Stable Isotope Ratios of Marijuana. I. Carbon and Nitrogen Stable Isotopes Describe Growth Conditions*

Abstract: There remains significant uncertainty in illicit marijuana cultivation. We analyzed the δ¹³C and δ¹⁵N of 508 domestic samples from known U.S.A. counties, 31 seized from a single location, 5 samples grown in Mexico and Colombia, and 10 northwest border seizures. For a subset, inflorescences and leaves were analyzed separately. These data revealed a strong correspondence, with inflorescences having slightly higher δ¹³C and δ¹⁵N values than leaves. A framework for interpreting these results is introduced and evaluated. Samples identified as outdoor‐grown by δ¹³C were generally recorded as such by the Drug Enforcement Administration (DEA). DEA‐classified indoor‐grown samples had the most negative δ¹³C values, consistent with indoor cultivation, although many were also in the outdoor‐grown domain. δ¹⁵N indicated a wide range of fertilizers across the dataset. Samples seized at the single location suggested multiple sources. Northwest border δ¹³C values suggested indoor growth, whereas for the Mexican and Colombian samples they indicated outdoor growth.     

Comparative analysis of 1-phenyl-2-propanone (P2P), an amphetamine-type stimulant precursor, using stable isotope ratio mass spectrometry: Presented in part as a poster at the 2nd meeting of the Joint European Stable Isotope User Meeting (JESIUM), Giens, France, September 2008

The isotope ratios of amphetamine type stimulants (ATS) depend as well on the precursor as the synthetic pathway. For clandestine production of amphetamine and methamphetamine, 1-phenyl-2-propanone (P2P, benzylmethylketone) is a commonly used precursor.Our aim was to determine the variation of the isotope ratios within precursor samples of one manufacturer and to compare seized samples of unknown sources to these values. δ¹³C_V-PDB, δ²H_V-SMOW and δ¹⁸O_V-SMOW isotope ratios were determined using elemental analysis (EA) and gas chromatography (GC) coupled to an isotope ratio mass spectrometer (IRMS). The comparison of all seized samples to the data of the samples of one manufacturer revealed considerable differences. The results show that IRMS provides a high potential in differentiating between precursors from different manufacturers for the clandestine production of ATS and identifying corresponding sources.     

Australian Federal Police seizures of illicit crystalline methamphetamine ('ice') 1998-2002: impurity analysis

Nineteen crystalline methamphetamine ('ice') seizures captured by the Australian Federal Police (AFP) at the Australian border between 1998 and 2002 were analysed. Using a modified gas chromatograph-mass spectrometry (GC-MS) impurity profiling approach of these samples we have identified >30 compounds associated with methamphetamine and/or its synthetic route. Major impurities detected include 1,2-dimethyl-3-phenylaziridine 8, dimethylamphetamine 14, N-formylmethamphetamine 24, N-acetylmethamphetamine 25, 1,3-dimethyl-2-phenylnaphthalene 32, 1-benzyl-3-methylnaphthalene 33 and methamphetamine dimer 34. These data are suggestive of ephedrine/pseudoephedrine as the main precursor of the 'ice' samples seized during 1998-2002. Additionally the two naphthalenes 32 and 33 further identified that 15 items in 9 seizures were produced via the more specific ephedrine/hydriodic acid/red phosphorus method. One sample comprised 75% dimethylamphetamine and 9.7% methamphetamine, representing the first Australian seizure of imported dimethylamphetamine reported.     

Population data on the 11 STR loci in the Upper Silesia (Poland)

Smuggling of methamphetamine is affected by enforced regulation and international situation, resulting in changes of precursors and synthetic methods used. Enantiomer ratio of methamphetamine can provide information concerning its precursor and synthetic method. This information is useful for the prevention of smuggling methamphetamine and its precursor, and resultant reduction of methamphetamine abuse. In the present study, we investigated on the enantiomer ratios of 433 crystalline methamphetamine samples seized in Korea from 1994 to 2005. Excluding 17 samples of low purity, 416 samples were used for enantiomer profiling. The methamphetamine samples were derivatized with (S)-(+)-alpha-methoxy-alpha-(trifluoromethyl)phenylacetyl chloride ((S)-(+)-MTPACl), and the derivatives were analyzed by GCMS in selected ion monitoring (SIM) mode. The enantiomer ratios of the samples were calculated from the standard calibration curves of each enantiomer, both of which showed good linearity in the range of 0-1.2 microg. Most of the seizures were pure S(+)-enantiomer, but 21% (95 of 416 samples) contained R(-)-enantiomer above 1%. They began to appear from 1997, and increased continuously up to 50% in the year 2005 (55 of 111 samples). From this study, we could find out that alternative precursors have been used recently for the illicit manufacture of methamphetamine seized in Korea.     

