stage 1) in cats.
Plasma unbound cortisol concentration is an accurate measure of its physiological activity. As unbound cortisol is excreted from plasma into saliva, it may be possible to non-invasively predict plasma unbound cortisol levels using salivary cortisol levels. However, part of the salivary cortisol is converted to cortisone, which can affect such predictions. Therefore, correlation between salivary cortisol and plasma unbound cortisol was analyzed in healthy subjects (n=9) and the effect of cortisol-to-cortisone conversion on this correlation was investigated. The correlation equation between salivary cortisol concentration (range, 0.26–3.22 ng/mL) and plasma unbound cortisol concentration (range, 0.31–2.74 ng/mL) for all subjects was determined to be y=0.5311x+0.5117 (r=0.51); however, it showed low correlation. Then, as individual differences in the slope of the correlation equation ranged from 0.62 to 2.62, we classified subjects into two groups based on a mean slope value of 1.10 and re-evaluated the correlation. The resulting equations yielded better correlations (y=2.0416x+0.0279, r=0.65, for two subjects and y=0.7417x+0.1195, r=0.77, for seven subjects), and the observed variation in slope was attributed to individual differences in salivary 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) activity. Importantly, mean absolute percentage error in predicting plasma cortisol using salivary cortisol levels using the two correlation equations was 0.6%. Thus, these results suggest that deriving and classifying salivary cortisone/cortisol ratio, along with relevant correlation equations, can be used to non-invasively predict plasma unbound cortisol concentration.
Many types of oral kinase inhibitors, including imatinib and gefitinib, have become clinically available and have significantly contributed to improving the survival outcomes in cancer patients. Therapeutic drug monitoring of some oral kinase inhibitors is important for ensuring the efficacy and safety of these drugs. Liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) has been used for the simultaneous quantification of these drugs in the human plasma. However, the development of a simultaneous LC-MS/MS analytical method is difficult owing to the differences in mass spectrometry (MS) sensitivity and the therapeutic range of each drug. In this study, we investigated the linear range shifts of calibration curves by adjusting the collision energy defect, in-source collision-induced dissociation, secondary product ion selected reaction monitoring, and isotopologue selected reaction monitoring to develop a simultaneous quantification method for 20 oral kinase inhibitors and active metabolite of sunitinib. Although these four techniques could easily adjust the number of ions introduced to the MS when used individually, it was difficult to adapt 21 analytes using only one technique. Therefore, it is important to utilize multiple techniques, considering the therapeutic concentration range of each drug, in order to develop a method for the simultaneous quantification of oral kinase inhibitors and active metabolites in molecular-targeted therapy.
Triage® DOA is widely used for the on-site screening of drugs of abuse. However, it often provides false positive results for amphetamine due to interference by putrefactive amines, such as 2-phenethylamine, produced by saprogenic bacteria in moderately-to-heavily decomposed bodies. In the present study, we evaluated the performance of five drug screening devices: Triage® TOX Drug Screen, SIGNIFYTM ER, IVeX-screen M-1, Status DS10 and DRIVEN-FLOW M8-Z. A total of 19 forensic autopsy urine samples, which were positive for amphetamines by Triage® DOA, were analyzed with the five drug screening devices and liquid chromatography tandem mass spectrometry. Only DRIVEN-FLOW M8-Z had no false positive or false negative results for methamphetamines. Triage® TOX Drug Screen and IVeX-screen M-1 each had one false positive result for methamphetamines. Other devices, including Triage® TOX Drug Screen, had multiple false positive and false negative results for amphetamines and methamphetamines. These results suggest that DRIVEN-FLOW M8-Z is more useful than other screening devices for screening of methamphetamines in the presence or absence of 2-phenethylamine, while none of the tested devices detected amphetamines precisely. It is necessary to develop platforms that can precisely detect both amphetamines and methamphetamines.