Simultaneous determination of tryptamine analogues in designer drugs using gas chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry

In recent years, a large number of tryptamine-based designer drugs have been encountered in forensic samples. We have developed simultaneous analytical methods for 14 tryptamine analogues using gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–tandem mass spectrometry (LC–MS–MS). Trimethylsilyl (TMS) derivatives of the analytes were separated on a DB-1ms column within 15 min. The structural isomers could be differentiated by electron ionization GC–MS. LC–MS–MS with a C₁₈ column could separate structural isomers of tryptamines except for a combination of 5-methoxy-N,N-diethyltryptamine and 5-methoxy-N-methyl-N-isopropyltryptamine. Higher collision energy gave different product ion spectra between the structural isomers. The results indicate that GC–MS is the first choice for identification of tryptamines, preferably after TMS derivatization, and LC–MS–MS can be used as a complementary approach for the unequivocal differentiation of tryptamine isomers.     

Differentiation of regioisomeric fluoroamphetamine analogs by gas chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry

In recent years, a large number of clandestinely produced controlled-substance analogs (designer drugs) of amphetamine with high structural variety have been detected in forensic samples. Analytical differentiation of regioisomers is a significant issue in forensic drug analysis because, in most cases, legal controls are placed only on one or two of the three isomers. In this study, we used gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) for the differentiation of regioisomers of fluoroamphetamine analogs (fluoroamphetamines and fluoromethamphetamines), which were synthesized in our laboratories. Free bases and their acylated and silylated derivatives were subjected to GC–MS analysis using DB-1ms, DB-5ms, and DB-17ms capillary columns. The separation of free bases was incomplete on all columns. Trifluoroacetyl derivatives of 3- and 4-positional isomers showed slight separation on DB-1ms and DB-5ms. On the other hand, trimethylsilyl derivatization enabled baseline separation of six fluoroamphetamine analogs on DB-1ms and DB-5ms columns, which was sufficient for unequivocal identification. For LC–MS/MS, a pentafluorophenyl column was able to separate six regioisomeric fluoroamphetamine analogs but a conventional C18 column could not achieve separation between 3- and 4-positional isomers. These results show that a suitable choice of derivatization and analytical columns allows the differentiation of regioisomeric fluoroamphetamine analogs.     

Identification and quantitation of JWH-213, a cannabimimetic indole, as a designer drug in a herbal product

In our survey of designer drugs in the Japanese market, a cannabimimetic indole was identified as a new active compound in a herbal product. The structure of this compound was elucidated by liquid chromatography–photodiode array–mass spectrometry (LC–PDA–MS), gas chromatography–mass spectrometry (GC–MS), high-resolution MS, and nuclear magnetic resonance (NMR) analyses. The compound was finally identified as (4-ethyl-1-naphthalenyl)(2-methyl-1-pentyl-1H-indol-3-yl)methanone (JWH-213), an indole-based cannabinoid receptor ligand. To our knowledge, this is the first finding of JWH-213 as a designer drug in a herbal product. The quantitative LC–PDA analysis showed that the JWH-213 content in the product was 252 mg/pack.     

MR Diagnosis of a Pulmonary Embolism: Comparison of P792 and Gd-DOTA for First-Pass Perfusion MRI and Contrast-Enhanced 3D MRA in a Rabbit Model

Objective

To compare P792 (gadomelitol, a rapid clearance blood pool MR contrast agent) with gadolinium-tetraazacyclododecanetetraacetic acid (Gd-DOTA), a standard extracellular agent, for their suitability to diagnose a pulmonary embolism (PE) during a first-pass perfusion MRI and 3D contrast-enhanced (CE) MR angiography (MRA).

Materials and Methods

A perfusion MRI or CE-MRA was performed in a rabbit PE model following the intravenous injection of a single dose of contrast agent. The time course of the pulmonary vascular and parenchymal enhancement was assessed by measuring the signal in the aorta, pulmonary artery, and lung parenchyma as a function of time to determine whether there is a significant difference between the techniques. CE-MRA studies were evaluated by their ability to depict the pulmonary vasculature and following defects between 3 seconds and 15 minutes after a triple dose intravenous injection of the contrast agents.

Results

The P792 and Gd-DOTA were equivalent in their ability to demonstrate PE as perfusion defects on first pass imaging. The signal from P792 was significantly higher in vasculature than that from Gd-DOTA between the first and the tenth minutes after injection. The results suggest that a CE-MRA PE could be reliably diagnosed up to 15 minutes after injection.

