Search

Purpose The present study aims to develop a forensic toxicological library to identify 56 natural toxic substances by liquid chromatography–quadrupole time-of-flight tandem mass spectrometry (LC–QTOF-MS/MS). Methods For setting up the library of product ion spectra, individual substances (31 plant toxins, 7 mushroom toxins, 5 marine toxins, 5 frog venoms, 4 mycotoxins, and 4 substances derived from plants) were analyzed by LC–QTOF-MS/MS with positive and negative ionization. The product ion spectra were acquired at the collision energies (CEs) of 20, 35, and 50 eV in single enhanced product ion mode and then in collision energy spread mode in which the CE ramp range was set to 35 ± 15 eV. Results To test the performance of the library, human blood plasma samples were spiked with a mixture of lycorine and domoic acid, extracted by acetonitrile deproteinization and analyzed by LC–QTOF-MS/MS. Identification by our library search could be achieved for these toxins at the purity scores of 79.1 and 67.2, respectively. The method was also applied to postmortem blood from a death case with an aconite intake, and showed that four toxins in an aconite could be identified in the blood sample at the purity scores of 54.6–60.3. Conclusions This library will be more effective for the screening of natural toxic substances in routine forensic toxicological analysis. To our knowledge, there are no reports dealing with development of library for natural toxic substances by LC–QTOF-MS/MS.

Because the number of studies on ambient ionization techniques and their applications continues to increase, especially with respect to the life sciences, a systematic understanding of numerous emerging ambient ionization techniques becomes necessary. In this chapter, we briefly outline ambient ionization techniques to improve readability for readers. First, we discuss the definition of ambient ionization by referring to previously reported papers on this topic and then classify numerous ambient ionization methods into six groups on the basis of the principle of each method, as follows: (1) spray desorption/ionization-based methods; (2) laser ablation/desorption-based methods; (3) thermal desorption-based methods; (4) plasma-based methods; (5) substrate-based methods; and (6) hybrid/other methods. According to such classification, we briefly explain the mechanisms and features of the representative methods in each group. We lastly present the objective of this book and provide a brief explanation of each chapter; additionally, we introduce some useful review articles on ambient ionization techniques for reference.

In this chapter, the principle of probe electrospray ionization (PESI) and its applications in the life sciences are introduced. In the first section, the unique nature of PESI and the development of commercially available instruments supplied by Shimadzu Corporation are briefly explained. PESI was developed by Hiraoka et al. in 2007, where an ultrathin needle probe (the tip diameter is ordinarily under 1 μm) was used as a sampling and ionization unit. Owing to such a thin needle probe, PESI achieves low-invasive sampling from a microscopic area, which, in fact, leads to various applications of PESI. In the second section, applications that use PESI, especially the combinational use of PESI with highly selective mass spectrometers such as tandem mass spectrometry or high-resolution mass spectrometry are introduced. In particular, previously reported studies on single-cell analysis, real-time monitoring of a living mouse, imaging mass spectrometry, and forensic applications are reviewed. Following the above contents, other ambient ionization techniques similar to PESI, such as wooden-tip ESI and robotic surface analysis mass spectrometry (RoSA-MS), are introduced, and future perspectives of PESI are discussed.

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.

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 © 2016 John Wiley & Sons, Ltd.

Recent improvements in ambient ionization techniques combined with mass spectrometry has enabled to achieve real-time monitoring of analytes of interest, even for biogenic molecules in living animals. Here, we demonstrate a newly developed system for in vivo real-time monitoring of metabolites in a living mouse brain. It consists of a semiautomated manipulation system and a unique probe electrospray ionization unit, which uses an extremely thin solid needle (tip dia.: 700 nm) for direct sampling and ionization, coupled to a conventional tandem mass spectrometer. The system successfully monitored 8 cerebrum metabolites related to central energy metabolism in an isoflurane-anesthetized mouse in real time with a 20 s interval. Moreover, our system succeeded in capturing dynamics of energy metabolism in a mouse administered with cannabinoid type-1 receptor agonist, which is known to disrupt cerebrum energy metabolism. The present system now opens the door to the next stage of cutting-edge technique in achieving in vivo real-time monitoring.

