Forensic Drug Analysis Research Articles

Enantiomeric Identification of Pregabalin by GC-MS via Methylation and S-TPC Chiral Derivatization.

Pregabalin is a Schedule V controlled substance which is defined as the (S) enantiomer of 3-(aminomethyl)-5-methylhexanoic acid. It is used legitimately to treat neuropathy in patients with diabetes as well as for epilepsy and fibromyalgia. Pregabalin is an amino acid and an amphoteric compound, which makes it difficult to analyze using the conventional GC-MS instrumentation found in most forensic drug analysis laboratories. Problems associated with the traditional GC-MS analysis of pregabalin include selective solubility, ring closure to the corresponding lactam in the GC injection port and/or the MS transfer line and difficulty with chiral derivatization due to the presence of a carboxylic acid moiety. Here, we show that these challenges can be overcome by methylating (capping) the carboxylic acid portion of the pregabalin molecule and converting to the corresponding methyl ester. Once the methyl ester is synthesized, chiral derivatization at the amine can be achieved to identify the controlled (S) enantiomer of pregabalin via GC-MS.

Microextraction Techniques for Forensic Drug Analysis in Saliva

Microextraction Techniques for Forensic Drug Analysis in Saliva

Applications of electrochemical sensors: Forensic drug analysis

Summary Electrochemical-based sensors are a powerful analytical tool which can be exploited by the forensic community particularly for the analysis of illicit substances from street and biological samples. This report presents a brief overview on selected recent publications related to the application of electrochemical sensors to forensic drug analysis and gives opinions on the technical developments as well as the future applications in this field.

Analytical differentiation of quinolinyl- and isoquinolinyl-substituted 1-(5-fluoropentyl)-1H-indole-3-carboxylates: 5F-PB-22 and its ten isomers

Differentiation among regioisomers of synthetic cannabinoids in forensic drug analysis is a crucial issue, since all isomers are not regulated by law. New equivalent analogs obtained via minor modification of their preexisting molecules keep on emerging. Isomers formed via substitutional exchange are also a cause for concern. This study is focused on the isomeric molecules that stem from minor modifications of 5F-PB-22. The analytical properties of these molecules and methods of differentiation are reported. Scan mode analysis using gas chromatography–electron ionization-mass spectrometry (GC–EI-MS) was performed using the authentic 5F-PB-22 standard, five regioisomeric quinolinyl ester indoles, and five regioisomeric isoquinolinyl ester indoles. Because it was not possible to separate 5F-PB-22 from the 5-hydroxyquinoline isomer using GC and all analytes showed similar EI mass spectra, liquid chromatography (LC)–tandem mass spectrometry analysis was performed. Using LC, a successful separation of 5F-PB-22 from all isomers could be achieved. Based on the electrospray ionization-mass spectra, the protonated molecular ion at m/z 377.2 was selected as the precursor ion for the regioisomeric and structural isomeric differentiation. Collision-induced dissociation provides relative intensity differences in the product ions among the isomers, enabling mass spectrometric differentiation of the isomers. To our knowledge, this is the first report on mass spectrometric differentiation of 5F-PB-22 and its ten isomers.

Differentiation of ring‐substituted regioisomers of amphetamine and methamphetamine by supercritical fluid chromatography

Chromatographic differentiation of the ring-substituted regioisomers of amphetamine (AMP) and methamphetamine (MA) was performed by supercritical fluid chromatography (SFC). The behaviour of the retention against the changes of column temperature and co-solvent proportion was studied. The obtained information facilitated the optimization of the each regioisomer. As a result, 2-, 3-, and 4-ring-substituted analogues of AMP and MA with methyl, methoxy, fluoro, chloro, and bromo groups were separated, generally within 6 min. In addition, we found that the separation pattern of the examined regioisomers was classified into two, which depended on the electron donating/withdrawing effect of the substituent. Our results indicate that SFC could be used in forensic drug analysis for fast, reliable identification of structurally similar drugs of abuse. Copyright © 2016 John Wiley & Sons, Ltd.

