Analytical characterization of three hallucinogenic N‐(2‐methoxy)benzyl derivatives of the 2C‐series of phenethylamine drugs

This publication reports analytical properties of three new hallucinogenic substances identified in blotter papers seized from the drug market, namely 25D‐NBOMe [2‐(2,5‐dimethoxy‐4‐methylphenyl)‐N‐(2‐methoxybenzyl)ethanamine], 25E‐NBOMe [2‐(4‐ethyl‐2,5‐dimethoxyphenyl)‐N‐(2‐methoxybenzyl)ethanamine] and 25G‐NBOMe [2‐(2,5‐dimethoxy‐3,4‐dimethylphenyl)‐N‐(2‐methoxybenzyl)ethanamine]. These substances are N‐(2‐methoxy)benzyl derivatives of the 2C‐series of phenethylamine drugs. The applied procedure covered a variety of analytical methods, including gas chromatography with electron impact mass spectrometry (GC‐EI‐MS; without derivatization and after derivatization with trifluoroacetic anhydride (TFAA)), liquid chromatography‐electrospray ionization‐quadrupole time of flight mass spectrometry (LC‐ESI‐QTOF‐MS), Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR), which made it possible to identify the active components unequivocally. The GC‐MS spectra of analyzed compounds were very similar, with dominant ions observed at m/z = 150, 121, and 91. The remaining ions were analogous to those observed for parent substances, namely 2C‐D, 2C‐E, 2C‐G, but their intensities were low. Derivatization allowed determination of molecular masses of the investigated substances. Their exact masses and chemical formulas were confirmed by LC‐QTOF‐MS experiments and the fragmentation patterns of these compounds following ESI were determined. The tandem mass spectrometry (MS/MS) experiments confirmed that the studied substances were N‐(2‐methoxy)benzyl derivatives of the 2C‐series compounds. Final elucidation of the structures was performed by NMR spectroscopy. The substances were also characterized by FTIR spectroscopy to corroborate the identity of the compounds. Copyright © 2012 John Wiley & Sons, Ltd.     

Synthesis of [2,3,4,5,6‐2H5]phenyl glucosinolate

Starting from commercially available [2,3,4,5,6‐²H₅]benzoic acid, [2,3,4,5,6‐²H₅]phenyl glucosinolate was synthesized. Under negative‐ion electrospray‐ionization mass spectrometric conditions, this compound affords a peak at m/z 399. Since this m/z value is not known from the ions derived from natural glucosinolates, the [2,3,4,5,6‐²H₅]phenyl glucosinolate reported here is useful as an internal standard for the quantification of glucosinolates by negative‐ion mass spectrometry (MS) and liquid chromatography (LC)/MS techniques. Copyright © 2007 John Wiley & Sons, Ltd.     

Caution in the use of 2‐iminothiolane (Traut's reagent) as a cross‐linking agent for peptides. The formation of N‐peptidyl‐2‐iminothiolanes with bombesin (BN) antagonist (d‐Trp6,Leu13‐ψ[CH2NH]‐Phe14)BN6−14 and d‐Trp‐Gln‐Trp‐NH2

