Quantification of risperidone and paliperidone by liquid chromatography-tandem mass spectrometry in biological fluids and solid tissues obtained from two deceased using the standard addition method.

Risperidone (RIS) is an atypical antipsychotic agent and its 9-hydroxylated metabolite named paliperidone (PAL) also has pharmacological properties similar to that of RIS. Quantifications of RIS and PAL in authentic human biological fluids and solid tissues by liquid chromatography (LC)-tandem mass spectrometry (MS/MS) have not been reported yet although those in plasma (and blood) were reported abundantly. In the present work, a quantification method for RIS and PAL based on the standard addition method was devised and validated for the human fluid and solid tissue specimens. RIS and PAL in biological fluids were quantified only after their dilution and deproteinization. The concentrations of RIS and PAL in the heart whole blood, pericardial fluid, stomach contents, bile, urine, liver, kidney and cerebrum were determined for a deceased who had been treated with RIS therapeutically, and also a deceased who had ingested RIS with other drugs intentionally. To our knowledge, this is the first report on the quantification of RIS and PAL by LC-MS/MS in the authentic human tissues and biological fluids.

Quantification of olanzapine and its three metabolites by liquid chromatography-tandem mass spectrometry in human body fluids obtained from four deceased, and confirmation of the reduction from olanzapine N-oxide to olanzapine in whole blood in vitro.

PURPOSE: Quantification of olanzapine (OLZ) and its metabolites such as N-desmethylolanzapine (DM-O), 2-hydroxymethylolanzapine (2H-O) and olanzapine N-oxide (NO-O) in five kinds of human body fluids including whole blood by liquid chromatography (LC)-tandem mass spectrometry (MS/MS) has been presented; the quantification methods were carefully devised and validated using the matrix-matched calibration and standard addition methods. METHODS: OLZ and its three metabolites were extracted from 40 µL each of body fluids by two-step liquid-liquid separations. The samples and reagents were pre-cooled in a container filled with ice for the extraction because of the thermal instability of OLZ and its three metabolites especially in whole blood. RESULTS: The limits of quantification (LOQs) of OLZ and 2H-O were 0.05 ng/mL and those of DM-O and NO-O were 0.15 ng/mL in whole blood and urine, respectively. The concentrations of OLZ and its metabolites in heart whole blood, pericardial fluid, stomach contents, bile and urine were determined for two cadavers and those in whole blood and urine for the other two cadavers. The reduction from NO-O to OLZ was observed at 25 â„ƒ in whole blood in vitro. CONCLUSIONS: To our knowledge, this is the first report on the quantification of metabolites of olanzapine in the authentic human body fluids by LC-MS/MS as well as on the confirmation of in vitro reduction from NO-O to OLZ in whole blood that seems to have induced the quick decrease of NO-O.

Long-term stability of 24 synthetic cannabinoid metabolites spiked into whole blood and urine for up to 168 days, and the comparable study for the 6 metabolites in non-spiked real case specimens stored for 1-5 years.

PURPOSE: The aim of this study is to investigate the stabilities of the 24 synthetic cannabinoid metabolites (SCMs) in blood and urine at various temperatures from - 30 to 37 ℃ stored for 1-168 days. In addition, experiments of stabilities at lower temperatures and for much longer duration have been performed as described below. METHODS: The quantification was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The blank blood and urine spiked with SCMs and non-spiked real case (authentic) specimens were incubated at 37 ℃ up to 56 days and at 22, 4 or - 30 ℃ up to 168 days. The non-spiked authentic blood and urine specimens were also stored at - 30 or - 80 ℃ for 1, 3 or 5 years to investigate stabilities during very long time frames. RESULTS: All the 24 SCMs were much more stable in urine than in blood at 37, 22 or 4 ℃. All 24 SCMs spiked into blood or urine were stable at - 30 ℃ for up to 168 days. The 6 SCMs in the authentic specimens exhibited long stabilities at - 30 or - 80 ℃ for 3-5 years. Some tendencies were observed according to the relation between the structures of SCMs and their stabilities. CONCLUSIONS: The long-term stabilities of 24 SCMs in spiked samples and those of 6 SCMs in the authentic specimens were examined using LC-MS/MS. SCMs were largely very stable and usable several years after storage at - 30 or - 80 ℃.