Electrochemiluminescent detection of methamphetamine and amphetamine

Direct detection of amphetamine type stimulants (ATSs) including methylamphetamine (MA) in street samples and biological matrices without the need for pretreatment or extraction is a great challenge for forensic drug analysis. Electrochemical techniques, such as electrochemiluminescence (ECL), are promising tools for this area of analysis. This contribution focuses on the electrochemical and photochemical properties of [Ru(bpy)3]2+ Nafion composite films and their subsequent use for the detection of ATS in particular MA. Under optimised conditions, the response linearly increased with the concentration over the concentration range 50pM≤[MA]≤1mM while an equivalent dynamic range was obtained for amphetamine with a correlation coefficient of 0.9903 and 0.9948 respectively. The ECL signal was monitored at ∼620nm, representing the λmax for the [Ru(bpy)3]2+ Nafion composite films. This wavelength is shifted by approximately 15nm compared to the photoexcited λmax for the same system. The modified films were formed by direct interaction with the electrode surface without the need for surface modification or chain linkers. This is a major advantage for the fabrication of any sensor as it reduces the synthesis times resulting in more economically and cheaper production costs. This technique is simple, rapid, selective and sensitive, and shows potential for the high-throughput quantitation of ATS as well as possibilities for adaptation with other techniques such as FIA or LC systems.

Differentiation of regioisomeric chloroamphetamine analogs using gas chromatography-chemical ionization-tandem mass spectrometry.

In recent years, a large number of clandestinely synthesized new psychoactive substances with high structural variety have been detected in forensic samples. Analytical differentiation of regioisomers is a significant issue in forensic drug analysis, because, in most cases, legal controls are placed on only one or two of the conceivable isomers. In this study, gas chromatography–tandem mass spectrometry (GC–MS–MS) was used to differentiate the regioisomers of chloroamphetamine analogs (chloroamphetamines and chloromethamphetamines) synthesized in the authors′ laboratories. Free bases, trifluoroacetyl derivatives, and trimethylsilyl derivatives were subjected to GC–MS–MS using DB-1ms, DB-5ms, and DB-17ms capillary columns, respectively. The regioisomers of chloroamphetamine analogs in all forms were well separated on the DB-5ms column. The electron ionization mass spectra of the chloroamphetamine analogs gave very little structural information for differentiation among these analogs, even after trifluoroacetyl and trimethylsilyl derivatization of the analytes. Characteristic product ions of the 2-positional isomers were observed by electron ionization-MS–MS. In contrast, chemical ionization-MS–MS of the free bases provided more structural information about chloride position on the aromatic ring when [M+H–HCl]+ was selected as a precursor ion. The results suggest that a combination of chromatographic analysis and MS–MS supports differentiation for regioisomers of chloroamphetamine analogs.

The role of ultra high performance liquid chromatography with time of flight detection for the identification of synthetic cannabinoids in seized drugs

Separation and mass spectrometric techniques are integral parts of forensic drug analysis for both screening and confirmation. The Scientific Working Group for the Analysis of Seized Drugs (SWGDRUG), which is responsible for setting standards for drug analysis, requires for drug identification a Category A test such as mass spectrometry with an additional test from either Category B or C. If a Category A method is not used at least two uncorrelated tests from Category B must be included, for which separation techniques such as gas chromatography and liquid chromatography would qualify. The utility and validity of using ultra high performance liquid chromatography (UHPLC) and time-of-flight (TOF) mass spectrometry (MS) for the analysis of synthetic cannabinoids is presented. The separation of 32 solutes, including 23 controlled substances and nine non-controlled positional isomers of JWH-018, are compared using UHPLC with TOF detection and capillary GC with electron ionization (EI). For these solutes, the reversed phase UHPLC separation on three different 2.1mm×150mm×2.7μm superficially porous (SPP) columns (C18, Phenyl-Hexyl and Dimethylpentafluorophenylpropyl (PFP)) compared favorably with the capillary gas chromatography (GC) separation using an Elite-5MS column 0.25mm×30m×0.25μm. Principal component analysis revealed that all three UHPLC separations for the separation of the controlled substances are orthogonal to the capillary GC separation. It was also revealed by principal component analysis that the separation of JWH-018 and the nine non-controlled positional isomers for the various techniques were significantly more correlated than the separation of the controlled substances. Although most of the controlled synthetic cannabinoids gave unique TOF in-source collision-induced dissociation MS spectra and EI spectra, it was not possible to discriminate among the geometric isomers (CP47, 497, Epi CP47, 497; Cp47, 497 C8 homologue, Epi CP47, 497 C8 homologue). JWH-018 could be distinguished from the non-controlled isomers based on its EI spectra. In contrast, several of the non-controlled JWH-018 isomers give identical TOF in-source collision-induced dissociation MS spectra to JWH-018.

