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.     

Validation of benzodiazepine β-glucuronide primary reference materials for hydrolysis and quality assurance controls

The major metabolite of hydroxylated benzodiazepines is the β-glucuronide metabolite, thus a hydrolysis step is included in benzodiazepine analyses in urine. We evaluated the new Alltech (R,S) oxazepam and lorazepam β-glucuronide primary reference materials as hydrolysis controls and for use in proficiency testing. Using HPLC analysis and GC/MS analysis following acid and β-glucuronidase hydrolysis, we found that the lorazepam β-glucuronide material was >95% pure, but the oxazepam β-glucuronide material was only 54.0% pure. Recovery after hydrolysis with β-glucuronidase at 60 °C for two hours and 22 °C for 24 h was compared. There was no difference in the recovery of either drug incubated under both conditions. Using HPLC analysis to separate the R and S isomers of the glucuronides, we found that the S isomer hydrolyzes faster than the R isomer, but at optimal conditions hydrolysis of both isomers is complete. Alltech's glucuronide materials can be valuable as hydrolysis controls in method development and routine benzodiazepine analyses, but each laboratory must validate the purity of the materials and determine the acceptable references ranges for in-house controls made from these materials. The reference materials were also evaluated for possible use as quality control material for urine drug screening. The material had to be added at 130% of the reported cutoff concentration to produce a positive result on the Triage^™ Panel for Drugs of Abuse. For the Triage^™ Plus TCA Panel for Drugs of Abuse lorazepam β-glucuronide was added at 154% of the cutoff concentration, but addition of the oxazepam β-glucuronide at >200% of the reported cutoff concentration did not produce a positive result. Discrepancies may be due to preferential binding of antibodies to the S isomer, because the Alltech glucuronide materials do not contain the isomers in the same ratio as that excreted in human urine.     

A review of impurity profiling and synthetic route of manufacture of methylamphetamine, 3,4-methylenedioxymethylamphetamine,amphetamine, dimethylamphetamine and p-methoxyamphetamine

Amphetamine-type substances (ATS), like other synthetically derived compounds, can be produced by a multitude of synthetic pathways using a variety of precursors and reagents, resulting in a large number of possible contaminants (by-products, intermediates and impurities). This review article describes the common contaminants found in preparations of methylamphetamine (MA), 3,4-methylenedioxymethylamphetamine (MDMA), amphetamine (AP), N,N-dimethylamphetamine (DMA) and p-methoxyamphetamine (PMA) synthesised via common synthetic pathways including reductive amination, Leuckart method, Nagai method, Emde method, Birch reduction, “Moscow” method, Wacker process, “Nitrostyrene” method and the Peracid oxidation method.Contaminants can facilitate identification of the synthetic route, origin of precursors and may suggest information as to the location of manufacture of these illicit drugs. Contaminant profiling can provide vital intelligence for investigations in which linking seizures or identifying the synthetic pathway is essential. This review article presents an accessible resource; a compilation of contaminants resulting from a variety of manufacturing methods used to synthesise the most common ATS. It is important for research in this field to continue as valuable information can be extracted from illicit drug samples, increasing discrimination amongst ATS, and in turn, leading to an increase in evidential value and forensic drug intelligence from forensic drug samples.     

GC-MS and GC-IRD analysis of 2-, 3- and 4-methylmethamphetamine and 2-, 3- and 4-methylamphetamine

4-Methylmethamphetamine has been detected in samples submitted for analysis in several states throughout Australia. Six ring substituted methyl isomers of methamphetamine and amphetamine were synthesised and analysed. As the regioisomeric 2-, 3- and 4-methylmethamphetamine and 2-, 3- and 4-methylamphetamine have virtually identical mass spectra, the use of MS is an ineffective technique to discriminate between these closely related compounds. We set out to determine whether the regioisomers could be differentiated by a combination of GC-MS, acetyl derivatisation and GC-IRD. We demonstrate that the three isomers of methylmethamphetamine and methylamphetamine can be separated by GC, and a combination of acetyl derivatisation and vapour phase IR can identify the specific ring substituted compound.     

