Authentic Specimens Research Articles

The authenticity of ‘I’iwi Drepanis coccinea (G. Forster, 1781) skins from Cook's third voyage: what taxidermy can add to the discussion

Summary. The ‘I’iwi Drepanis coccinea was discovered during James Cook's third circumnavigation (1776–80) and described by G. Forster in 1781. Several possibly authentic specimens and data sources linked to the original expedition exist. However, investigations into preparation style of the various ‘I’iwi specimens in question identified five different workshops and thus provenances. Only one specimen (at Gottingen, Germany) can unequivocally be considered authentic Cook material.

Sensitive quantification of 5F-PB-22 and its three metabolites 5F-PB-22 3-carboxyindole, B-22 N-5-hydroxypentyl and PB-22 N-pentanoic acid in authentic urine specimens obtained from four individuals by liquid chromatography–tandem mass spectrometry

Urine is the most suitable specimen to collect from individuals because of noninvasiveness and relatively large volumes obtainable. In authentic urine specimens, however, synthetic cannabinoids having the structures of quinolinyl ester indoles, such as 5F-PB-22, PB-22 and BB-22, in unchanged forms as well as their metabolites, have not been quantified yet. Therefore, the aim of this study was to establish a sensitive analytical method for the quantification of 5F-PB-22 and its three metabolites 5F-PB-22 3-carboxyindole, PB-22 N-5-hydroxypentyl and PB-22 N-pentanoic acid in authentic urine samples in four cases. These compounds were extracted from β-glucuronide-hydrolyzed and unhydrolyzed urine via liquid-liquid extraction. The identification and quantification were performed using the QTRAP type of a liquid chromatography–tandem mass spectrometer. The limits of detection were 3–30 pg/mL and their summed quantitation range was 10–10,000 pg/mL. The devised method was applied to quantify these compounds in authentic urine specimens obtained from four individuals. The levels of 5F-PB-22 were 5.1, 13.6, 94.7 and 470 pg/mL; those of 5F-PB-22 3-carboxyindole were 8.25, 3.39, 23.2 and 880 ng/mL; and those of PB-22 N-pentanoic acid were 12.0, 57.4, 959 and 2090 pg/mL, respectively, in four unhydrolyzed urine samples, and the levels of PB-22 N-5-hydroxypentyl could be quantified as 29.9 and 131 pg/mL in two unhydrolyzed urine samples. The 5F-PB-22 and its metabolites PB-22 N-5-hydroxypentyl have been detected from authentic human urine samples for the first time. Also, this is the first report dealing with the quantification of the three metabolites in human urine samples.

Situation of type specimens of Cordyceps and allies described by Dr Kobayasi

ABSTRACTInformation about authentic specimens of Cordyceps spp. that were described by Dr Kobayasi were summarised. Dr Kobayasi, National Museum of Nature and Science, has described/proposed about 28% of the scientific names for the major two genera of entomopathogenic fungi (Cordyceps s. l. and Torrubiella s. l.) with Mr Shimizu before 2007. In total 44 authentic specimens were discovered at present: 19 in Torrubiella, 6 in Cordyceps spp. on Elaphomyces, 19 in Cordyceps spp. on cicada. Although the colour iconography books published by Dr Kobayasi and Mr Shimizu did not explain the information about the illustrated specimens, 22 among the discovered specimens have been illustrated in colour showing their fresh condition in the books.

Rediscovery of Tragopogon dshimilensis (Asteraceae), endemic to Turkey

Tragopogon dshimilensis K. Koch is endemic to Turkey. This neglected species had been known from the original collection and was not included in the Flora of Turkey. Recently it was recollected from its type locality (A8 Rize: Cimil) and several adjacent localities. Based on two authentic specimens deposited at P and B, as well as new collections from Eastern Anatolia, an emended description and typification of T. dshimilensis are presented here, and its conservation status is also indicated.

