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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.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.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.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 ℃.

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 ℃ 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.

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). 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. 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 ℃. To our knowledge, this is the first report for quantification of unchanged tetramethrin and resmethrin in human specimens obtained in a fatal case.

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 β-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.

The aim of this study is to characterize the metabolites of EAM-2201 in human hepatocytes obtained in vitro and those in liver and urine specimens obtained in vivo from the autopsy of an EAM-2201 abuser. EAM-2201 was incubated with human hepatocytes for 3 h in a CO2 incubator and the metabolites of EAM-2201 were produced. The human liver specimen was homogenized and the metabolites were extracted. The urine specimen was hydrolyzed first with β-glucuronidase and the metabolites were extracted. The tentative detection and identification of those metabolites were performed using liquid chromatography (LC)–quadrupole-ion trap tandem mass spectrometry (MS/MS) and high-resolution LC–Orbitrap-MS/MS, respectively. Twelve metabolites of EAM-2201 could be characterized in hepatocytes, liver and urine. They were produced by defluorination, hydroxylation, carboxylation, dehydrogenation, N-dealkylation and/or glucuronidation of EAM-2201. The most detectable metabolite obtained from the in vitro hepatocyte incubation was that monohydroxylated at the 4-ethylnaphthalene moiety of EAM-2201 after dehydrogenation. However, only a trace amount of the same metabolite was detected in the liver tissue obtained postmortem from an EAM-2201 abuser. In the liver tissue, the highest metabolite was 5-carboxypentyl derivative of EAM-2201, which was monohydroxylated at the 4-ethylnaphthalene moiety, followed by monohydroxylpentyl and monohydroxy-4-ethylnaphthalenyl derivative of EAM-2201 without dehydrogenation. Twelve metabolites of a synthetic cannabinoid, EAM-2201, were tentatively identified in human hepatocytes in vitro, and in human liver and urine specimens in vivo for the first time. There was a distinct difference in metabolism profile between the in vitro and in vivo results.

A simple and high-throughput analytical method for determining clitidine in Paralepistopsis acromelalga using hydrophilic interaction liquid chromatography–tandem mass spectrometry (LC–MS/MS) was established. To determine clitidine in the mushrooms, a simple procedure including dilution with methanol solution and filtering by cartridge was employed just before quantification by LC–MS/MS for high-throughput analysis. In this report, concentrations of clitidine in mushrooms were determined by the standard addition method. The present established method was successfully applied to the analysis of fruit bodies of P. acromelalga, which were obtained from five different locations in Japan. Results on concentrations of clitidine in each stem and cap of P. acromelalga specimens tested showed that their concentrations were quite different, not only between stems and caps, but also among locations and strains; the concentrations of clitidine in stems and caps ranged from 1.41 to 9.30 mg/g and 3.17 to 14.4 mg/g, respectively. This is the first report to present a detailed quantitative analysis of clitidine by MS and the distribution of clitidine in stems and caps of P. acromelalga. This analytical method for clitidine was thought to be useful in P. acromelalga poisoning cases to identify the causative toxic mushroom.

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.

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 46.9 μg/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.

A synthetic cannabinoid 5F-NNEI and its metabolites in authentic human specimens have not been reported yet. The aim of this study is, firstly, to establish a sensitive quantification method of 5F-NNEI in human serum and urine specimens, and, secondly, the characterization of its metabolites in authentic urine specimens obtained from three individuals. These compounds were extracted from β-glucuronide-hydrolyzed and unhydrolyzed urine and/or serum specimens 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 metabolites was done using LC–high-resolution-MS/MS. The limits of detection for unchanged 5F-NNEI were 6 pg/mL with the quantification range of 10–2000 pg/mL in serum, and 3 pg/mL with the quantification range of 5–1000 pg/mL in urine. The devised method was applied to quantify 5F-NNEI in authentic serum and/or urine specimens from three individuals. The levels of 5F-NNEI were 11.6 and 3680 pg/mL in serum specimens of cases 1 and 2, respectively, and were 5.07 and 7.90 pg/mL in urine specimens of cases 2 and 3, respectively. Several metabolites of 5F-NNEI produced by amide hydrolysis, defluorination, hydroxylation, carboxylation and/or glucuronization were tentatively identified in the urine specimens by the LC–high-resolution-MS/MS. A synthetic cannabinoid, 5F-NNEI, was identified and quantified in its unchanged form, and its several metabolites were tentatively identified in authentic specimens obtained from three individuals, for the first time to our knowledge.

Urine is the most suitable specimen to collect from individuals because of noninvasiveness and relatively large volumes obtainable. In authentic urine specimens, however, synthetic cannabinoids having the structures of quinolinyl ester indoles, such as 5F-PB-22, PB-22 and BB-22, in unchanged forms as well as their metabolites, have not been quantified yet. Therefore, the aim of this study was to establish a sensitive analytical method for the quantification of 5F-PB-22 and its three metabolites 5F-PB-22 3-carboxyindole, PB-22 N-5-hydroxypentyl and PB-22 N-pentanoic acid in authentic urine samples in four cases. These compounds were extracted from β-glucuronide-hydrolyzed and unhydrolyzed urine via liquid-liquid extraction. The identification and quantification were performed using the QTRAP type of a liquid chromatography–tandem mass spectrometer. The limits of detection were 3–30 pg/mL and their summed quantitation range was 10–10,000 pg/mL. The devised method was applied to quantify these compounds in authentic urine specimens obtained from four individuals. The levels of 5F-PB-22 were 5.1, 13.6, 94.7 and 470 pg/mL; those of 5F-PB-22 3-carboxyindole were 8.25, 3.39, 23.2 and 880 ng/mL; and those of PB-22 N-pentanoic acid were 12.0, 57.4, 959 and 2090 pg/mL, respectively, in four unhydrolyzed urine samples, and the levels of PB-22 N-5-hydroxypentyl could be quantified as 29.9 and 131 pg/mL in two unhydrolyzed urine samples. The 5F-PB-22 and its metabolites PB-22 N-5-hydroxypentyl have been detected from authentic human urine samples for the first time. Also, this is the first report dealing with the quantification of the three metabolites in human urine samples.