Identification and quantitation of 3,4-methylenedioxy-N-methylamphetamine (MDMA,ecstasy) in human urine by 1H NMR spectroscopy. Application to five cases of intoxication

Identification of 3,4-methylenedioxy-N-methylamphetamine (MDMA, ecstasy) in five cases of intoxication using nuclear magnetic resonance (NMR) spectroscopy of human urine is reported. A new water suppression technique PURGE (Presaturation Utilizing Relaxation Gradients and Echoes) was used. A calibration curve was obtained using spiked samples. The method gave a linear response (correlation coefficient of 0.992) over the range 0.01–1 mg/mL. Subsequently, quantitation of the amount of MDMA present in the samples was performed. The benefit and reliability of NMR investigations of human urine for cases of intoxication with MDMA are discussed.     

Simultaneous separation of different types of amphetamine and piperazine designer drugs by capillary electrophoresis with a chiral selector

The recent emergence of a new class of piperazine-type compounds has brought about the need for laboratory screening methods for both seized drugs and toxicological samples. These piperazine compounds, which include 1-benzylpiperazine (BZP) and 1-(3-trifluoromethylphenyl)piperazine (TFMPP), exhibit comparable physiological effects and can be substituted for the classic amphetamine-type drugs. We have optimized a chiral capillary electrophoresis (CE) separation that detects a set of 6 piperazine and 4 chiral amphetamine compounds in under 23 min using a 200 mM phosphate buffer at a pH = 2.8 with 20 mM hydroxypropyl- beta-cyclodextrin (HPbeta3CD). In addition to the above compounds, a series of "clandestine" BZP diHCl samples were also analyzed using this method to assess the ruggedness of the procedure. The novel CE separation was tailored to simultaneously detect these piperzine compounds in addition to amphetamine-type drugs. Distinct migration time and UV-spectral data were obtained for all compounds of interest.     

Postmortem Blood Concentrations of R‐ and S‐Enantiomers of Methadone and EDDP in Drug Users: Influence of Co‐Medication and P‐glycoprotein Genotype

Abstract: We investigated toxicological and pharmacogenetic factors that could influence methadone toxicity using postmortem samples. R‐ and S‐methadone were measured in femoral blood from 90 postmortem cases, mainly drug users. The R‐enantiomer concentrations significantly exceeded that of the S‐enantiomers (Wilcoxon’s test, p < 0.001). The samples were divided into four groups according to other drugs detected (methadone only, methadone and strong analgesics, methadone and benzodiazepines, or methadone and other drugs). There was no significant difference in any of the R‐methadone/total methadone ratios among the four groups. The median R/S ratio was 1.38, which tends to be higher than that reported for the plasma of living subjects. In addition, we investigated whether small nucleotide polymorphisms in the MDR1 gene that encode the drug transporter P‐glycoprotein were associated with the concentrations of R‐ and S‐methadone and its metabolite 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine. No significant association was detected.     

Impurities,adulterants and cutting agents in cocaine as potential candidates for retrospective mining of GC-MS data

Cocaine is one of the most widely used illicit drugs worldwide. Cocaine powders seized by the Police may contain numerous other substances besides the drug itself. These can be impurities originating from the coca plant or the production process, or be purposely added to the drug formulation as adulterants and cutting agents. In forensic laboratories, identification of cocaine is routinely done through GC-MS analysis, but other components are often ignored even if the method allows for their detection. Yet, they can provide valuable insight into the history of a seizure and its potential connection to other samples. To explore this idea, an extensive review of common impurities and adulterants encountered in cocaine is presented. Based on their incidence, concentration in the end product and compatibility with GC-MS methods, their overall usefulness as candidates for the statistical investigation of existing forensic data is evaluated. The impurities cis- and trans-cinnamoylcocaine, tropacocaine, norcocaine and N-benzoylnormethylecgonine as well as the adulterants lidocaine, procaine, tetracaine, benzocaine, caffeine, acetylsalicylic acid, phenacetin, ibuprofen, levamisole, hydroxyzine and diltiazem are promising candidates to provide additional forensic intelligence. Future research on optimized routine GC-MS methods, signal reproducibility, comparison, statistics and databases is suggested to facilitate this concept. Ultimately, such an approach may significantly advance the amount of information that is extracted from routine casework data, elucidate developments in the cocaine markets in the past and facilitate Police work in the future. Preliminary assessment of existing data from the forensic laboratory of the Amsterdam Police has been included to show that the detection of the identified target impurities is feasible, and that small adjustments to the analysis method could significantly increase the detectability of these analytes in prospective drug screenings. Forensic intelligence based on retrospective data mining of cocaine containing casework samples may thus be realized with minimal additional laboratory efforts by using already available instrumentation, samples and data.     