High-resolution mass spectrometry and accurate mass measurement by liquid chromatography/quadrupole-time of flight mass spectrometry (LC/Q-TOFMS) was applied to postmortem plasma and urine specimens from an autopsy of a fatal case involving synthetic cannabinoid use, resulting in the detection of three synthetic cannabinoids: MAM-2201, AM-1220, and AM-2232. We searched for their metabolites existing in postmortem plasma or urine by LC/Q-TOFMS and were able to detect N-dealkylated metabolites, defluorinated and further oxidized metabolites of MAM-2201, and some hydroxylated metabolites. Postmortem plasma concentrations of the parent drugs, N-dealkylated metabolites, and fluorinated and further oxidized metabolites of MAM-2201 were measured, and quantitation results revealed site differences between heart and femoral postmortem plasma concentrations of parent drugs and some metabolites, suggesting postmortem redistribution of the synthetic cannabinoids and their metabolites. Quantitation results suggest that defluorination is a major metabolic pathway for MAM-2201, and N-dealkylation is a common but minor pathway for the naphthoylindole-type synthetic cannabinoids in human.

Like many new designer drugs of abuse, synthetic cannabinoids (SC) have structural or positional isomers which may or may not all be regulated under law. Differences in acute toxicity may exist between isomers which impose further burden in the fields of forensic toxicology, medicine and legislation. Isomer differentiation therefore becomes crucial from these standpoints as new designer drugs continuously emerge with just minor positional modifications to their preexisting analogs. The aim of this study was to differentiate the positional isomers of JWH-081. Purchased standard compounds of JWH-081 and its positional isomers were analyzed by gas chromatography-electron ionization-mass spectrometry (GC-EI-MS) first in scan mode to investigate those isomers who could be differentiated by EI scan spectra. Isomers with identical or near-identical EI spectra were further subjected to GC-tandem mass spectrometry (MS/MS) analysis with appropriate precursor ions. EI scan was able to distinguish 3 of the 7 isomers: 2-methoxy, 7-methoxy and 8-methoxy. The remaining isomers exhibited near-identical spectra; hence, MS/MS was performed by selecting m/z 185 and 157 as precursor ions. 3-Methoxy and 5-methoxy isomers produced characteristic product ions that enabled the differentiation between them. Product ion spectrum of 6-methoxy isomer resembled that of JWH-081; however, the relative ion intensities were clearly different from one another. The combination of EI scan and MS/MS allowed for the regioisomeric differentiation of the targeted compounds in this study.

A new high-throughput method was developed for analysis of valproate in human plasma samples by QuEChERS extraction and gas chromatography–tandem mass spectrometry (GC-MS/MS). Plasma samples (0.2 ml) spiked with valproate and secobarbital- d 5 (internal standard) were diluted with 1.3 ml of distilled water. Acetonitrile (1 ml) was added followed by 0.4 g MgSO 4 and 0.1 g NaOAC. After a centrifugation step (2000 g for 10 min), 1 ml of the supernatant was transferred to a dispersive-solid phase extraction (dSPE) tube containing 150 mg MgSO 4 and 50 mg C 18 . This mixture was vortexed and centrifuged at 3000 g for 5 min, and then the upper layer was evaporated to dryness under a stream of nitrogen. The residue was dissolved in 40 μl ethyl acetate, and a 1-μl aliquot was injected into the GC-MS/MS. The GC separation of the compounds was achieved on a fused-silica capillary column Rxi-5Sil MS (30 m × 0.25 mm i.d.; 0.25-µm film thickness) and detected by MS/MS operating in electron ionization ion source mode. The regression equations showed excellent linearity ( r > 0.9997) from 50 to 5000 ng/ml for plasma, with limit of detection of 10 ng/ml. The extraction efficiency of valproate for plasma ranged between 71.2%–103.5%. The coefficient of variation was

