Carbon and nitrogen isotopic analysis of morphine from opium and heroin samples originating in the four major heroin producing regions

The forensic analysis of stable isotopes is a valuable tool to geo-source natural or semisynthetic drugs such as cocaine and heroin. The present study describes a novel methodology to isolate morphine from opium for isotopic analysis. Furthermore, this isotopic data from regional sources is corroborated with morphine data obtained from seized heroin (deacetylated to morphine) from the same regions. All five primary alkaloids of opium, namely, morphine, codeine, thebaine, noscapine, and papaverine, were quantified using high performance liquid chromatography with photodiode array (PDA) detector before the preparative experiment to gather a complete major alkaloidal profile. Morphine fractions of authentic opium submissions from Mexico, South America, Southwest Asia, and Southeast Asia were isolated and collected using preparative high performance liquid chromatography, and the collected morphine samples were subsequently analyzed by isotope ratio mass spectrometry. Carbon and nitrogen isotope data are presented. The data demonstrate that nitrogen ratios are capable of differentiating samples from Mexico and South America while carbon ratios are able to distinguish Southwest Asian samples from other source regions. Analogous results have routinely been observed (as part of Heroin Signature Program analysis) for morphine obtained from deacetylated authentic heroin samples from the same source regions. The results suggest that the poppy growing region has a greater influence on the carbon and nitrogen isotope values than the heroin manufacturing processes employed. When utilized in conjunction with existing signature methodologies, carbon and nitrogen isotope ratio data can enhance the ability to geo-source heroin.     

Cathinones: Isotopic profiling as an aid to linking seizures

The number of cathinone derivatives available to the street market has increased steadily since 2008. Many of these compounds have proven to be potent psychostimulants and fatalities have occurred through their recreational use. The method of manufacture is essentially the same for each cathinone, i.e., (i) selection of the appropriate β‐ketoarylalkane, (ii) bromination alpha to the keto group, followed by (iii) amination using the desired amine. The cathinone derivatives are usually prepared at a very high purity and little information is available from an organic manufacturing by‐products profile because the product is so pure. To provide law enforcement agencies with a tool that would enable links to be identified between samples from the same production batch, the carbon, hydrogen, and nitrogen stable isotope ratios in a number of cathinones were investigated. The aim was to determine if sufficient diversity existed in the light element stable isotope ratios of cathinones to allow the isotopic ratios to be used to discriminate between different seizures and to assist in linking samples from the same seizure. Careful measurement of the δ¹³C, δ¹⁵N, and δ²H values in each sample revealed that the stable isotope ratios for a particular cathinone analogue vary from one seizure to another. In the seizures studied, carbon, hydrogen, and nitrogen stable isotope ratios were found to vary from ‐32.8‰ to ‐26.1‰, ‐152‰ to +72‰, and ‐16.6‰ to ‐2.7‰, respectively. Copyright © 2015 John Wiley & Sons, Ltd.     

Profiling ephedrine prepared from N‐methylalanine via the Akabori‐Momotani reaction

Novel methods for synthesising methylamphetamine precursors are appearing in clandestine laboratories within Australia. One such laboratory involved the synthesis of ephedrine from N‐methylalanine and benzaldehyde via the Akabori‐Momotani reaction. This article presents chiral and stable isotope ratios of ephedrine synthesised via this method, along with a chemical profile of methylamphetamine produced from this ephedrine. Based on the chiral results and the δ¹³C, δ¹⁵N, and δ²H values, it is possible to distinguish ephedrine made via the Akabori‐Momotani reaction from ephedrine of a “natural”, “semi‐synthetic”, or “fully‐synthetic” origin. Methylamphetamine and ephedrine samples synthesised from benzaldehyde having an enriched δ²H value (ie, > 0‰), via the Akabori‐Momotani reaction, had an isotopic profile which set them apart from all other methylamphetamine samples. It was noted, however, that using stable isotope ratios alone to determine the precursor of methylamphetamine is limited; they could not with confidence differentiate between methylamphetamine and ephedrine synthesised from benzaldehyde having a depleted δ²H value (ie, <0‰) from other ephedrine sources and phenyl‐2‐propanone based methylamphetamine samples profiled.     