Non-fentanyl-derived synthetic opioids emerging during recent years.

PURPOSE: Since the appearance of fentanyl followed by its many kinds of analogues around 1988, North America has been exposed to fierce synthetic opioid pandemic resulting in more than 130,000 deaths due to their overdoses until May 2019, when China declared to prohibit the licit fentanyl analog production. However, the Chinese announcement did not go into force in USA due to the adroit strategies of tough traffickers. Thus, contrary to the expectation, the number of synthetic opioid products and their poisoning cases in USA has increased by about 30%; especially, various benzimidazole synthetic opioids have revived on the illicit drug market during a recent few years. In this article, the recent abrupt changes in the situations of illicit synthetic opioid market and their current abuses are described. METHODS: Various databases, such as SciFinder, Google, and Google Scholar, were utilized to collect relevant reports referring old but newly appearing synthetic opioids. RESULTS: At the present time, there are several families of new synthetic opioids, which are not fentanyl derivatives; MT-45 and its analogs, benzamide and 2-phenylacetamide opioids (U-series opioids), and benzimidazole opioids. Most of the above substances had been developed in 1950s to 1970s, but had never been used as analgesic medicines, because of their severe adverse effects, such as respiratory depression, physical dependence, and resulting deaths. However, there is possibility that these drugs will become main illicit synthetic opioids in place of the fentanyl analogs during coming several years from this time. CONCLUSIONS: All of the above non-fentanyl-derived families had been developed 50-70 years ago to establish them as analgesic medicines, but had been unsuccessful. These drugs largely appeared in the illicit drug markets in North America, Europe, and Australia, during recent years. Pharmacological, toxicological, and metabolic studies are insufficient for benzamide and 2-phenylacetamide opioids, and are very scant especially for benzimidazole opioids. This time we should start studying pharmacotoxicology of the newly emerging synthetic opioids to alert forensic toxicologists in the world and to suppress their rapid and wide spread in the world.

A fatal case involved in pyrethroid insecticide ingestion: quantification of tetramethrin and resmethrin in body fluids of a deceased by LC-MS/MS.

PURPOSE: The quantification of parent molecules of pyrethroids tetramethrin and resmethrin in human specimens by a mass spectrometry (MS) technique has not been reported yet. A woman in her 60s was found dead in a wasteland. At the scene, an empty beer can and a spray for insecticides containing tetramethrin and resmethrin were found. Therefore, the concentrations of tetramethrin and resmethrin in postmortem specimens and the methanol solution used for rinsing the inside of the beer can were determined using liquid chromatography (LC)-tandem mass spectrometry (MS/MS). METHODS: The quantification method by LC-MS/MS for intact parent molecules of tetramethrin and resmethrin in whole blood and urine has been devised and validated in this work. The method was applied to the quantification of tetramethrin and resmethrin in whole blood, urine and stomach contents obtained from a cadaver at autopsy. RESULTS: The limits of detection of tetramethrin and resmethrin were 0.06 and 0.03 ng/mL; limits of quantification were 0.2 and 0.1 ng/mL in blood and urine, respectively. The concentrations of tetramethrin of the deceased were 11.1 ± 1.2 and 0.425 ± 0.017 ng/mL for stomach contents and urine, respectively; the concentration of resmethrin in stomach contents was 1.77 ± 0.18 ng/mL. The tetramethrin and resmethrin were unstable in blood and urine at room temperature; they should be kept at not higher than 4 â„ƒ. CONCLUSIONS: To our knowledge, this is the first report for quantification of unchanged tetramethrin and resmethrin in human specimens obtained in a fatal case.

Non-fentanyl-derived synthetic opioids emerging during recent years.

