Isotopic fractionation of carbon and nitrogen during the illicit processing of cocaine and heroin in South America

The forensic application of stable isotope analysis to cocaine and heroin for geolocation of exhibits must take into account the possible enrichment and/or depletion of 13C and 15N during the illicit manufacturing process. Continuous-flow elemental analysis-isotope ratio mass spectrometry was utilized to measure changes in the stable isotope ratios of carbon and nitrogen for both cocaine (N = 92) and heroin/morphine (N = 81) exhibits derived from illicit manufacturing processes utilized by South American clandestine chemists. In controlled settings in South America, there was no siginficiant carbon isotope fractionation during the conversion of cocaine base to cocaine HCI using current illict methodologies. In contrast, nitrogen isotope fractionation for this conversion was 1 per thousand. There was a kinetic carbon isotope ratio fractionation during the acetylation of Colombian morphine to heroin and as a result heroin exhibits will almost always have more negative delta13C values than the original morphine. There was an isotopic fractionation against 15N during the acetylation of morphine base to heroin base, but this effect was not expressed since all of the heroin base was precipitated during the manufacturing process. However, the clandestine process of converting a single batch of heroin base usually involved two consecutive crops of heroin HCl and the latter crop was isotopically depleted as expected from a Rayleigh distillation process. When heroin was deacetylated to morphine, the morphine produced resulted in delta13C values that were indistinguishable from the original morphine. The kinetic carbon isotope fractionation factor for the South American process of morphine acetylation was -1.8 per thousand, allowing calculation of the delta13C values of the acetic anhydride from deacetylated heroin delta13C values.     

Signature Profiling and Classification of Illicit Heroin by GC-MS Analysis of Acidic and Neutral Manufacturing Impurities

Abstract:  The illicit manufacture of heroin results in the formation of trace level acidic and neutral impurities. These impurities are detectable in illicit heroin and provide valuable information about the manufacturing process used. The isolation, derivatization, and semiquantitative analysis of neutral and acidic heroin manufacturing impurities by programmed temperature vaporizing injector-gas chromatography-mass spectrometry (PTV-GC-MS) is described. Trace acidic and neutral heroin impurities were isolated from basic fractions using liquid–liquid extraction. Extracted impurities were treated with N -Methyl- N -trimethylsilyltrifluoroacetamide followed by PTV-GC-MS analyses. Semiquantitative data were obtained using full scan mass spectrometry utilizing unique ions or ion combinations for 36 trace impurities found in crude and/or highly refined heroin samples. Minimum detection limits for acidic and neutral impurities were estimated to be at the 10⁻⁷ level relative to total morphine. Over 500 authentic heroin samples from South America, Mexico, Southwest Asia, and Southeast Asia were analyzed. Classification of illicit heroin based on the presence or absence and relative amounts of acidic and neutral impurities is presented.     

Isotope ratio mass spectrometry as a tool for forensic investigation (examples from recent studies).

The versatility of isotope ratio mass spectrometry is demonstrated by reference to diverse case studies. Variations in the natural isotopic composition of non-biological, organic materials are compared as a means by which samples may be associated or discriminated. These techniques may be used to augment or compliment conventional forensic methodologies. delta13C analysis was used to demonstrate that different masking tape had been recovered in two, apparently similar cases, involving the smuggling of money. Visually similar ecstasy tablets were compared by consideration of the delta13C and delta15N composition of MDMA extracted from the tablets. Although only a limited number of tablets were analysed, the isotopic similarity between two different seizures was sufficient to induce a guilty plea from a person suspected of possessing both. A combination of delta2H, delta13C, delta15N and delta18O together with GC-MS analyses were applied to small samples of seized heroin. Although GC-MS analysis indicated differences between the chemical composition of two of the heroin samples, isotopic analysis suggested similarities, which were confirmed by further delta2H, delta13C and delta18O isotopic analysis of the clingfilm in which the samples were wrapped.     