Stable isotope ratio profiling of testosterone preparations

Gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) is the preferred method of confirming the administration of exogenous testosterone by athletes. This relies on synthetic testosterone preparations being depleted in (13) C compared to natural testosterone. There is concern, however, about the existence of synthetic testosterone products that are unexpectedly (13) C-enriched and which may allow athletes to circumvent the current GC-C-IRMS test. Further to the reported studies of legitimate pharmaceutical-grade testosterone products, a detailed analysis of seized materials from border-level seizures was required to obtain intelligence concerning trends in 'black market' testosterone manufacture and distribution. The sample set collected for this study between 2006 and 2009 inclusive provided a δ(13) C range (n = 266) of -22.9‰ to -32.6‰ with mean and median values of -28.4‰ and -28.6‰, respectively. Within this distribution there were 24 samples (9%) confirmed to have δ(13) C values in the range reported for endogenous urinary steroid metabolites (≥ -25.8‰). The benefit of δ(13) C profiling for testosterone preparations was demonstrated by the ability to identify specific seized products that can be target tested for future intelligence purposes. In addition, the potential of stable hydrogen isotope ratio ((2) H/(1) H; δ(2) H) discrimination to complement δ(13) C analysis was investigated. Methodologies for the determination of δ(2) H values by gas chromatography-thermal conversion-isotope ratio mass spectrometry (GC-TC-IRMS) were developed to provide a δ(2) H range (n = 173) of -177‰ to -268‰ with mean and median values of -231‰ and -234‰, respectively.     

δ34S and δ18O of dissolved sulfate as biotic tracer of biogeochemical influences on arsenic mobilization in groundwater in the Hetao Plain,Inner Mongolia,China

Environmental isotopology of sulfur and oxygen of dissolved sulfate in groundwater was conducted in the Hetao Plain, northwestern China, aiming to better understand the processes controlling arsenic mobilization in arsenic-rich aqueous systems. A total of 22 groundwater samples were collected from domestic wells in the Hetao Plain. Arsenic concentrations ranged from 11.0 to 388 μg/L. The δ³⁴S-_SO4 and δ¹⁸O-_SO4 values of dissolved sulfate covered a range from +1.48 to +22.4 ‰ and +8.17 ‰ to +14.8 ‰ in groundwater, respectively. The wide range of δ³⁴S-_SO4 values reflected either an input of different sources of sulfate, such as gypsum dissolution and fertilizer application, or a modification from biogeochemical process of bacterial sulfate reduction. The positive correlation between δ³⁴S-_SO4 and arsenic concentrations suggested that bacteria mediated processes played an important role in the mobilization of arsenic. The δ¹⁸O-_SO4 values correlated non-linearly with δ³⁴S-_SO4, but within a relatively narrow range (+8.17 to +14.8 ‰), implying that complexities inherent in the sulfate-oxygen (O-SO₄ ^2?) origins, for instance, water-derived oxygen (O-H₂O), molecular oxygen (O-O₂) and isotope exchanging with dissolved oxides, are accounted for oxygen isotope composition of dissolved sulfate in groundwater in the Hetao Plain.     

A Targeted UHPLC-MS/MS Method Validated for the Quantification of Ergot Alkaloids in Cereal-Based Baby Food from the Belgian Market

Following pending new legislation in the European Union setting a maximum of 20 ng g⁻¹ for the total sum of ergot alkaloids in dry cereal-based baby food, a new UHPLC-MS/MS method was developed. It is suitable for the quantification of six ergot alkaloids: Ergocornine, ergocristine, ergometrine, ergosine, ergotamine, α-ergocryptine, and their corresponding epimers. The method is able to reliably detect individual ergot alkaloids at a level as low as 0.5 ng g⁻¹. The method uses a modified QuEChERS extraction approach before UHPLC-MS/MS analysis. The method showed good sensitivity, accuracy, and precision. It has been applied to 49 samples from the Belgian market. In 26 samples, not a single ergot alkaloid was detected while in 23 out of 49 samples at least one ergot alkaloid was detected with 2 samples containing 12 ergot alkaloids. Ergometrine was the alkaloid most frequently detected i.e., 16 out of 49 samples. Only one sample, testing positive for all 12 ergot alkaloids, would be non-conforming to the newly proposed Maximum Residue Level (MRL).     