Purpose: Since the appearance of fentanyl followed by its many kinds of analogues around 1988, North America has been exposed to fierce synthetic opioid pandemic resulting in more than 130,000 deaths due to their overdoses until May 2019, when China declared to prohibit the licit fentanyl analog production. However, the Chinese announcement did not go into force in USA due to the adroit strategies of tough traffickers. Thus, contrary to the expectation, the number of synthetic opioid products and their poisoning cases in USA has increased by about 30%; especially, various benzimidazole synthetic opioids have revived on the illicit drug market during a recent few years. In this article, the recent abrupt changes in the situations of illicit synthetic opioid market and their current abuses are described. Methods: Various databases, such as SciFinder, Google, and Google Scholar, were utilized to collect relevant reports referring old but newly appearing synthetic opioids. Results: At the present time, there are several families of new synthetic opioids, which are not fentanyl derivatives; MT-45 and its analogs, benzamide and 2-phenylacetamide opioids (U-series opioids), and benzimidazole opioids. Most of the above substances had been developed in 1950s to 1970s, but had never been used as analgesic medicines, because of their severe adverse effects, such as respiratory depression, physical dependence, and resulting deaths. However, there is possibility that these drugs will become main illicit synthetic opioids in place of the fentanyl analogs during coming several years from this time. Conclusions: All of the above non-fentanyl-derived families had been developed 50-70 years ago to establish them as analgesic medicines, but had been unsuccessful. These drugs largely appeared in the illicit drug markets in North America, Europe, and Australia, during recent years. Pharmacological, toxicological, and metabolic studies are insufficient for benzamide and 2-phenylacetamide opioids, and are very scant especially for benzimidazole opioids. This time we should start studying pharmacotoxicology of the newly emerging synthetic opioids to alert forensic toxicologists in the world and to suppress their rapid and wide spread in the world. Supplementary Information: The online version contains supplementary material available at 10.1007/s11419-022-00624-y.

In Vivo Metabolites of AB-PINACA in Solid Tissues Obtained from Its Abuser: Comparison with In Vitro Experiment.

In this study, solid tissues such as the lung, liver, kidney and urine were highlighted to profile the AB-PINACA in vivo metabolites in a fatal abuse case, although such metabolite analysis is usually made with urine specimens. We compared the relative peak intensities of in vivo metabolites of AB-PINACA in lung, liver, kidney and urine specimens collected at the autopsy of its abuser with its in vitro metabolites in human hepatocytes. The metabolites of AB-PINACA in tissues were extracted after homogenization. The urine specimen and portions of the extracted metabolites from tissues were firstly hydrolyzed with ß-glucuronidase, and the metabolites were extracted. For in vitro experiment, AB-PINACA was incubated with human hepatocytes for 3 h to produce its metabolites. The identification of the in vivo and in vitro metabolites was performed using liquid chromatography (LC)-high-resolution Orbitrap-tandem mass spectrometry (MS-MS), and the relative intensities of these metabolites were measured using low resolution LC-quadrupole-ion trap-MS-MS. Thirteen metabolites of AB-PINACA were characterized in vivo in several human specimens and in in vitro human hepatocytes. They were produced by the terminal amide hydrolysis to carboxylic acid, hydroxylation, carbonyl formation and/or glucuronidation. The most detectable metabolite in the hepatocytes, lung or liver was the one produced by the terminal amide hydrolysis, whereas the top metabolite in the kidney or urine was the one produced by hydroxylation or carbonyl formation on the pentyl side chain after the terminal amide hydrolysis, respectively. At least 12 metabolites of AB-PINACA were detected in authentic human lung, liver or kidney specimen from a cadaver. It is concluded that the postmortem metabolite profiling of AB-PINACA can be fulfilled with solid tissues, and the lung and kidney were most recommendable especially when urine specimen is not available.

Quantification of Major Metabolites of AB-FUBINACA in Solid Tissues Obtained from an Abuser.

