Forensic Research using Isotope Intra-Molecular Experiments: Development and use of quantitative 13C and 15N NMR spectrometry – FRIIME

The FRIIME project addresses the general question: «can our ability to detect the origin of homemade explosives and products involved in drug trafficking be significantly improved by using data on intramolecular 'position-specific' isotope distribution ? «. For this purpose it is useful to answer the following scientific and technical questions:
(i) NMR is a tool capable of determining the isotope contents for each atom from a molecule. This has been achieved successfully for deuterium and more recently for carbon-13 in CEISAM unit at the University of Nantes. What are the conditions for a methodological transfer to the measurement of the position-specific content of nitrogen-15?
(ii) What kind of new information collected by the methodologies developed in the FRIIME project, using NMR applied to determine the 13C and / or 15N contents of each carbon and / or nitrogen atom constituting the molecule, could provide on the characterization of the origin of the products studied?
(iii) What is the contribution of this new approach (intramolecular isotopic measurement by NMR) to the investigative work of the Police at the tactical and strategic level?
(iv) How does this new framework apply to actual cases of seized illegal drugs (cutting agents, drugs) and what are the benefits compared to the usual tools during a police investigation?

The first tests on the application of NMR to the study of isotopic nitrogen-15 distribution have focused on very innovative development on increasing sensitivity while maintaining an accuracy of 0.1%. As described in the project, a strong increase in sensitivity can be achieved through the use of NMR methodological developments. A proof of concept was obtained using the adiabatic INEPT sequence (developed for carbon 13). A double innovation has been demonstrated: (i) use of a polarization transfer sequence via long-distance couplings (instead of a sensitivity transfer related to the directly hydrogen-bonded atoms) and (ii) Obtaining a repeatable quantitative response to the ‰ level permitting the observation never previously obtained of both normal and inverse nitrogen isotopic fractionation in the same molecule.
In parallel, the classical 13C-NMR analytical protocol could be used for the study of the target molecules: paracetamol, caffeine, levamisole, phenacetin. Taking advantage of the INEPT 15N long-distance development, the same approach has been validated in carbon-13. This leads to an advantage, hitherto inaccessible, of obtaining the isotopic profile 13C of all the carbons, including quaternaries, of the molecule, but with an increase in the sensitivity allowing to work with a few tens of mg instead of hundreds of mg. This significant step makes it possible to work on real samples requiring complex separations.
The last point discussed led to the testing of separations / purifications of the target molecules, not unsing hundreds of mg but a target value of 40 mg. The production of pure molecules is greatly facilitated allowing easy automation on the chromatography flash system (acquired within the framework of this project). Once the protocol has been validated, it will be used on the actual samples.

Stable isotopes have become an essential tool for detecting the origin of food products. In forensic science, stable isotopes can also be used to determine the origin of the molecules studied. Tracing is therefore possible on the basis of isotopic signatures depending on the raw materials, the synthesis process and the purification process used. However, the usual isotopic analysis method, mass spectrometry (CG-SMRI), is limited to volatile molecules and provides only global information on the isotopic changes of the molecule. The FRIIME project will add a new analytical method by NMR to ensure tracing in larger molecules. This tracing will be finer because it will show effects at each position (carbon and / or nitrogen) of the molecule. This new tool will be tested on real samples of drug for which the cutting agents will be analyzed to have an isotopic profile marker of their origin. Then new information on networks will be obtained.

A poster was presented at an international congress:
«Development of irm-15N NMR for position-specific isotope analysis.« To post. Valentin Joubert, Virginie Silvestre, Serge Akoka, Gerald S. Remaud. International Symposium on Isotopomers (ISI2016), 3-6 October 2016, La Cité des Congrès, Nantes, France

Other presentations will take place in 2017 and the first articles will also be submitted for publication.

The FRIIME project addresses the question: ‘Can the intramolecular stable isotope composition be used to enhance the Forensic Intelligence capabilities to track the origin of home-made explosives and psychoactive substances?’
In the battle against the illicit drugs market, methodologies have been developed to address origin determination and trafficking route dismantlement for various scheduled molecules: this is the concept of drug profiling. Profiling methods can rely on synthetic impurities, residual solvents, etc. In FRIIME, the same objective will be pursued but using the powerful intrinsic marker of the isotopic content of the molecules in question. In recent years, stable isotope strategies have proved to be a valuable resource in various forensic fields, including explosives and illicit drugs. However, these prior studies were conducted using isotope ratio measured by Mass Spectrometry (irm-MS), which only allows the isotopic characterisation to be applied to the whole molecule of interest.
FRIIME, by resorting to a cutting edge isotopic nuclear magnetic resonance (NMR) methodology, will investigate the position-specific distribution of isotopes in two common explosives, two well-known cutting agents used with illicit drugs and one new psychoactive substance. Isotopic 13C NMR will be applied to target molecules in order to refine the data obtained by irm-MS, as has already been demonstrated for counterfeiting of medicines. Furthermore, isotopic 15N NMR will be developed as a very pertinent contribution to the analyses of explosives. By collecting a larger number of parameters characteristic of the studied products than are currently obtainable, a new strategic and tactical paradigm will be made available to Law Enforcement Agencies enabling models of greater sophistication and predictive power to be developed, including the origins of raw materials and chemical reaction pathways used to manufacture targeted products.
FRIIME is an ambitious and breakthrough project proposing a new approach made possible by uniting analytical chemists specialised in isotope analysis with scientists specialised in forensic applications. Innovative spin-offs from FRIIME can be expected in the following areas:
• Rationalization of the connection between forensic science and intelligence-led policing: more data on position-specific isotope, 13C and 15N composition of home-made explosives (ammonium nitrate and urea nitrate) from different manufacturing processes, on psychoactive drugs (meta-chlorophenylpiperazine) , and on different cutting agents (caffeine, phenacetin). These data will serve to build refined models with which to combat crime.
• An improved, more robust methodology through the high-precision isotopic characterization of a drug or an explosive for determining the origin of the raw materials and/or the manufacturing process used.
• A protocol for isotopic NMR that can be adapted to other forensic fields, such as the environment (ground water or soil contamination), food control and toxicology, or counterfeit medicines.
• Applications of a new instrumental methodology to other research domains: metabolomics, fluxomics and isotopomics.
The success of the FRIIME project requires the interaction of scientific skills in quantitative NMR, Isotope Analytical Chemistry and Forensic investigation. To fulfil its purposes, FRIIME is built as a Collaborative Research Project associating two public partners: 1) the academic research team of the Isotope Analysis Group (EBSI) of CEISAM (Nantes), providing its innovative NMR platform, and 2) the French Police Forensic Institute: INPS (Lyon), providing a long-term experience in illicit drug profiling, including the use of its own irm-MS. Thanks to its expertise and experience in the areas of illicit drugs and explosives, INPS is in a unique position to provide a collection of real samples, thus guaranteeing an operational outcome, in accordance with the expectations of the law enforcement agencies.