Koutaro Hasegawa

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.

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 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.

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.

GC/MS with Post-Column Switching for Large Volume Injection of Headspace Samples: Sensitive Determination of Volatile Organic Compounds in Human Whole Blood and Urine

When volatile or semivolatile compounds are measured by headspace (HS) gas chromatography (GC)/mass spectrometry (MS), the maximum gas volume to be injected is usually 0.5-1.0 mL; over the volume, the MS detector automatically shuts down due to impairment of the vacuum rate of the MS ionization chamber. To overcome the problem, we modified the gas flow routes of a new type of GC/MS instrument to create a postcolumn switching system, which can eliminate the large volume of gas before introduction of target compounds into the MS ionization chamber. Our HS-GC/MS system enabled injection of as large as 5 mL of HS gas without any disturbance. As the first example analysis, we tried to establish the analysis of naphthalene and p-dichlorobenzene in human whole blood and urine by this method with large volume injection. The limits of detection for both compounds in whole blood and urine were as low as about 10 and 5 pg/mL, respectively. The validation data and actual measurements were also demonstrated. The new GC/MS system has great potential to analyze any type of volatile or semivolatile organic compounds in biological matrixes with very high sensitivity and full automation.

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.

Diphenidine and its metabolites in blood and urine analyzed by MALDI-Q-TOF mass spectrometry

New psychoactive substances that are different from synthetic cannabinoids or cathinone derivatives have appeared very recently on illicit drug markets. In 2014, three reports were published on diphenidine distribution in Japan. The first study characterized diphenidine and 1-benzylpiperidine in a powdered product called ‘‘fragrance powder’’ [1], while the second determined diphenidine and 5-fluoro-AB-PINACA in a dubious herbal product [2]. The third study dealt with a fatal case in which a victim smoked a herbal product containing diphenidine together with a herbal product containing AB-CHMINACA and 5-fluoroAMB [3]. The postmortem distributions of these substances determined by gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–tandem mass spectrometry (LC–MS-MS) indicated that the main cause of death was diphenidine poisoning [3]. To date, the toxicity and metabolism of diphenidine have not been studied in any scientific context because it is a research chemical [4] with very little history of use in humans [5]. To tentatively identify unreported molecules, such as metabolites, their exact masses with sensitivities better than 0.001 Da are necessary [6]. Matrix-assisted laser desorption ionization (MALDI) can achieve a softer ionization than electrospray ionization under some conditions, and hence is suitable for the detection of intact molecules. Therefore, in the present work we examined diphenidine and its metabolites by MALDI quadrupole time-of-flight mass spectrometry (Q-TOF-MS) with a resolution better than 0.001 Da. Diphenidine [1-(1,2-diphenyl-ethyl)piperidine]-HCl, acetonitrile (ACN), and ethanol that were suitable for LC– MS, and other chemicals of analytical grade were obtained from Wako Pure Chemicals (Osaka, Japan). a-Cyano-4hydroxycinnamic acid (CHCA) was obtained from SigmaAldrich (St. Louis, MO, USA), and amitriptyline, to be used as internal standard (IS) for diphenidine quantitation, was obtained from Astellas Pharma (Tokyo, Japan). Pure water with a specific resistance of 18 MX cm was used (Millipore, Bedford, MA, USA). Blood samples from healthy volunteers under permission were used as blank samples, and those spiked with several amounts of diphenidine were used as positive samples. The postmortem blood and urine samples of a victim intoxicated mainly with diphenidine were obtained at autopsy performed in our laboratory [3]. Individual stock solutions of diphenidine and amitriptyline were prepared separately by dissolving the appropriate amount of each compound in ethanol at 1 mg/ ml. The solutions were stored at -20 C. Working calibration solutions and quality control solutions were prepared daily by diluting the stock solution of diphenidine with blank blood at 1–100 ng/ml. Amitriptyline at 50 ng/ ml in blood was used as IS for the determination. Diphenidine and its metabolites in a sample were extracted and detected together. One microliter of IS and 59 ll of water were added to 20 ll of blood or urine placed in a tube (Eppendorf AG, Hamburg, Germany) using a MICROMAN M10 pipette (Gilson S.A.S., Villiers le Bel, France), and the mixture was centrifuged at 10,000 g for 3 min. The supernatant (72 ll) was placed in a new tube, & Kayoko Minakata kminakat@hama-med.ac.jp

Human brain microsomes: their abilities to metabolize tetrahydrocannabinols and cannabinol

In spite of the psychedelic action of Δ9-tetrahydrocannabinol (Δ9-THC) in the brain, no report on its metabolism by human brain microsomes has been published. In this study, the metabolism of Δ8-THC, Δ9-THC and cannabinol (CBN) was studied using human brain microsomes. The metabolites formed were analyzed by gas chromatography–mass spectrometry after trimethylsilylation. The three cannabinoids were biotransformed to two main metabolites by human brain microsomes. Δ8- and Δ9-THCs were mainly oxidized at the allylic positions. The main metabolites of Δ8-THC were 7α-hydroxy- and 11-hydroxy-Δ8-THCs, whereas those of Δ9-THC were 8α-hydroxy- and 11-hydroxy-Δ9-THCs. CBN was metabolized to 8-hydroxy- and 11-hydroxy-CBNs. Although the primary metabolic pathways of the THCs and CBN in brain microsomes are different from those in liver microsomes for other mammalian species, those in human brain microsomes were similar to those in human liver microsomes.

Simple analysis of naphthalene in human whole blood and urine by headspace capillary gas chromatography with large-volume injection

A very simple method for analysis of naphthalene in human whole blood and urine by headspace gas chromatography (GC) is presented. It does not require solid-phase microextraction or cryogenic trapping devices, but needs only a conventional capillary GC instrument with flame ionization detection. The advantage of the method is that as much as 5 ml of headspace vapor can be injected into a GC instrument in splitless mode for sensitive detection. After heating a diluted whole blood or urine sample containing naphthalene and 1-methylnaphthalene (internal standard, IS) in a 7.0-ml vial at 80°C for 30 min, 5 ml of the headspace vapor was drawn with a glass syringe and injected into the gas chromatograph. Before injection, the column temperature was set at 50°C to trap the analytes, and then the oven temperature was programmed up to 300°C. Sharp peaks were obtained for both analyte and IS, and only a few impurity peaks appeared, which did not interfere with the test peaks, mainly for whole blood samples. The detection limit (signal-to-noise ratio. 3) were about 0.05 and 0.01 μg/ml for whole blood and urine, respectively. Precision and linearity were also examined to confirm the reliability. Such a simple headspace GC technique with large-volume injection will be applicable to other low-volatility compounds in biological matrices, and will be useful in forensic toxicological analysis.

A double-suicide autopsy case of potassium poisoning by intravenous administration of potassium aspartate after intake of some psychopharmaceuticals

We report a curious double-suicide autopsy case of both male and female who died of potassium poisoning by intravenous administration of concentrated potassium aspartate solution. The plasma concentrations of potassium of the male and female subjects were as high as 49.7 and 62.8 mEq/L, respectively. In addition to the high concentrations of potassium, toxic levels of phenobarbital, promethazine and chlorpromazine, and relatively low levels of etizolam and brotizolam were also detected from whole blood and urine specimens of both cadavers. Twenty empty plastic bottles (10-mL capacity) labeled ‘ASPARA® Potassium Injection 10 mEq’ were found at the suicide spot. To our knowledge, this is the first description for suicidal death by potassium aspartate; in all of the previous literature, they used potassium chloride intravenously or per os.

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 ± 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.