Conclusion

P792 is superior to Gd-DOTA for the MR diagnosis of PE.     

Comparison of first-pass Gd-DOTA and FAIRER MR perfusion imaging in a rabbit model of pulmonary embolism

PURPOSE: To compare the sensitivity of contrast-enhanced magnetic resonance imaging (MRI) and arterial spin labeling to perfusion deficits in the lung. MATERIALS AND METHODS: A rabbit model of pulmonary embolism was imaged with both flow-sensitive alternating inversion recovery with an extra radiofrequency pulse (FAIRER) arterial spin labeling and Gd-DOTA enhanced MRI. The signal-to-noise ratio (SNR) was measured in the area of the perfusion deficit and the normal lung for both techniques. RESULTS: The defect was readily visible in all images. The normal lung had an average of 3.8 +/- 1.2 times the SNR of the unperfused lung with the arterial spin labeling technique, and approximately 13.7 +/- 3.3 times the SNR with the contrast-enhanced technique. CONCLUSION: Gd-DOTA enhanced MRI provides higher SNR in pulmonary perfusion imaging; however, arterial spin labeling is also adequate and may be used when repeated studies are indicated.     

Calcium binding by gadolinium-based MR contrast agents

PURPOSE: This study was conducted to characterize the alterations in ionic sodium, potassium, and calcium by gadolinium-based MR contrast agents. MATERIALS AND METHODS: An electrolyte solution (ES) containing 1.2 mM/L calcium ions,120 mM/L sodium, and 4.0 mM/L potassium were diluted with various gadolinium compounds and alterations in ionized electrolytes were measured using an ion-specific electrometer. Gadolinium compounds including Gd-DTPA, Gd-DOTA, gadoteridol, gadodiamide, meglumine/sodium diatrizoate (76% Urografin), and isotonic saline as a control were investigated. The dilution ranged from 5% (ES/test solution = 100/5) to 100%. Alterations of ionic electrolytes were measured. Calcium-binding capacities caused by each gadolinium compound also were measured. RESULTS: The alterations of ionic sodium and potassium by gadolinium compound were similar to those of isotonic saline. A significant reduction in ionized calcium was observed with Gd-DTPA and Gd-DOTA in comparison with gadoteridol and gadodiamide. CONCLUSION: Ionic gadolinium compounds induced significant reductions of calcium ions in vitro compared with non-ionic gadolinium compounds.     

Comprehensive analytical and structural characteristics of methyl 3,3-dimethyl-2-(1-(pent-4-en-1-yl)-1H-indazole-3-carboxamido)butanoate (MDMB-4en-PINACA)

Purpose

The purpose of the study was to evaluate a complete analytical and structural characterization of methyl 3,3-dimethyl-2-(1-(pent-4-en-1-yl)-1H-indazole-3-carboxamido)butanoate (MDMB-4en-PINACA), a novel synthetic cannabinoid being the analogue of 5F-ADB.

Methods

The compound was analyzed by gas chromatography–mass spectrometry (GC–MS), high-resolution liquid chromatography–mass spectrometry (LC–MS), X-ray diffraction and spectroscopic methods, such as nuclear magnetic resonance (NMR) and Fourier-transform infrared (FTIR) spectroscopies. To derive MDMB-4en-PINACA molecular geometry and to assign infrared absorption bands, quantum calculations with the employment of density functional theory were also used.

Results

We present a wide range of chromatographic and spectroscopic data supported with theoretical calculations allowing to identify MDMB-4en-PINACA.

Conclusions

To our knowledge, this is the first report presenting a comprehensive analytical and structural characterization of MDMB-4en-PINACA obtained by 1D and 2D NMR, GC–MS, LC–MS(/MS), attenuated total reflection-FTIR spectroscopy, powder X-ray diffraction and quantum chemical calculations. The presented results not only broaden the knowledge about this psychoactive substance but also are useful for forensic and clinical purposes.