Metabolism of novel opioid agonists U-47700 and U-49900 using human liver microsomes with confirmation in authentic urine specimens from drug users.

Recently, the number of adverse events, including death, involving novel opioids has continued to increase, providing additional and sustained challenges for forensic and medical communities. Identification of emerging novel opioids can be challenging, compounded by detection windows and unknown metabolic profiles. In this study, human liver microsomes were used for the generation of in vitro metabolic profiles of U-47700 and U-49900. Generated metabolites were analyzed via a SCIEX TripleTOF® 5600+ quadrupole time-of-flight mass spectrometer and resulting data files were processing using MetabolitePilot™. Characterized metabolites were verified in vivo by analysis of authentic human urine specimens collected after analytically confirmed cases of overdose involving U-47700 or U-49900. In total, four metabolites were identified and present in urine specimens for U-47700, and five metabolites for U-49900. N-Desmethyl-U-47700 was determined to be the primary metabolite of U-47700. Parent U-47700 was identified in all urine specimens. N-Desmethyl-U-47700 and N,N-didesmethyl-U-47700 were structurally confirmed for the first time during this study following acquisition of standard reference material. N-Desethyl-U-49900 was determined to be the primary metabolite of U-49900 following microsomal incubations, while N,N-didesethyl-N-desmethyl-U-49900 was the most abundant in a urine specimen. Similarities in metabolic transformation were identified between U-47700 and U-49900, resulting in a common metabolite and isomeric species. These phenomena should be considered in cases involving U-47700 or U-49900. This study is the first to map the metabolic profiles of U-47700 and U-49900 using human liver microsomes, as well as the first to report any literature involving U-49900 and analysis of case specimens.

Identification and quantification of predominant metabolites of synthetic cannabinoid MAB-CHMINACA in an authentic human urine specimen.

An autopsy case in which the cause of death was judged as drug poisoning by two synthetic cannabinoids, including MAB-CHMINACA, was investigated. Although unchanged MAB-CHMINACA could be detected from solid tissues, blood and stomach contents in the case, the compound could not be detected from a urine specimen. We obtained six kinds of reference standards of MAB-CHMINACA metabolites from a commercial source. The MAB-CHMINACA metabolites from the urine specimen of the abuser were extracted using a QuEChERS method including dispersive solid-phase extraction, and analyzed by liquid chromatography-tandem mass spectrometry with or without hydrolysis with β-glucuronidase. Among the six MAB-CHMINACA metabolites tested, two predominant metabolites could be identified and quantified in the urine specimen of the deceased. After hydrolysis with β-glucuronidase, an increase of the two metabolites was not observed. The metabolites detected were a 4-monohydroxycyclohexylmethyl metabolite M1 (N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-((4-hydroxycyclohexyl)methyl)-1H-indazole-3-carboxamide) and a dihydroxyl (4-hydroxycyclohexylmethyl and tert-butylhydroxyl) metabolite M11 (N-(1-amino-4-hydroxy-3,3-dimethyl-1-oxobutan-2-yl)-1-((4-hydroxycyclohexyl)methyl)-1H-indazole-3-carboxamide). Their concentrations were 2.17±0.15 and 10.2±0.3ng/mL (n=3, each) for M1 and M11, respectively. Although there is one previous in vitro study showing the estimation of metabolism of MAB-CHMINACA using human hepatocytes, this is the first report dealing with in vivo identification and quantification of MAB-CHMINACA metabolites in an authentic human urine specimen.