Identification of impurities and statistical classification of methamphetamine tablets (Ya-Ba) seized in Thailand

Impurity profiles of methamphetamine tablets seized in Thailand have been investigated. The samples are extracted with small amounts of ethyl acetate under alkaline condition and the extracts are analyzed by capillary gas chromatography. Nine compounds (1,2-dimethyl-3-phenylaziridine, ephedrine, methylephedrine, N-formylmethamphetamine, N-acetylmethamphetamine, N-formylephedrine, N-acetylephedrine, N,O-diacetylephedrie, methamphetamine dimer) are identified as impurities in methamphetamine tablet. Caffeine and ethyl vanillin are also detected as diluents and/or adulterants, and acetylcodeine monoacetylmorphine and diacetylmorphine are contained in many samples. In addition, trans-3,4-dimethyl-5-phenyl-2-oxazolidone is newly found as an impurity. For characterization and comparison of methamphetamine tablet exhibits, intensely and commonly detectable nine peaks are selected as the factor for multivariate analysis. The procedures reported here permit classification of 250 analyzed exhibits into five groups and characterization of classified groups.     

Case of Fatal Starvation: Can Stable Isotope Analysis Serve to Support Morphological Diagnosis and Approximate the Length of Starvation?

The diagnosis of death as a result of starvation is established on anthropological measurements, visual appearance of the deceased on external and internal examination, microscopic analysis, laboratory testing, and exclusion of other causes of death. Herein, we present our findings on a case of 95‐year‐old man who died of starvation. After the diagnosis of starvation was established by traditional forensic medicine methods, we have conducted retrospective segmental analysis of stable carbon (δ¹³C) and nitrogen (δ¹⁵N) isotope ratios in hair sample. This method reveals periods of starvation through decrease in δ¹³C and increase in δ¹⁵N along the strand of hair. Our analysis revealed the decrease of 0.6 ‰ in δ¹³C during the last 10–12 weeks prior to death, similar as reported in other investigations. Also, a decrease of 0.7 ‰ in δ¹⁵N during the last 8–10 weeks prior to death was determined that was different than observed in previous studies.     

Discrimination of Geographical Origin of Asian Garlic Using Isotopic and Chemical Datasets under Stepwise Principal Component Analysis

Isotopic compositions of δ²H, δ¹⁸O, δ¹³C, and δ¹⁵N and concentrations of 22 trace elements from garlic samples were analyzed and processed with stepwise principal component analysis (PCA) to discriminate garlic's country of origin among Asian regions including South Korea, Vietnam, Taiwan, and China. Results indicate that there is no single trace‐element concentration or isotopic composition that can accomplish the study's purpose and the stepwise PCA approach proposed does allow for discrimination between countries on a regional basis. Sequentially, Step‐1 PCA distinguishes garlic's country of origin among Taiwanese, South Korean, and Vietnamese samples; Step‐2 PCA discriminates Chinese garlic from South Korean garlic; and Step‐3 and Step‐4 PCA, Chinese garlic from Vietnamese garlic. In model tests, countries of origin of all audit samples were correctly discriminated by stepwise PCA. Consequently, this study demonstrates that stepwise PCA as applied is a simple and effective approach to discriminating country of origin among Asian garlics.     

The impurity characteristics of methamphetamine synthesized by Emde and Nagai method

Impurity profiling and classification of seized methamphetamine may play an important role in the interpretation of analytical results, the determination of the synthetic method employed, and the criminal investigations of drug traffic routes. Our study is focused on classifying seized methamphetamine samples according to the groups sorted by the types and quantities of impurities present in illicit methamphetamine samples. The samples (100 mg) were dissolved in 2 mL of potassium phosphate buffer (pH 7.0), extracted with 200 μL of ethyl acetate under basic condition, and then analyzed by gas chromatography-mass spectrometry (GC–MS) with a DB-1 capillary column (30 m × 0.25 mm i.d., 0.25 μm). Five impurities are used as criteria for the classification of seized methamphetamine samples by Emde and Nagai method. A total of fifty-two samples of seized methamphetamine were analyzed by GC–MS and classified by five organic impurities, and then sorted into four groups, which are Nagai type, Emde Type, Undetermined I type, and Undetermined II type.     