     

Günther vena caval filter: results of long-term follow-up

A Günther vena caval filter was implanted in the inferior vena cava in 59 patients to prevent pulmonary embolism. This newly available device, which can be inserted percutaneously via a 10-French introduction system, has three filtering planes. No complications occurred at the puncture site. Follow-up included clinical examinations (54 patients), plain radiographs (50 patients), and CT scans (41 patients); these examinations were performed up to 21 months after implantation. Caudal migration of the filter occurred in 35 (70%) of the 50 patients who had radiographs, but no cranial or oblique movement occurred. Occlusion of the filter was noted in three (7%) of 41 patients who had CT examinations. Thromboemboli were seen inside the filter in 16 (39%) of the 41 patients who had CT scans. Recurrent pulmonary embolism was not observed after filter implantation. The Günther vena caval filter seems to be a satisfactory device for preventing pulmonary embolism.     

Mass spectrometric differentiation of the isomers of mono-methoxyethylamphetamines and mono-methoxydimethylamphetamines by GC–EI–MS–MS

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.     

The effects of gadolinium-DTPA and -DOTA on neural tissue metabolism.

Gadolinium DTPA and DOTA are being used extensively for imaging blood-brain barrier lesions. This study was performed to determine clinically relevant blood, cerebrospinal fluid (CSF), and neural tissue concentrations of these agents, and to determine if they alter neural tissue glucose metabolism. Bolus injections of 0.2 mmol Gd-DTPA/kg were made in rabbits, and blood, CSF, and neural tissue Gd concentrations were measured using atomic emission. Rat hippocampus slices were incubated for 6 hours in solutions of Gd-DTPA and Gd-DOTA, and effects on the production of carbon-14-labeled CO2 from glucose determined. Plasma concentrations reached a peak of 2.46 mmol at 1 minute postinjection, and dropped to 50% of peak in 6 minutes. The highest CSF concentration observed was approximately 0.1 mmol, and the mean lumbar cord concentration was approximately 8.5 mumol/g. Gd-DTPA and Gd-DOTA concentrations greater than 1.0 mmol caused significant increases in CO2 production. In areas of blood-brain barrier lesions, Gd-DTPA and Gd-DOTA may cause changes in tissue metabolism; however, in other areas it is much less likely.     

A fatal poisoning case of acetone cyanohydrin and citalopram

Acetone cyanohydrin (ACH) is a readily available source of cyanide and is widely used in basic and applied sciences. In toxicology, ACH is classified as extremely hazardous as it readily decomposes on contact with water, with the potential rapid release of highly toxic hydrogen cyanide (HCN). We report the case of a young woman found dead from the intentional ingestion of ACH and citalopram, an antidepressant of the selective serotonin reuptake inhibitor class. The autopsy findings included bright reddish-purple hypostasis and mild pulmonary edema. As ACH can decompose to acetone and HCN, we quantified the concentration of each compound and thiocyanate separately in various body fluids and organs and determined their whole-body distributions by using gas chromatography–mass spectrometry (GC–MS). We observed high concentrations of both acetone and cyanide in the blood (0.63 mg/mL and 17.99 mM, respectively) and gastric contents (9.76 mg/mL and 472.44 mM). The whole-body distributions of acetone and cyanide were similar (i.e., the concentration of each compound was the highest in the lung, followed by the heart, and then the liver). Our results suggest that not only the route of administration but also the dose taken could greatly affect the body distributions of cyanide in humans. In addition, as toxicological screening detected citalopram, which was not prescribed to the deceased, we performed a chiral analysis by using liquid chromatography–tandem mass spectrometry (LC–MS/MS). We determined that only (S)-citalopram was ingested antemortem; its concentration was 0.36 μg/mL, which is in the toxic range.     

Identification and quantitation of a new cathinone designer drug PV9 in an “aroma liquid” product,antemortem whole blood and urine specimens,and a postmortem whole blood specimen in a fatal poisoning case

A couple bought “aroma liquid” and “bath salt” type drugs at a dubious drug shop. Both of them orally took the liquid type drug; although the male subject showed no symptoms, the female subject suffered shivering, convulsions, and low levels of consciousness. The woman was taken to an emergency hospital to receive intensive medical treatment, but died about 20 h after admission. The aroma liquid solution, and the antemortem blood and urine collected during medical treatment at the hospital were brought to our laboratory by the police for analysis of the causative drug(s). In addition, a sample of postmortem femoral vein blood was collected from the cadaver. After some screening tests, we finally identified PV9 (α-POP) in all specimens by gas chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry (LC–MS–MS). The concentration of PV9 was 18.3 mg/ml in the aroma liquid solution, 45.7 ng/ml in the antemortem blood, 20.3 ng/ml in the antemortem urine, and 180 ng/ml in the postmortem femoral vein blood. The concentrations in antemortem blood and urine and in postmortem blood were greatly lowered by dilution during the intensive medical treatment, including intravenous drip infusion of a large volume of solution. The probable coexistence of a β-hydroxyl metabolite was also investigated by mass chromatography and analysis of fragment ions of the product ion spectrum obtained by LC–MS–MS. To our knowledge, this is the first reported identification and quantitation of PV9 in human specimens in a fatal PV9 poisoning case.     