AB-FUBINACA M3 was reported to be a major metabolite of the drug, but its in vivo concentration in authentic human solid tissues has not been quantified yet. Another metabolite AB-FUBINACA M4 did not receive much attention previously and also has not been quantified yet in any authentic human specimens. The aims of this study are to establish a sensitive method for quantification of M3 and M4 in solid tissues and to compare the metabolite profile of AB-FUBINACA in authentic human specimens in vivo with that produced by human hepatocytes in vitro. The quantification was performed by liquid chromatography (LC)-quadrupole-ion trap-tandem mass spectrometry (MS-MS), and the characterization by LC-quadrupole Orbitrap MS-MS The limits of quantification of M3 were 10 pg/mL and 60 pg/g, and those of M4 were 100 pg/mL and 600 pg/g in urine and tissues, respectively. In the present work, M3 and M4 were identified and quantified in human lung, liver and kidney obtained from a cadaver for the first time; the concentrations of M3 were 226, 255, 202 and 155 pg/mL or g, and those of M4 14,400, 768, 637 and 1,390 pg/mL or g in urine, lung, liver and kidney, respectively. The peak intensity profiles of seven metabolites in these specimens were compared with that produced by human hepatocytes; the top three metabolites in urine specimen were completely different from those of hepatocytes. M3 was reported as the predominant metabolite in several previous works and M4 was listed as a minor metabolite in only one work, but, in this work, M4 has been found to be the major metabolite in all of the authentic urine, lung, liver and kidney specimens. The M3 plus M4 metabolites in lung or kidney were found most recommendable to prove AB-FUBINACA consumption, when urine specimen is lacking.

Postmortem distribution/redistribution of buformin in body fluids and solid tissues in an autopsy case using liquid chromatography-tandem mass spectrometry with QuEChERS extraction method.

An autopsy for a suicidal case of a male in his 40s, who had died of poisoning due to ingestion of a large amount of buformin, was performed at our department. Buformin is biganide class agent used for patients of diabetes mellitus, which can occasionally cause severe lactic acidosis. The autopsy was performed about 10 days after his death, and the direct cause of his death was judged as asphyxia due to the aspiration of stomach contents into the airway. The nine body fluids and eight solid tissues specimens were dealt with for investigating postmortem distribution/redistribution of buformin in a whole body; femoral vein blood, right and left heart blood, pericardial fluid, urine, bile, stomach contents, small intestine contents, cerebrospinal fluid, the brain, lung, heart muscle, liver, spleen, kidney and skeletal muscle were examined. For extracting buformin from specimens, a modified QuEChERS method including dispersive solid-phase extraction was employed, followed by the analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS). Buformin in various kinds of human matrices were quantified by the standard addition method in this study, which can overcome the matrix effects and recovery rates without use of blank human matrices. All concentrations of buformin in specimens examined in this case were extremely higher than those of previously reported poisoning cases. The concentrations of buformin in left and right heart blood and femoral vein blood specimens of this case were 399, 216 and 261µg/mL, respectively; although the direct cause of his death was judged as asphyxia due to occlusion of airway with stomach contents, the vomiting was thought to be provoked by buformin poisoning. In this study, marked differences of buformin concentrations between brain tissue and cerebral spiral fluids, and other specimens were observed, which suggested that its distribution was influenced also by blood-brain-barrier. Although a number of buformin poisoning cases were published so far, they gave sporadic data on its concentrations and/or distribution in some limited human specimens. This study is the first to describe detailed distribution/redistribution of buformin in a whole human body quantified by using LC-MS/MS.

Sensitive quantification of BB-22 and its metabolite BB-22 3-carboxyindole, and characterization of new metabolites in authentic urine and/or serum specimens obtained from three individuals by LC-QTRAP-MS/MS and high-resolution LC-Orbitrap-MS/MS.