Sourcing explosives: A multi-isotope approach

Although explosives are easily identified with current instrumental techniques, it is generally impossible to distinguish between sources of the same substance. To alleviate this difficulty, we present a multi-stable isotope (δ¹³C, δ¹⁵N, δ¹⁸O, δD) approach for appraising the possibility of discriminating explosives. The results from 30 distinct PETN, TNT and ANFO samples show that the different families of explosives are clearly differentiated by both their specific isotope signatures and their combination with corresponding element concentrations. Coupling two or more of the studied isotope systematics yields an even more precise differentiation on the basis of their raw-material origin and/or manufacturing process.     

Can Stable Isotopes and Radiocarbon Dating Provide a Forensic Solution for Curbing Illegal Harvesting of Threatened Cycads?

Cycads in South Africa are facing an extinction crisis due to the illegal extraction of plants from the wild. Proving wild origin of suspect ex situ cycads to the satisfaction of a court of law is difficult, limiting law enforcement efforts. We investigated the feasibility of using multiple stable isotopes to identify specimens removed from the wild. Relocated and wild specimens from two species in the African genus Encephalartos (E. lebomboensis and E. arenarius) were sampled. ¹⁴C analysis indicated that a ± 30‐year chronology could be reliably obtained from the cycads. For E. arenarius, pre‐relocation tissue was consistent with a wild origin, whereas tissue grown post‐relocation was isotopically distinct from the wild for ⁸⁷Sr/⁸⁶Sr and δ¹⁵N. For E. lebomboensis, δ³⁴S, δ¹⁸O, and ⁸⁷Sr/⁸⁶Sr were different between relocated and control plants, consistent with the >30 years since relocation. Our findings demonstrate the potential for a forensic isotope approach to identify illegal ex situ cycads.     

Determination of impurities in illicit methamphetamine samples seized in Iran

In this study fifty samples of crystalline methamphetamine obtained from antinarcotics police of Iran seized during the year 2010 were analyzed. In order to determine the chemical characteristics of these samples, anion test, Gas Chromatography-Mass Spectrometry (GC-MS) and Liquid Chromatography-Mass Spectrometry (LC-MS) were carried out on the samples. All of the samples containing methamphetamine tested positive for chloride anion. The range of methamphetamine hydrochloride content in these samples was 33-95%. One sample out of 50 contained no methamphetamine. The fact that 1,2-dimethyl-3-phenylaziridine was the most frequently found impurity in the analyzed samples, indicates that most of the methamphetamine samples seized in Iran have been synthesized from pseudoephedrine as starting material.     

Isolation and Identification of Three By‐products Found in Methylamphetamine Synthesized by the Emde Route

This article describes the isolation and structural elucidation of three compounds produced during the synthesis of methylamphetamine by the so‐called “Emde” procedure. The “Emde” procedure involves the preparation of the intermediate chloropseudoephedrine or chloroephedrine from ephedrine or pseudoephedrine, respectively. The intermediates are then reduced to methylamphetamine with hydrogen under pressure in the presence of a catalyst. The by‐product compounds were isolated from methylamphetamine by column chromatography and liquid chromatography (LC). Proton nuclear magnetic resonance spectroscopy (¹H NMR), carbon nuclear magnetic resonance spectroscopy (¹³C NMR), and nanospray quadrupole‐time of flight‐mass spectrometry (Q‐TOF‐MS) were used to identify them as two stereoisomers of the compound N, N′‐dimethyl‐3,4‐diphenylhexane‐2,5‐diamine and N‐methyl‐1‐{4‐[2‐(methylamino)propyl]phenyl}‐1‐phenylpropan‐2‐amine.     

The application of capillary electrophoresis for enantiomeric separation of N,N-dimethylamphetamine and its related analogs: intelligence study on N,N-dimethylamphetamine samples in crystalline and tablet forms

This paper reports a new capillary zone electrophoresis method for the simultaneous chiral determination of the enantiomers of N,N-dimethylamphetamine (DMA), methamphetamine (MA), ephedrine (E), pseudoephedrine (PE) and methylephedrine (Me-E). In this study, a number of electophoretic parameters were examined and optimized including the choice of chiral selectors, the use of short-chain tetraalkylammonium cations, the effect of chiral selector concentration, buffer concentration, applied voltage and capillary temperature. Heptakis(2,6-di-O-methyl)-beta-cyclodextrin (DM-beta-CD) and tetrabutylammonium (TBA) being the best chiral selector and buffer cation, respectively, the optimized electrophoretic conditions were found to be: 20 mM DM-beta-CD, 50 mM TBAPO(4) at pH 2.5, applied voltage at 30 kV and temperature at 25 degrees C. Under the optimized conditions, all analytes were well separated with resolution factors between 3.3 and 24.0 achieved. Using phentermine as an internal standard, the intra-day (n=8) precisions for the relative migration times and peak areas of all analytes were below 1.09%, while the inter-day precisions (n=12, 6 days) for the relative migration times and peak areas of all analytes were under 3.77%. This method has been applied to the measurement of the enantiomeric purities of the seized DMA samples. The result of the measurement could provide important clues about the possible synthetic pathways of these samples.     