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

Urinary metabolites of α-pyrrolidinovalerophenone (α-PVP) in humans were investigated by analyzing urine specimens obtained from abusers. Unambiguous identification and accurate quantification of major metabolites were realized using gas chromatography–mass spectrometry and liquid chromatography-tandem mass spectrometry with newly synthesized authentic standards. Two major metabolic pathways were revealed: (1) the reduction of the β-keto moiety to 1-phenyl-2-(pyrrolidin-1-yl)pentan-1-ol (OH-α-PVP, diastereomers) partly followed by conjugation to its glucuronide, and (2) the oxidation at the 2″-position of the pyrrolidine ring to α-(2″-oxo-pyrrolidino)valerophenone (2″-oxo-α-PVP) via the putative intermediate α-(2″-hydroxypyrrolidino)valerophenone (2″-OH-α-PVP). Of the metabolites retaining the structural characteristics of the parent drug, OH-α-PVP was most abundant in most of the specimens examined.

A high-throughput method for analysis of ramelteon, agomelatine, and melatonin in human plasma by ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS–MS) is presented. The LC system, MS–MS system, and separation column used were Waters Acquity UPLC, Acquity TQD, and Poroshell 120 EC-C18, respectively. For extraction of the target compounds, solid-phase extraction was performed with Oasis HLB cartridges. All compounds were detected with retention times of

Mass spectrometric differentiation of the six isomers of mono-methoxyethylamphetamines (MeO-EAs) and mono-methoxydimethylamphetamines (MeO-DMAs) by gas chromatography–electron ionization–tandem mass spectrometry (GC–EI–MS–MS) was investigated. Based on their EI-mass spectra, the fragment ions at m/z 121 and 72 were selected as precursor ions for their regioisomeric and structurally isomeric differentiation, respectively. Collision-induced dissociation provides intensity differences in product ions among the isomers, enabling mass spectrometric differentiation of the isomers. Furthermore, high reproducibility of the product ion spectra at the optimized collision energy was confirmed, demonstrating the reliability of the method. To our knowledge, this is the first report on mass spectrometric differentiation of the six isomers of MeO-EAs and MeO-DMAs by GC–EI–MS–MS. Isomeric differentiation by GC–EI–MS–MS has a high potential to discriminate isomers of newly encountered designer drugs, making GC–MS–MS a powerful tool in the forensic toxicology field.

β-Keto derivatives of 3,4-methylenedioxyphenylalkylamines (bk-MDPAs), especially 2-methylamino-1-(3,4-methylenedioxyphenyl)propan-1-one (methylone), 2-methylamino-1-(3,4-methylenedioxyphenyl)butan-1-one (bk-MBDB), and 2-ethylamino-1-(3,4-methylenedioxyphenyl)propan-1-one (bk-MDEA), are abused as substitutes for 3,4-methylenedioxyphenylalkylamines in some countries, causing increased social problems. With the widespread abuse of bk-MDPAs, the analysis of bk-MDPAs and their metabolites in human specimens is quite important in forensic and clinical toxicology. In this review, the metabolisms of bk-MDPAs and simultaneous analytical methods for bk-MDPAs and their metabolites by gas chromatography–mass spectrometry, liquid chromatography–mass spectrometry, and liquid chromatography–tandem mass spectrometry are presented.