PURPOSE: A synthetic cannabinoid BB-22 and its metabolite BB-22 3-carboxyindole have not yet been quantified in human urine. The aim of this study is to establish a sensitive analytical method for the quantification of BB-22 and its 3-carboxyindole in human serum and urine specimens, and the characterization of the unreported metabolites of BB-22 in authentic urine specimens from three individuals. METHODS: These compounds were extracted from ß-glucuronide-hydrolyzed and unhydrolyzed urine and/or serum via liquid-liquid extraction. The identification and quantification were performed using liquid chromatography (LC)-QTRAP-tandem mass spectrometry (MS/MS) and the characterization of the new metabolites was made by high-resolution LC-MS/MS. RESULTS: The limits of detection of BB-22 and BB-22 3-carboxyindole were 3 and 30 pg/mL in urine, respectively. The devised method was applied to quantify these compounds in authentic serum and urine obtained from two drug abusers and in urine from one drug abuser. The serum levels of BB-22 were 149 and 6680 pg/mL, and those of BB-22 3-carboxyindole were 0.755 and 38.0 ng/mL in cases 1 and 2, respectively. The urine levels of BB-22 were 5.64, 5.52 and 6.92 pg/mL and those of BB-22 3-carboxyindole were 0.131, 21.4 and 5.15 ng/mL in cases 1, 2 and 3, respectively. New monohydroxyl metabolites retaining the structure of BB-22 were found in the urine specimens. CONCLUSIONS: The synthetic cannabinoid BB-22 and its metabolite BB-22 3-carboxyindole were identified and quantified in authentic human serum and urine specimens for the first time, and new metabolites of BB-22 were tentatively identified in authentic urine specimens obtained from three drug users in this study.

A case of intoxication with a mixture of synthetic cannabinoids EAM-2201, AB-PINACA and AB-FUBINACA, and a synthetic cathinone α-PVP.

We report a case of intoxication with a mixture of three synthetic cannabinoids and a synthetic cathinone, which have been disclosed by a highly sensitive progressing technology. A man was found dead, and his forensic autopsy was performed at our department. After further examinations of his specimens, EAM-2201 and α-PVP have been newly found in his lung. The concentrations of EAM-2201 have not been reported yet in any authentic human specimens although its existence (not quantified) in blood was reported in 2015. Therefore, a sensitive quantitation method of these compounds in blood and solid tissues has been devised using the sensitive instrument. The limits of detection of these compounds were in the range of 3-10 pg/ml with their quantification range of 10-1000 pg/ml in blood. The femoral vein blood levels of EAM-2201 and AB-PINACA were 56.6 ±â€¯4.2 and 12.6 ±â€¯0.1 pg/ml, respectively, and AB-FUBINACA could be detected but not quantifiable in the blood specimens; α-PVP could not be detected. The standard addition method was employed for the quantification of these compounds in the lung, liver and kidney specimens. The lung levels of EAM-2201, AB-PINACA, AB-FUBINACA and α-PVP were 348 ±â€¯34, 355 ±â€¯30, 124 ±â€¯12 and 59.0 ±â€¯7.4 pg/g, respectively. In conclusion, in this study, the concentrations of EAM-2201 in authentic human specimens including blood and solid tissues and those of AB-PINACA and AB-FUBINACA in solid tissue specimens were quantified for the first time to our knowledge.

Fatal zolpidem poisoning due to its intravenous self-injection: Postmortem distribution/redistribution of zolpidem and its predominant metabolite zolpidem phenyl-4-carboxylic acid in body fluids and solid tissues in an autopsy case.

We experienced a curious fatal case, in which a male in his 20s self-administered zolpidem intravenously. The victim was found dead lying on floor of his apartment room, with a tourniquet band and new injection marks on his right forearm. Nearby the body, a medical disposal syringe containing small-volume solution dissolving crushed zolpidem tablets was found. The postmortem interval was estimated at about two days. The direct cause of his death was judged as asphyxia due to the aspiration of stomach contents into the trachea and bronchi. The specimens dealt with were body fluids and solid tissues including femoral vein blood, right and left heart blood, pericardial fluid, urine, bile, stomach contents, the brain, lung, heart muscle, liver, spleen, kidney, pancreas and skeletal muscle. For the extractions of zolpidem, zolpidem phenyl-4-carboxylic acid, deuterated internal standards zolpidem-d7 and zolpidem phenyl-4-carboxylic acid-d4, a modified QuEChERS method was used, followed by the analysis by liquid chromatography-tandem mass spectrometry. Because this study included various kinds of human matrices with quite different properties, the standard addition method was most preferable to overcome the matrix effects and recovery rates, and also did not need to use blank human matrices for validation experiments. The concentration of zolpidem and its phenyl-4-carboxylic acid metabolite in various specimens tested were generally extreme higher than those of reported fatal cases, supporting that the victim had died of intravenous zolpidem injection. The concentrations of zolpidem in femoral vein blood and right and left heart blood specimens in the present case were 9.55, 28.5 and 46.9µg/mL, respectively, which far exceeded estimated fatal levels. The present study also showed the postmortem distribution/redistribution of zolpidem and its phenyl-4-carboxylic acid metabolite in 15 body fluid and solid tissue specimens including stomach contents. Although a number of published literatures dealt with zolpidem poisoning cases due to oral ingestion of the drug, this is the first report on fatal intravenous zolpidem injection case and postmortem distribution of zolpidem and its predominant metabolite.