Neutral heroin impurities from tetrahydrobenzylisoquinoline alkaloids

Laudanosine, reticuline, codamine, and laudanine are members of the tetrahydrobenzylisoquinoline family of natural products. These alkaloids are present in the opium poppy, Papaver somniferum, and are subsequently found as impurities in clandestinely processed morphine. Morphine is then synthesized to heroin using hot acetic anhydride. During the course of this study, it was determined that these four tetrahydrobenzylisoquinolines undergo degradation to a series of 18 neutral impurities when subjected to hot acetic anhydride. Based on the degradation pathway, these new impurities were categorized into two sets of impurities called the C1-acetates compounds and the stilbene compounds. Synthesis, isolation, and structural elucidation information is provided for the tetrahydrobenzylisoquinoline alkaloids, and the new neutral impurities have been studied. Several hundred authentic heroin samples were analyzed using an established heroin signature program method. This methodology features the detection of trace neutral impurities present in heroin samples. It was determined that all 18 new impurities were detected in various quantities in four different types of heroin samples. Analytical results featuring these new impurities are reported for South American-, Southwest Asian-, Mexican-, and Southeast Asian-type heroin samples. These new impurities, coupled with other established forensic markers, enhance the ability to classify illicit heroin samples.     

Chemical profiling of heroin recovered from the North Korean merchant vessel Pong Su

Heroin samples, seized from the North Korean merchant vessel Pong Su in Australian waters, were analyzed to determine geographic origin. Duplicate samples were analyzed by the National Measurement Institute's Australian Forensic Drug Laboratory and the United States Drug Enforcement Administration's Special Testing and Research Laboratory. Alkaloid ratios were determined by both liquid chromatography-diode array detection (LC-DAD) and capillary electrophoresis-diode array detection (CE-DAD) techniques. Acid/neutral manufacturing by-products were determined by solvent extraction followed by gas chromatography-mass spectrometry (GC-MS). Solvents, trapped in the heroin particles during manufacture, were detected by both static headspace GC-MS and purge and trap GC-MS. The alkaloid ratios obtained were consistent with heroin of a Southeast Asian (SEA) origin and principal component analysis of the alkaloid results demonstrated the presence of at least four subgroupings within the seizure. The solvent analysis detected diethyl ether and ethyl acetate, solvents typically seen in SEA heroin. However, the acid/neutral analysis revealed compounds not normally seen in heroin of a SEA origin. Furthermore, sterol-like molecules, always detected in the acid/neutral analysis of SEA heroin, were absent from the Pong Su samples. The Pong Su heroin, although similar to SEA heroin, has sufficient differences to classify it as having an unknown origin at the time of this writing.     

Qualitative and quantitative analysis of seized street drug samples and identification of source

In this paper we describe the identification of constituents of the illicit drugs seized from different regions of eastern India by GC-MS. The constituents were identified to be heroin, acetyl morphine, morphine and acetyl codeine. Quantitative estimation of the constituents were made by GC-MS and HPTLC. In view of non-availability of the authentic samples of drugs of different origin, nothing positive can be said about the origin of illicit drug samples. The possibility of isotopic substitution, an important method for identification of source, was examined from the comparison of the intensity of different (ion) peaks 369 (heroin, m/z=369), 370, 371 and 372 using selective ion monitoring mode. No isotopic substitution in the constituents was observed. Attempts were made to identify the source of the illicit samples from heroin/acetylcodeine ratios in the way described in the literature.     

Hmong folk remedies: limited acetylation of opium by aspirin and acetaminophen.

The traditional folk medicine of the Hmong and other Southeast Asian refugees has accompanied them during their immigration in this country over the past two decades. In two recent cases involving Hmong defendants, unknown solids, resembling charcoal in consistency and purported to be "backache remedies," were analyzed and found to be complex mixtures of aspirin, acetaminophen, caffeine and partly acetylated opium. In particular, significant amounts of acetylacetaminophen, 3-O-acetylmorphine, 6-O-acetylcodeine, 6-O-acetylmorphine, and heroin were identified by gas chromatography/mass spectrometry. Heating approximately equal weights of solid opium, aspirin, and acetaminophen at 130 degrees C for several hours produced a mixture of compounds showing a similar acetylation pattern.     