Rapid enantiomeric analysis of zopiclone in serum by supercritical fluid chromatography–tandem mass spectrometry

Zopiclone (ZOP) is a hypnotic drug prescribed to treat insomnia. Due to the chiral nature of ZOP, the psychologically active S-form and inactive R-form need to be determined enantiomerically in a forensic drug analysis. In the present study, a supercritical fluid chromatography (SFC) method was designed with a faster analysis ability than that of previously reported techniques. The SFC–tandem mass spectrometry (SFC–MS/MS) method was optimized using a column with a chiral polysaccharide stationary phase (Trefoil CEL2). ZOP was extracted from pooled human serum using solid-phase extraction (Oasis HLB) and analyzed. The developed SFC–MS/MS method achieved the baseline separation of S-ZOP and R-ZOP within 2 min. The fit-for-purpose method validation indicated that the optimized solid-phase extraction achieved near complete recovery and approximately 70% of the matrix effect. Both the retention time and peak area showed sufficient precision. The lower and upper limits of quantification (LOQ) were 5.7 × 10⁻² ng/mL and 25 ng/mL for R-ZOP, and 5.2 × 10⁻² ng/mL and 25 ng/mL for S-ZOP. The calibration line was linear in the range from lower LOQ to upper LOQ. The stability test indicated that ZOP in serum stored in a refrigerator (4°C) degraded and about 55% remained in 31 days. The quick analysis of the SFC–MS/MS method makes it a valid option for the enantiomeric analysis of ZOP.     

Degradation in soil of precursors and by-products associated with the illicit manufacture of methylamphetamine: implications for clandestine drug laboratory investigation

Key precursors and by-products in the Leuckardt, Nagai and dissolving metal reductive syntheses of methylamphetamine undergo degradation in soil as a result of biotic and abiotic processes. Furthermore, methylamphetamine is a product of the degradation of 1-(1',4'-cyclohexadienyl)-2-methylaminopropane and N-formylmethylamphetamine. These findings have implications for the forensic assessment of buried residues recovered from clandestine laboratory sites because markers used to infer the synthetic methods used might be absent as a result of degradation and because methylamphetamine might be present in residues as a result of degradation rather than as a direct result of its manufacture in the laboratory.     

Elemental Analysis of Variably Contaminated Cremains Using X‐ray Fluorescence Spectrometry

Analyzing and identifying skeletal remains becomes increasingly difficult when remains have been cremated, especially in cases where the cremated material may have been intentionally contaminated with nonskeletal material. This study examined the potential of X‐ray fluorescence spectrometry (XRF) to detect the presence of nonskeletal contaminants in samples of cremains. Eleven samples of cremains were variably combined with concrete mix and analyzed using XRF. Photon counts of elements in each sample were analyzed, and the coefficient of determination (R²) using unweighted linear regression as a function of percent cremains was calculated. Results showed that with changes in the proportion of skeletal material and contaminant, there were significant (R² > 0.90) changes in detected levels of phosphorus, potassium, zinc, aluminum, and sulfur. The use of XRF is concluded to be a valid approach in the identification of the presence of nonskeletal material in potentially contaminated cremains.     

Entomological identification of the post-mortem colonization of wolf cadavers in different decomposition stages

Human-driven biodiversity loss is progressively becoming a problem with dramatic consequences for the conservation of vital ecosystems. The increasing number of illegal killings of the grey wolf (Canis lupus, Linnaeus, 1758), a threatened species, displays the need for investigation and prosecution of such offences. Forensic entomology makes use of the knowledge about necrophagous insects to estimate a minimum time-since-death interval of the deceased person or animal, which can give important information on a possible perpetrator. The cadaver fauna along five decomposition stages of wolves in Germany was investigated in the period 2014–2021. The insects from 70 wolf cadavers, originating from all over Germany, were provided by the Leibniz Institute for Zoo and Wildlife Research Berlin. The accumulated degree day (ADD) model was applied for the post-mortem interval estimation on wolf cadavers for the first time. A total of 20 coleopteran species and 14 different dipteran species were discovered and identified. Almost 99 % of all insect specimens were from the order of Diptera, and beetles (Coleoptera) accounted for only 1 % of the cadaver fauna. The blowflies (Calliphoridae) are of particular importance for forensic issues, accounting for about 66 % of all families. Carrion beetles (Silphidae) were found as the second most abundant family (about 21 %). In addition, combining all cases, a steadily increasing insect species richness S was detected from early decay to advanced decay (fresh S = 8; bloated S = 12; active decay S = 21; advanced decay S = 34). In the following remains stage, the species number decreased again (S = 24). However, no significant difference in the number of species was found between the stages of decay when the cases were considered individually. The temporal pattern of insect appearance was found to be congruent with those of previous studies. Furthermore, a time of death was determined for each case and compared to the pathologist's estimates. This study provides insights into the arthropod fauna of wolf remains for the first time, applies the ADD-Model for post-mortem interval estimation, and discusses the suitability of forensic entomology for wildlife death investigations.     