Variation of alkaloid contents and antimicrobial activities of Papaver rhoeas L. growing in Turkey and northern Cyprus

Context: Papaver rhoeas L. (Papaveraceae) corn poppy, widely distributed in Turkey, is used to make a cough syrup for children, as a tea for disturbed sleep, for pain relief and as a sedative in folk medicine.

Objective: Samples of P. rhoeas collected from eight different locations in Turkey and three from northern Cyprus were investigated for their alkaloid content and screened for their antimicrobial activities.

Materials and methods: From the aerial parts of P. rhoeas samples, alkaloids were isolated by column and preparative thin-layer chromatography. The alkaloids were identified by comparing their spectral data (UV, IR and ¹H-NMR) and TLC Rf values with those of authentic samples. The antimicrobial study was carried out by microbroth dilution technique against six strains of bacteria and three strains of fungi.

Results: Twelve different alkaloids belonging to proaporphine (mecambrine), aporphine (roemerine), promorphinan (salutaridine), protopine (coulteropine and protopine) and rhoeadine (epiglaucamine, glaucamine, glaudine, isorhoeadine, isorhoeagenine, rhoeadine and rhoeagenine) groups were isolated. The most significant activity was observed with the alkaloid extract of P8 against Staphylococcus aureus with a MIC value of 1.22?μg/mL and against Candida albicans with a MIC value of 2.4?μg/mL.

Discussion: The results indicate that P. rhoeas samples (P8 and P9), which contain roemerine as their major alkaloid, were the most active extracts.     

海南萝芙木季胺碱化学成分的研究

从海南萝芙木生产“降压灵”的母液,经盐析得到生物碱部分,用硅胶柱层,制备薄层和葡聚糖凝胶过滤法,分得六种生物碱。碱Ⅰ(ajmaline)、碱Ⅱ(vellosimine)、碱Ⅲ(spegatrine)、碱Ⅳ(verticillatine)为已知碱,碱Ⅴ和碱Ⅵ为两个新的二聚体生物碱。根据红外、紫外、质谱、氢谱及碳谱等光谱分析,推定碱Ⅴ的化学结构为1式,命名为双斯配加春(dispegatrine)。并用半合成方法进一步确证了其结构.碱Ⅵ的结构待定.药理研究表明碱Ⅴ和碱Ⅵ均具有α-肾上腺素能受体阻断作用。     

Alkaloids in ergot found on different Gramineae in The Netherlands

The alkaloid content and the composition of the alkaloid complex of thirteen samples of ergot sclerotia from different gramineous host species collected in The Netherlands were investigated. Two samples collected in France were also examined. Ergot ofGlyceria fluitans (L.) R.Br. did not contain alkaloids. The total alkaloid content of ergot found on the other wild grasses was more than 0.2%. The ratio between the contents of water soluble and water insoluble alkaloids of the investigated ergot sclerotia varies between 1∶10 and 1∶20. The composition of the alkaloid complex in the sclerotia was studied by quantitative thin-layer chromatography. The sclerotia could be grouped into three main categories:

sclerotia ofLolium perenne L.,Festuca arundinacea Schreb. andArrhenatherum elatius (L.) P.B. ex J. et C. Presl, containing predominantly ergocornine, α- and β-ergokryptine and ergosine;

sclerotia ofDactylis glomerata L.,Phalaris arundinacea L.,Alopecurus geniculatus L. andHolcus mollis L., containing predominantly ergosine, ergocristine and ergotamine;

sclerotia ofMolinia caerulea (L.) Moench, containing predominantly ergocristine, ergosine and ergqmetrine.

A great similarity was found between the composition of the alkaloid complex in ergot ofLolium perenne L. from different locations and also in ergot ofMolinia caerulea (L.) Moench from different locations. This similarity was less in ergot ofDactylis glomerata L. collected from different locations.     