Confirmation of metabolites of the neuroleptic drug prothipendyl using human liver microsomes, specific CYP enzymes and authentic forensic samples—Benefit for routine drug testing

Metabolism of the tricyclic azaphenothiazine neuroleptic drug prothipendyl was investigated with in vitro studies using human liver microsomes but also specific isoforms of cytochrome P450 (CYP) enzymes. Identification and analysis of metabolites was done by liquid chromatography (LC) coupled with quadrupole time of flight mass spectrometry (LC-QTOF-MS) as well as triple quadrupole mass spectrometry (LC-QQQ-MS).Results of the herein presented study revealed the proof of various demethylated and oxidized metabolites (-CH2, -C2H4, four derivatives of prothipendyl +O and three derivatives of prothipendyl -CH2+O). Metabolic reactions of prothipendyl were mainly catalyzed by CYP enzymes CYP1A2, CYP2D6, CYP2C19 and CYP3A4. N-demethyl-prothipendyl was predominantly formed by isoforms CYP2C19 and CYP1A2, while particularly the CYP isoenzyme 3A4 was responsible for the formation of prothipendyl sulfoxide.To confirm the formation of previously identified metabolites in vivo, cardiac blood samples that were tested positive for prothipendyl during routine drug testing and serum and urine samples, collected after a voluntary intake of prothipendyl, were analyzed by LC-QQQ-MS. All metabolites of prothipendyl were proven in these authentic specimens. Neither in serum samples nor in urine samples, a prolonged detectability of metabolites in comparison to prothipendyl could be demonstrated.

Detection of the recently emerged synthetic cannabinoid 5F–MDMB‐PICA in ‘legal high’ products and human urine samples

Indole or indazole-based synthetic cannabinoids (SCs) bearing substituents derived from valine or tert-leucine are frequently abused new psychoactive substances (NPS). The emergence of 5F-MDMB-PICA (methyl N-{[1-(5-fluoropentyl)-1H-indol-3-yl]carbonyl}-3-methylvalinate) on the German drug market is a further example of a substance synthesized in the context of scientific research being misused by clandestine laboratories by adding it to 'legal high' products. In this work, we present the detection of 5F-MDMB-PICA in several legal high products by gas chromatography-mass spectrometry (GC-MS) analysis. To detect characteristic metabolites suitable for a proof of 5F-MDMB-PICA consumption by urine analysis, pooled human liver microsome (pHLM) assays were performed and evaluated using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QToF-MS) techniques to generate reference spectra of the in vitro phase I metabolites. The in vivo phase I metabolism was investigated by the analysis of more than 20 authentic human urine specimens and compared to the data received from the pHLM assay. Biotransformation of the 5-fluoropentyl side chain and hydrolysis of the terminal methyl ester bond are main phase I biotransformation steps. Two of the identified main metabolites formed by methyl ester hydrolysis or mono-hydroxylation at the indole ring system were evaluated as suitable urinary biomarkers and discussed regarding the interpretation of analytical findings. Exemplary analysis of one urine sample for 5F-MDMB-PICA phase II metabolites showed that two of the main phase I metabolites are subject to extensive glucuronidation prior to renal excretion. Therefore, conjugate cleavage is reasonable for enhancing sensitivity. Commercially available immunochemical pre-tests for urine proved to be unsuitable for the detection of 5F-MDMB-PICA consumption. Copyright © 2017 John Wiley & Sons, Ltd.