Analysis of Ecstasy Tablets Using Capillary Electrophoresis with Capacitively Coupled Contactless Conductivity Detection

A method for the identification of 3,4‐methylenedioxymethamphetamine (MDMA) and meta‐chlorophenylpiperazine (mCPP) was developed employing capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C⁴D). Sample extraction, separation, and detection of “Ecstasy” tablets were performed in <10 min without sample derivatization. The separation electrolyte was 20 mm TAPS/Lithium, pH 8.7. Average minimal detectable amounts for MDMA and mCPP were 0.04 mg/tablet, several orders of magnitude lower than the minimum amount encountered in a tablet. Seven different Ecstasy tablets seized in Rio de Janeiro, Brazil, were analyzed by CE‐C⁴D and compared against routine gas chromatography‐mass spectrometry (GC‐MS). The CE method demonstrated sufficient selectivity to discriminate the two target drugs, MDMA and mCPP, from the other drugs present in seizures, namely amphepramone, fenproporex, caffeine, lidocaine, and cocaine. Separation was performed in <90 sec. The advantages of using C⁴D instead of traditional CE‐UV methods for in‐field analysis are also discussed.     

A collaborative genetic study on the STR system FGA in two Austrian population samples

Population genetic data of the short tandem repeat system FGA were determined by PCR analysis in two Austrian population samples, one population north of the Alps and one population south of the Alps. A total of 15 different alleles could be observed in 500 unrelated individuals. No significant differences were found between the phenotype frequencies in the two populations, as determined by R×C contingency test, so the populations could be pooled for further analysis. Both the single populations and the pooled population are in accordance with Hardy–Weinberg equilibrium. FGA proves to be very efficient for both stain analysis and paternity testing. The presented allele and genotype data allow the statistical interpretation of this system for Austrians.     

Some principles of Pānini's grammargrammar

The following principles are seen to operate in the rules Pānini provides for Sanskrit grammar.

1. The obvious principle that the introduction of affixes and augments which condition sound replacements necessarily precede the latter.
2. Bracketing, whereby an operation whose condition is internal relative to a condition causing another operation applies prior to the latter.
3. The derivational prehistory of a form is pertinent to the operations which apply to it.
4. Blocking: a rule R₂ is said to block an R₁ if, in a given domain, R₁ tentatively applies (and would apply in the absence of R₂) wherever R₂ can apply, while R₂ would be vacuous if R₁ applied.
5. Limited blocking, which obtains where R₁ and R₂ overlap but also have independent domains of application.

These principles account for correct derivations in cases where post-Paninian grammarians invoked rule order: where two rules conflict, that one takes precedence which is stated later in the grammar. This principle is not generally tenable, since some derivations require that a rule stated previously in the grammar take precedence. Hence, the grammarians who invoke such rule ordering in cases of conflict must admit that only a knowledge of the correct results to be obtained by applying the rules allows one to correctly apply the rules in the first place. It is argued that Pānini avoided this weakness.     

Confronting Religion: Veiled Witnesses,the Right to a Fair Trial and the Supreme Court of Canada's Judgment in R v N.S.

The Supreme Court of Canada's decision in R v N.S. is significant because the majority seems to endorse an understanding of confrontation that assumes a defendant's right to a fair trial is imperilled by a witness who seeks to give evidence while wearing the niqab. The case is of interest because it permits reflection upon the interrelationship between the right to a fair trial and the right to confront witnesses enshrined in Article 6 of the European Convention on Human Rights. Given that the European Court of Human Rights conceptualises confrontation in epistemic terms, it is argued that it would be unlikely to find that a conviction based upon evidence from a niqab‐wearing witness would infringe the right to a fair trial. This note examines the value of demeanour evidence and whether the majority in R v N.S. was correct that the abrogation of the ability to assess demeanour evidence necessarily undermines trial fairness.     