MR imaging of rat brain glioma: Gd-DTPA versus Gd-DOTA

The enhancement properties of gadolinium diethylenetriaminepentaacetic acid (DTPA) and gadolinium tetraazacyclododecanetetraacetic acid (DOTA) were compared using a rat glioma model. In vitro analysis included the calculation of T1 relaxivity and determination of characteristic curves. Enhancement of the intracerebral glioma was studied in 23 rats approximately 2 weeks after glioma implantation with Gd-DTPA in 12 rats and Gd-DOTA in 11 rats. Six rats were also studied 1 week after implantation. Gd-DTPA exhibited a slightly greater T1 relaxivity in vitro than Gd-DOTA. Enhancement of the glioma was also greater with Gd-DTPA than with Gd-DOTA (P less than .05).     

SPME–GC–MS analysis of α-pyrrolidinovaleorophenone in blood in a fatal poisoning case

We provided toxicological analytical support for a fatal case of abuse of α-pyrrolidinovaleorophenone (α-PVP). Solid-phase microextraction (SPME) and capillary gas chromatography coupled to mass spectrometry (GC–MS) was employed to quantify the drug in whole blood. The whole blood concentration of the drug in the heart was 486 ng/ml. This is the first report of α-PVP intoxication as ascertained by mass spectrometric identification of α-PVP in whole blood.     

Contrast enhanced magnetic resonance imaging of the brain using gadolinium-DTPA

This report concerns a clinical trial with gadolinium-DTPA (Gd-DTPA) as an intravenous contrast medium for magnetic resonance imaging (MRI) in patients with disorders of the central nervous system. Fifty patients, 30 females and 20 males, were examined without and with Gd-DTPA. The contrast medium was well tolerated by all patients. The results of MRI scanning without and with Gd-DTPA and those obtained with computed tomography (CT) using intravenous contrast enhancement were compared. This investigation comprised mainly patients with intracranial tumors, multiple sclerosis, and nasopharyngeal tumors. The results may be summarized as follows: 1) MRI with Gd-DTPA (MRI+) gave better results than MRI without Gd as regards delineation of the lesion, blood vessels and edema in cerebral tumors, pituitary adenomas and acute forms of multiple sclerosis (MS). 2) MRI+ was better than CT in 32 of the 50 cases examined; with intracerebral tumors it was better in 15 out of 18 cases. 3) MRI+ was always better than CT in patients with MS. In 3 out of 7 cases MRI demonstrated the acute MS lesions. 4) MRI+ seemed to have advantages also in nasopharyngeal tumors as ascertained from this limited experience.     

Characterization of the designer benzodiazepine pyrazolam and its detectability in human serum and urine

In 2012, online shops selling so-called research chemicals started offering pyrazolam, a new benzodiazepine that differs from phenazepam and etizolam, which have also recently appeared on the “gray market”, in that it is not marketed by pharmaceutical companies anywhere in the world. This article describes the characterization of pyrazolam (8-bromo-1-methyl-6-pyridin-2-yl-4H-[1,2,4]triazolo[4,3–a][1, 4]benzodiazepine) using gas chromatography-mass spectrometry, liquid chromatography-tandem mass spectrometry (LC–MS–MS), liquid chromatography quadrupole time-of-flight mass spectrometry (LC–Q–TOF–MS), and nuclear magnetic resonance spectroscopy. In addition, a study was carried out in which one of the authors ingested two 0.5-mg pyrazolam tablets. Serum and urine samples were then obtained to investigate the metabolism of pyrazolam and to obtain preliminary results for the elimination half-life and the detectability of a 1-mg dose in serum and urine using a highly sensitive LC–MS–MS method and immunoassays. The results showed an elimination half-life of about 17 h and no detectable metabolism. The parent compound was detected with the described LC–MS–MS method in serum for more than 50 h and in urine for approximately 6 days. Immunoassays showed cross-reactivity, but poor detection in the study samples demonstrated that consumption or administration of this presumably potent drug could go undetected unless instrumental analytical techniques are also used.     

