Maiko Kusano

Comparison of the Volatile Organic Compounds from Different Biological Specimens for Profiling Potential

Previous work has demonstrated the ability to differentiate individuals based on the analysis of human scent hand odor chemicals. In this paper, a range of forensic biological specimens are shown to also have the ability to differentiate individuals based upon the volatile organic compounds (VOCs) present. Human VOC profiles from hand odor, oral fluid, breath, blood, and urine of 31 individuals were analyzed by solid‐phase microextraction–gas chromatography–mass spectrometry (SPME‐GC‐MS) and combined methods of chromatogram comparison, Spearman rank correlation comparison, and principal component analysis. Intra‐specimen comparisons demonstrated the distinguishability of individuals above 99%. Inter‐specimen VOC profiles from the same individual were found to be too different to be used for scent‐matching purposes, with Spearman rank coefficients below 0.15. A 6‐month VOC profile monitoring of two individuals demonstrated the consistency of VOC profiles over time across specimens.

Application of metabolomics to toxicology of drugs of abuse: A mini review of metabolomics approach to acute and chronic toxicity studies

Metabolomics has been widely applied to toxicological fields, especially to elucidate the mechanism of action of toxicity. In this review, metabolomics application with focus on the studies of chronic and acute toxicities of drugs of abuse like stimulants, opioids and the recently-distributed designer drugs will be presented in addition to an outline of basic analytical techniques used in metabolomics. Limitation of metabolomics studies and future perspectives will be also provided.

Intact Endogenous Metabolite Analysis of Mice Liver by Probe Electrospray Ionization/Triple Quadrupole Tandem Mass Spectrometry and Its Preliminary Application to in Vivo Real-Time Analysis

Probe electrospray ionization (PESI) is a recently developed ionization technique that enables the direct detection of endogenous compounds like metabolites without sample preparation. In this study, we have demonstrated the first combination use of PESI with triple quadrupole tandem mass spectrometry (MS/MS), which was then applied to intact endogenous metabolite analysis of mice liver, achieving detection of 26 metabolites including amino acids, organic acids, and sugars. To investigate its practicality, metabolic profiles of control and CCl4-induced acute hepatic injury mouse model were measured by the developed method. Results showed clear separation of the two groups in score plots of principal component analysis and identified taurine as the primary contributor to group separation. The results were further validated by the established gas chromatography/MS/MS method, demonstrating the present methods usefulness. In addition, we preliminarily applied the method to real-time analysis of an intact liver of a living mouse. We successfully achieved monitoring of the real-time changes of two tricarboxylic acid cycle intermediates, α-ketoglutaric acid and fumaric acid, in the liver immediately after pyruvic acid injection via a cannulated tube to the portal vein. The present method achieved an intact analysis of metabolites in liver without sample preparation, and it also demonstrates future possibility to establish in vivo real-time metabolome analysis of living animals by PESI/MS/MS.

Regioisomeric differentiation of the alkyl-substituted synthetic cannabinoids JWH-122 and JWH-210 by GC-EI-MS/MS

Synthetic cannabinoids (SCs) are known to have structural or positional isomers. While regulations on synthetic drugs like synthetic cathinones and SCs have been placed worldwide for the ever-growing variety of new designer drugs, laws may not necessarily be applicable to their isomers. Toxicological differences may also exist among isomers for which most new designer drugs are still uninvestigated; thus, isomer differentiation becomes of forensic importance. The aim of this study was to differentiate the regioisomers of alkyl-substituted naphthoylindole-type SCs JWH-122 and JWH-210. Reference standards of the two drugs and their regioisomers were analyzed by gas chromatography–electron ionization-mass spectrometry (GC–EI-MS) first in full scan mode. Isomers that produced identical EI spectra were further analyzed by GC-tandem mass spectrometry (MS/MS) by selecting appropriate precursor ions. For JWH-210, comparison of the product ion spectra and the relative ion intensity ratios obtained from precursor ions at m/z 312 and 183 enabled differentiation between all seven regioisomers. Complete isomeric differentiation by MS/MS analysis was not attainable for JWH-122; however, combining chromatographic results with MS/MS analysis results enabled differentiation for all isomers. Two basic fragmentation pathways were speculated for both SCs; for JWH-210, fragmentation pathway tendencies differed among the isomers, resulting in their distinguishability. Our results demonstrated that the difference between the methyl (JWH-122) and ethyl (JWH-210) group substituents contributed to fragmentation pathway tendency differences and further distinguishability between the regioisomers. Functional group differences, especially their stereochemistries, were indicated to be critical factors in positional isomer differentiation by GC-MS/MS.