Abstract: During a study aimed at generating a bispecific molecule between BN antagonist (d ‐Trp⁶,Leu¹³‐ψ[CH₂NH]‐Phe¹⁴)BN_6‐14 (Antag1) and mAb22 (anti‐FcγRI), we attempted to cross‐link the two molecules by introducing a thiol group into Antag1 via 2‐iminothiolane (2‐IT, Traut's reagent). We found that reaction of Antag1 with 2‐IT, when observed using HPLC, affords two products, but that the later eluting peptide is rapidly transformed into the earlier eluting peptide. To understand what was occurring we synthesized a model peptide, d ‐Trp‐Gln‐Trp‐NH₂ (TP1), the N‐terminal tripeptide of Antag1. Reaction of TP1 with 2‐IT for 5 min gave products 1a and 3a ; the concentration of 1a decreased with reaction time, whereas that of 3a increased. Thiol 1a , the expected Traut product, was identified by collecting it in a vial containing N‐methylmaleimide and then isolating the resultant Michael addition product 2a , which was confirmed by mass spectrometry. Thiol 1a is stable at acidic pH, but is unstable at pH 7.8, cyclizes and loses NH₃ to give N‐TP1‐2‐iminothiolane ( 3a ), ES‐MS (m/z) [602.1 (M+H)⁺], as well as regenerating TP1. Repeat reaction with Antag1 and 2‐IT allowed us to isolate N‐Antag1–2‐iminothiolane ( 3b ), FAB‐MS (m/z) [1212.8 (M+H)⁺] and trap the normal Traut product 1b as its N‐methylmaleimide Michael addition product 2b , ES‐MS (m/z) [1340.8 (M+H)⁺]. Thiol 1b is also stable at acidic pH, but when neutralized is unstable and cyclizes, forming 3b and Antag1.     

In‐source collision‐induced dissociation (IS‐CID): Applications,issues and structure elucidation with single‐stage mass analyzers

A discussion of the definition, advantages, and issues with the formation of ions in the transition region between an electrospray ionization (ESI) source and the ion optics of a mass analyzer is presented. The various types of ions formed in the so‐called in‐source collision‐induced dissociation (IS‐CID) process are illustrated. Applications of IS‐CID with single‐stage mass analyzers, such as structure elucidation and quantitation, are demonstrated. The discussion is illustrated by examples of the in‐source fragmentation of ginkgolides, which are marker compounds found only in Ginkgo biloba. Supercritical fluid chromatography (SFC) with non‐aqueous eluents was used to achieve a fast resolution of the ginkgolides without the hydrolysis reactions possible with aqueous high‐performance liquid chromatography (HPLC) eluents. In‐source ion generation occurs at relatively high pressures (ca. 1–3 torr) compared to the low pressure normally observed in collision chambers of tandem mass spectrometry (MS/MS). As a result, the fragmentation process is complex and often generates ions other than the fragments observed with classic CID or the same ions at different intensities. The objective of the current tutorial is to illustrate the conditions under which single‐stage, quadrupole or time‐of‐flight mass analyzers with electrospray or in‐air (direct analysis in real time; DART) ionization can be used for quantitation and structure elucidation in a manner similar to that observed with MS/MS. While the low m/z (≤ [M±H]^±) ions formed in‐source often duplicate the ions observed in MS/MS systems, it is the focus of this discussion to illustrate the utility of in‐source generated fragment ions that may not be observed or observed at different intensities than in the collision cells of MS/MS instruments.     

Identification of the designer steroid Androsta‐3,5‐diene‐7,17‐dione in a dietary supplement

A liquid chromatography‐mass spectrometry (LC–MS) screen for known anabolic‐androgenic steroids in a dietary supplement product marketed for “performance enhancement” detected an unknown compound having steroid‐like spectral characteristics. The compound was isolated using high performance liquid chromatography with ultraviolet detection (HPLC–UV) coupled with an analytical scale fraction collector. After the compound was isolated, it was then characterized using gas chromatography with simultaneous Fourier Transform infrared detection and mass spectrometry (GC–FT–IR–MS), liquid chromatography–high resolution accurate mass–mass spectrometry (LC–HRAM–MS) and nuclear magnetic resonance (NMR). The steroid had an accurate mass of m/z 285.1847 (error?0.57 ppm) for the protonated species [M + H]⁺, corresponding to a molecular formula of C₁₉H₂₄O₂. Based on the GC–FT–IR–MS data, NMR data, and accurate mass, the compound was identified as androsta‐3,5‐diene‐7,17‐dione. Although this is not the first reported identification of this designer steroid in a dietary supplement, the data provided adds information for identification of this compound not previously reported. This compound was subsequently detected in another dietary supplement product, which contained three additional active ingredients.     