Identification and quantification of predominant metabolites of synthetic cannabinoid MAB-CHMINACA in an authentic human urine specimen.

An autopsy case in which the cause of death was judged as drug poisoning by two synthetic cannabinoids, including MAB-CHMINACA, was investigated. Although unchanged MAB-CHMINACA could be detected from solid tissues, blood and stomach contents in the case, the compound could not be detected from a urine specimen. We obtained six kinds of reference standards of MAB-CHMINACA metabolites from a commercial source. The MAB-CHMINACA metabolites from the urine specimen of the abuser were extracted using a QuEChERS method including dispersive solid-phase extraction, and analyzed by liquid chromatography-tandem mass spectrometry with or without hydrolysis with ß-glucuronidase. Among the six MAB-CHMINACA metabolites tested, two predominant metabolites could be identified and quantified in the urine specimen of the deceased. After hydrolysis with ß-glucuronidase, an increase of the two metabolites was not observed. The metabolites detected were a 4-monohydroxycyclohexylmethyl metabolite M1 (N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-((4-hydroxycyclohexyl)methyl)-1H-indazole-3-carboxamide) and a dihydroxyl (4-hydroxycyclohexylmethyl and tert-butylhydroxyl) metabolite M11 (N-(1-amino-4-hydroxy-3,3-dimethyl-1-oxobutan-2-yl)-1-((4-hydroxycyclohexyl)methyl)-1H-indazole-3-carboxamide). Their concentrations were 2.17 ± 0.15 and 10.2 ± 0.3 ng/mL (n = 3, each) for M1 and M11, respectively. Although there is one previous in vitro study showing the estimation of metabolism of MAB-CHMINACA using human hepatocytes, this is the first report dealing with in vivo identification and quantification of MAB-CHMINACA metabolites in an authentic human urine specimen.

Identification and quantification of metabolites of AB-CHMINACA in a urine specimen of an abuser.

We experienced an autopsy case in which the cause of death was judged as poisoning by multiple new psychoactive substances, including AB-CHMINACA, 5-fluoro-AMB and diphenidine [Forensic Toxicol. 33 (2015): 45-53]. Although unchanged AB-CHMINACA could be detected from 8 solid tissues, it could neither be detected from blood nor urine specimens. In this article, we obtained eight kinds of reference standards of AB-CHMINACA metabolites from a commercial source. The AB-CHMINACA metabolites from the urine specimen of the abuser were extracted by a modified QuEChERS method and analyzed by liquid chromatography-tandem mass spectrometry before and after hydrolysis with ß-glucuronidase. Among the eight AB-CHMINACA metabolites tested, only 2 metabolites could be identified in the urine specimen of the deceased. After hydrolysis with ß-glucuronidase, the concentrations of the two metabolites were not increased, suggesting that the metabolites were not in the conjugated forms. The metabolites detected were 4-hydroxycyclohexylmethyl AB-CHMINACA (M1), followed by N-[[1-(cyclohexylmethyl)-1H-indazol-3-yl]carbonyl]-l-valine (M3). Their concentrations were 52.8 ± 3.44 and 41.3 ± 5.04 ng/ml (n=10) for M1 and M3, respectively. Although there is one preceding report showing the estimations of metabolism of AB-CHMINACA without reference standards, this is the first report dealing with exact identification using reference standards, and quantification of M1 and M3 in an authentic urine specimen.