Application of mixed-mode ultra high performance liquid chromatography to forensic drug analysis

Methodology is presented for the rapid analysis of drugs of abuse using a single UHPLC column with orthogonal separation methods using different combinations of the same solutions in the solvent reservoir.

P54 Capillary electrophoresis – mass spectrometry for the forensic analysis of cathinone drugs and metabolites

(For research use only, not for use in diagnostic procedures). In forensic drug analysis there is always a need for increased sensitivity to provide better detection of new synthetic substances in challenging casework. The capability to resolve very complex mixtures and provide confirmation of the identity of polar drugs and metabolites is the strength of Capillary Electrophoresis integrated with Mass Spectrometry. In this work we apply a sheathless interface for Capillary Electrophoresis-ESI-Mass Spectrometry (CESI-MS) for screening of 25 cathinone related designer drugs and metabolites in biofluids. The sample preparation included a pre-treatment step using only a simple dilution of urine. The CE used a 90 cm barefused silica, 150 μm o.d., 30 μm i.d. capillary, at 200-300 V/cm, 2.4 to 2.8 μamps at 25 °C. The background electrolyte (BGE) and conductive liquid were both 50 mM Ammonium Formate, pH 2.85. Sample introduction was either Hydrodynamic; 16s at 34.5 mBar or Electrokinetic; 10kV for 16s. The CE Instrument was a Beckman Coulter CESI 8000 coupled to an AB SCIEX 5600 TripleTOF® mass spectrometer through a sheathless interface High Sensitivity Porous Sprayer (HSPS). The ESI Voltage applied to the interface was 1.2 to 1.8 kV. The capillaries were initially conditioned with MeOH, water, 1N NaOH, water and BGE. A sheathless HSPS was used to interface CE and ESI-MS. The drugs tested included 25 cathinone drugs and metabolites in urine. Acquired MS/MS spectra were searched against an accurate mass drug library for confident compound identification. Combining high resolution MS and MS/MS acquisition with Capillary Electrophoresis separation, allows for retrospective compound identification of the cathinones with highest confidence based on accurate mass product ion specificity, mass spectral library matching as well as mass error, isotopic profile and molecular formula finding. The LOQ values for all components were found to be less than 5 ng/mL. Linearity was achieved between 5-1000 ng/mL for most drugs and % RSDs for different analytes are reported to be less than 10 % in most cases. A sheathless interface for Capillary Electrophoresis- ESI-Mass Spectrometry (CESI-MS) has shown to be a viable alternative for screening of 25 cathinone related designer drugs and metabolites in biofluids.

Recent Advances in Forensic Drug Analysis by Dart-MS

Mass spectrometry methods play a major role in many forensic applications. While gas chromatography-mass spectrometry methods are commonly used in crime laboratories and enforcement agencies, a variety of advanced techniques are now available that can improve upon standard methods and address emerging issues in forensic science. New mass spectrometry technologies include more versatile ionization sources, allowing the next generation of instrumentation to be more multipurpose and adaptable to the needs of the discipline. Direct analysis in real-time mass spectrometry is an ambient ionization method that allows direct testing of gas, liquid and solid samples without the need for any preparation or extraction, based on thermal desorption and ionization directly from the sample surface. This Review will provide an in-depth description of direct analysis in real-time time-of-flight mass spectrometry as applied to samples relevant to forensic science, with a focus on analysis and characterization related to forensic drug chemistry.