A new approach is described for imaging mass spectrometry (IMS) of methamphetamine (MA) incorporated into human hair using matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF) and MALDI-Fourier transform ion cyclotron resonance (FTICR). A longitudinal section of a lengthwise manually-cut single human hair shaft from a chronic MA user was directly analyzed by MALDI-TOF-IMS after deposited with α-Cyano-4-hydroxycinnamic acid matrix. A barcode-like image, which was most probably generated with repeated intakes of MA, was for the first time obtained by monitoring MA-specific product ion in the selected reaction monitoring mode. Laser beam scan lengthwise-cut hair shafts gave only poor mass spectra of MA, probably due to the loss of MA and/or the thermal denaturation of hair. The identity of MA detected in hair was further confirmed by MALDI-FTICR mass spectrometry. A combination with ultra-high resolution mass spectrometry by FTICR provided indisputable identification of MA. The MALDI-FTICR-IMS of another hair shaft from the same MA user also provided a barcode-like image by monitoring the protonated molecule of MA with ultra-high resolution. The two barcode-like images exhibited a close resemblance. Thus, MALDI-IMS can offer a new perspective: 'imaging hair analyses for drugs'.

Comprehensive analytical method to identify 11 kinds of synthetic cannabinoids has been investigated by thin layer chromatography (TLC), gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). The analytes used in this study have already been detected from various herbal-type designer drugs: 8 kinds of aminoalkylindoles (AAIs) (JWH-015, JWH-018, JWH-073, JWH-081, JWH-200, JWH-250, JWH-251 and JWH-398), two kinds of cyclohexylphenols (CPs) (CP 47,497 and Cannabicyclohexanol), and a Δ9-tetrahydrocannabinol analog (HU-210). Although specific color changes were observed for the cannabinoids using Marquis reagent, identification of each analyte based on Rf values was difficult to be obtained by TLC. On the other hand, GC/MS and LC/MS/MS were appropriate for their qualitative analyses because of their chromatographic and mass spectral differentiation. A semi-polar capillary column DB-5MS showed the best separation and retention properties of the targeted cannabinoids among the tested GC column phases. Also, characteristic fragment ions were observed in each electron ionization-mass spectrum. The observed fragment ions were mainly derived from α-cleavage of ketone and α-cleavage of amine for AAIs, simple cleavage for CPs, and McLafferty rearrangements for HU-210. Based on the ionization efficiency of the target analytes using LC/MS/MS, electrospray ionization positive mode was selected for AAIs, and negative mode for CPs and HU-210. All analytes were completely separated by gradient elution of ammonium formate aqueous solution-acetonitrile mobile phase on a C18 (ODS) separation column. In addition, characteristic fragment ions were observed in product ion spectra of AAIs and second generation product ion spectra of CPs and HU-210, enabling reliable confirmation. These results provide useful information not only for simultaneous analyses of the targeted cannabinoids but also for structural assignment of future cannabimimetic compounds that may appear in the illicit drug market.

5-Methoxy-N,N-dialkyltryptamines are tryptamine derivatives that possess strong hallucinogenic effects. Because of their escalating popularity and potent physiological effects, an increasing number of acute poisoning cases have been reported in various countries. For their metabolism in humans, only a few studies have been reported. Thus, based on previous studies, the authors forecasted and synthesized authentic standards of their expected metabolites, which retained the structural characteristics of the parent drugs. Using these authentic standards, several urine specimens from abusers and rats were analyzed by gas chromatography mass spectrometry, high-performance liquid chromatography mass spectrometry, and high-performance liquid chromatography tandem mass spectrometry. The present study reveals that four metabolic pathways of great quantitative significance for 5-Methoxy-N,N-dialkyltryptamines to four characteristic metabolites, which retain structural characteristics identifiable with the parent compound, exist in humans and rats. The finding in the present study will be of great importance in the study on the metabolism of other psychotomimetic tryptamine-derived drugs of abuse and in forensic toxicologic analyses.