Tropical ulcer plant treatments used by Papua New Guinea's Apsokok nomads

Ethnopharmacological relevanceThe tropical ulcer is a debilitating bacterial infection that is common in Papua New Guinea. Deploying healthcare infrastructure to remote and inaccessible rainforest locations is not practical, therefore local plants may be the best treatment option. Here we present an ethnobotanical survey of the tropical ulcer plant medicines used by the semi-nomadic Apsokok who roam the remote central mountains of Papua New Guinea's West New Britain Province. In vitro biological activity in assays relevant to tropical ulcer wound healing is also presented. Materials and methodsFocus groups and semi-structured interviews were used to acquire information on the uses of plants, vouchers of which were identified by comparison with authentic herbarium specimens. Antibacterial disc diffusion assays with Staphylococcus aureus and Fusobacterium ulcerans, MMP-9 enzyme inhibition and dermal fibroblast stimulation assays were carried out on plant saps and aqueous extracts of plant material. LC-MS was used to identify known plant metabolites. ResultsThe ethnobotanical survey identified sixteen species that were used to treat tropical ulcers, all of which were applied topically. A subset of twelve species were investigated further in vitro. Four species produced zones of inhibition with S. aureus, all 12 species provided low level inhibition of MMP-9 and 8 species stimulated dermal fibroblast proliferation, although cytotoxicity occurred at higher concentrations. The extract of Homalium foetidum Benth. inhibited S. aureus and MMP-9 while at lower sub-cytotoxic concentrations stimulated fibroblast proliferation. Trans-3-O-p-coumaroylquinic acid cis-3-O-p-coumaroylquinic acid were detected in the aqueous extract of H. foetidum. ConclusionsTopical application of plant saps to wounds results in very high localised concentrations of plant metabolites which is likely to result in inhibition of MMP proteases. H. foetidum is a candidate plant for tropical ulcer treatment in remote areas.

Typifications of Verbascum adrianopolitanum and V. banaticum, and a New Hybrid Name Verbascum × stribrnyi (Scrophulariaceae)

Verbascum adrianopolitanum and V. banaticum occur in the Balkans and are welldefined from the taxonomical point of view, but their typification was unclear and lectotypes are here designated for nomenclatural stability. This is particularly important for V. banaticum because of the large number of authentic specimens appearing in botanical collections all over the world. Verbascum × stribrnyi Murbeck ex Sutorý is a new name for a hybrid between V. adrianopolitanum and V. densiflorum.

Development and validation of a liquid chromatography tandem mass spectrometry method for the determination of cannabinoids and phase I and II metabolites in meconium

A liquid chromatography–tandem mass spectrometry (LC–MSMS) method was developed and fully validated for the determination of Δ9-tetrahydrocannabinol (THC), 11-hydroxyTHC (OHTHC), 11-nor-9-carboxyTHC (THCCOOH), 8-β-11-dihydroxyTHC (diOHTHC), cannabinol, cannabidiol, and THC and THCCOOH glucuronides in 0.25±0.02g meconium. Samples were homogenized in methanol and subjected to cation exchange solid-phase extraction. Chromatographic separation was performed on a Kinetex C18 column (50 mm×2.1mm, 2.6μm) at 35°C, with a gradient of 0.1% formic acid in water and acetonitrile at a flow rate of 0.3 mL/min; total run time was 10min. Two transitions per analyte were monitored in MRM mode. The method was specific and sensitive; LOD was from 1 to 2ng/g, and LOQ from 4 to 10ng/g; linearity ranged from 4 to 400 ng/g for all the analytes, except for THC glucuronide (10–400ng/g); intra-assay, inter-assay and total imprecision were <11.2%, <13.45% and <15.6%, respectively; accuracy ranged from 93.9% to 109.0% of the target concentration; matrix effect, extraction and process efficiency ranged from −26.4% to −71.4%, 49.9% to 69.5% and 14.3% to 45.0%, respectively. The inclusion of THC and THCCOOH glucuronides avoided the need for the hydrolysis process, thus facilitating sample pretreatment. Application of the method to 19 authentic meconium specimens from uncontrolled pregnancies or women suspicious of drug consumption revealed fetal cannabis exposure in 4 newborns. THCCOOH (24.1–288.8ng/g), diOHTHC (53.2–332.4ng/g), THC (4.2–7.7ng/g), CBN (30.7–93.3ng/g) and CBD (7.1–251.5ng/g) were detected in all cases; THCCOOH glucuronide (190.2–306.8ng/g) in 3 cases; and OHTHC (11.9ng/g) in the remaining one; however, THC glucuronide was not identified in any specimen.