Forensic drug analysis and microfluidics

The analysis of drugs of abuse in microfluidic devices has the potential to provide solutions to today's on-site analysis challenges. The use of such devices has not been limited to miniaturising conventional analytical methods used routinely in forensic laboratories; new and interesting approaches have been implemented in microfluidics and benefit from the ability to control minute amounts of liquids in the small channels. The microfluidic platforms developed so far have been used successfully to carry out single or multiple analytical processes and offer a great opportunity for new technologies for on-site drug testing.

Differentiation of regioisomeric fluoroamphetamine analogs by gas chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry

In recent years, a large number of clandestinely produced controlled-substance analogs (designer drugs) of amphetamine with high structural variety have been detected in forensic samples. Analytical differentiation of regioisomers is a significant issue in forensic drug analysis because, in most cases, legal controls are placed only on one or two of the three isomers. In this study, we used gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) for the differentiation of regioisomers of fluoroamphetamine analogs (fluoroamphetamines and fluoromethamphetamines), which were synthesized in our laboratories. Free bases and their acylated and silylated derivatives were subjected to GC–MS analysis using DB-1ms, DB-5ms, and DB-17ms capillary columns. The separation of free bases was incomplete on all columns. Trifluoroacetyl derivatives of 3- and 4-positional isomers showed slight separation on DB-1ms and DB-5ms. On the other hand, trimethylsilyl derivatization enabled baseline separation of six fluoroamphetamine analogs on DB-1ms and DB-5ms columns, which was sufficient for unequivocal identification. For LC–MS/MS, a pentafluorophenyl column was able to separate six regioisomeric fluoroamphetamine analogs but a conventional C18 column could not achieve separation between 3- and 4-positional isomers. These results show that a suitable choice of derivatization and analytical columns allows the differentiation of regioisomeric fluoroamphetamine analogs.

Toxicokinetics of Cocaine and Metabolites: The Forensic Toxicological Approach

Cocaine is one of the most used psychomotor stimulants all over the world. On this basis, the interest for the pharmacological activity and the pharmacodynamic and pharmacokinetic aspects of this drug is very prominent in both clinical and forensic toxicological environments. The review presents and discusses 65 scientific publications covering all the aspects of cocaine toxicokinetic, including absorption, distribution, metabolism and elimination of the drug. Particular attention has been dedicated to the studies on the disposition of the drug in alternative biological matrices, such as oral fluid, hair, fetus fluids and tissues, and sweat. In fact, in the last years the use of these matrices has been proposed in clinical and forensic drug analysis in order to obtain additional information to that which can be obtained by analyzing the traditional biological matrices, such as blood and urine.

Rapid Separation and Characterization of Cocaine and Cocaine Cutting Agents by Differential Mobility Spectrometry–Mass Spectrometry

Forensic drug laboratories are inundated with cases requiring time-consuming GC- or LC-based chromatographic separations of submitted samples. High-throughput analytical methods would be of great practical utility within forensic drug analysis. Recently developed ion-mobility-based separation methods combined with mass spectrometry can often be used without chromatography, suppress chemical interferents of similar mass, and operate in seconds. We have evaluated differential mobility spectrometry-mass spectrometry (DMS-MS) for performance on adulterated cocaine mixtures. The DMS interface is only a few centimeters in length, operates in seconds, and can be adapted to any MS system using atmospheric pressure ionization. Drug cutting agents, typical targets such as cocaine, and drug metabolites are rapidly separated by the DMS ion prefilter. Tests demonstrated characterization of complex mixtures, such as isolation of levamisole, an adulterant with alarming side effects, from a 13-component mixture. DMS-MS holds great potential for the analysis of drug samples submitted for forensic analysis.

Evaluation and Optimization of Capillary Zone Electrophoresis for Common Drugs of Forensic Interest in Aqueous Matrix

Capillary zone electrophoresis (CZE) is one of the primary analytical tools in forensic drug analysis. The purpose of this study was to evaluate and optimize the role of deionized water and diluted running buffer solutions as reconstitution solvents for CZE application. Eighteen common basic drug analytes of forensic interest were selected for the experiments, and the detection sensitivity and assay reproducibility were measured and analyzed. It was concluded that CZE detection sensitivity could be optimized using deionized water as the reconstitution solvent and injection condition of 10 kV/18s or 8 kV/18s. Diluted phosphate buffer solutions ranging from 1–10 mM reduced CZE detection sensitivity. CZE assay reproducibility could be improved by selecting electrokinetic injection of 10kV/18s for deionized water samples; assay variations were smaller in diluted buffer solutions. Results obtained from this systematic evaluation of aqueous samples were beneficial in directing and optimizing CZE characteristics in routine forensic specimen preparation and operation.