Metabolism of the tryptamine‐derived new psychoactive substances 5‐MeO‐2‐Me‐DALT, 5‐MeO‐2‐Me‐ALCHT,and 5‐MeO‐2‐Me‐DIPT and their detectability in urine studied by GC–MS,LC–MSn,and LC‐HR‐MS/MS

Many N,N‐dialkylated tryptamines show psychoactive properties and were encountered as new psychoactive substances. The aims of the presented work were to study the phase I and II metabolism and the detectability in standard urine screening approaches (SUSA) of 5‐methoxy‐2‐methyl‐N,N‐diallyltryptamine (5‐MeO‐2‐Me‐DALT), 5‐methoxy‐2‐methyl‐N‐allyl‐N‐cyclohexyltryptamine (5‐MeO‐2‐Me‐ALCHT), and 5‐methoxy‐2‐methyl‐N,N‐diisopropyltryptamine (5‐MeO‐2‐Me‐DIPT) using gas chromatography–mass spectrometry (GC–MS), liquid chromatography coupled with multistage accurate mass spectrometry (LC–MSⁿ), and liquid chromatography‐high‐resolution tandem mass spectrometry (LC‐HR‐MS/MS). For metabolism studies, urine was collected over a 24 h period after administration of the compounds to male Wistar rats at 20 mg/kg body weight (BW). Phase I and II metabolites were identified after urine precipitation with acetonitrile by LC‐HR‐MS/MS. 5‐MeO‐2‐Me‐DALT (24 phase I and 12 phase II metabolites), 5‐MeO‐2‐Me‐ALCHT (24 phase I and 14 phase II metabolites), and 5‐MeO‐2‐Me‐DIPT (20 phase I and 11 phase II metabolites) were mainly metabolized by O‐demethylation, hydroxylation, N‐dealkylation, and combinations of them as well as by glucuronidation and sulfation of phase I metabolites. Incubations with mixtures of pooled human liver microsomes and cytosols (pHLM and pHLC) confirmed that the main metabolic reactions in humans and rats might be identical. Furthermore, initial CYP activity screenings revealed that CYP1A2, CYP2C19, CYP2D6, and CYP3A4 were involved in hydroxylation, CYP2C19 and CYP2D6 in O‐demethylation, and CYP2C19, CYP2D6, and CYP3A4 in N‐dealkylation. For SUSAs, GC–MS, LC‐MSⁿ, and LC‐HR‐MS/MS were applied to rat urine samples after 1 or 0.1 mg/kg BW doses, respectively. In contrast to the GC–MS SUSA, both LC–MS SUSAs were able to detect an intake of 5‐MeO‐2‐Me‐ALCHT and 5‐MeO‐2‐Me‐DIPT via their metabolites following 1 mg/kg BW administrations and 5‐MeO‐2‐Me‐DALT following 0.1 mg/kg BW dosage. Copyright © 2017 John Wiley & Sons, Ltd.     

Preparation and characterization of the ‘research chemical’ diphenidine,its pyrrolidine analogue,and their 2,2‐diphenylethyl isomers