Economic botany collections: A source of material evidence for exploring historical changes in Chinese medicinal materials

Ethnopharmacological relevanceMany Chinese medicinal materials (CMMs) have changed over centuries of use, particularly in terms of their botanical identity and processing methods. In some cases, these changes have important implications for safety and efficacy in modern clinical practice. As most previous research has focused on clarifying the evolution of CMMs by analyzing traditional Chinese materia medica (“bencao”) literature, assessments of historical collections are needed to validate these conclusions with material evidence. Aim of the studyHistorical collections of Chinese medicines reveal the market materials in circulation at a given moment in time, and represent an underexploited resource for analyzing the evolution of Chinese herbal medicines. This study compares specimens from a rare collection of CMMs from the 1920s with contemporary market materials; by highlighting examples of changes in botanical identity and processing that remain relevant for safe clinical practice in the modern era, this work aims to stimulate further research into previously unexplored historical collections of Chinese medicines. Materials and methods620 specimens of CMMs that were collected from Chinese pharmacies in the Malay peninsula in the 1920s were examined macroscopically and compared with current pharmacopoeia specifications and authentic contemporary samples. These historical specimens, which are stored in the UK in the Economic Botany Collections (EBC) of Royal Botanic Gardens Kew, were morphologically examined, photographed, and compared to authentic CMMs stored at the Bank of China (Hong Kong) Chinese Medicines Center at Hong Kong Baptist University, as well as authentic herbarium-vouchered specimens from the Leon Collection (LC) at the Kew EBC. Case studies were selected to illustrate examples of historical changes in botanical identity, used plant parts, and processing methods. ResultsThis investigation confirmed that confusion due to shared common names and regional variations in the botanical identity of certain CMMs has been a persistent issue over time. Additionally, historical changes in processing methods and the plant parts used were observed for some CMMs. In some cases, these changes have direct implications for the safe clinical practice of Chinese medicine. ConclusionsThis preliminary assessment illustrated the significant potential of collections for clarifying historical changes in CMMs. More research is needed to investigate pre-modern collections of CMMs, including a more comprehensive assessment of the holdings in the Kew EBC and other European collections that have not yet been explored from the perspective of Chinese medicine.

Detection of metabolites of two synthetic cannabimimetics, MDMB-FUBINACA and ADB-FUBINACA, in authentic human urine specimens by accurate mass LC–MS: a comparison of intersecting metabolic patterns

The structures of two indazole-derived synthetic cannabimimetics, a methyl ester, MDMB-FUBINACA, and an amide, ADB-FUBINACA, differ only in the terminal groups on the side chains. Based upon liquid chromatography–quadrupole time-of-flight-mass spectrometry analysis of urine and blood samples collected from patients who were admitted to hospital with suspected drug intoxications and from postmortem forensic investigations, 38 metabolites were tentatively identified. Hydrolysis of the terminal groups (methyl ester and amide for MDMB-FUBINACA and ADB-FUBINACA, respectively) was found to be a common metabolic pathway for both compounds, leading to the formation of other common metabolites. Hydrolysed metabolites undergo subsequent monohydroxylation, dihydrodiol formation, fluorobenzyl loss and dehydrogenation. Most of the metabolites of MDMB-FUBINACA were products of ester hydrolysis. Metabolites formed by hydrolysis, additional monohydroxylation, dihydrodiol formation and fluorobenzyl loss were also detected in forms of glucuronides. Two unhydrolysed metabolites were identified as products of hydroxylation and fluorobenzyl loss with subsequent glucuronidation. In the case of ADB-FUBINACA, products of mono- and dihydroxylation, dihydrodiol formation, dihydrodiol formation combined with monohydroxylation, monohydroxylation combined with dehydrogenation and fluorobenzyl loss with monohydroxylation were identified. Monohydroxylated and dihydrodiol metabolites were also detected in the form of glucuronides. The most abundant metabolites were products of ester hydrolysis in free and glucuronidated forms (for MDMB-FUBINACA) and of dihydrodiol formation (for ADB-FUBINACA). These compounds are recommended for toxicological screening. To our knowledge, there are no reports dealing with the detection of metabolites of MBDB-FUBINACA and ADB-FUBINACA in authentic human urine specimens.