An autopsy case in which the cause of death was judged as drug poisoning by two synthetic cannabinoids, including MAB-CHMINACA, was experienced. Although unchanged MAB-CHMINACA could be detected from solid tissues, blood and stomach contents in the case, the compound could not be detected from the urine specimen. In this article, 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 by a QuEChERS method including dispersive solid-phase extraction, and analyzed by liquid chromatography-tandem mass spectrometry with or without hydrolysis with β-glucuronidase. Among the six MAB-CHMINACA metabolites tested, 2 predominant metabolites could be identified and quantified in the urine specimen of the deceased. After hydrolysis with β-glucuronidase, 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-carbo-xamide) of MAB-CHMINACA, and a dihydroxyl (4-hydroxycyclohexylmethyl and tert-butyl hydroxyl) metabolite M11 (N-(1-amino-4-hydroxy-3,3-dimethyl-1-oxobutan-2-yl)-1-((4-hydroxycyclohexyl)methyl)-1H-indaz-ole-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 preceding 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.

Human urine samples are easier to obtain than human blood samples due to noninvasiveness. The urine levels of synthetic cannabinoids (SCs) in unchanged forms, however, are usually much lower than their blood and tissue levels and cannot be detected in most cases. Therefore, in the present work a sensitive analytical method was devised for the determination of urine levels of six SCs in unchanged forms such as N-(1-amino-3-methy-1-oxobutan-2-yl)-1-pentyl-1H-indazole-3-carboxamide (AB-PINACA), N-(1-amino-3-methy-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamide (AB-FUBINACA), N-[(1S)-1-(1-aminocarbonyl)-2-methyl-propyl]-1-(cyclohexylmethyl)-1H-indazole-3-carboxamide (AB-CHMINACA), N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-(cyclohexylmethyl)-1H-indazole-3-carboxamide (MAB-CHMINACA), methyl-2-[1-(5-fluoropentyl)-1H-indazole-3-carboxamido]-3-methylbutanoate (5F-AMB) and methyl-2-[1-(5-fluoropentyl)-1H-indazole-3-carboxamido]-3,3-dimethylbutanoate (5F-ADB). These SCs were extracted from urine via liquid–liquid extraction. The identification and quantitation were performed by a relatively new type of an instrument for liquid chromatography–tandem mass spectrometry. The limits of detection were as low as 3–8 pg/mL, and the quantitation range was 10–1000 pg/mL using 400 μL of urine. The urine levels of AB-PINACA and AB-FUBINACA of victim 1 were 23 and 10 pg/mL, those of AB-CHMINACA and 5F-AMB of victim 2 were 239 and 19 pg/mL, and those of MAB-CHMINACA and 5F-ADB of victim 3 were 229 and 19 pg/mL, respectively. To our knowledge, this is the first report dealing with successful analysis of low levels of parent synthetic cannabinoids in authentic human urine specimens.

In this work, the application of matrix-assisted laser desorption ionization (MALDI)-quadrupole time-of-flight (QTOF)-tandem mass spectrometry (MS/MS) to the quantitation of the anticholinergic/antiparkinson drug biperiden in human whole blood, and the estimation of new phase I metabolites of biperiden in human urine, were performed for the first time. The limit of detection of biperiden in human whole blood was 0.3 ng/mL with a quantitation range of 1–100 ng/mL using 20 μL each of whole blood. The developed method was successfully applied to the determination of biperiden in whole blood obtained from two deceased subjects having a history of biperiden treatment. The concentrations of biperiden in the whole blood samples were 1.7 and 17.5 ng/mL. The MALDI-QTOF-MS/MS method was also used to estimate the metabolites of biperiden in urine obtained from the subjects. Six new metabolites formed by hydroxylation and/or dehydration were detected.

Application of matrix-assisted laser desorption ionization (MALDI) quadrupole time-of-flight (QTOF)-mass spectrometry (MS) to the determination of timiperone and estimation of its phase I metabolites were performed for the first time. The limit of detection of timiperone in human whole blood was 1 ng/mL with a quantitation range of 2–100 ng/mL using 20 μL each of whole blood. The developed method was successfully applied to the determination of timiperone in whole blood obtained from a deceased subject that had a history of timiperone treatment. The concentration of timiperone in whole blood of the victim was 3.98 ng/mL. Moreover, the MALDI-QTOF-MS/MS method was also used for detection of new metabolites of timiperone in urine also obtained from this victim. Five new metabolites were detected, which were formed via exchange of S for O, deletion of S, carbonyl reduction, hydroxylation, and a combination of them. Most likely, some of the newly detected metabolites seem pharmacologically active because they retain butyrophenone structures.

A rapid and sensitive method using matrix-assisted laser desorption ionization (MALDI)-quadrupole time-of-flight (QTOF)-mass spectrometry (MS) was developed for the analysis of six tricyclic antidepressants (ADs) and their related drugs, such as amitriptyline, carbamazepine, clomipramine, imipramine, nortriptyline, quetiapine, and two tetracyclic ADs, mianserin and mirtazapine, because these eight drugs are commonly observed medicines in poisoning cases in Japan. In the present MALDI-MS method, the limits of detection were 0.3–2 ng/mL. The present method provided a simple and high throughput simultaneous analysis of these drugs. This is the first report dealing with quantitation of ADs and their related drugs in biological samples by MALDI-MS. In addition, we applied the MALDI-MS to two authentic cases, and three new metabolites of amitriptyline and three new metabolites of quetiapine could be detected in blood and urine samples in the two cases when regioisomers were counted as one metabolite.

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.

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 ± 4.2 and 12.6 ± 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 ± 34, 355 ± 30, 124 ± 12 and 59.0 ± 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.

We recently encountered an autopsy case in which methamphetamine (MA) was involved. The postmortem interval was estimated to be as long as 6–7 days. We collected the blood specimens from 9 locations of the heart and blood vessels, and specimens from pericardial fluid, bile, stomach contents and 9 solid tissues (in total 21 specimens), which were subjected to analysis of MA and the metabolite amphetamine by liquid chromatography–tandem mass spectrometry after a modified QuEChERS extraction. The highest concentration of MA was found for the stomach contents, suggesting the oral ingestion of MA. Among the blood specimens, their MA concentrations varied from 268 to 911 ng/mL. The concentration of MA in the left heart blood was higher than that in the right heart blood. In spite of this, the MA concentration in the pulmonary vein was much lower than that in the left heart blood. The comparison of MA concentrations in venous blood vessels showed that MA concentration was highest in the vena cava, followed by the iliac vein, and was lowest in the femoral vein, suggesting that MA concentration is decreased by diffusion across the thin walls of venous blood vessels during the postmortem interval. The partial loss of a xenobiotic by diffusion via the thin wall of the femoral vein should be taken into consideration especially when the postmortem interval is relatively long.