Substances with the diphenylethylamine nucleus represent a recent addition to the product catalog of dissociative agents sold as ‘research chemicals’ on the Internet. Diphenidine, i.e. 1‐(1,2‐diphenylethyl)piperidine (1,2‐DEP), is such an example but detailed analytical data are less abundant. The present study describes the synthesis of diphenidine and its most obvious isomer, 1‐(2,2‐diphenylethyl)piperidine (2,2‐DEP), in order to assess the ability to differentiate between them. Preparation and characterization were also extended to the two corresponding pyrrolidine analogues 1‐(1,2‐diphenylethyl)‐ and 1‐(2,2‐diphenylethyl)pyrrolidine, respectively. Analytical characterizations included high‐resolution electrospray mass spectrometry (HR‐ESI‐MS), liquid chromatography ESI‐MS/MS, gas chromatography ion trap electron and chemical ionization MS, nuclear magnetic resonance spectroscopy (NMR) and infrared spectroscopy. Differentiation between the two isomeric pairs was possible under GC‐(EI/CI)‐MS conditions and included the formation of distinct iminium ions, such as m/z 174 for 1,2‐DEP and m/z 98 for 2,2‐DEP, respectively. The pyrrolidine counterparts demonstrated similar phenomena including the expected mass difference of 14 Da due to the lack of one methylene unit in the ring. Two samples obtained from an Internet vendor provided confirmation that diphenidine was present in both samples, concurring with the product label. Finally, it was confirmed that diphenidine (30 μM) reduced N‐methyl‐D ‐aspartate‐mediated field excitatory postsynaptic potentials (NMDA‐fEPSPs) to a similar extent to that of ketamine (30 μM) when using rat hippocampal slices. The appearance of 1,2‐ diphenylethylamines appears to reflect the exploration of alternatives to arylcyclohexylamine‐type substances, such as methoxetamine, PCP and PCPy‐based analogues that also show NMDA receptor activity as demonstrated here for diphenidine. Copyright © 2014 John Wiley & Sons, Ltd.     

Regioselective syntheses of [13C]4‐labelled sodium 1‐carboxy‐2‐(2‐ethylhexyloxycarbonyl)ethanesulfonate and sodium 2‐carboxy‐1‐(2‐ethylhexyloxycarbonyl)ethanesulfonate from [13C]4‐maleic anhydride

The entitled monohydrolysis products, also known as α‐ethylhexyl and β‐ethylhexyl sulfosuccinate (EHSS), of the surfactant diisooctyl sulfosuccinate (DOSS) were synthesized in stable isotope‐labelled form from [¹³C]₄‐maleic anhydride. Sodium [¹³C]₄‐1‐carboxy‐2‐(2‐ethylhexyloxycarbonyl)ethanesulfonate (α‐EHSS) was prepared by the method of Larpent by reaction of 2‐ethylhexan‐1‐ol with [¹³C]₄‐maleic anhydride followed by regioselective conjugate addition of sodium bisulfite to the resulting monoester (38% overall yield). The regiochemical outcome of bisulfite addition was confirmed by a combination of ¹³C/¹³C (incredible natural abundance double quantum transfer) and ¹H/¹³C (heteronuclear multiple‐bond correlation (HMBC)) NMR spectral correlation experiments. Sodium [¹³C]₄‐2‐carboxy‐1‐(2‐ethylhexyloxycarbonyl)ethanesulfonate (β‐EHSS) was prepared in four steps by reaction of 4‐methoxybenzyl alcohol with [¹³C]₄‐maleic anhydride, regioselective sodium bisulfite addition, N,N′‐dicyclohexylcarbodiimide‐mediated esterification with 2‐ethylhexan‐1‐ol, and p‐methoxybenzyl ester deprotection with trifluoroacetic acid (13% overall yield). The regiochemical outcome of the second synthesis was confirmed by a combination of ¹J_CC scalar coupling constant analysis and ¹H/¹³C (HMBC) NMR spectral correlation. The materials prepared are required as internal standards for the liquid chromatography–mass spectrometry (LC‐MS)/MS trace analysis of the degradation products of DOSS, the anionic surfactant found in Corexit, the oil dispersant used during emergency response efforts connected to the Deepwater Horizon oil spill of April 2010. Copyright © 2014 John Wiley & Sons, Ltd.     

UPLC‐PDA‐TOF/MS coupled with multivariate statistical analysis to rapidly analyze and evaluate Ginkgo biloba leaves from different origin