Six novel hallucinogens classed as (tetrahydrobenzodifuranyl) aminoalkanes or (benzodifuranyl)aminoalkanes, which are known by the common names of “FLY” and “DragonFLY,” respectively, were synthesized. These compounds were simultaneously analyzed by gas chromatography (GC)-mass spectrometry (MS), liquid chromatography (LC)-MS, and LC-MS-MS. GCMS analysis of their free bases was not satisfactory for both mass spectral and chromatographic measurements, and thus trifluoroacetyl (TFA) derivatization was employed. However, it was found that the usual TFA derivatization procedure using trifluoroacetic anhydride caused dehydrogenation of FLYs to the corresponding DragonFLYs. Therefore, TFA derivatization of FLYs was reinvestigated; the presence of triethylamine could almost inhibit such dehydrogenation. LC separation of the analytes was successfully achieved by using a phenyl-type semimicro column with methanol gradient elution, while 1-(8-bromo-2,3,6,7-tetrahydrobenzo[1,2-b;4,5-b′]difuran-4-yl)-2-methylaminopropane (N-methyl-DOB-FLY) and 1-(8-bromo-2,3,6,7-tetrahydrobenzo[1,2-b;4,5-b′]difuran-4-yl)-2-aminopropane (DOB-FLY) were not separated on an octadecylsilica (ODS)-type column. Specific product ion spectra for all compounds were also obtained using LC-MS-MS, which enabled sensitive and reliable identification.

Very recently, β-keto derivatives of 3,4-methylenedioxyamphetamines (MDAs) have appeared on the illicit drug market. In the present study, we synthesized three isomers of β-keto derivatives of MDAs, 2-methylamino-1-(3,4-methylenedioxyphenyl)butan-1-one (bk-MBDB), 2-ethylamino-1-(3,4-methylenedioxyphenyl) propan-1-one (bk-MDEA), and 2-dimethylamino-1-(3,4-methylenedioxyphenyl)propan-1-one (bk-MDDMA), and measured their electron ionization mass spectra without and with trifluoroacetyl (TFA) derivatization using gas chromatography-mass spectrometry (GC-MS). Although the spectral profiles of the three isomers were very similar to each other in both the free and TFA-derivatized forms, there were characteristic peaks at m/z 44 and 140, for bk-MDEA without and with TFA derivatization, respectively; a peak at m/z 110 for bk-MBDB-TFA was also characteristic. These peaks are useful for discrimination of an isomer from others. All isomers could be well separated in both free and TFA-derivatized forms using a slightly polar fused-silica capillary GC column DB-5MS. The present data are likely to be very useful for actual identification and quantitation of β-keto analogues of MDAs by GC-MS, because abuse of these materials is expected to spread worldwide in the near future.

The urinary concentrations of the main metabolites of 3,4-methylenedioxymethamphetamine (MDMA; Ecstasy), specifically 4-hydroxy-3-methoxymethamphetamine sulfate (HMMA-Sul) and 4-hydroxy-3-methoxymethamphetamine glucuronide (HMMA-Glu), have been directly measured in both MDMA users and rats by an established liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) procedure. The concentrations of these conjugates in urine from MDMA users (n = 25) ranged from 6.5 to 202 microM (from 1.8 to 55.6 microg ml(-1)) for HMMA-Sul and from 1.3 to 87.0 microM (from 0.5 to 32.3 microg ml(-1)) for HMMA-Glu, and the ratio of HMMA-Sul to HMMA-Glu ranged from 1.6 to 9.9 (3.1 +/- 1.8). These results demonstrate that the sulfation is quantitatively more significant than the glucuronidation for HMMA in humans. In rats, in contrast, almost all the conjugated HMMA (>99%) was excreted as the glucuronide. These findings indicate that hydrolysis should be carefully made in urine analysis by gas chromatography (GC) or gas chromatography-mass spectrometry (GC-MS) by using either an acid or an enzyme possessing both sulfatase and beta-glucuronidase activities. It is concluded that a considerable interspecies variation exists in the conjugation of HMMA between humans and rats.