Simple and sensitive determination of α- and β-amanitin by liquid chromatography–quadrupole time-of-flight mass spectrometry

Amatoxins, including aand b-amanitins, are highly toxic cyclic octapeptide compounds belonging to the Amanita species; these toxins exert gastroenteric symptoms in the early stage, and lead to hepatic failure and renal damage by inhibiting RNA polymerase activity in hepatocytes [1–3]. The Amanita species are considered to be responsible for more than 90 % of the lethal cases of mushroom toxin poisoning [4, 5]. It is thus important to detect amatoxins with high sensitivity. Recently, liquid chromatography (LC)–mass spectrometry (MS) [6–13] and tandem mass spectrometry (MS/MS) [14–18] or matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI–TOFMS) [19] have been increasingly applied to detect and identify these compounds from body fluids or mushroom specimens. In this Letter, we present a new method for detecting and identifying aand b-amanitin using a novel LC–quadrupole time-of-flight mass spectrometer (Q-TOFMS) with high sensitivity and specificity. Amanitins were purchased from Sigma (St. Louis, MO, USA); other chemicals used were of the highest purity commercially available. Urine samples were collected from a healthy volunteer after obtaining informed consent. Analyses by Q-TOFMS were performed on an AB SCIEX Triple TOF 5600 system (AB SCIEX, Framingham, MA, USA) coupled to a Shimadzu Prominence XR LC system (Shimadzu, Kyoto, Japan). For LC separation, a Scherzo SM-C18 column (2 9 100 mm, particle size 3 lm, Imtakt, Kyoto, Japan) was used; the column temperature was kept at 40 C and the flow rate was 0.5 ml/ min. The gradient program was started at 90 % mobile phase A (5 mM ammonium formate in distilled water) and 10 % mobile phase B (methanol) for 1 min; it was changed linearly to 5 % A and 95 % B over 4 min and maintained with a 2-min hold. The MS conditions were as follows: ionization mode, electrospray ionization (ESI) positive mode; turbo gas temperature, 500 C; spray voltage, 5,000 V. In the MS mode, ions were scanned in the range from m/z 850 to 1,000; the declustering potential (DP) and collision energy (CE) were 80 V and 10 V, respectively. In the MS/MS mode, four conditions for production ion monitoring were settled as follows: precursor ions were 919.4 for a-amanitin and 920.4 for b-amanitin, and product ions were scanned in the range of m/z 200–1,000 (DP, 80 V; CE, 50 V); and precursor ions were 919.4 for a-amanitin and 920.4 for b-amanitin, and product ions were scanned in the range of m/z 900–1,600 (DP, 80 V; CE, 10 V). Measurements in MS and MS/MS modes were employed alternatively, for 100 ms in each condition; the total cycle time was 550 ms. Authentic amanitins were dissolved in methanol or added to purified urine samples to assess the sensitivity of the instrument and evaluate matrix effects. In the samples diluted in methanol, the concentrations of both amanitins were in the range of 0.001–5 ng/ll. In urine samples, 200 ll of urine was mixed with 100 ll of acetonitrile and centrifuged at 5,000 rpm for 10 min. The supernatant was mixed with 700 ll of distilled water and A. Ishii (&) M. Kusano K. Zaitsu Department of Legal Medicine and Bioethics, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan e-mail: akishii@med.nagoya-u.ac.jp

Laser Desorption/Ionization Mass Spectrometry (LDI-MS) of Lipids with Iron Oxide Nanoparticle-Coated Targets.