Chiral identification and determination of ephedrine,pseudoephedrine, methamphetamine and metecathinone by gas chromatography and nuclear magnetic resonance

The enantiomers of the related substances methamphetamine, ephedrine, pseudoephedrine and methcathinone were determined by both gas chromatography after derivatization and by nuclear magnetic resonance using a chiral solvating agent. For GC the substances were derivatized with (R)-(+)-α-methoxy-α-(trifluoromethyl)phenylacetic acid (MTPA) to give diasteromeric derivatives. Resolution (baseline) of at least 1.6 was obtained between all derivatives. NMR determination of the enantiomers was conducted in a chiral environment by the addition of the chiral solvating agent, (R)-(+)-1,1′-bi-2-naphthol, to NMR solutions of the substances. Racemization of methcathinone was demonstrated to be facile by exposure to alkaline solutions for varying periods of time. Enantiomeric ratios of some products derived from the oxidation of ephedrine were determined.     

Levels of cocaine and its metabolites in washed hair of demonstrated cocaine users and workplace subjects

In a study of subjects in drug rehabilitation programs, cocaine and cocaine metabolite levels were determined in the hair of 75 subjects who had produced cocaine-positive urine results. The hair was analyzed after being washed with the 3.75 h wash procedure developed by this laboratory. In addition, results of testing 73 non-users are presented, as well as levels of cocaine, benzoylecgonine (BE), cocaethylene, and norcocaine from workplace population samples. The data support a recommendation of reporting as positive a sample with cocaine of 500 pg/mg hair and either a 5% ratio of benzoylecgonine (BE) to cocaine in samples, or the presence of cocaethylene at 50 pg/mg hair, or norcocaine at 50 pg/mg hair for samples < or =2000 pg cocaine/mg hair. For samples with cocaine present at >2000 pg/mg hair, the data indicate that a ratio of 5% BE may be an overly conservative approach. In appropriately washed hair samples, cocaine users can produce hair levels of <5% BE and thus a minimum BE cutoff in lieu of a ratio could be considered.     

A survey of bacteria associated with various life stages of primary colonizers: Lucilia sericata and Phormia regina

Blow flies are common primary colonizers of carrion, play an important role in the transfer of microbes between environments, and serve as a vector for many human pathogens. While some investigation has begun regarding the bacteria associated with different life stages of blow flies, a well replicated study is currently not available for the majority of blow flies. This study investigated bacteria associated with successive life stages of blow fly species Lucilia sericata and Phormia regina. A total of 38 samples were collected from four true replicates of L. sericata and P. regina. Variable region four (V4) of 16S ribosomal DNA (16S rDNA) was amplified and sequenced on MiSeq FGx sequencing platform using universal 16S rDNA primers and dual-index sequencing strategy. Bacterial communities associated with different life stages of L. sericata and P. regina didn't differ significantly from each other. In both blow fly species, Bacilli (e.g., Lactococcus) and Gammaproteobacteria (e.g., Providencia) constituted >95% of all bacterial classes across all life stages. At the genus level, Vagococcus and Leuconostoc were present at relatively high abundances in L. sericata whereas Yersinia and Proteus were present at comparatively high abundances in P. regina. Overall, information on bacterial structures associated with various life stages of blow flies can help scientists in better understanding or management of vector-borne pathogen dispersal and in increasing the accuracy of microbial evidence based postmortem interval (PMI) prediction models.     

Progress Toward the Determination of Correct Classification Rates in Fire Debris Analysis,,

Principal components analysis (PCA), linear discriminant analysis (LDA), and quadratic discriminant analysis (QDA) were used to develop a multistep classification procedure for determining the presence of ignitable liquid residue in fire debris and assigning any ignitable liquid residue present into the classes defined under the American Society for Testing and Materials (ASTM) E 1618‐10 standard method. A multistep classification procedure was tested by cross‐validation based on model data sets comprised of the time‐averaged mass spectra (also referred to as total ion spectra) of commercial ignitable liquids and pyrolysis products from common building materials and household furnishings (referred to simply as substrates). Fire debris samples from laboratory‐scale and field test burns were also used to test the model. The optimal model's true‐positive rate was 81.3% for cross‐validation samples and 70.9% for fire debris samples. The false‐positive rate was 9.9% for cross‐validation samples and 8.9% for fire debris samples.