Applicability of ultra‐high performance liquid chromatography‐quadrupole‐time of flight mass spectrometry for cocaine profiling

For the first time in China, the chemical profiling of cocaine specimens was performed at the National Narcotics Laboratory. An ultra‐high performance liquid chromatography‐quadrupole‐time of flight mass spectrometry (UHPLC‐QTOF‐MS) method was developed and validated for simultaneous analysis of 14 cocaine alkaloids and 5 main adulterants. Among them, ecgonine methyl ester, ecgonine, benzoylecgonine, and norcocaine were identified by comparing with the standard materials; tropacocaine, 3,4,5‐trimethoxycocaine, cis‐/trans‐cinnamoylcocaine were tentatively identified based on the exact masses of protonated molecules and product ions; six unidentified alkaloids of 182/1.47, 316/9.54, 659/9.85, 316/9.87, 420/10.34, and 420/10.85 were marked with ‘extracted mass/retention time’ for convenience. Minimum sample preparation and analysis time were required, which was suitable for routine analysis. Based on the semi‐quantitative data set of 14 alkaloid impurities in 131 linked/unlinked cocaine samples, 50 combinations of pretreatment methods and distance/correlation measurements were tested for their potential discrimination power for cocaine profiling, and Logarithm/Pearson exhibited the best result. After hierarchical cluster analysis (HCA), 183 cocaine samples collected from 2011 to 2015 were classified into 7 major groups. Moreover, 37 groups of linked samples were found within and between provinces, which provide intelligence for the case connection and revealing of the distribution networks. Our results highlighted the practical utilities of drug profiling, especially to support the investigation through operational intelligence and to improve the knowledge related to the drug trafficking through strategic intelligence. Copyright © 2016 John Wiley & Sons, Ltd.     

Stable carbon isotope ratio profiling of illicit testosterone preparations – domestic and international seizures

Gas chromatography‐combustion‐isotope ratio mass spectrometry (GC‐C‐IRMS) is now established as a robust and mature analytical technique for the doping control of endogenous anabolic androgenic steroids in human sport. It relies on the assumption that the carbon isotope ratios of naturally produced steroids are significantly different to synthetically manufactured testosterone or testosterone prohormones used in commercial medical or dietary supplement products. Recent publications in this journal have highlighted the existence of black market testosterone preparations with carbon isotope ratios within the range reported for endogenous steroids (i.e. δ¹³C ≥ ?25.8 ‰). In this study, we set out to profile domestic and international law enforcement seizures of illicit testosterone products to monitor the prevalence of ‘enriched’ substrates – which if administered to human subjects would be considered problematic for the use of current GC‐C‐IRMS methodologies for the doping control of testosterone in sport. The distribution of δ¹³C values for this illicit testosterone sample population (n = 283) ranged from ?23.4 ‰ to ?32.9 ‰ with mean and median of ?28.6 ‰ – comparable to previous work. However, only 13 out of 283 testosterone samples (4.6 %) were found to display δ¹³C values ≥ ?25.8 ‰, confirming that in the vast majority of cases of illicit testosterone administration, current GC‐C‐IRMS doping control procedures would be capable of confirming misuse. Copyright © 2014 John Wiley & Sons, Ltd.     

UHPLC–MS/MS quantitation of porphyroxine in opium and application of porphyroxine‐acetylated products as signature markers for heroin

Porphyroxine, a trace alkaloid in opium, was identified in the early 1800s and isolated/characterized in the 1960s. Recently, two significant porphyroxine‐related byproducts found in the acidic and neutral extracts of illicit heroin were characterized by this laboratory as the N‐acetyl‐O¹⁴‐desmethyl‐epi‐porphyroxine ( B ) and N,O⁸‐diacetyl‐O¹⁴‐desmethyl‐epi‐porphyroxine ( C ). The prevalence of the B and C compounds has been consistent in the following order of abundance for the thousands of authentic heroin samples analyzed: Southwest Asia (SWA) > South America (SA) > Southeast Asia (SEA) > Mexico (MEX). In this research, a rapid and efficient ultra‐high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was developed to determine the content of porphyroxine and five primary alkaloids (morphine, codeine, thebaine, noscapine, and papaverine) in opium after extraction with methanol/water (50/50). The method was validated in terms of linearity, accuracy, recovery, and precision for porphyroxine. The limit of quantitation (LOQ) for porphyroxine was 2.5 ng/mL. The developed method was successfully applied to a total of 114 authentic opium samples from the major poppy‐growing regions. The amount of porphyroxine was determined at the level of part per thousand (‰) and the relative concentrations to morphine were in the range of 1x10^?4 and 1x10^?2 with an order of SWA > SEA, SA > MEX for its average abundance, which is consistent with the order of the average abundance of its acetylated products ( B , C ) in illicit heroin. This study reveals the significance of porphyroxine and its acylated compounds in classifying heroin and opium samples to major geographical regions of production.     