Simultaneous extraction and determination of monoamine neurotransmitters in human urine for clinical routine testing based on a dual functional solid phase extraction assisted by phenylboronic acid coupled with liquid chromatography-tandem mass spectrometry.

The major monoamine neurotransmitters, serotonin (5-HT) and catecholamines (i.e., norepinephrine (NE), epinephrine (E), and dopamine (DA)), are critical to the nervous system function, and imbalances of the neurotransmitters have been connected to a variety of diseases, making their measurement useful in a clinical setting. A simple, rapid, robust, sensitive, and specific LC-MS/MS method has been developed and validated for the simultaneous quantitation of urinary serotonin and catecholamines with low cost, which is ideal for routine clinical applications. A simple extraction from complex urine was accomplished using tailored solid phase extraction incorporating phenylboronic acid complexation on a 96-well HLB microplate for the sample extraction and resulted in significantly improved throughput, selectivity, and extraction recovery. Compared to 1-10mL of urine typically used, this method required only 10μL. A rapid chromatographic elution with a total cycle time of 6min per sample compared to reported run times of 19-75min was achieved on a PFP column. The sensitivity of l and 2ngmL-1 for the detection of low abundant E and NE combined with the high coverage of 1024ngmL-1 for DA enabled the multi-analyte detection of these biogenic amines in a single run. Good linearity (2.0-512, 1.0-512, 4.0-1024, and 4.0-1024ngmL-1 for NE, E, DA, and 5-HT, respectively), accuracy (87.6-104.0%), precision (≤8.0%), extraction recovery (69.6-103.7%), and matrix effect (87.1-113.1% for catecholamines and 63.6-71.4% for 5-HT) were obtained. No autosampler carryover was observed. The analytes were stable for 5days at 20°C, 14days at 4°C, and 30days at -20°C and five freeze-thaw cycles. The easy sample preparation, rapid LC, and multi-analyte MS detection allow two 96-well plates of samples to be extracted within 2h and analyzed on an LC-MS/MS system within 24h. The applicability and reliability of the assay were demonstrated by assessment of the reference interval for authentic urine specimens from 90 healthy individuals. Graphical abstract A simple, rapid, robust, sensitive and specific LC-MS/MS method combined with a dual functional solid phase extraction has been developed and validated for the simultaneous extraction and quantitation of monoamine neurotransmitters in human urine with low cost.

Sensitive identification and quantitation of parent forms of six synthetic cannabinoids in urine samples of human cadavers by liquid chromatography–tandem mass spectrometry

Human urine samples are easier to obtain than human blood samples due to noninvasiveness. The urine levels of synthetic cannabinoids (SCs) in unchanged forms, however, are usually much lower than their blood and tissue levels and cannot be detected in most cases. Therefore, in the present work a sensitive analytical method was devised for the determination of urine levels of six SCs in unchanged forms such as N-(1-amino-3-methy-1-oxobutan-2-yl)-1-pentyl-1H-indazole-3-carboxamide (AB-PINACA), N-(1-amino-3-methy-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamide (AB-FUBINACA), N-[(1S)-1-(1-aminocarbonyl)-2-methyl-propyl]-1-(cyclohexylmethyl)-1H-indazole-3-carboxamide (AB-CHMINACA), N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-(cyclohexylmethyl)-1H-indazole-3-carboxamide (MAB-CHMINACA), methyl-2-[1-(5-fluoropentyl)-1H-indazole-3-carboxamido]-3-methylbutanoate (5F-AMB) and methyl-2-[1-(5-fluoropentyl)-1H-indazole-3-carboxamido]-3,3-dimethylbutanoate (5F-ADB). These SCs were extracted from urine via liquid–liquid extraction. The identification and quantitation were performed by a relatively new type of an instrument for liquid chromatography–tandem mass spectrometry. The limits of detection were as low as 3–8 pg/mL, and the quantitation range was 10–1000 pg/mL using 400 μL of urine. The urine levels of AB-PINACA and AB-FUBINACA of victim 1 were 23 and 10 pg/mL, those of AB-CHMINACA and 5F-AMB of victim 2 were 239 and 19 pg/mL, and those of MAB-CHMINACA and 5F-ADB of victim 3 were 229 and 19 pg/mL, respectively. To our knowledge, this is the first report dealing with successful analysis of low levels of parent synthetic cannabinoids in authentic human urine specimens.