Diagnostic approach to drug-screening tests for fatal diabetic ketoacidosis: Forensic autopsy of a methamphetamine abuser

To diagnose the cause of death in autopsy cases, systematic examinations, such as macroscopic, pathological, biochemical, and toxicological are important. In this case report, drug examinations also gave very useful information to diagnose the cause of death, fatal diabetic ketoacidosis (DKA). A female methamphetamine abuser in her forties was found dead lying on a hotel bed. Diagnosing her cause of death was difficult only from the macroscopic findings because there was no fatal and/or serious injury or disease. On toxicological examination, acetone was detected at a high concentration (682 μg/mL in blood, 887 μg/mL in urine) using gas chromatography (GC). Using gas chromatography–mass spectrometry (GC–MS), methamphetamine was detected in the blood, urine, hair, and visceral organs; however, these concentrations were low. At the same time, GC–MS examination revealed a high glucose peak. From the results of the biochemical examination of urine, acetoacetic acid was 1940 μmol/L, β-hydroxybutyric acid was 14,720 μmol/L, and glucose was 4620 mg/dL. Histologically, Langerhans’ islets in the pancreas were fibrotic and atrophic, and no insulin-immunoreactive cells were observed. The subsequent police investigation also revealed that she had contracted diabetes mellitus type 1; therefore, we concluded that her cause of death was DKA, due to a lack of insulin injection.     

Fatal sarin poisoning in Syria 2013: forensic verification within an international laboratory network

During the United Nations fact-finding mission to investigate the alleged use of chemical warfare agents in the Syrian Arab Republic in 2013, numerous tissues from a deceased female victim, who had displayed symptoms of cholinergic crisis, were collected. The Organisation for the Prohibition of Chemical Weapons (OPCW) authorized two specialized laboratories in the Netherlands and Germany for forensic analysis of these samples. Diverse modern mass spectrometry (MS)-based procedures in combination with either liquid chromatography (LC) or gas chromatography (GC) separation were applied. A variety of biotransformation products of the nerve agent sarin was detected, including the hydrolysis product O-isopropyl methylphosphonic acid (IMPA) as well as covalent protein adducts with e.g., albumin and human butyrylcholinesterase (hBChE). IMPA was extracted after sample acidification by solid-phase extraction and directly analyzed by LC–tandem-MS with negative electrospray ionization (ESI). Protein adducts were found, either by fluoride-induced reactivation applying GC–MS techniques or by LC–MS-based detection after positive ESI for proteolyzed proteins yielding phosphonylated tyrosine residues or a specific phosphonylated hBChE-derived nonapeptide. These experimental results provided unambiguous evidence for a systemic intoxication and were the first proving the use of sarin in the ongoing bellicose conflict. This scenario underlines the requirement for qualified and specialized analytical laboratories to face repeated violation of the Chemical Weapons Convention.     

Spectroscopic characterization and crystal structures of two cathinone derivatives: 1-(4-chlorophenyl)-2-(1-pyrrolidinyl)-pentan-1-one (4-chloro-α-PVP) sulfate and 1-(4-methylphenyl)-2-(dimethylamino)-propan-1-one (4-MDMC) hydrochloride salts,seized on illicit drug market

Purpose

Two compounds newly found in the seizures by drug enforcement agencies were identified and characterized by various instrumental analytical methods.

Methods

The obtained powder samples were analyzed by gas chromatography–mass spectrometry (GC–MS), liquid chromatography–mass spectrometryⁿ (LC–MSⁿ), nuclear magnetic resonance (NMR) spectroscopy, infrared and Raman spectroscopy and X-ray crystallography.

Results

The two compounds were tentatively identified as 4-chloro-α-PVP and 4-MDMC by GC–MS, and LC–MS/MS. The confirmation of the results was made by NMR spectroscopy. The X-ray crystallography gave information that 4-chloro-α-PVP and 4-MDMC were in salted forms with sulfate and hydrochloride, respectively; in addition, both compounds existed as racemic mixtures.

Conclusions

We could identify 4-chloro-α-PVP and 4-MDMC in the seizure powder samples by various analytical methods. X-ray crystallography was especially useful for identifying the salted forms and enantiomeric forms.