In order to prove the intake of a newly encountered designer drug, Methylone, a sensitive and useful method, which allows us to simultaneously detect Methylone and its metabolites in human urine, has been established by means of a combination of gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography-electrospray ionization mass spectrometry (LC-ESI MS). GC-MS accompanied with trifluoroacetyl (TFA) derivatization and LC-MS analyses were performed following acid hydrolysis and liquid-liquid extraction with a chloroform-2-propanol mixture (3:1, v/v). Methylone and its metabolites, 3,4-methylenedioxycathinone (MDC), 4-hydroxy-3-methoxymethcathinone (HMMC) and 3-hydroxy-4-methoxymethcathinone (3-OH-4-MeO-MC) could be satisfactorily separated on a semi-micro ODS column using linear gradient elution with a binary mobile phase of methanol and 10 mM ammonium formate buffer (pH 3.5). The detection limits of the four analytes by GC-MS were over the range of 5-25 ng/ml in a scan mode, and 0.5-2.5 ng/ml in a selected ion monitoring (SIM) mode. Upon applying the LC-ESI MS technique, the linear calibration curves were obtained using the SIM mode in the range of 25-500 ng/ml for Methylone, HMMC and 3-OH-4-MeO-MC, and 50-1000 ng/ml for MDC. The detection limits were over the range of 25-100 ng/ml in the scan mode, and 2.5-25 ng/ml in the SIM mode. Because of its high sensitivity, this analytical procedure will be applicable for proof of Methylone intake in forensic toxicology and clinical chemistry.

5-Methoxy-N,N-diisopropyltryptamine (5-MeO- DIPT), a psychotomimetic tryptamine derivative, and its relevant metabolites have been determined in eleven urine specimens from six 5-MeO-DIPT users, and their excretion profiles have been investigated by gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS). Three metabolites, 5-hydroxy-N,N- diisopropyltryptamine (5-OH-DIPT), 6-hydroxy- 5-methoxy-N,N-diisopropyltryptamine (6-OH-5- MeO-DIPT), an d5 -methoxy-N-isopropyltryptamine (5-MeO-NIPT) were determined in the urine speci- mens. Urinary conjugated metabolites, both sulfates and glucuronides of 5-OH-DIPT and 6-OH-5-MeO- DIPT, were hydrolyzed completely by the use of Helix pomatia sulfatase/β-glucuronidase. Degradation of 6-OH-5-MeO-DIPT during incubation for hydrolysis was successfully prevented by the addition of ascor- bic acid. The hydrolysis treatment increased the detection amounts of 5-OH-DIPT and 6-OH-5-MeO- DIPT in most of the specimens, and the increase in 6-OH-5-MeO-DIPT was more drastic than that in 5-OH-DIPT. The concentrations of 5-MeO-DIPT (< 1.7µg/ml) and 5-MeO-NIPT (< 3.5µg/ml) were lowe rt ha nt hose of 5-OH-DIPT (0.01-47µg/ml) and 6-OH-5-MeO-DIPT (< 69µg/ml) detected after hydrolysis (the totals of their free and conjugated forms). These metabolites were detectable over longer periods post intake than the parent drug; 35hr for 5-MeO-DIPT, 80hr for 5-OH-DIPT, and ∗ To whom correspondence should be addressed: Forensic Sci

In order to investigate the incorporation of drugs into hair, matrix-assisted laser desorption/ionization-time-of-flight tandem mass spectrometry (MS/MS) imaging was performed on the longitudinal sections of single scalp hair shafts sampled from volunteers after a single oral administration of methoxyphenamine (MOP), a noncontrolled analogue of methamphetamine. Hair specimens were collected by plucking out with the roots intact, and these specimens were prepped by an optimized procedure based on freeze-sectioning to detect the drug inside the hair shaft and hair root. Time-course changes in the imaging results, with confirmatory quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for each 1-mm segment of single hair strands, revealed a substantial concentration of the drug first onto the hair bulbs after ingestion, while only a small portion appeared to be incorporated into the hair matrix, forming a 2-3 mm distinctive drug band with tailing. Comparable amount of the drug also appeared to be incorporated into the keratinized hair shaft in the upper dermis zone, forming another distinct drug band of about 2 mm, which both moved toward the distal side, following the strand's growth rate. These findings provide forensically crucial information: there are two major drug incorporation sites, at least for MOP, which cause overlap of the recordings and deteriorates its chronological resolution down to about 11 days or perhaps longer.