Investigating Residential History Using Stable Hydrogen and Oxygen Isotopes of Human Hair and Drinking Water

The relationship between isotopic signals in human hair and geographic region has potential forensic applications for identifying unknown individuals' place of recent residence. This study analyzes δ²H and δ¹⁸O isotopes in residential tap water and bulk hair samples from 17 volunteers representing 12 locations in Ontario, Canada. There is a strong correlation (R² = 0.9) between δ²H and δ¹⁸O values of the water samples. In contrast, the δ²H and δ¹⁸O values of the hair samples are weakly correlated (R² = 0.3), and the greater variability in the data is linked to dietary factors. This study demonstrates that the δ²H and δ¹⁸O values of hair and drinking water can be used to help identify potential place of residence in forensic cases, particularly in relation to proximity to large bodies of water such as the Great Lakes, but interpretations are complicated by the contribution of both water and diet to δ²H and δ¹⁸O values in hair.     

A test to identify cyanide origin by isotope ratio mass spectrometry for forensic investigation

Cyanide is one of the common poisons in murders. When cyanide has been used, to identify the origin of cyanide may be necessary in the forensic investigation. We have examined the possibility of distinguishing different commercial cyanide samples through the δ(13)C and δ(15)N values and developed a protocol for the isotope analysis of cyanide extracted from several matrices as food and medicine. Several cyanide precipitates were tested for the isotope analysis. The results show that cupric ferrocyanide Cu(2)[Fe(CN)(6)] is the most appropriate precipitate for the analysis. Thirteen batches of KCN and nine batches of NaCN chemicals were randomly chosen from different suppliers. The cyanides were converted to cupric ferrocyanide and then analysed by isotope ratio mass spectrometry coupled to elemental analysis (EA-IRMS). The isotopic signature of the commercial samples varied from -51.96 to -25.77 ‰ for δ(13)C and from -4.51 to +3.81 ‰ for δ(15)N, highlighting the potential of applying EA-IRMS technique to identify cyanide from different batches and sources. The influence of the cyanide extraction and isolation from spiked matrix on the isotopic analysis was also studied. Three matrices: orange juice, yogurt drink and a medicine were tested. In many cases, the isotopic analysis results obtained from the original cyanides precipitates and those isolated from the matrices showed a good accordance, especially for δ(15)N. In some matrices, the (13)C analysis was interfered by co-precipitates. With carefully elaborated working protocol, determining the isotope ratio of N and C in cyanide by EA-IRMS is a promising method for forensic investigations.     

Analysis of the impurities in the methamphetamine synthesized by three different methods from ephedrine and pseudoephedrine

Organic impurities of methamphetamine may show different patterns by synthesis. In the present study, we tried to find the impurities reflecting the conditions of synthesis by comparing impurity patterns of the methamphetamine samples synthesized by different methods. Sixteen methamphetamine samples were synthesized from ephedrine and pseudoephedrine by the three different manufacturing methods of Emde, Nagai and Moscow. The synthesized samples were extracted with ethyl acetate containing four internal standards, and the patterns of the organic impurities were investigated by GC-MS and GC-FID . Through the investigation, we found 10 peaks appearing in the latter part of GC chromatograms are characteristic to synthesis. The areas of the selected peaks were converted to the variables suitable for the statistical calculation, and the synthesized samples could be classified into four groups through the resultant cluster analysis.     

GC/MS检验不同案件中摇头丸

目的 分析不同案件缴获的“摇头丸”主要成分和添加物,为确定“摇头丸”的合成路线、非法来源提供依据.方法 以GC/MS检测法为主要检测方法.结果 检验的14个“摇头丸”主要成分为甲基苯丙胺和咖啡因,其中含有少量的其他杂质如麻黄碱和氯胺酮等.结论 检测的14个“摇头丸”样本其中12个含甲基苯丙胺,实为冰毒成分,1个为咖啡因均不属于摇头丸.     