Morphine to codeine concentration ratio in blood and urine as a marker of illicit heroin use in forensic autopsy samples

A morphine to codeine ratio greater than unity (M/C>1) has been suggested as an indicator of heroin use in living individuals. The aim of this study was to examine the morphine to codeine ratio in a large population (N=2438) of forensically examined autopsy cases positive for 6-monoacetylmorphine (6-MAM) and/or morphine in blood and/or urine. Blood and urine concentrations of 6-MAM, morphine and codeine were examined using GC-MS and LC-MS/MS methods. In 6-MAM positive samples, the M/C ratio was greater than unity in 98% (N=917) of the blood samples and 96% (N=665) of the urine samples. Stratification of 6-MAM negative cases by M/C above or below unity revealed similarities in morphine and codeine concentrations in cases where M/C>1 and 6-MAM positive cases. Median blood and urine morphine concentrations were 8-10 times greater than codeine for both groups. Similarly to 6-MAM positive cases, 25-44 year-old men prevailed in the M/C>1 group. In comparison to cases where M/C ≤ 1, the M/C ratio was a hundred times higher in both 6-MAM positive and M/C>1 cases. The range of morphine concentration between the lowest and the highest quintile of codeine in M/C>1 cases was similar to that in 6-MAM positive cases. This range was much higher than for M/C ≤ 1 cases. Moreover, linear regression analyses, adjusted for age and gender, revealed a strong positive association between morphine and codeine in 6-MAM positive and M/C>1 cases. The M/C ratio appeared to be a good marker of heroin use in post-mortem cases. Both blood and urine M/C>1 can be used to separate heroin users from other cases positive for morphine and codeine.     

Levels of trace and major elements in illicit heroin

Ten elements, aluminum (Al), barium (Ba), calcium (Ca), cadmium (Cd), copper (Cu), iron (Fe), magnesium (Mg), manganese (Mn), lead (Pb), and zinc (Zn) were analyzed in 44 illicit heroin samples from Southeast Anatolia, Turkey. Illicit heroin samples were dissolved in nitric acid using microwave oven and were quantified by electrothermal atomic absorption spectrometry (Cd and Pb) and inductively coupled plasma-atomic emission spectrometry (Al, Ba, Ca, Cu, Fe, Mg, Mn, and Zn). The most abundant element was calcium, 4050 to 14,200 microg/g, which could be ascribed to the use of lime in the manufacturing process and/or as diluting agent. Iron (180 to 1470 microg/g), aluminum (42 to 2280 microg/g), and zinc (160 to 210 microg/g) were found at moderately high levels, possibly because of the use of metal pots in the acetic anhydride cooking process and also for storage. Cadmium and lead concentrations were at the lowest measured levels. The amounts of magnesium, manganese, barium, and copper were in the range of 100 to 800 micro/g, 3 to 17 microg/g, 4 to 30 microg/g, and 2 to 46 microg/g, respectively.     

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.     

Quantitative determination of amphetamines, cocaine, and opiates in human hair by gas chromatography/mass spectrometry

Hair of young subjects (N = 36) suspected for drug abuse was analysed for morphine, codeine, heroin, 6-acetylmorphine, cocaine, methadone, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), and 3,4-methylenedioxyethylamphetamine (MDEA). The analysis of morphine, codeine, heroin, 6-acetylmorphine, cocaine, and methadone in hair included incubation in methanol, solid-phase extraction, derivatisation by the mixture of propionic acid anhydride and pyridine, and gas chromatography/mass spectrometry (GC/MS). For amphetamine, methamphetamine, MDA, MDMA, and MDEA analysis, hair samples were incubated in 1M sodium hydroxide, extracted with ethyl acetate, derivatised with heptafluorobutyric acid anhydride (HFBA), and assayed by GC/MS. The methods were reproducible (R.S.D. = 5.0-16.1%), accurate (85.1-100.6%), and sensitive (LoD = 0.05-0.30ng/mg). The applied methods confirmed consumption of heroin in 18 subjects based on positive 6-acetylmorphine. Among these 18 heroin consumers, methadone was found in four, MDMA in two, and cocaine in two subjects. Cocaine only was present in two, methadone only in two, methamphetamine only in two, and MDMA only in seven of the 36 subjects. In two out of nine coloured and bleached hair samples, no drug was found. Despite the small number of subjects, this study has been able to indicate the trend in drug abuse among young people in Croatia.     