High-resolution mass spectrometry and accurate mass measurement by liquid chromatography/quadrupole-time of flight mass spectrometry (LC/Q-TOFMS) was applied to postmortem plasma and urine specimens from an autopsy of a fatal case involving synthetic cannabinoid use, resulting in the detection of three synthetic cannabinoids: MAM-2201, AM-1220, and AM-2232. We searched for their metabolites existing in postmortem plasma or urine by LC/Q-TOFMS and were able to detect N-dealkylated metabolites, defluorinated and further oxidized metabolites of MAM-2201, and some hydroxylated metabolites. Postmortem plasma concentrations of the parent drugs, N-dealkylated metabolites, and fluorinated and further oxidized metabolites of MAM-2201 were measured, and quantitation results revealed site differences between heart and femoral postmortem plasma concentrations of parent drugs and some metabolites, suggesting postmortem redistribution of the synthetic cannabinoids and their metabolites. Quantitation results suggest that defluorination is a major metabolic pathway for MAM-2201, and N-dealkylation is a common but minor pathway for the naphthoylindole-type synthetic cannabinoids in human.

The urinary metabolites of 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) in humans have been investigated by analyzing urine specimens from its users. For the unequivocal identification and accurate quantification of its major metabolites, careful analyses were conducted by gas chromatography/mass spectrometry, liquid chromatography/mass spectrometry, and liquid chromatography-tandem mass spectrometry, using authentic standards of each metabolite synthesized. Three major metabolic pathways were revealed as follows: 1) side chain degradation by O-demethylation to form 5-hydroxy-N,N-diisopropyltryptamine (5-OH-DIPT), which would be partly conjugated to its sulfate and glucuronide; 2) direct hydroxylation on position 6 of the aromatic ring of 5-MeO-DIPT, and/or methylation of the hydroxyl group on position 5 after hydroxylation on position 6 of the aromatic ring of 5-OH-DIPT, to produce 6-hydroxy-5-methoxy-N,N-diisopropyltryptamine (6-OH-5-MeO-DIPT), followed by conjugation to its sulfate and glucuronide; and 3) side chain degradation by N-deisopropylation, to the corresponding secondary amine 5-methoxy-N-isopropyltryptamine (5-MeO-NIPT). Of these metabolites, which retain structural characteristics of the parent drug, 5-OH-DIPT and 6-OH-5-MeO-DIPT were found to be more abundant than 5-MeO-NIPT. Although the parent drug 5-MeO-DIPT was detectable even 35 h after dosing, no trace of its N-oxide was detected in any of the specimens examined.

The urinary metabolites of methylone in humans and rats were investigated by analysing urine specimens from its abuser and after administrating to rats with gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-electrospray ionization mass spectrometry (LC-ESI MS), using authentic standards. The time-course excretion profiles of methylone and its three metabolites in rats were further investigated after a single intraperitoneal dosing of 5 mg kg-1 methylone hydrochloride. Two major metabolic pathways were revealed for both humans and rats as follows: (1) side-chain degradation by N-demethylation to the corresponding primary amine methylenedioxycathinone (MDC), partly conjugated; and (2) demethylenation followed by O-methylation of either a 3- or 4-OH group on the benzene ring to produce 4-hydroxy-3-methoxymethcathinone (HMMC) or 3-hydroxy-4-methoxymethcathinone (3-OH-4-MeO-MC), respectively, mostly conjugated. Of these metabolites, HMMC was the most abundant in humans and rats. The cumulative amount of urinary HMMC excreted within the first 48 h in rats was approximately 26% of the dose, and the amount of the parent methylone was not more than 3%. These results demonstrate that the analysis of HMMC will be indispensable for proof of the use of methylone in forensic urinalysis.