Diphenidine [1-(1,2-diphenyl-ethyl)piperidine] in the tissue sections of the adipose, brain, heart, kidney, and liver obtained from a human cadaver in a poisoning case was directly analyzed as the first trial by matrix-assisted laser desorption ionization (MALDI)-quadrupole time-of-flight mass spectrometry (QTOF-MS). The frozen tissue block of approximately 5 × 5 × 5 mm3 was sectioned to a thickness of 10 or 20 μm at −20 or −35 °C. For quantitation, the reference standard solutions of diphenidine were placed at 0, 0.1, 1, and 10 ng/well on the wells of a MALDI sample plate firstly, and about 20 tissue sections (ca. 5 × 5 mm2 each) were placed on the sample plate secondly, followed by detection by MALDI-MS/MS after desiccation of the tissue sections and spraying α-cyano-4-hydroxycinnamic acid as the matrix. The quantitation was performed by the standard addition method without the use of blank human tissues. The correlation coefficients between the concentrations of diphenidine and the relative signal intensities were from 0.937 to 0.994 for five kinds of tissue. The suppression of signals by tissue was increased in the order of adipose tissue

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.

Police officials brought three different packages of herbal blends, with brand names “AL 37”, “AP 31”, and “GM sapphire”, to our laboratory for drug testing. Using our in-house high-resolution mass spectrometric analysis, we were able to estimate the presence of 5-fluoro-ADB-PINACA and MAB-CHMINACA in them without their reference standards. After obtaining the reference standards, we compared the mass spectra of the extracts of the herbal blends with those of the reference standards using both gas chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry. The mass spectra of the herbal blend extracts coincided with those of the reference standards, disclosing the presence of 5-fluoro-ADB-PINACA in “AL 37” and “AP 31”, and MAB-CHMINACA in “GM sapphire”. We then quantitated the concentrations of both compounds in the herbal blends using the standard addition method. The concentrations of 5-fluoro-ADB-PINACA were 19.4 ± 0.55 and 19.0 ± 0.47 mg/g (mean ± standard deviation of triplicate determinations) for herbal product brands “AL 37” and “AP 31”, respectively, and that of MAB-CHMINACA was 133 ± 4.5 mg/g for the “GM sapphire” herbal product. To our knowledge, this is the first study to demonstrate the identification and quantitation of the newest synthetic cannabinoids 5-fluoro-ADB-PINACA and MAB-CHMINACA in herbal blend products.

In our previous study, we identified PV9 [1-phenyl-2-(pyrrolidin-1-yl)octan-1-one] in human blood and urine in a fatal poisoning case. The victim was an 18-year-old woman. After ingesting “aroma liquid” solution, the victim showed various symptoms including low levels of consciousness, and was taken to a hospital emergency department. Although the victim received intensive medical treatment including an intravenous drip infusion of a large volume of transfusion solution, she was pronounced dead about 20 h after admission. In this study, we carefully examined the postmortem distribution of PV9 in nine solid tissues of the victim collected at forensic autopsy. The extraction of PV9 and internal standard (IS) PV8 [1-phenyl-2-(1-pyrrolidinyl)-1-heptanone] was performed by acetonitrile deproteinization, followed by modified QuEChERS dispersive solid-phase extraction and filtration through Captiva ND Lipids cartridges. Anaysis was performed by liquid chromatography–tandem mass spectrometry. Because this study dealt with various kinds of human matrices, we used the standard addition method to overcome matrix effects. After thorough validations, such as checking the product ion mass spectra, selected reaction monitoring chromatograms, linearity of the standard addition calibration curves, the intraday and interday repeatability, matrix effects, and recovery rates for the method, the concentrations of PV9 in nine solid tissue specimens were measured using PV8 as IS. The highest level of PV9 was found in the kidney at 907 ± 19.5 ng/g followed by the skeletal muscle, pancreas, adipose tissue, liver, lung, spleen, heart muscle, and brain. The lowest level of PV9 in the brain was 212 ± 11.9 ng/g. The high level of PV9 in the kidney suggests that this drug tends to be rapidly excreted into urine via the kidney as was the case for α-pyrrolidinovalerophenone. The low concentration of PV9 in the brain was unexpected, because this drug is a psychotropic drug with a long hydrophobic side chain, and is considered to cross the blood–brain barrier very easily. To our knowledge, this is the first demonstration of the distribution of the new pyrrolidinophenone derivative PV9 in human solid tissues in a poisoning case.

Since late September 2014, there have been approximately 10 cases in Japan where people have died from inhaling smoke from herbal blends containing a newly emerged synthetic cannabinoid. Although the drug was tentatively identified by some drug-monitoring institutions as 5-fluoro-ADB, to our knowledge, its data have not been published in any scientific context. We recently encountered an autopsy case in which 5-fluoro-ADB was involved. The deceased was a 34-year-old man who was found dead in his room. The postmortem interval was estimated at 35–40 h. The direct cause of the death was asphyxia due to aspiration of stomach contents into the trachea, which likely took place during vomiting under low-consciousness conditions provoked by inhalation of the 5-fluoro-ADB smoke. The cadaver was subjected to autopsy at our department. Femoral vein blood, right heart blood, left heart blood, urine, stomach contents, and nine solid tissues including the adipose tissue were collected and frozen until analysis. The extraction of 5-fluoro-ADB and internal standard 5-fluoro-AMB was performed using a modified QuEChERS method plus filtration through Captiva ND Lipids cartridges, followed by liquid chromatography–tandem mass spectrometry (LC–MS–MS) analysis. Because this study dealt with various kinds of human matrices, we used the standard addition method for quantitation to overcome the matrix effects. The levels of 5-fluoro-ADB in the cadaver specimens were generally low; it could not be detected from blood or urine specimens. The levels of 5-fluoro-ADB in solid tissues were 1.17–7.95 ng/g. Because the highest levels were found for the adipose tissue and heart muscle, the final extracts of the adipose tissue and/or heart muscle were concentrated 10- and 200-fold to obtain product ion mass spectra of 5-fluoro-ADB using LC–MS–MS and its mass spectrum by gas chromatography–mass spectrometry, respectively. Both spectra completely coincided with those obtained from the reference standard 5-fluoro-ADB, confirming that the target compound was 5-fluoro-ADB. The quantitative results obtained by selected reaction monitoring of LC–MS–MS showed the highest level, at 7.95 ng/g, in the adipose tissue, followed by stomach contents, brain, heart muscle, pancreas, and spleen. For the lung, liver, kidney, and skeletal muscle, levels were below the quantitation limit (about 0.5 ng/g), although very small peaks above the detection limit (about 0.1 ng/g) could be observed for all of the above solid tissues. The low levels of 5-fluoro-ADB in the solid tissues were likely as a result of only a small amount of 5-fluoro-ADB incorporated into the body via the lungs due to the short period from the beginning of smoking the herb to the fatal asphyxia resulting from aspiration of a massive amount of stomach contents into the trachea under low-consciousness conditions. In addition, we measured the content of 5-fluoro-ADB in three packages, all of which were opened, that were found under a pillow near the deceased; their levels of 5-fluoro-ADB were 49.2 mg/g, 12.2 μg/g, and 0.77 μg/g. To our knowledge, this is the first reported identification and quantitation of 5-fluoro-ADB in human specimens and herbal products.