Chemical signature of ecstasy volatiles by comprehensive two-dimensional gas chromatography

A method for ecstasy volatiles 'signature' analysis based on two-dimensional gas chromatography separation and time-of-flight mass spectrometry detection (GC×GC-TOFMS) is presented. Organic impurity volatiles were extracted by head space solid phase microextraction (HS-SPME). The final column phase choice of the four different column combinations tested was a low-polarity 5% phenyl polysilphenylene-siloxane coupled with a polyethylene glycol phase, which best displayed the complex impurity profile. Second dimension ((2)D) retention time reproducibility was found to be about 1% RSD, and area reproducibility of SPME sampling was just over 5% RSD for compounds with S/N ratio of about 100. High similarity of TOFMS spectra of impurities was obtained against commercial MS libraries. 16 components from the two-dimensional profiles were selected for comparison of the 24 ecstasy tablets, most of which proved to be benzodioxole derived compounds. All tablets were correctly classified in eight groups according to their post-tabletting characteristics, when appropriate data pre-treatment was applied. Principal component analysis revealed clustering of samples according to the country of origin. Samples from Macedonia were elevated in N-formyl-MDMA and N-acetyl-MDMA while samples from Australia were elevated in 3,4-methylenedioxypropane and 3,4-methylenedioxyacetophenone. Furthermore, three components were found to be unique for one of the source countries. The additional separation of components on the (2)D column, increased response due to modulation, high acquisition rate with full mass spectra using TOFMS detection, and MS deconvolution extend the possibility of detecting additional markers and route-specific components, especially of low abundant, polar components.     

Chromatographic and spectroscopic profiles of Cannabis of different origins: Part I

High-resolution capillary gas chromatography with flame ionization detection and mass spectrometry (GC and GC/MS) and high-performance liquid chromatography (HPLC) were used to establish complex chemical profiles (chemical signatures) of Cannabis samples of known origin. Over 100 compounds could be differentiated, including noncannabinoids (terpenes, alkanes) as well as minor and major cannabinoids and their acids. A characteristic peak pattern was found within a limited number of specimens of identical origin. Correlation studies on the basis of peak area ratios [A(x)/A(i.s.)] showed the feasibility of tracing Cannabis chemically to its country of origin. Several forensic science applications for the chromatographic and spectroscopic profiles of confiscated Cannabis samples are discussed, such as detection of additives (phencyclidine), differentiation of chemotypes, and monitoring of tetrahydrocannabinol (THC) potency.     

海洛因来源鉴定的新方法

毒品来源的鉴定是指对毒品样品中包含的来源信息进行提取拜分析,这有助于打击毒品犯罪.海洛因是危害我国的主要毒品,它的来源鉴定更引起人们的关注.本文介绍了国际上研究海洛因来源的三种方法,即研究海洛因样品中的各种有机杂质及其相对比例;研究海洛因样品中的痕量无机元素;研究海洛因及吗啡的稳定同位素相对比值.第三种方法能够反映样品的地域性特点,因而在来源鉴定方面有突出的优势.气相色谱-燃烧-同位素比值质谱法是同位素比值测定方法中用于海洛因来源鉴定的最佳手段.这一技术还可用于其他微量物证(如炸药、药品、油品等)的来源鉴别,因而在法庭科学领域内显示了广阔的应用前景.     