Triclisia sacleuxii (Pierre) Diels (Menispermaceae), a potential source of acetylcholinesterase inhibitors

Objectives To search for compounds possibly useful for the treatment of Alzheimer's disease. Methods Alkaloid fractions from the roots, stems and leaves of Triclisia sacleuxii (Menispermaceae) and pure bisbenzylisoquinoline alkaloids isolated from the roots (phaeanthine, N‐methylapateline, 1,2‐dehydroapateline and gasabiimine) were assessed for acetylcholin‐esterase inhibitory activity. Key findings All extracts and compounds tested inhibited acetylcholinesterase to varying degrees; the leaf tertiary alkaloid fractions and the root quaternary alkaloid fractions exhibited the strongest inhibitory potential (90% at 0.1 mg/ml). The leaf tertiary alkaloid fraction was selected for further analysis (the quaternary alkaloids, which are too polar for absorption and brain distribution, were presumed to be clinically uninteresting). TLC bioautography using Ellman's reagent allowed the detection of acetylcholinesterase inhibitors and the isolation of the major active constituent, which was identified as lindoldhamine, a one‐bridged bisbenzylisoquinoline alkaloid. Lindoldhamine displayed high acetylcholinesterase inhibitory activity with a 50% inhibition concentration in the micromolar range. Conclusions All T. sacleuxii alkaloid fractions tested exhibited anti‐acetylcholinesterase activity; isolated bisbenzylisoquinoline alkaloids showed weak‐to‐high inhibition depending on their structural features. Structure modification could provide interesting derivatives with enhanced anti‐acetylcholinesterase activity.     

云南萝芙木生物碱的研究

从云南萝芙木(Rauwolfia yunnaensis Tsiang)根的总弱碱性生物碱部分分离出十一种生物碱。其中四种已有报道从同种植物中得到ajmalicine(生物碱Ⅰ)、ajmaline(生物碱Ⅱ)、reserpine(生物碱Ⅲ)和serpentinine(生物碱Ⅶ);两种为初次从该植物中分离出的已知的吲哚生物碱tetraphyllicine(生物碱Ⅸ)和vellosimine(生物碱Ⅺ),两种为新的吲哚生物碱生物碱Ⅳ和生物碱Ⅷ。并根据光谱分析和生物活性比较,提出了它们的化学结构,推论了它们的立体构型,生物碱Ⅳ为19-epi-ajmalicine,生物碱Ⅷ为pseudoreserpine 16、17位立体异构体。药理实验表明,生物碱Ⅷ具有类似利血平的降压作用,但作用弱、显效快且无中枢镇静作用。其它三种生物碱(生物碱Ⅹ、生物碱Ⅵ和生物碱Ⅴ)的化学结构有待进一步鉴定。     

宾川乌头中的生物碱及其结构的研究

从云南药用草乌之一的宾川乌头(Aconitum duclouxii Levl)根中分得两种结晶性生物碱,称为碱A及碱B。经鉴定,碱B为乌头碱(aconitine)。碱A分子式为C₃₄H₄₇O₁₀N,熔点168～169℃,[α]_D^28.1+28.3(C,1.43,氯仿),此系一新生物碱,命名为宾乌碱(duclouxine),经光谱分析,初步推定其化学结构式为12-β-羟基查斯乌头碱(12-β-hydroxy chasmaconitine)。宾乌碱经初步药理试验说明有镇痛作用。     

Box-Behnken效应面法优化龙葵中总生物碱的提取工艺

目的 优选龙葵中总生物碱的提取工艺。方法 采用单因素试验结合Box-Behnken 效应面法,以总生物碱提取率为指标,考察乙醇体积分数、料液比、提取时间、提取次数等因素对提取工艺的影响,采用酸性染料比色法,利用紫外分光光度计测定总生物碱的含量。结果 确定龙葵中总生物碱提取的最佳工艺条件：乙醇体积分数为63.72%,料液比为1∶41.23,提取时间为54.93 min,提取2次,总生物碱提取率为1.40 mg·g^-1。结论 Box-Behnken 效应面法用于龙葵中总生物碱提取工艺条件的优选是可行的,模型预测效果较好。     