In the present study, an ultra performance liquid chromatography coupled with photodiode array detector and time‐of‐flight mass spectrometry (UPLC‐PDA‐TOF/MS) was proposed and validated for rapidly analyzing and evaluating Ginkgo biloba leaves from different origins by using multivariate statistical analysis. Batches of these kinds of G. biloba leaves were subjected to UPLC‐PDA‐TOF/MS analysis, the datasets of retention time (RT)‐m/z pairs, ion intensities and sample codes were further processed with orthogonal partial least squared discriminant analysis (OPLS‐DA) to holistically compare the difference between these G. biloba leaves, and to generate an S‐plot. Those compounds correlated to the points at the two ends of S were regarded as the most differentiating components. By comparing the mass/UV spectra and retention times with those of reference compounds and/or tentatively assigned by matching empirical molecular formulae with those of the known compounds published in the literatures, these differentiating components were finally characterized as kaempferol 3‐O‐[2‐O‐(6‐O‐p‐hydroxy‐trans‐cinnamoyl)‐β‐D‐glucosyl)‐α‐L‐rhamnoside], kaempferol 3‐O‐[2‐O,6‐O‐bis(α‐L‐rhamnosyl)‐β‐D‐glucoside], ginkgolide C, kaempferol 3‐O‐[2‐O‐(β‐D‐glucosyl)‐α‐L‐rhamnoside], and bilobetin. These compounds would be the potential chemical markers for the two kinds of leaves. The results suggested that this newly established approach could be used to rapidly evaluate the quality of herbs from different origin, and to provide good strategy for further rectify and standardize the herb market. Copyright © 2013 John Wiley & Sons, Ltd.     

The identification and analytical characterization of 2,2′‐difluorofentanyl

New psychoactive substances (NPS) have expanded their distribution and become widely available in the global market in recent years. The illicit use of fentanyl and its analogs has become an important worldwide concern linked to their high potency and risk of fatal overdose. This study describes the analytical characterization of a new fentanyl derivative N‐(1‐(2‐fluorophenethyl)‐4‐piperidinyl)‐N‐(2‐fluorophenyl)propionamide (2,2′‐difluorofentanyl). Identification was based on ultra‐high‐performance liquid chromatography–quadrupole time‐of‐flight–mass spectrometry (UHPLC–QTOF–MS), gas chromatography–mass spectrometry (GC–MS), Fourier transform infrared (FTIR) spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy. To our knowledge, this study is the first to report on analytical data for this compound. The most abundant fragment ion in the electrospray ionization (ESI) mass spectrum under collision‐induced dissociation (CID) mode was formed by the cleavage between the piperidine ring and the N‐phenyl‐amide moiety of the protonated molecule. Two diagnostic ions in the electron ionization (EI) mass spectrum were formed by the loss of a tropylium ion (M‐91), and by the degradation of the piperidine ring and dissociate of the COC₂H₅ moiety altogether, respectively.     

The characterization of isobaric product ions of fentanyl using multi‐stage mass spectrometry,high‐resolution mass spectrometry and isotopic labeling

This study uses a combination of multi‐stage mass spectrometry (MSⁿ), accurate mass measurements – with high‐resolution mass spectrometry (HRMS) – and isotopic labeling to characterize the fragmentation behavior of fentanyl and 4‐ANPP. By understanding the fragmentation behavior of fentanyl and its analogs in more detail, toxicologists and seized drug analysts will be better poised to identify new and emerging fentalogs, which are increasingly common and deadly adulterants in the growing opioid crisis. Throughout the literature the product ion at m/z 188 is often the most abundant fragment in the mass spectrometric analysis of fentanyl and fentanyl analogs, and this fragment is used for both qualitative and quantitative determinations. Our work shows there are at least three different structures for the isobaric fentanyl product ions at m/z 188, and they each form and fragment via different pathways. The development of fragmentation mechanisms to explain the observed fragmentation pathways of fentanyl and its main precursor 4‐ANPP helps contribute to the advancement of knowledge about fentanyl fragmentation and could provide important information for the identification of future fentanyl analogs.     