Iron oxide nanoparticle (NP)-coated target plates were employed for the direct detection and analysis of low molecular weight lipids by laser desorption/ionization (LDI) mass spectrometry (MS). We have demonstrated that the use of the iron oxide NP-coated target provides a simple, direct, and rapid detection method for lipid standards and epidermal surface lipids without any cumbersome sample pretreatment as well as mass spectra that are free of background matrix peaks. Lipid standards (1-stearoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycerol, 1-palmitoyl-2-oleoyl-3-linoleoyl-rac-glycerol, 1,2-distearoyl-sn-glycero-3-phosphocholine) were detected as either protonated or cationated species. Clean MS/MS spectra for each lipid were also successfully obtained. Pre-MS surface cleaning of the target plates with UV-ozone treatment successfully removed organic contaminants that would interfere with the mass spectra especially in the low molecular weight region. Preliminary application of the presented target plate to the detection of endogenous lipids in latent fingerprints showed promising results and for potential use in the visualization and chemical composition determination of latent fingerprints by nanoparticle assistance.

Development of a mass spectrometric hydroxyl-position determination method for the hydroxyindole metabolites of JWH-018 by GC-MS/MS.

One of the many issues of designer drugs of abuse like synthetic cannabinoids (SCs) such as JWH-018 is that details on their metabolism has yet to be fully elucidated and that multiple metabolites exist. The presence of isomeric compounds poses further challenges in their identification. Our group has previously shown the effectiveness of gas chromatography-electron ionization-tandem mass spectrometry (GC-EI-MS/MS) in the mass spectrometric differentiation of the positional isomers of the naphthoylindole-type SC JWH-081, and speculated that the same approach could be used for the metabolite isomers. Using JWH-018 as a model SC, the aim of this study was to differentiate the positional isomers of its hydroxyindole metabolites by GC-MS/MS. Standard compounds of JWH-018 and its hydroxyindole metabolite positional isomers were first analyzed by GC-EI-MS in full scan mode, which was only able to differentiate the 4-hydroxyindole isomer. Further GC-MS/MS analysis was performed by selecting m/z 302 as the precursor ion. All four isomers produced characteristic product ions that enabled the differentiation between them. Using these ions, MRM analysis was performed on the urine of JWH-018 administered mice and determined the hydroxyl positions to be at the 6-position on the indole ring. GC-EI-MS/MS allowed for the regioisomeric differentiation of the hydroxyindole metabolite isomers of JWH-018. Furthermore, analysis of the fragmentation patterns suggests that the present method has high potential to be extended to hydroxyindole metabolites of other naphthoylindole type SCs in identifying the position of the hydroxyl group on the indole ring. Copyright

Identification and quantitation of mifepristone and its N-demethyl metabolite in the plasma of an aborted fetus by liquid chromatography-quadrupole-time-of-flight-mass spectrometry (LC-Q-TOFMS) and ultra-performance liquid chromatography- tandem mass spectrometry (UPLC-MS-MS)