Murdelisest tegelaskõnest "Tõe ja õiguse" I osas / Dialect in the Speech of Characters in A. H. Tammsaare’s Truth and Justice Part I

Murdelisest tegelaskõnest "Tõe ja õiguse" I osas / Dialect in the Speech of Characters in A. H. Tammsaare’s Truth and Justice Part I

Stability of 21 Cocaine, Opioid and Benzodiazepine Drug Analytes in Spiked Meconium at Three Temperatures.

In this study, the stability of 21 cocaine, opioid and benzodiazepine analytes in spiked meconium was investigated at three storage temperatures: 4°C, room temperature (RT), and 37°C (body temperature). The drugs/metabolites included were hydrocodone, hydromorphone, codeine, morphine, 6-acetylmorphine (6-AM), oxycodone, oxymorphone, cocaine, cocaethylene, benzoylecgonine, m-hydroxybenzoylecgonine, diazepam, oxazepam, temazepam, nordiazepam, chlordiazepoxide, lorazepam, alprazolam, alpha-hydroxyalprazolam, clonazepam, 7-aminoclonazepam, midazolam, alpha-hydroxymidazolam and zolpidem. Drug testing was performed using mass spectrometry methods that were validated for clinical use. After 2 weeks of storage, a substantial loss was observed in the concentrations of 7-aminoclonazepam (48.4% at 4°C and 71.5% at RT), and chlordiazepoxide (59.5% at RT). A slight decrease was observed in the concentrations of alprazolam (20.9% at 4°C), clonazepam (24.5% at 4°C), chlordiazepoxide (23.5% at 4°C), midazolam (20.8% at 4°C), nordiazepam (22.8% at RT), and alpha-hydroxyalprazolam (20.7% at 4°C). At 37°C, the concentrations of chlordiazepoxide, 7-aminoclonazepam, lorazepam, oxazepam, nordiazepam and temazepam decreased by 81.4%, 86.8%, 56.5%, 59.9%, 45.4% and 31.7%, respectively, after 2 weeks. 6-AM was observed to be unstable regardless of storage temperatures. For morphine, a 33.3% increase at 4°C and a 23.4% increase at RT were observed after 2 weeks, respectively, possibly due to 6-AM degradation, while no changes ≥20% were observed at 37°C. All other analytes were stable up to 2 weeks at all three storage temperatures (concentration changes <20%). The stability of select drug analytes in authentic clinical meconium specimens was consistent with that observed in spiked meconium. In conclusion, some drugs in meconium may not be stable for long periods of time. Sample storage conditions are an important consideration in the context of detection windows and interpreting drug-testing results in meconium. To the best of our knowledge, this is the first stability study of cocaine, opioids and benzodiazepines in meconium concerning the effects of storage temperatures.