Synthesis and characterization of tritium labeled N‐((R)‐1‐((S)‐4‐(4‐chlorophenyl)‐4‐hydroxy‐3,3‐dimethylpiperidin‐1‐yl)‐3‐methyl‐1‐oxobutan‐2‐yl)‐3‐sulfamoylbenzamide

N‐((R)‐1‐((S)‐4‐(4‐chlorophenyl)‐4‐hydroxy‐3,3‐dimethylpiperidin‐1‐yl)‐3‐methyl‐1‐oxobutan‐2‐yl)‐3‐sulfamoylbenzamide is a potent C‐C chemokine receptor 1 (CCR1) antagonist. The compound, possessing benzamide functionality, successfully underwent tritium/hydrogen (T/H) exchange with an organoiridium catalyst (Crabtree's catalyst). The labeling pattern in the product was studied with liquid chromatography–mass spectrometry, time‐of‐flight mass spectrometry, and ³H‐NMR. Overall, multiple labeled species were identified. In addition to the anticipated incorporation of tritium in the benzamide moiety, tritium labeling was observed in the valine portion of the molecule including substitution at its chiral carbon. Using authentic standards, liquid chromatography analysis of the labeled compound showed complete retention of stereochemical configuration.     

Synthesis and characterization of radiolabeled 17β‐estradiol conjugates

The use of radioactive tracers for environmental fate and transport studies of emerging contaminants, especially for those that are labile, offers convenience in tracking study compounds and their metabolites, and in calculating mass balances. The aim of this study was to synthesize radiolabeled glucuronide and sulfate conjugates of 17 β ‐estradiol (17 β ‐E2). The conjugates 17 β ‐[4‐¹⁴C]estradiol‐3‐glucuronide ([¹⁴C]17 β ‐E2‐3‐G) and 17 β ‐[4‐¹⁴C]estradiol‐17‐sulfate ([¹⁴C]17 β ‐E2‐17‐S) were synthesized utilizing immobilized enzyme and chemical syntheses, respectively. Microsomal proteins from the liver of a phenobarbital induced pig (Sus scrofa domestica) were harvested and used to glucuronidate [¹⁴C]17 β ‐E2. Synthesis of [¹⁴C]17 β ‐E2‐17‐S consisted of a three‐step chemical process – introducing a blocking group at the C‐3 position of [¹⁴C]17 β ‐E2, sulfation at C‐17 position, and subsequent deblocking to yield the desired synthetic product. Successful syntheses of [¹⁴C]17 β ‐E2‐3‐G and [¹⁴C]17 β ‐E2‐17‐S were achieved as verified by liquid chromatography, radiochemical analyses, quadrupole‐time‐of‐flight (QTOF) mass spectrometry, and ¹H and ¹³C nuclear magnetic resonance spectroscopy. Radiochemical yields of 84 and 44% were achieved for 17 β ‐E2‐3‐G and 17 β ‐E2‐17‐S, respectively. Synthetic products were purified using high‐performance liquid chromatography and radiochemical purities of 98% or greater were obtained. Copyright © 2011 John Wiley & Sons, Ltd.     

AMT (3‐(2‐aminopropyl)indole) and 5‐IT (5‐(2‐aminopropyl)indole): an analytical challenge and implications for forensic analysis

5‐(2‐Aminopropyl)indole (5‐IT) and 3‐(2‐aminopropyl)indole (α‐methyltryptamine, AMT) are isomeric substances and their differentiation can be a challenge under routine analytical conditions, especially when reference material is unavailable. 5‐IT represents a very recent addition to the battery of new psychoactive substances that are commercially available from online retailers. This report illustrates how subtle differences observed under mass spectral and UV conditions can help to facilitate the differentiation between the two isomers. Analyses included ¹ H and ¹³C NMR, GC‐EI/CI ion trap MS, applications of several U/HPLC‐DAD and HPLC‐MS methods. Investigations currently underway also highlight the confirmation that AMT was detected in a number of fatal intoxications. These findings also demonstrate that there is a potential risk of misidentification when dealing with both substances. Copyright © 2012 John Wiley & Sons, Ltd.     