Mifepristone, also known as RU 486, is a derivative of norethindrone, a synthetic 19-nor-steroid; it acts as an antagonist to progestational and glucocorticoid functions by its binding to progesterone as well as glucocorticoid receptors [1]. Mifepristone is useful for terminating early pregnancy and for emergency contraception [1–4]. Mifepristone is approved in the US, EU countries, Israel, and China, but is not approved in Japan. In Japan, mifepristone has been ‘privately’ or secretly imported; the Ministry of Health, Labour and Welfare warned about its private import in 2004 because of possible complications, e.g., massive hemorrhaging [5]. Despite the warning, it has still been imported and used. It is, thus, important to detect mifepristone and its metabolites with high sensitivity. Several analytical methods have been reported for the measurement of mifepristone, such as the radioimmunoassay [6, 7], the radioreceptor assay [8, 9], voltammetry [10] and high performance liquid chromatography (LC) with an ultraviolet detector [11–17]. Recently, LC– tandem mass spectrometry (MS/MS) [18, 19] has been applied to detect and identify mifepristone and its metabolites from body fluids. In this Letter, we describe the determination of mifepristone and its metabolite in a plasma sample of an aborted fetus using a novel LC–quadrupole–time-of-flight– mass spectrometer (Q–TOFMS) with high sensitivity and specificity and an ultra-performance liquid chromatography–tandem mass spectrometer (UPLC–MS–MS). A woman in her twenties consulted an ER due to her poor health conditions; she then confessed her delivery and that she discarded the dead fetus. She also admitted that she had administered mifepristone and misoprostol prior to her delivery, although the time of administration and the amounts of the compounds were not clear. Around one day after delivery, autopsy of the fetus was performed. Autopsy findings were as follows: a male fetus whose height and weight were 30 cm and 505 g, respectively. No apparent injuries or malformations were observed. A cephalic hematoma-like finding was obtained. The lungs seemed liver-like, although histopathological observations detected some alveolar enlargement. Based on the above findings, the possibility that the fetus had been alive at the delivery was not fully excluded. The fetus was estimated to be at approximately 20–24 weeks gestation. Mifepristone and misoprostol were purchased from Sigma (St. Louis, MO, USA); N-demethyl mifepristone and alfaxalone were from Toronto Research Chemicals (Ontario, Canada) and Tocris (Bristol, UK), respectively. Drug free plasma was purchased from Tennessee Blood Services (Memphis, TN, USA). Other chemicals used were of the highest purity commercially available. A plasma specimen was collected from the femoral blood of the fetus. Mifepristone and its metabolites were analyzed based on the method by Homer et al. [19] with appropriate modifications. Screening of mifepristone and quantitation of the drug and its metabolites by UPLC–MS–MS were employed on an ACQUITY UPLC-TQD system (Waters, & Akira Ishii akishii@med.nagoya-u.ac.jp

GAS CHROMATOGRAPHY | Forensic Applications

Gas Chromatography (GC) is one of the most widely used techniques in forensic science. Major applications include identification of drugs, toxicology, trace analysis, arson and explosion investigations and environmental forensics. Extraction procedures are commonly employed prior to GC analysis for preconcentration of analytes and to remove extraneous matrix interferences. Following extraction and GC separation, a variety of detectors are employed. However, the most important detector in forensic science is the mass spectrometer (MS) used in the electron impact (EI) mode. The purpose of this article is to review the major applications of GC in forensic science.

Metabolome analysis of the serotonin syndrome rat model: Abnormal muscular contraction is related to metabolic alterations and hyper-thermogenesis

Aims: Serotonin syndrome (SS) is an adverse outcome of selective serotonin reuptake inhibitors, though its mechanism is not understood and there is no specific clinical biomarker. In this article, metabolic profiles of the SS model rats and causes of metabolome disruption were investigated. Main methods: Gas chromatography‐tandem mass spectrometry (GC/MS/MS)‐based metabolomics, clinical biomarker measurements and qRT‐PCR analysis for UCP‐3 in skeletal muscles were performed. Key findings: Metabolome analysis demonstrated that 55, 22, 49 and 41 of those were significantly altered in plasma, liver, gastrocnemius muscle, and trapezius, respectively. In particular, lactic acid significantly accumulated in the gastrocnemius muscle of the model, while the branched chain amino acids were not consumed in the trapezius, suggesting site differences in abnormal muscular contractions in the model. This result was supported by UCP‐3 expression analysis. Alteration of the urea cycle was observed in the liver of the model, attributed mainly to catabolism of proteins and/or amino acids from excess skeletal muscle activity, which was supported by plasma BUN: BUN levels in the model were significantly higher than those in the control. In contrast, almost all metabolites including amino acids and TCA‐cycle intermediates significantly increased in plasma of the model, suggesting these were not consumed in some parts of the muscle due to acceleration of anaerobic respiration. Significance: Metabolic profiling revealed that abnormal muscular contractions occurred in specific skeletal muscles and enhanced energy production by up‐regulation of anaerobic respiration, followed by excess expression of UCP‐3, which contributes to the hyper‐thermogenesis observed in the SS model.