Isolation and purification of heroin from heroin street samples by preparative high performance liquid chromatography

The present study established a novel method using preparative high performance liquid chromatography to isolate and purify heroin·HCl from heroin street samples to be used as a reference standard. Different kinds of mobile phases and columns were used, ultimately the mobile phase consisting of hexane-isopropanol-methanol (65:28:7, v/v) and the SIL preparative column prepared in laboratory were selected as the final condition. Heroin was further purified by the drowning-out crystallization method using isopropanol-methanol (50:1, v/v) and hexane as drowning-out anti-solvents and salting-out agents, respectively. The purity was assessed by analytical high performance liquid chromatography and the confirmation of the chemical structure was performed by IR and NMR. About 110.7mg of heroin·HCl at a purity of over 99.52% was obtained from 180mg of heroin street samples which contained 156.15mg of heroin·HCl component by preparative high performance liquid chromatography. This method is suitable for preparing heroin standards in forensic science area.     

Noscapine as an adulterant in illicit heroin samples

In this short report the evidence is given (based on the analyses of 22 case samples) that noscapine can be used as an adulterant in illicit heroin samples. In this context, the appearance of illicit heroin samples characterised by a high noscapine content (up to 61%) and a high noscapine/whole morphine ratio (up to 3.5) is highlighted. All samples discussed in the paper (132) were seized in Slovenia, in the period from 1997 to 1999 and were analysed by gas chromatography-mass spectrometry.     

Unconjugated morphine in blood by radioimmunoassay and gas chromatography/mass spectrometry

Morphine, the active metabolite of heroin, is rapidly inactivated by glucuronidation at the 3 carbon. Unconjugated (pharmacologically active) morphine was measured in postmortem blood by radioimmunoassay using an antibody-coated tube kit. The kit shows less than 0.2% cross-reactivity with codeine and morphine-glucuronide. Unconjugated morphine concentrations were confirmed by gas chromatography/mass spectrometry (GC/MS) using deuterated morphine as the internal standard. The blood was precipitated with 10% trichloroacetic acid (TCA) and concentrated hydrochloric acid (HCl), centrifuged, and decanted. The supernatant was then either diluted (unhydrolyzed) or heated to 100 degrees C, 30 min (hydrolyzed), followed by a wash with 4:1 chloroform:isopropranol. The upper aqueous layer was then saturated with sodium bicarbonate (NaHCO3) and extracted with 4:1 chloroform:isopropranol. The organic layer was evaporated, derivatized with trifluoroacetic anhydride (TFA), and analyzed by selected ion monitoring (SIM) GC/MS. Comparison of the results for unconjugated morphine by radioimmunoassay and unhydrolyzed morphine by GC/MS gave a correlation coefficient of r = 0.98, n = 100. Unconjugated morphine ranged from 0 to 100% of total morphine with a mean of 42%, n = 200, for heroin or morphine involved deaths. Review of 56 putative rapid deaths gave a mean of 68% unconjugated morphine with a range of 26 to 100%. The ratio of unconjugated to total morphine was found to be stable in postmortem blood after more than a year of storage at room temperature, within the precision of the method.     

Illicitly imported heroin products: some physical and chemical features indicative of their origin

Samples taken from over 200 seizures of imported illicit heroin preparations of known geographical origin have been examined by gas liquid chromatography (GLC). The chromatographic characteristics were considered in conjunction with the physical appearance of the materials and it was found possible in many instances to discriminate between samples of different origin. Thus by carrying out GLC and HPLC and a visual inspection on a sample of unknown provenance, it may be possible to give an opinion as to its geographical origin.     

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.     

Investigation of trace inorganic elements in street doses of heroin

Sixteen trace elements found in 309 street heroin samples, piped water and contaminated water were determined using inductively coupled plasma-mass spectrometry. All the street heroin samples were found to contain high levels of sodium, a reflection of the use of sodium bicarbonate during heroin synthesis. Additionally, this element was also found to be one of the potential contaminants acquired from the piped water. Calcium could be derived from lime while iron, aluminum and zinc could have come from the metallic container used in the processing/cutting stage. The levels of these elements remained low in the heroin and it could be due to the dilution effects from the addition of adulterants. Statistical validation was performed with six links of related heroin samples using principal component analysis to find the best pretreatment for sample classification. It was obtained that normalization followed by fourth root showed promising results with 8% errors in the sample clustering. The technique was then applied to the case samples. Finally, the result suggested that the case samples could have originated from at least two major groups respectively showing unique elemental profiles at the street level.