Return of the lysergamides. Part II: Analytical and behavioural characterization of N6‐allyl‐6‐norlysergic acid diethylamide (AL‐LAD) and (2’S,4’S)‐lysergic acid 2,4‐dimethylazetidide (LSZ)

Lysergic acid N ,N ‐diethylamide (LSD) is perhaps one of the most intriguing psychoactive substances known and numerous analogs have been explored to varying extents in previous decades. In 2013, N ⁶‐allyl‐6‐norlysergic acid diethylamide (AL‐LAD) and (2’S ,4’S )‐lysergic acid 2,4‐dimethylazetidide (LSZ) appeared on the ‘research chemicals’/new psychoactive substances (NPS) market in both powdered and blotter form. This study reports the analytical characterization of powdered AL‐LAD and LSZ tartrate samples and their semi‐quantitative determination on blotter paper. Included in this study was the use of nuclear magnetic resonance (NMR) spectroscopy, gas chromatography‐mass spectrometry (GC‐MS), low and high mass accuracy electrospray MS(/MS), high performance liquid chromatography diode array detection and GC solid‐state infrared analysis. One feature shared by serotonergic psychedelics, such as LSD, is the ability to mediate behavioural responses via activation of 5‐HT_2A receptors. Both AL‐LAD and LSZ displayed LSD‐like responses in male C57BL/6 J mice when employing the head‐twitch response (HTR) assay. AL‐LAD and LSZ produced nearly identical inverted‐U‐shaped dose‐dependent effects, with the maximal responses occurring at 200 µg/kg. Analysis of the dose responses by nonlinear regression confirmed that LSZ (ED₅₀ = 114.2 nmol/kg) was equipotent to LSD (ED₅₀ = 132.8 nmol/kg) in mice, whereas AL‐LAD was slightly less potent (ED₅₀ = 174.9 nmol/kg). The extent to which a comparison in potency can be translated directly to humans requires further investigation. Chemical and pharmacological data obtained from NPS may assist research communities that are interested in various aspects related to substance use and forensic identification. Copyright © 2016 John Wiley & Sons, Ltd.     

Synthesis of [1,3, NH2‐15N3] (5′S)‐8,5′‐cyclo‐2′‐deoxyguanosine

To facilitate NMR studies and low‐level detection in biological samples by mass spectrometry, [1,3, NH₂‐¹⁵N₃] (5′S)‐8,5′‐cyclo‐2′‐deoxyguanosine was synthesized from imidazole‐4,5‐dicarboxylic acid in 21 steps. The three ¹⁵N isotopes were introduced during the chemo‐enzymatic preparation of [1,3, NH₂‐¹⁵N₃]‐2′‐deoxyguanosine using an established procedure. The ¹⁵N‐labeled 2′‐deoxyguanosine was converted to a 5′‐phenylthio derivative, which allowed the 8‐5′ covalent bond formation via photochemical homolytic cleavage of the C–SPh bond. SeO₂ oxidation of C‐5′ followed by sodium borohydride reduction and deprotection gave the desired product in good yield. The isotopic purity of the [1,3, NH₂‐¹⁵N₃] (5′S)‐8,5′‐cyclo‐2′‐deoxyguanosine was in excess of 99.94 atom% based on liquid chromatography–mass spectrometry measurements. Copyright © 2013 John Wiley & Sons, Ltd.     

Gas chromatography−mass spectrometry analysis of non‐hydrolyzed sulfated steroids by degradation product formation