A rapid and sensitive detection method using matrix-assisted laser desorption ionization (MALDI)–quadrupole time-of-flight–mass spectrometry (Q-TOF–MS) was developed for the analysis of six pyrrolidino cathinone derivatives, α-pyrrolidinopentiothiophenone (PVT), 4′-fluoro-α-pyrrolidinopentiophenone (F-PVP), 4′-methyl-α-pyrrolidinohexanophenone (MPHP), α-pyrrolidinoheptanophenone (PV8), α-pyrrolidinooctanophenone (PV9) and 4′-fluoro-α-pyrrolidinooctanophenone (F-PV9), that were newly designated as illegal drugs in Japan. In this method, α-cyano-4-hydroxycinnamic acid was used as the matrix to assist the ionization of cathinones. MS spectra of these cathinones showed a protonated molecule [M+H]+ and iminium, respectively, and hence the main ions of MALDI–MS were a mixture of the main ions detected by liquid chromatography–electrospray ionization–mass spectrometry and that by gas chromatography–electron ionization–mass spectrometry. The quantification of these cathinones was performed using α-pyrrolidinopentiophenone as the internal standard. The limit of detection was 1 ng/ml and the quantification range was 2–100 ng/ml for these cathinones using 20 μl of blood. We encountered a fatal poisoning case where PV9 was abused. The PV9 levels in postmortem blood samples from the right heart, left heart and femoral vein were 198, 209 and 163 ng/ml, respectively. The metabolites of PV9 were detected, and the peak height of an oxidized metabolite was higher than those of the protonated molecule and the other metabolites.

We encountered an autopsy case in which the cause of death was judged as poisoning by multiple drugs, including AB-CHMINACA, 5-fluoro-AMB, and diphenidine. The deceased was a 30-year-old man. The postmortem interval to autopsy was estimated to be 3.5 days. Femoral vein blood, right heart blood, left heart blood, urine, and eight solid tissues including adipose tissue were collected and frozen until analysis. Extraction of the three drugs, and internal standards phencyclidine and 5-fluoro-AB-PINACA was performed by a modified QuEChERS method, followed by analysis by liquid chromatography–tandem mass spectrometry. Because this study dealt with various kinds of human matrices, we used the standard addition method to overcome the matrix effects. Quantitation of all three compounds was only achieved for the adipose tissue, whereas the levels of 5-fluoro-AMB were below the lower limit of quantitation (about 1 ng/g or ml) in all other samples. AB-CHMINACA was quantitated for all solid tissues, but not for all body fluid specimens. Diphenidine showed high concentrations in all specimens; it was highest in the adipose tissue (11,100 ± 1,120 ng/g), an order of magnitude lower in other solid tissue specimens, and two orders of magnitude lower in body fluid samples. The results suggest that adipose tissue is the best specimen for detection of lipophilic drugs, such as AB-CHMINACA, 5-fluoro-AMB, and diphenidine, in their unchanged forms. In this poisoning case, diphenidine appeared to have played the major role in the cause of death, because the concentrations of diphenidine were much higher than those of the synthetic cannabinoids in all specimens tested. To our knowledge, this is the first report to document the presence of AB-CHMINACA, 5-fluoro-AMB, and diphenidine in actual postmortem specimens in a fatal poisoning case.

A rapid and sensitive detection method using matrix-assisted laser desorption ionization time-of-flight mass spectrometry was developed for the analysis of amphetamine (A), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), and 3,4-methylenedioxymethamphetamine (MDMA). In this method, α-cyano-4-hydroxy cinnamic acid was used as the matrix to assist the ionization of amphetamines. The MS spectra of these amphetamines showed protonated molecules [M + H]+ and fragment ions with comparable or higher intensities. The quantifications of A and MA were performed using A-d 7 and MA-d 5 as the internal standard, respectively, and those of MDA and MDMA were performed using MDMA-d 5 as the internal standard. The limit of detection and the quantification range using 20 μl of blood were about 10 ng/ml and 30–500 ng/ml for A and MDA, respectively, and about 1 ng/ml and 3–50 ng/ml for MA and MDMA, respectively. In two cases of poisoning in which MA was abused, the levels of A in blood and urine were from 0.050 to 3.24 μg/ml and the levels of MA were from 0.231 to 25.1 μg/ml.

A rapid and sensitive detection method using matrix-assisted laser desorption ionization (MALDI) — time-of-flight (TOF) — mass spectrometry (MS) was developed for the analysis of four pyrrolidino cathinones, such as α-pyrrolidinopropiophenone (PPP), 4’-methyl-α-pyrrolidinopropiophenone (MPPP), α-pyrrolidinobutiophenone (PBP), and α-pyrrolidinovalerophenone (PVP). In this method, α-cyano-4-hydroxy cinnamic acid was used as the matrix to assist the ionization of cathinones. Each MALDI-TOF-MS spectrum of these cathinones showed not only protonated molecular ion [M + H]+, but also several fragment ions having comparable intensities to that of [M + H]+. Hence MPPP and PBP could be clearly discriminated by the mass spectra alone, although they have almost the same mass numbers in their protonated molecular ions. The quantification of MPPP, PBP or PVP was performed using PPP as internal standard, respectively, and that of PPP was performed using PBP as internal standard. The limit of detection was 1 ng/ml, and the quantification range was 2 – 200 ng/ml for the four cathinones using 20 μl of blood. In a fatal poisoning case in which PVP was abused, the PVP levels in whole blood samples obtained from the right heart, left heart and femoral vein were 0.597, 0.635 and 0.580 μg/ml, respectively. We recommend the MALDI-TOF-MS method without any chromatography for both identification and quantification of the pyrrolidino cathinones in various matrices in forensic toxicological analysis, because of the simplicity, rapidness and reliability of the method.

