Itaru Yamagishi

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.

Determination of iodide in urine using electrospray ionization tandem mass spectrometry

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.

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.

Sensitive determination of arsenite and arsenate in plasma by electrospray ionization tandem mass spectrometry after chelate formation

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.

Determination of Urine Luck in urine using electrospray ionization tandem mass spectrometry

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+.

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 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

Application of thermoresponsive HPLC to forensic toxicology: determination of barbiturates in human urine

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

Trace analysis of platinum in blood and urine by ESI-MS-MS

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.