Validation of Thin Layer Chromatography with AccuTOF-DART™ Detection for Forensic Drug Analysis*

Thin layer chromatography (TLC) is a technique that is commonly employed in the forensic drug analysis of pharmaceutical preparations. Detection is typically accomplished using various visualization spray reagents. Conventional gas chromatography-mass spectrometry (GC-MS) analysis is typically performed to confirm the TLC results. Depending on the drugs tested and the instrument conditions required, this confirmation can take up to an hour to complete. Direct analysis in real time (DART™) is an ionization source, coupled to an accurate-mass time-of-flight mass spectrometer that has the capability to ionize materials under ambient conditions. To streamline analysis, the combination of TLC with DART™ detection is proposed to screen and subsequently identify drug compounds, all from the same TLC plate. DART™ confirmations of TLC analyses take <10 min to complete and compare favorably to GC-MS in sensitivity and selectivity. This study validates the use of TLC-DART in the forensic identification of the components of several pharmaceutical preparations.

Purification of pharmaceutical preparations using thin‐layer chromatography to obtain mass spectra with Direct Analysis in Real Time and accurate mass spectrometry

Forensic analysis of pharmaceutical preparations requires a comparative analysis with a standard of the suspected drug in order to identify the active ingredient. Purchasing analytical standards can be expensive or unattainable from the drug manufacturers. Direct Analysis in Real Time (DART™) is a novel, ambient ionization technique, typically coupled with a JEOL AccuTOF™ (accurate mass) mass spectrometer. While a fast and easy technique to perform, a drawback of using DART™ is the lack of component separation of mixtures prior to ionization. Various in-house pharmaceutical preparations were purified using thin-layer chromatography (TLC) and mass spectra were subsequently obtained using the AccuTOF™- DART™ technique. Utilizing TLC prior to sample introduction provides a simple, low-cost solution to acquiring mass spectra of the purified preparation. Each spectrum was compared against an in-house molecular formula list to confirm the accurate mass elemental compositions. Spectra of purified ingredients of known pharmaceuticals were added to an in-house library for use as comparators for casework samples. Resolving isomers from one another can be accomplished using collision-induced dissociation after ionization. Challenges arose when the pharmaceutical preparation required an optimized TLC solvent to achieve proper separation and purity of the standard. Purified spectra were obtained for 91 preparations and included in an in-house drug standard library. Primary standards would only need to be purchased when pharmaceutical preparations not previously encountered are submitted for comparative analysis. TLC prior to DART™ analysis demonstrates a time efficient and cost saving technique for the forensic drug analysis community. Copyright © 2011 John Wiley & Sons, Ltd.

The General Chemical State Laboratory of Greece and its role in forensic drug analysis

The General Chemical State Laboratory of Greece and its role in forensic drug analysis

Convenient Method for Synthesis of l-Methamphetamine

Enantioselective analysis of methamphetamine (MA) and amphetamine (AP) is necessary in forensic drug analysis. In general, standard materials are required for performing forensic investigation and also for developing new analytical methods. However, l-MA is not commercially available in Japan and only a few reports are available on the enantioselective synthesis of l-MA. We developed a new and convenient method for the synthesis of l-MA using d-norephedrine (d-NE) as a starting material. d-NE was treated with 1,1′-carbonyldiimidazole to produce the corresponding cyclic carbonate, and the product was treated with sodium hydride and iodomethane to form mono-N-methylated amine derivative, which was treated with palladium on activated carbon in hydrogen atmosphere for catalytic reduction. After the addition of aqueous hydrogen chloride (HCl), l-MA was obtained as its HCl salt (total yield 58%).