We previously established a sensitive gas chromatography–mass spectrometry method for analysis of ethylene glycol (EG), propylene glycol (PG), and diethylene glycol (DEG), and disclosed the presence of appreciable amounts of EG, PG, and DEG in fresh whole blood and urine specimens obtained from nonoccupational healthy humans. These results led us to analyze EG and PG in specimens taken from three human cadavers. EG and PG concentrations in the postmortem blood and solid tissues were much higher than those found in fresh whole blood; their concentrations in many postmortem specimens were more than tenfold those in fresh whole blood specimens, suggesting the postmortem production of EG and PG. Therefore, we examined the time courses of EG and PG concentrations in blood specimens in the absence and presence of saprogenic blood (10 % volume) and/or glucose (3 mg/ml) in vitro, which were left at room temperature for 7 days. EG concentration in fresh blood without any addition decreased slightly during the 7 days. EG concentration in the blood with addition of glucose, and PG concentrations in the blood with and without addition of glucose did not change appreciably during the 7 days. EG and PG concentrations in the blood after addition of 10 % saprogenic blood increased 3.1-fold and 3.5-fold after 7 days, respectively; those after addition of saprogenic blood plus glucose increased 9.1-fold and 11.9-fold after 7 days, respectively. These results show that microorganisms present in the saprogenic blood caused the postmortem production of EG and PG, and the addition of glucose further enhances the EG and PG concentrations, probably acting as the substrate for glycol production by the microorganisms. To our knowledge, this is the first report to describe postmortem production of EG and PG in human specimens.

In a previous study, we found that appreciable amounts of ethylene glycol (EG), propylene glycol (PG), and diethylene glycol (DEG) were present in the blood of nonoccupational healthy humans. In this study, we measured the three glycols in the urine of healthy subjects by the same isotope dilution gas chromatography–mass spectrometry method, and found the concentrations to be much higher than those in blood. The concentrations of EG, PG, and DEG in urine samples (mean ± standard deviation) obtained from 23 subjects at random were 604 ± 360, 5,450 ± 9,290, and 59.0 ± 49.3 ng/ml, respectively. These values were 9.44, 30.1, and 5.31 times higher than those in whole blood samples, respectively. The much higher concentrations of the three glycols found in urine samples of nonoccupational healthy humans suggest that the three glycols, once incorporated into the human body, are rapidly excreted into urine. To our knowledge, this is the first demonstration of the presence of relatively high concentrations of EG, PG, and DEG in urine of healthy human subjects.

Ethylene glycol (EG), propylene glycol (PG), and diethylene glycol (DEG) are widely used as components of antifreeze liquids for automobiles and in many other products. They occasionally cause severe poisonings when they are ingested in high doses. In this study, we established a detailed procedure for highly sensitive and simultaneous determination of EG, PG, and DEG in human whole blood by isotope dilution gas chromatography–mass spectrometry (GC–MS). A 0.25-ml aliquot of whole blood containing the glycols was mixed with 100 ng each of deuterated EG, PG, and DEG as internal standards. After centrifugation, the supernatant fraction was evaporated to dryness, derivatized with heptafluorobutyric anhydride, and the derivative was extracted with n-hexane; 1 μl of the n-hexane layer was subjected to GC–MS analysis. Contrary to our expectation, appreciable amounts of EG, PG, and DEG were found, even in human whole blood samples obtained from nonoccupational healthy subjects. Because such results have never been reported, in the early stages of this study, we suspected that our results were caused by carry-over of the glycols during the GC–MS procedure; thus, we cleaned the injection port, installed a new GC column, and washed the ion-source chamber. Despite these efforts, the clear peaks were detected at the same retention times as those of the authentic glycols for the extracts of healthy subjects, which led us to confirm the presence of the glycols in the blood of healthy subjects. Because the blank whole blood samples from healthy subjects already contained glycols, we constructed a standard addition calibration curve for each sample; the horizontal intercept point, where the straight calibration line intersected the horizontal concentration axis, showed the background concentration of the glycol. The calibration curves showed linearity in the range of 0.4–400 ng/ml for EG and DEG, and in the range of 4–2,000 ng/ml for PG, with correlation coefficients larger than 0.99. The limits of detection and limits of quantitation could not be exactly determined, because of the unavailability of blank human whole blood without each glycol. However, the values were estimated to be around or lower than 1 ng/ml from the signal-to-noise ratios of peaks for each glycol at the lowest concentrations obtained by selected ion monitoring. To validate the method, intraday and interday repeatability was tested; the percent relative standard deviations were 2.5–12.2 % and 0.8–8.5 %, respectively. Recoveries of the three glycols using whole blood samples of two subjects obtained by a unique method were 61.6–80.9 %. The concentration ranges (mean) of EG, PG, and DEG in whole blood obtained from ten subjects were 39.1–97.0 (64.0), 49.1–689 (181), and 8.08–22.9 (11.1) ng/ml, respectively. To test the effect of oral intake of PG, two volunteers ingested 100 ml of a commercially available energy drink containing 33.7 mg of PG, the safety of which was accredited by the Japanese Government. The PG levels in the blood of the two subjects increased by 74.0 and 158 % at 1 and 0.5 h, respectively, suggesting that the glycols present in human blood are largely derived from food. To our knowledge, the analytical method for the glycols presented in this article is the most sensitive among those so far reported. In addition, this is the first description of the presence of the glycols in healthy human subjects, which should be useful for setting cutoff levels of the glycols in poisoning cases.

In our previous article, we reported the analysis of ibotenic acid and muscimol in Amanita mushrooms by liquid chromatography–tandem mass spectrometry (LC–MS–MS). The levels of ibotenic acid and muscimol in the mushroom were as high as 210 and 107 μg/g, respectively. We have since tried to measure the same toxins in human serum obtained from a poisoned subject, who ingested the Amanita mushrooms, by the same method. However, the levels of the toxins in the human serum were about three orders of magnitude lower than those in Amanita mushrooms. In addition, the recovery rates for ibotenic acid and muscimol in human serum were found to be much lower than those in the previous study for the mushrooms. Therefore, we optimized the solid-phase extraction procedure again, and reevaluated the data for validation at much lower levels of ibotenic acid and muscimol in human serum. A 100-μl aliquot of human serum containing the target toxins was mixed with 100 ng of acivicin as internal standard (IS), 200 μl of distilled water, and 100 μl of 0.5 % ammonium hydroxide in distilled water, and vortexed well for 10 s. The mixture was loaded on an Oasis MAX 3cc (60 mg) extraction cartridge. The cartridge was washed with 0.5 ml of distilled water and 1.0 ml of methanol. The target compounds and IS were eluted with 4 ml of 0.05 % trifluoroacetic acid in methanol. The eluate was evaporated to dryness and reconstituted in methanol, and its small volume was subjected to LC–MS–MS analysis with the same TSK-GEL Amide-80 separation column. The LC elution was made in the gradient and isocratic modes. The selected reaction monitoring chromatograms showed clear peaks at 5.3, 3.5, and 3.6 min for ibotenic acid, muscimol, and IS, respectively; the blank serum sample without the target compounds or IS gave no peaks at the respective retention times except for an impurity peak at 6.5 min. There was good linearity from 10 to 1,000 ng/ml for both ibotenic acid and muscimol with correlation coefficients not