Postmortem distribution of MAB-CHMINACA in body fluids and solid tissues of a human cadaver.

During the latter part of 2014, we experienced an autopsy case in which 5-fluoro-ADB, one of the most dangerous synthetic cannabinoids, was identified and quantitated in solid tissues and in three herbal blend products [Forensic Toxicol (2015) 33:112-121]. At that time, although we suspected that there may be some drug(s) other than 5-fluoro-ADB in the herbal products, all trials to find it/them were unsuccessful. Subsequently, we carefully re-examined the presence of other synthetic cannabinoid(s) in the above herbal blend products using accurate mass spectrometry and found two new compounds, 5-fluoro-ADB-PINACA and MAB-CHMINACA (Forensic Toxicol. doi: 10.1007/s 11419-015-0264-y). In the present communication, we report the distribution of MAB-CHMINACA in body fluids and solid tissue specimens collected from the same deceased individual (kept frozen at -80 °C) as described above for demonstration of 5-fluoro-ADB. Unexpectedly, unchanged MAB-CHMINACA could be identified and quantitated in whole blood and in pericardial fluid specimens, but it was below the detection limit (0.1 ng/ml) in the urine specimen. A higher concentration of MAB-CHMINACA could be found in all of the nine solid tissues; the highest concentration of MAB-CHMINACA was found in the liver (156 ng/g), followed by the kidney, pancreas and so on. The compounds were detected in all nine solid tissues; their levels were generally higher than those in the whole blood and pericardial fluid. Contrary to expectations, the concentration of MAB-CHMINACA in the adipose tissue was relatively low. Our results show that the victim smoked one of the three herbal blend products containing both MAB-CHMINACA and 5-fluoro-ADB, resulting in the coexistence of both compounds. It should be concluded that 5-fluoro-ADB and MAB-CHMINACA synergically exerted their toxicities, leading to death after a short interval. The differences in the distribution of 5-fluoro-ADB and MAB-CHMINACA among the cadaver specimens were also discussed in view of the structures of both compounds. To our knowledge, this is the first report to demonstrate MAB-CHMINACA in biological/human specimens.

Postmortem distribution of flunitrazepam and its metabolite 7-aminoflunitrazepam in body fluids and solid tissues in an autopsy case: Usefulness of bile for their detection.

We experienced an autopsy case of a woman in her 70s, in which the direct cause of her death was judged as asphyxia due to the occlusion of food in the trachea. The postmortem interval was estimated at about 2days. The specimens dealt with were femoral vein blood, right heart blood, left heart blood, bile, brain, lung, heart muscle, liver, spleen, kidney, pancreas, skeletal muscle, and adipose tissue. By tentative drug screening, we found a high concentration of 7-aminoflunitrazepam in the femoral vein blood, which lead us to examine the postmortem distribution of flunitrazepam and its metabolite 7-aminoflunitrazepam in her body fluids and solid tissues. The extraction of flunitrazepam, 7-aminoflunitrazepam and internal standard nimetazepam was performed by a modified QuEChERS method, followed by the analysis by liquid chromatography-tandem mass spectrometry. Because this study included various kinds of human matrices with quite different properties, we used the standard additional method to overcome the matrix effects. The concentration of 7-aminoflunitrazepam were generally much higher than those of the parent drug flunitrazepam for most specimens except for the adipose tissue, showing that flunitrazepam is readily metabolized to its 7-amino metabolite after absorption into the body both antemortem and postmortem. The outstandingly highest concentration of 7-animoflunitrazepam was found in the bile, followed by the kidney, pancreas, left heart blood, spleen and liver. Although a majority of flunitrazepam was converted to 7-aminoflunitrazepam, the flunitrazepam concentration was highest in the pancreas, followed by the spleen, bile, left heart blood, and brain. In contrast to the results on synthetic cannabinoids, the levels of flunitrazepam and 7-animoflunitrazepam in the adipose tissue were relatively low. The present study showed that the bile may be a useful specimen for detection of unchanged benzodiazepines/their metabolites to be collected at autopsy.