Steroid detection and identification remain key issues in toxicology, drug testing, medical diagnostics, food safety control, and doping control. In this study, we evaluate the capabilities and usefulness of analyzing non‐hydrolyzed sulfated steroids with gas chromatography?mass spectrometry (GC–MS) instead of the conventionally applied liquid chromatography?mass spectrometry (LC–MS) approach. Sulfates of 31 steroids were synthesized and their MS and chromatographic behavior studied by chemical ionization?GC?triple quadrupole MS (CI?GC‐TQMS) and low energy?electron ionization?GC?quadrupole time‐of‐flight?MS (LE?EI?GC?QTOF?MS). The collected data shows that the sulfate group is cleaved off in the injection port of the GC–MS, forming two isomers. In CI, the dominant species (ie, [MH – H₂SO₄]⁺ or [MH – H₄S₂O₈]⁺ for bis‐sulfates) is very abundant due to the limited amount of fragmentation, making it an ideal precursor ion for MS/MS. In LE?EI, [M – H₂SO₄]^.+ and/or [M – H₂SO₄ – CH₃]^.+ are the dominant species in most cases. Based on the common GC–MS behavior of non‐hydrolyzed sulfated steroids, two applications were evaluated and compared with the conventionally applied LC–MS approach; (a) discovery of (new) sulfated steroid metabolites of mesterolone and (b) expanding anabolic androgenic steroid abuse detection windows. GC–MS and LC–MS analysis of non‐hydrolyzed sulfated steroids offered comparable sensitivities, superseding these of GC–MS after hydrolysis. For non‐hydrolyzed sulfated steroids, GC–MS offers a higher structural elucidating power and a more straightforward inclusion in screening methods than LC–MS.     

Synthesis and bioevaluation of 4‐chloro‐2‐tert‐butyl‐5‐[2‐[[1‐[2‐[18F]fluroethyl]‐1H‐1,2,3‐triazol‐4‐yl]methyl]phenylmethoxy]‐3(2H)‐pyridazinone as potential myocardial perfusion imaging agent with PET

This study reports the synthesis and characterization of 4‐chloro‐2‐tert‐butyl‐5‐[2‐[[1‐[2‐[¹⁸F]fluroethyl]‐1H‐1,2,3‐triazol‐4‐yl]methyl]phenylmethoxy]‐3(2H)‐pyridazinone ([¹⁸F]Fmp2) for myocardial perfusion imaging (MPI). The tosylate precursor and non‐radioactive compound [¹⁹F]Fmp2 were synthesized and characterized by infrared, ¹H‐NMR, ¹³C‐NMR, and mass spectra (MS). The radiotracer [¹⁸F]Fmp2 was obtained by one‐step nucleophilic substitution of tosyl with ¹⁸F, and evaluated as an MPI agent in vitro and in vivo. Starting from [¹⁸F]KF/K₂₂₂ solution, the typical decay‐corrected radiochemical yield (RCY) was 38 ± 8.8% with high radiochemical purity (>98%). The specific activity was calculated as 10 GBq/µmol at the end of synthesis determined by HPLC analysis. In the mice biodistribution, [¹⁸F]Fmp2 showed very high initial heart uptake (53.35 ± 5.47 %ID/g at 2 min after injection) and remarkable retention. The heart/liver, heart/lung, and heart/blood ratios were 7.98, 8.20, and 53.13, respectively at 2 min post‐injection. In the Positron Emission Tomography (PET) imaging study of Chinese mini‐swine, the standardized uptake value of the liver decreased modestly during the 2 h post‐injection, while the heart uptake and heart/liver ratios continued to increase with time. [¹⁸F]Fmp2 exhibited good stability, high heart uptake and low lung uptake in mice and Chinese mini‐swine. It may be worthy of further modification to improve liver clearance for MPI in the future.     

Identification of the anabolic steroid 6β‐chlorotestosterone in a dietary supplement

Capsules that were labeled to be performance‐enhancing dietary supplements obtained during an investigation were found to contain an unrecognized steroid‐like substance. This compound was isolated by liquid chromatography (LC) fraction collection and characterized using several qualitative analytical techniques, including ultraviolet (UV) spectroscopy, gas chromatography–mass spectrometry (GC–MS), liquid chromatography‐high resolution accurate mass‐mass spectrometry (LC–HRAM–MS), as well as ¹H, ¹³C, and two‐dimensional nuclear magnetic resonance (NMR) spectrometry. This multi‐technique analytical approach was used to identify the designer steroid as 6β‐chloro‐4‐androsten‐17β‐ol‐3‐one (6β‐chlorotestosterone), an analog of testosterone about which little has been published.