A rapid screening method was developed for the determination of phenothiazines by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF–MS). In this method, α-cyano-4-hydroxy cinnamic acid (CHCA) was used as the matrix to assist the ionization of phenothiazines. The identification of 11 phenothiazines with heavy side chains was achieved by observing their protonated molecular ions [M+H]+ at m/z 340–447. Quantification was achieved by using triflupromazine at m/z 353 as the internal standard (IS). The relative ionization efficiencies of 11 phenothiazines and IS on MALDI-TOF–MS were different from those achieved by ESI-MS, but the product ion spectra from MALDI-MS–MS were quite similar to those from ESI-MS–MS except in the case of flupentixol. The limit of detection was 0.3 ng/ml with a quantification range of 1–50 ng/ml urine in the best case; the limit of detection was 5 ng/ml with a quantification range of 10–100 ng/ml urine in the worst case for 10 phenothiazines except thiethylperazine. The present method provides a simple and high-throughput method for the screening of these phenothiazines using only 20 μl of urine. To our knowledge, this study is the first trial to analyze phenothiazines by MALDI-TOF–MS (–MS).

A rapid and sensitive method was developed for simultaneous determination of perchlorate (ClO4 −), thiocyanate (SCN−), and iodide (I−). The ions were reacted with tris(1,10-phenanthroline)iron(II) chelating cation, [Fe(phen)3]2+, to produce their ternary complexes, [Fe(phen)2ClO4]+, [Fe(phen)2SCN]+, and [Fe(phen)2I]+ that were extracted together with nitrobenzene. Five microliters of the extract was injected directly into an electrospray ionization tandem mass spectrometer. Quantification of ClO4 −, SCN−, and I− was performed by selected reaction monitoring of the product ion [Fe(phen)O2]+ at m/z 268 that derived from the precursor ion [Fe(phen)2ClO4]+ at m/z 515, the product ion [Fe(phen)SCN]+ at m/z 294 that derived from precursor ion [Fe(phen)2SCN]+ at m/z 474, and the product ion [Fe(phen)I]+ at m/z 363 that derived from precursor ion [Fe(phen)2I]+ at m/z 543, respectively. Perchlorate, SCN−, and I− were measured in the range of 0.1–10 μM with the limits of detection at 0.03 μM by using a 50-μl sample aliquot containing the three ions within 10 min. The SCN− level in gastric fluid of a victim of NaCN poisoning was 137 ± 23 μM, which is slightly higher than the results from control subjects (n = 7), 72 ± 39 μM.

We have established the most modern method for analysis of ibotenic acid and muscimol in toxic Amanita mushrooms by liquid chromatography–tandem mass spectrometry (LC–MS–MS). Acivicin, an ibotenic acid analog and antitumor agent, was used as internal standard (IS). A target mushroom sample was homogenized in water/methanol (1:1) and mixed with a fixed concentration of IS. The target compounds and IS were purified with an Oasis MAX 3cc (60 mg) extraction cartridge. The eluate was subjected to hydrophilic interaction (TSK-GEL Amide-80 3 μm, 150 × 2.0 mm i.d. column) LC–MS–MS. The quantitation was made by multiple reaction monitoring (MRM). The ion transitions were: m/z 179→133.1 for IS, m/z 159→113.1 for ibotenic acid, and m/z 115→98.1 for muscimol. The elution was made in the gradient mode with 0.5 % formic acid aqueous solution (A) and 0.5 % formic acid in acetonitrile (B) from 90 % B to 80 % B in 1.85 min, and then in the isocratic elution mode with 20 % A/80 % B up to 10 min. The MRM chromatograms gave clear and symmetrical peaks for ibotenic acid, muscimol, and IS. Their recovery rates were 84.6–107 %. There was good linearity from 10 to 500 μg/g for both ibotenic acid and muscimol with correlation coefficients not

A rapid and sensitive method was developed for analysis of α-amanitin, β-amanitin, and phalloidin by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). In this method, α-cyano-4-hydroxy cinnamic acid was used as the matrix to assist the ionization of toxins. The identification of α-amanitin, β-amanitin, and phalloidin was achieved through their sodium adducts [M+Na]+ at m/z = 941, 942, and 811, and quantification of the three toxins was also achieved using microcystin RR at m/z = 1038 as internal standard. For all toxins, the limit of detection was 5 ng/ml, and all calibration curves were linear in the range of 10–500 ng/ml using 0.4 ml of urine. The sensitivity for identification was increased about tenfold when the tandem MS (MS–MS) mode was used for detection. Because these quantifications could be achieved in the toxin concentration range of 4–200 ng, the present MALDI-TOF MS method can serve as the most sensitive method so far reported for the analysis of these mushroom toxins. To our knowledge, this study is the first trial to analyze amanitins and phalloidin by MALDI-TOF MS (-MS).

A rapid and sensitive mass spectrometric method was developed for the simultaneous determination of the coinage metals of copper (Cu), silver (Ag), and gold (Au). The metals in a wet-ashed tissue solution were complexed with diethyldithiocarbamate (DDC; C4H10NCS2) and were extracted with isoamyl alcohol. After acidification of the extract with oxalic acid, metals were quantified using their product ions, Cu(DDCH)+, Ag(DDCH)+, and Au(DDCH)+ that derived from the precursor ions Cu(DDC) 2 + , Ag(DDC) 2 + , and Au(DDC) 2 + , respectively, by electrospray ionization tandem mass spectrometry. The limits of detection were 0.6, 0.3, and 1 μg/l for Cu, Ag, and Au, and the quantification ranges were 2–100, 1–100, and 3–100 μg/l for Cu, Ag, and Au, respectively. Cu levels in spontaneously hypertensive osteogenic disorder rats at 6 weeks of age and 30 weeks of age were found to be 1.8 times and 5.1 times those of the normotensive osteogenic disorder rats, respectively, when using wet-ashed kidney solutions diluted 1000-fold.