MALDI-TOF mass spectrometric determination of eight benzodiazepines with two of their metabolites in blood.

A rapid and sensitive method was developed for the determination of benzodiazepines and benzodiazepine-like substances (BZDs) by matrix-assisted laser desorption ionization (MALDI)-time-of-flight (TOF)-mass spectrometry (MS). In this method, α-cyano-4-hydroxy cinnamic acid was used as the matrix to assist the ionization of BZDs. Determination of 8 BZDs (with two of their metabolites) belonging to top 12 medical drugs detected in poisonous cases in Japan, was performed using diazepam-d5 as the internal standard. The limit of detection of zolpidem was 0.07ng/ml with its quantification range of 0.2-20ng/ml in blood, in the best case, and the limit of detection of flunitrazepam was 2ng/ml with its quantification range of 6-200ng/ml in blood, in the worst case. The spectra of zopiclone in MALDI-MS and MS/MS were different from those in electrospray ionization MS and MS/MS. Present method provides a simple and high throughput method for the screening of these BZDs using only 20µl of blood. The developed method was successfully used for the determination of BZDs in biological fluids obtained from two victims.

Postmortem distribution of α-pyrrolidinobutiophenone in body fluids and solid tissues of a human cadaver.

We experienced an autopsy case of a 21-year-old male Caucasian, in which the direct cause of his death was judged as subarachnoid hemorrhage. There was cerebral arteriovenous malformation, which seemed related to the subarachnoid hemorrhage. The postmortem interval was estimated to be about 2days. By our drug screening test using gas chromatography-mass spectrometry, we could identify α-pyrrolidinobutiophenone (α-PBP) in his urine specimen, which led us to investigate the postmortem distribution of α-PBP in this deceased. The specimens dealt with were right heart blood, left heart blood, femoral vein blood, cerebrospinal fluid, urine, stomach contents and five solid tissues. The extraction of α-PBP and α-pyrrolidinovalerophenone (α-PVP, internal standard) was performed by a modified QuEChERS (quick, easy, cheap, effective, rugged and safe) method, followed by the analysis by liquid chromatography-tandem mass spectrometry. Because this study included various kinds of human matrices, we used the standard addition method to overcome the matrix effects. The highest concentration was found in urine, followed by stomach contents, the kidney, lung, spleen, pancreas and liver. The blood concentrations were about halves of those of the solid tissues. The high concentrations of α-PBP in urine and the kidney suggest that the drug tends to be rapidly excreted into urine via the kidney after its absorption into the blood stream. The urine specimen is of the best choice for analysis. This is the first report describing the postmortem distribution of α-PBP in a human to our knowledge.

MALDI-Q-TOF mass spectrometric determination of gold and platinum in tissues using their diethyldithiocarbamate chelate complexes.

A rapid determination method is presented for gold (Au(3+)) and platinum (Pt(4+)) in tissues using matrix-assisted laser desorption ionization quadrupole time-of-flight mass spectrometry (MALDI-Q-TOF-MS). Au and Pt ions in wet-ashed tissue solution were reacted with diethyldithiocarbamate (DDC), and the resulting chelate complex ions Au(DDC)2 (+) and Pt(DDC)3 (+) were detected by MALDI-Q-TOF-MS using α-cyano-4-hydroxycinnamic acid as a matrix. The limit of detection (LOD) was 0.8 ng/g tissue and the quantification range was 2-400 ng/g for Au, and the LOD was 6 ng/g tissue and the quantification range was 20-4,000 ng/g for Pt. The Pt levels detected by MALDI-Q-TOF-MS in several tissues of a patient overdosed with cisplatin were nearly the same as those detected by flow-injection electrospray ionization mass spectrometry. The LODs of Au and Pt were 0.04 pg per well (sample spot) and 0.3 pg per well, respectively. To our knowledge, this is the first attempt to quantify Au(3+) and Pt(4+) ions in tissues by MALDI-Q-TOF-MS.