Variation of δ2H, δ18O & δ13C in crude palm oil from different regions in Malaysia: Potential of stable isotope signatures as a key traceability parameter

A total of 33 crude palm oil samples were randomly collected from different regions in Malaysia. Stable carbon isotopic composition (δ¹³C) was determined using Flash 2000 elemental analyzer while hydrogen and oxygen isotopic compositions (δ²H and δ¹⁸O) were analyzed by Thermo Finnigan TC/EA, wherein both instruments were coupled to an isotope ratio mass spectrometer. The bulk δ²H, δ¹⁸O and δ¹³C of the samples were analyzed by Hierarchical Cluster Analysis (HCA), Principal Component Analysis (PCA) and Orthogonal Partial Least Square-Discriminant Analysis (OPLS-DA). Unsupervised HCA and PCA methods have demonstrated that crude palm oil samples were grouped into clusters according to respective state. A predictive model was constructed by supervised OPLS-DA with good predictive power of 52.60%. Robustness of the predictive model was validated with overall accuracy of 71.43%. Blind test samples were correctly assigned to their respective cluster except for samples from southern region. δ¹⁸O was proposed as the promising discriminatory marker for discerning crude palm oil samples obtained from different regions. Stable isotopes profile was proven to be useful for origin traceability of crude palm oil samples at a narrower geographical area, i.e. based on regions in Malaysia. Predictive power and accuracy of the predictive model was expected to improve with the increase in sample size. Conclusively, the results in this study has fulfilled the main objective of this work where the simple approach of combining stable isotope analysis with chemometrics can be used to discriminate crude palm oil samples obtained from different regions in Malaysia. Overall, this study shows the feasibility of this approach to be used as a traceability assessment of crude palm oils.     

Impurity profiling of methamphetamine hydrochloride drugs seized in the Philippines

Methamphetamine hydrochloride is one of the most widely used illicit drugs in the Philippines. In this study, we describe the application of cluster analysis of trace impurities in the profiling of the seized methamphetamine drug samples. Thirty milligrams of a homogenized drug sample were dissolved in 1 mL of pH 10.5 buffer solution and extracted with ethyl acetate containing three internal standards. The trace impurities were identified using gas chromatography-mass spectrometry (GC-MS) and quantified by gas chromatography with a flame ionization detector (GC-FID). Following previously reported methodologies, 30 impurity peaks were selected from the GC-FID chromatograms. The peak areas and retention times were referenced to the internal standards. The peak areas of the selected peaks were then grouped for cluster analysis. In order to check for consistency of clustering, two further cluster analyses were performed using 40 and 50 impurity peaks. Changes in clustering were observed in going from 30 to 40 impurity peaks, while analyses using 40 and 50 impurity peaks gave similar results. Thus, for the seized drug samples used in this study, cluster analysis using at least 40 impurity peaks showed better consistency of clustering as compared to analysis using 30 peaks only. Ten of the impurity peaks were identified, of which four were identified for the first time in methamphetamine drug samples. These are p-bromotoluene, N-benzyl amphetamine, N-ethyl amphetamine, and N-ethyl methamphetamine. The presence of phenyl-2-propanone (P2P), N,N-dimethyl amphetamine, and N-formyl amphetamine is indicative that these casework samples were synthesized using the Leuckart method.     

Refining Stable Oxygen and Hydrogen Isoscapes for the Identification of Human Remains in Mississippi,

Isoscape refinement is an essential component for accurately predicting region‐of‐origin in forensic investigations involving isotope analysis of unidentified human remains. Stable oxygen (δ¹⁸O) and hydrogen (δ²H) isotopes were measured from 57 tap water samples collected across Mississippi to model refined isoscapes for the state. A tap water conversion equation, δ¹⁸O_tw=1.64 δ¹⁸Op?31.35, was developed for the southeastern USA to test the prediction accuracy of the δ¹⁸O_tw isoscape using individuals with known residential histories. A local Mississippi resident (USAFA‐134) was assigned with 90% probability to the correct region‐of‐origin reported by the participant. Assignments for Georgia residents (USAFA‐118 and USAFA‐205) had variable results, predicting USAFA‐118 from Mississippi and USAFA‐205 as a nonlocal resident. Stable isotope values often overlap geographically and a multi‐isotope approach should be used when narrowing region(s)‐of‐origin(s). This study demonstrates the utility of refining isoscapes and the importance of tissue calibration in prediction assignments of human remains.