A rapid method was developed to identify and quantify the azide ion (N3 –) in gastric fluid and urine. N3 – in diluted biological fluids was reacted with NaAuCl4 to produce Au(N3)2 –, which was extracted with octanol. Five microliters of the extract were flow-injected into an electrospray ionization tandem mass spectrometric instrument. Quantification of N3 – was performed by selected reaction monitoring of the product ion Au(N)(N3) – at m/z 253, which was derived from the precursor ion Au(N3)2 – at m/z 281, using 50 μL of aqueous solution within 10 min. This method was found to be linear up to 10–5 M, to have a limit of quantification of 10–7 M, a limit of detection of 3.0 × 10–8 M, and a coefficient of variation of ≦10% at 10–7 M. In the case of urine, 50 μL of urine were spiked with N3 –, this was diluted 10-fold and passed through 1 mL of a resin, and finally diluted to 100-fold of the original. This method was linear up to 10–3 M, had a limit of quantification of 10–5 M, a limit of detection of 3.0 × 10–6 M, and coefficient of variation of ≦8.8% for an original urine concentration of 10–5 M. The practical applicability of this method was checked by diluting 1 μL of a suspected suicide victim’s gastric fluid 20,000-fold and 1 μL of the victim’s urine 5,000-fold and then measuring the N3 – levels. These levels were found to be (7.5 ± 1.0) × 10–2 M and (3.2 ± 0.4) × 10–3 M, respectively.

A rapid and reliable method was developed to identify and quantify the thiocyanate ion (SCN−) in blood. SCN− was reacted with NaAuCl4 to produce Au(SCN)2−, which was extracted with octanol. The extract was injected directly into an electrospray ionization tandem mass spectrometer. Quantification of SCN− was performed by selected reaction monitoring of the product ion SCN− at m/z 58 that derived from the Au(SCN)2− precursor ion (m/z 313). SCN− could be measured in the quantification range of 0.05–10 μM in aqueous solution with a limit of detection of 0.013 μM within 15 min. Using only 5 μl of blood, the SCN− level of a victim who ingested sodium cyanide was determined to be 32.7 ± 2.1 μM, indicating a quite small increase from the control level of 15.5 ± 8.7 μM.

Given that there are many autopsy cases in which erectile dysfunction (ED) treatment drugs can be detected from elderly men who are diagnosed to have died of cardiovascular disorders, the degree of cardiovascular risk posed by ED treatment drugs is still controversial. Moreover, counterfeit ED drugs or illegal dietary supplements containing ED drugs are also threats to public health. In this study, we established a detailed procedure for simultaneous analysis of typical ED drugs (sildenafil, vardenafil, tadalafil) and their metabolites in human blood and urine by isotope dilution liquid chromatography-tandem mass spectrometry (LC–MS–MS). Each sample of whole blood and urine containing the three ED treatment drugs, their metabolites, and deuterated internal standards (ISs) was diluted with alkalinized water, loaded onto an Oasis HLB cartridge, washed with dilute ammonium hydroxide solution, and eluted with chloroform. The eluate was acidified with methanol and concentrated HCl and evaporated to dryness. The resulting residue was reconstituted with methanol and mobile phase solution, and 5 μl of the solution was injected into an LC–MS–MS instrument. The determinations were made by multiple reaction monitoring using each product ion. The recovery rates of the drugs, metabolites, and ISs from whole blood and urine ranged from 80.7 to 127%. Good linearity was obtained for all drugs and their metabolites in the range of 1–100 ng/ml in whole blood and urine with correlation coefficients greater than 0.99. The detection limits (signal-to-noise ratio = 3) for all compounds were not higher than 0.05 ng/ml. Intraday and interday accuracy and precision data were also satisfactory for all compounds in whole blood and urine. Actual measurements of sildenafil and N-desmethyl sildenafil were also demonstrated by analysis of whole blood and urine specimens from two male volunteers after ingestion of a 25-mg tablet of sildenafil.

An electrospray ionization tandem mass spectrometric (ESI-MS-MS) method has been developed for the determination of cyanide (CN–) in blood. Five microliters of blood was hemolyzed with 50 μL of water, then 5 μL of 1 M tetramethylammonium hydroxide solution was added to raise the pH of the hemolysate and to liberate CN– from methemoglobin. CN– was then reacted with NaAuCl4 to produce dicyanogold, Au(CN) 2 – , that was extracted with 75 μL of methyl isobutyl ketone. Ten microliters of the extract was injected directly into an ESI-MS-MS instrument and quantification of CN– was performed by selected reaction monitoring of the product ion CN– at m/z 26, derived from the precursor ion Au(CN) 2 – at m/z 249. CN– could be measured in the quantification range of 2.60 to 260 μg/L with the limit of detection at 0.56 μg/L in blood. This method was applied to the analysis of clinical samples and the concentrations of CN– in the blood were as follows: 7.13 ± 2.41 μg/L for six healthy non-smokers, 3.08 ± 1.12 μg/L for six CO gas victims, 730 ± 867 μg for 21 house fire victims, and 3,030 ± 97 μg/L for a victim who ingested NaCN. The increase of CN– in the blood of a victim who ingested NaN3 was confirmed using MS-MS for the first time, and the concentrations of CN– in the blood, gastric content and urine were 78.5 ± 5.5, 11.8 ± 0.5, and 11.4 ± 0.8 μg/L, respectively.

A rapid and sensitive electrospray ionization (ESI) tandem mass spectrometry (MS-MS) procedure was developed for the determination of iodide (I(-)). A gold (Au) and I(-) complex was formed immediately after the addition of the chelating agent NaAuCl(4) to I(-) solution, and was extracted with methyl isobutyl ketone. One to five microliters of the extract were injected directly into an ESI-MS-MS instrument. I(-) quantification was performed by selecting reaction monitoring of the product ion I(-) at m/z 127 derived from the precursor ion (197)AuI(2)(-) at m/z 451. I(-) concentration was measured in the quantification range from 10(-7) to 10(-5) M using 50 microL of solution within 10 min. Iodate was reduced to I(-) with ascorbic acid and determined. I(-) concentration in reference urine 2670a was measured after treatments.

A very simple and sensitive method for the simultaneous analysis of naphthalene and p-dichlorobenzene in human whole blood and urine by headspace capillary gas chromatography-mass spectrometry (GC-MS) is presented. The advantages of the method were that as much as 1 mL of headspace vapor could be injected into a GC port in the splitless mode, and that the addition of deuterated naphthalene and p-dichlorobenzene as internal standards resulted in much better headspace extraction efficiencies, which resulted in high sensitivity. The detection limits for both naphthalene and p-dichlorobenzene were 1 ng mL(-1) for whole blood and 0.5 ng mL(-1) for urine. Validation data, such as the linearity of calibration curves, reproducibility and recovery rates, were all satisfactory. Using this method, both compounds could actually be detected from whole blood samples of a male volunteer after the inhalation of each gas of the compounds.