Eine neue Methode zur Bestimmung der Gesamtalkaloide und des Cephaelins in Ipecacuanha

A new Determination Method for Total Alkaloids and Cephaeline in Ipecacuanha Graf's procedure¹) to determine the total alkaloids of Ipecacuanha root using adsorption chromatography is revised and improved. Both the total alkaloid content and the cephaeline content can be determined directly in the chloroform eluate, total alkaloids by spectrophotometry at 286 nm, cephaeline by reaction with 2,6-dichloroquinone-chloroimide in presence of methanolic potassium hydroxide, followed by spectrophotometry at 595 nm.     

Effects of harvesting time and processing on the contents of five alkaloids in the herbaceous stems of Ephedra sinica

In the present study, we studied the changes of the contents of alkaloids in the herbaceous stems of Ephedra sinicaduring different harvest periods as well as before and after processing. The alkaloid contents of 39 batches of ephedra herb, prepared slices ephedra and honey-fried ephedra, 24 batches of ephedra herb with different harvesting periods, which were all collected from cultivation base in Inner Mongolia, and 38 batches of prepared slices ephedra purchased from the market were detected by taking norephedrine (NE), norpseudoephedrine (NPE), ephedrine (E), pseudoephedrine (PE) and methylephedrine (ME) as indicators by using HPLC method. The content of total alkaloid in prepared slices ephedra (1.71%–3.14%) was higher than that in ephedra herb (1.20%–2.53%) and honey-fried ephedra (1.52%–2.99%). Contents of different alkaloids in these three types of samples were significantly different. Prepared slices ephedra and honey-fried ephedra showed significant differences in the contents of NE, NPE and ME (P<0.05), and the contents of E were significantly different between ephedra herb and honey-fried ephedra (P<0.05). The total alkaloid content of ephedra herb was the highest in September (3.10%). Alkaloid contents of prepared slices ephedra collected in the market were uneven and 13%–91% lower than those collected from cultivation base. The results provided a basis for the quality evaluation of ephedra herb and its processed products, and had certain guiding significance for the selection of processed ephedra according to different drug purposes in clinical application. It also provided data support for the harvesting time of ephedra herb.     

A preliminary study on sulfate reduction bacteria behaviors in groundwater by sulfur and carbon isotopes: a case study in Jiaozuo City,China

Inorganic pollutants in groundwater, such as sulfate and nitrate, have been a serious problem in China for decades. These pollutants are difficult to be removed because of their high solubility and ease of transport in subsurface environment. It had been found that microorganism could be one of the most feasible methods for inorganic pollutant elimination. During the process of degradation, some microorganisms can utilize sulfur and nitrogen in sulfate and nitrate forms, respectively, as energy sources. Meanwhile, significant variations of sulfur stable isotope ratios happened. Therefore sulfur isotope can be used as a good indicator for pollutant degradation and microbial activities. Shallow groundwater (SGW), deep groundwater (DGW), and surface water (SFW) were investigated in alluvial plain in Jiaozuo City, China. The results of hydrochemical analysis indicated that K⁺, Na⁺, and HCO₃ ^? were dominant ions in DGW, Mg²⁺ and HCO₃ ^? were dominant ions in SGW, and Ca²⁺ and HCO₃ ^? were dominant in SFW except for LR sample. A wide variation of δ ³⁴S_SO4 values ranging from + 7.3 to +23.6 ‰ had been observed for all water samples, with a mean value of +20.7, +12.6 and +10.0 ‰ for DGW, SGW, and SFW respectively. At the same time, δ ¹³C values of dissolved inorganic carbon (DIC) ranged from ?12.4 to ?5.7 ‰, with a mean value of ?7.5, ?9.0, and ?9.6 ‰ for DGW, SGW, and SFW, respectively. The microbial degradation processes resulted in significant sulfur isotope fractionations in DGW. Organic carbon was utilized by bacteria and transferred into inorganic carbon, leading to negative fractionation of carbon isotopes. Thus the variations in stable isotope ratios of sulfur and carbon in groundwater can be used as good indicators for understanding of the relationship between bacteria behaviors and sulfate degradation.