Looking at flubromazolam metabolism from four different angles: Metabolite profiling in human liver microsomes, human hepatocytes, mice and authentic human urine samples with liquid chromatography high-resolution mass spectrometry

Flubromazolam is a triazolam benzodiazepine that recently emerged as a new psychoactive substance. Since metabolism data are scarce and good analytical targets besides the parent are unknown, we investigated flubromazolam metabolism in vitro and in vivo. 10μmol/L flubromazolam was incubated with human liver microsomes for 1h and with cryopreserved human hepatocytes for 5h. Mice were administered 0.5 or 1.0mg flubromazolam/kg body weight intraperitoneally, urine was collected for 24h. All samples, together with six authentic forensic human case specimens, were analyzed (with or without hydrolysis, in case it was urine) by UHPLC–HRMS on an Acquity HSS T3 column with an Agilent 6550 QTOF. Data mining was performed manually and with MassMetasite software (Molecular Discovery).A total of nine metabolites were found, all generated by hydroxylation and/or glucuronidation. Besides O-glucuronidation, flubromazolam formed an N+-glucuronide. Flubromazolam was not metabolized extensively in vitro, as only two monohydroxy metabolites were detected in low intensity in hepatocytes. In the mice samples, seven metabolites were identified, which mostly matched the metabolites in the human samples. However, less flubromazolam N+-glucuronide and an additional hydroxy metabolite were observed. The six human urine specimens showed different extent of metabolism: some samples had an intense flubromazolam peak next to a minute signal for a monohydroxy metabolite, others showed the whole variety of hydroxylated and glucuronidated metabolites. Overall, the most abundant metabolite was a monohydroxy metabolite, which we propose as α-hydroxyflubromazolam based on MSMS fragmentation. These metabolism data will assist in interpretation and analytical method development.

The recovering of breeding achievements of Populus × leningradensis bogd. and Populus × newensis bogd. Based on microsatellite analysis

The genotyping of 75 trees from poplar plantations in St. Petersburg and Leningrad oblast was conducted with microsatellite markers to identify the elite clonal varieties developed by P.L. Bogdanov in the period of 1938–1965. The information about the varieties was lost. The authentic herbarium specimens of poplar clonal varieties preserved at the St. Petersburg State Forest Technical University were used as reference genotypes. According to the results of DNA fingerprinting, we identified the clonal plantations of Populus × newesis Bogd. and Populus × leningradensis Bogd. from the Kartashevskii forest district and the arboretum of the St. Petersburg State Forest Technical University. The identified elite poplar hybrids have a higher frost resistant and a higher growth rate. They are recommended for plantation cultivation in the northwest of Russia.

In vitro and in vivo human metabolism of a new synthetic cannabinoid NM-2201 (CBL-2201).

In 2014, NM-2201 (CBL-2201), a novel synthetic cannabinoid (SC), was detected by Russian and United States laboratories. It was already added to the scheduled drugs list in Japan, Sweden and Germany. Unfortunately, no human metabolism data are currently available, making it challenging to confirm its intake because all previous investigated SCs were extensively metabolized. The present study aims to recommend appropriate marker metabolites by investigating NM-2201 metabolism in human hepatocytes and confirm the results in authentic human urine specimens. For the metabolic stability assay, 1 μM NM-2201 was incubated in human liver microsomes (HLMs) for up to 1 h; for metabolite profiling, 10 μM of NM-2201 was incubated in human hepatocytes for 3 h. Two authentic urine specimens from NM-2201 positive cases were analyzed after β-glucuronidase hydrolysis. Metabolite identification in hepatocyte samples and urine specimens was achieved with high-resolution mass spectrometry via information-dependent acquisition. NM-2201 was quickly metabolized in HLMs with an 8.0 min half-life. In human hepatocyte incubation samples, a total of thirteen NM-2201 metabolites were identified, generated mainly from ester hydrolysis and further hydroxylation, oxidative defluorination and subsequent glucuronidation. M13 (5-fluoro PB-22 3-carboxyindole) was the major metabolite. In the urine specimens, the parent drug NM-2201 was not detected; M13 was the predominant metabolite after β-glucuronidase hydrolysis. Therefore, based on our study, we recommend the M13 as a suitable urinary marker metabolite for confirming NM-2201 and/or 5F-PB-22 intake.