A high-performance liquid chromatography (HPLC) method has been developed for the assays of five barbiturates in human urine using a new thermoresponsive polymer separation column, which is composed of N-isopropylacrylamide polymer. According to elevating the column temperature from 10 ℃ to 50 ℃, five barbiturates, such as metharbital, primidone, phenobarbital, mephobarbital and pentobarbital, became well separated by this method. Five barbiturates showed good linearity in the range of 0.2-10 mg/ml. Good accuracy, precision and recoveries for these drugs were obtained at 1 and 5 μg/ml urine. The method with the new-type column seems to have high potential to be extensively used in forensic toxicology for analysis of many drugs and poisons by HPLC and HPLC-mass spectrometry (MS) (-MS).

A very simple method for the analysis of methamphetamine and amphetamine in human whole blood and urine by headspace gas chromatography (GC) has been presented. It neither needs solid-phase microextraction nor cryogenic trapping devices, but only a conventional capillary GC instrument with flame ionization detection (FID). The two special points to be mentioned in this method are the in-matrix derivatization of amphetamines for vaporization and the capability of injection of as large as 5 mL of the headspace vapor into a GC instrument in the splitless mode for sensitive detection. After heating a whole blood or urine sample containing amphetamines, α-methylbenzylamine (internal standard, IS) and heptafluoro-n-butyryl chloride under alkaline conditions in a 7.0-mL vial at 90 °C for 20 min, 5 mL of the headspace vapor was drawn with a glass syringe and injected into the gas chromatograph. During injection the column was at 40 °C to trap the analytes, and then the oven temperature was programmed up to 320 °C. Sharp peaks were obtained for each analyte and IS, and only a relatively small number of background impurity peaks for the whole blood and urine samples. The detection limits for each amphetamine were estimated to be 0.1 μg mL−1 for whole blood and 0.03 μg mL−1 for urine. Precision and linearity were also tested to confirm the reliability. Methamphetamine and amphetamine could be determined from whole blood and urine obtained at autopsy in three methamphetamine poisoning cases. The identity of each peak appearing in the gas chromatograms was confirmed by GC/mass spectrometry.

Inorganic arsenite (As3+) and arsenate (As5+) are well-known poisons, and the toxicity of As3+ is about ten times that of As5+. In this study, a simple, rapid, and sensitive method was developed for As3+ in plasma using electrospray ionization (ESI) tandem mass spectrometry (MS-MS). After washing plasma with trichloroethylene (TCE), As3+ in the aqueous layer was reacted with pyrrolidinedithiocarbamate (PDC, C4H8NCSS-), and the produced As(PDC)3 was extracted with methyl isobutyl ketone (MIBK); a 1-µl aliquot of the MIBK layer containing As(PDC)3 was introduced into the MS-MS instrument in the direct-flow injection mode. Other arsenic compounds such as As5+, monomethyl arsonic acid, dimethyl arsinic acid, arsenobetaine, arsenocholine, and tetramethyl arsonium did not produce As(PDC)3. Therefore, without liquid chromatographic separation, As3+ alone could be detected after washing with TCE followed by solvent extraction of As(PDC)3 with MIBK. Thus, inorganic As5+ was reduced to As3+ with thiosulfate, and then the total inorganic As was quantifi ed as As3+; As5+could be calculated by subtracting As3+from the total inorganic As. The MS-MS quantification was performed by selected reaction monitoring using a peak at m/z 114 of a product ion (C4H8NCS)+ formed by collision-induced dissociation from the precursor ion As(PDC)2 + at m/z 367. The mass spectral identification on MS-MS spectrum was possible even at 1 ng As3+/ml plasma. The calibration curve for As3+ showed linearity from 0.5 to 100 ng/ml plasma. The limits of detection by selected reaction monitoring were 0.3 ng/ml in water and 0.2 ng/ml in plasma. The analysis could be completed in less than 15 min, because chromatographic separation was not necessary before the MS-MS detection.

A simple, rapid and sensitive method using tandem mass spectrometry (MS-MS) has been developed for the determination of chromate Cr6+ in urine. Cr6+ is a substantial component of Urine Luck, which is used to conceal the presence of drugs in urine. Cr6+ was complexed with diethyldithiocarbamate (DDC) and extracted with isoamyl alcohol in the presence of citric acid. Then a 1-μl aliquot of isoamyl alcohol containing Cr-DDC complex was directly injected into an MS-MS instrument without chromatographic separation. The quantification was performed using selected reaction monitoring at m/z 363.8 of product ion CrO(DDC)2 + obtained by collision-induced dissociation from the precursor ion, CrOH(DDC)3 + at m/z 513.1. This method was validated with the analysis urine samples obtained from volunteers. A linear calibration curve could be obtained in the range of 0.18–100 ng/ml. The limits of detection and quantification of Cr6+ were 0.05 and 0.18 ng/ml, respectively, using only 10 μl of urine. Results could be obtained in less than 10 min for a sample. After oxidation of Cr3+ to Cr6+, near 100% recovery was confirmed using standard reference materials such as SRM 2670a (low level and high level) and SRM 1643e. The most outstanding advantage of this ESI-MS-MS method is that it gives excellent product ion mass spectra for identification of Cr6+.

A simple, rapid, and sensitive method has been developed for determination of platinum (Pt) in blood and urine by tandem mass spectrometry (MS-MS). Pt4+ in wet-ashed blood or acid-treated urine was complexed with diethyldithiocarbamate (DDC), extracted with isoamyl alcohol, and acidified with oxalic acid; a 1-μl aliquot of the isoamyl alcohol layer containing the Pt-DDC complex was directly injected into the MS-MS instrument without chromatographic separation. The quantitation was performed using selected reaction monitoring at m/z 491 of the product ion Pt(DDC)2+, which was produced by collision-induced dissociation from the precursor ion Pt(DDC)3+ at m/z 639. This method was validated for the analysis of blood and urine samples; the limits of quantitation were about 0.3 and 0.1 ng/ml for blood and urine, respectively, using only 10 μl of sample. The calibration curves for Pt in urine and blood showed linearity from 0.1 to 30 ng/ml. Because chromatographic separation is not required before MS-MS detection, the analysis can be completed in less than 10 min.

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 β-glucuronidase. Among the eight AB-CHMINACA metabolites tested, only 2 metabolites could be identified in the urine specimen of the deceased. After hydrolysis with β-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.