Isotope Ratio Determination: A Closer Look

1. Isotope Ratio Determination: A Closer Look Kevin Roberson Analytical Seminar November 23, 2009

2. Isotopic Composition • Life story of a molecule, plant, and animal • Effected by immediate environment http://u759.curie.u-psud.fr/images%20du%20site/imagesSIMS/N15.jpg http://www.fort.usgs.gov/resources/spotlight/shorebird_isotopes/images/plantlifecycle.gif Isotope ratios are heavily used as tracer in medical and biological studies for authenticity in traceability

3. On a Molecular Level • A molecule’s isotopic composition heavily depends on: • Starting material • Manner in which it’s prepared http://t2.gstatic.com/images?q=tbn:mfk65D6uZLf4BM:http://images.absoluteastronomy.com/images/encyclopediaimages/b/be/benzoic_acid-chemical-reaction-1.png

4. What’s the Big Deal? Operation Cyber Chase The Fight Against Counterfeiting Focus on the presence of impurities Chromatographic techniques Pattern recognition method Near-Infrared Raman Methods do not focus on the active pharmaceutical ingredient (API) • World’s largest counterfeit prescription drug ring • Shutdown 200 illegal e-pharmacies http://149.101.225.20/dea/pubs/pressrel/pr042005b.jpg

5. Stable Isotope Analysis • Probes the atomic composition of the molecules • Extremely valuable for the detection of a number of different types counterfeiting • Isotope Ratio Mass Spectrometry (IRMS) • 13C Stable Natural Isotope Fractionation-NMR (SNIF-NMR)

6. Stable Isotopic Composition of the Active Pharmaceutical Ingredient (API) Naproxen A. M. Wokovich, J. A. Spencer, B. J. Westenberger, L. F. Buhse, J. P. Jasper

7. Isotope Ratio Mass Spectrometry Pros Cons Only obtain global isotope distribution • Requires little compound • Fast acquisition • Relatively cheap • Analyze several nuclei simultaneously

8. Goal • To asses the potential of IRMS to distinguish the provenance of active pharmaceutical ingredients (APIs).

9. Experimental • India, Manufacturer A – Five lots • India, Manufacturer B – Five lots • Italy, Manufacturer C – Three lots • Italy, Manufacturer D – Three lots • Ireland, Manufacturer E – Seven lots • USA, Manufacturer F – Three lots

10. Instrumentation Elemental Analyzer Thermal Conversion Elemental Analyzer Pyrolysis Converts analyte to H2 and CO Furnace temp. 1350 oC Column temp. 85 oC • Oxidative combustion • Converts analyte to H2O and CO2 • Oxidative furnace temp. 1020 oC • Reductive furnace temp. 650 oC • Column temp. 60 oC Fig. 2. Schematic drawing of (a) a Finnigan elemental analyzer/ mass spectrometer (EAMS) and (b) a Finnigan thermal conversion elemental analyzer/mass spectrometer (TCEAMS)

11. Instrumentation (cont.) http://www.sahra.arizona.edu/programs/isotopes/methods/images/diagram.gif

12. Results Fig. 3. (a) Naproxen: δ13C vs. δD; (b) naproxen: δ13C vs. δ18O; (c) naproxen: δ18O vs. δD.

13. Results (cont.) Fig. 4. Naproxen isotope ratios. Plot made with raw isotope ration data normalize using Pt = (value−min)/range.

14. Results (cont.)

15. Conclusion • Plausible method for screening manufacturer of given API • No evidence of geographic identification • Isotopic variation may be due to thermodynamic and/or kinetic fractionation

16. Multiple Synthetic Routes Org. Process Res. Dev., 1997, 1 (1), pp 72–76

17. Multiple Synthetic Routes Org. Process Res. Dev., 1997, 1 (1), pp 72–76

18. Isotopic Composition of multistep-synthesized products • Isotopic composition of raw materials • Type of chemical reaction involved • The yield of each reaction • Purification step

19. Isotopic 13C NMR Spectrometry to Assess Counterfeiting of Active Pharmaceutical Ingredients: Site-Specific 13C Content of aspirin and Paracetamol Silvestre V., and Remaud G. S., et al.

20. 13C NMR Spectroscopy Pros Cons Large amounts of product Long analysis time Restricted to molecules with low molecular weight <300 • Site-specific isotope ratio determination • Separation of the signal of each molecular site • Quantification of the nuclei under each peak • Produces multiple parameters

21. Goal • Assess the effectiveness of quantitative isotopic 13C NMR as a tool to better characterize active pharmaceutical ingredients (APIs) and detect counterfeiting of medicines.

22. Experimental

23. Extraction-purification • 1 g of API was powdered and/or blended • Boiling ethanol (20 mL) was added • Suspension was filtered through a fritted glass funnel • Filtrate was evaporated to dryness using rotary evaporator • Residue was recrystalized with minimum ethanol • Crystals were collected by filtration through fritted glass funnel and dried overnight at 70 oC

24. IRMS measurements • EA-Delta-V Advantage • Sample size: 1 – 2.5 mg

25. NMR Spectroscopy Experiments Acetylsalicylic acid Acetaminophen 250 mg dissolved in DMSO-d6 (600 µL) Solution was then filtered into 5 mm o.d. tube • 300 mg dissolved in DMSO-d6 (400 µL) + CHCl3 (200 µL ) • Solution was then filtered into 5 mm o.d. tube Bruker DRX 500 http://www.kjemi.uio.no/nmr/dekor/DRX500magnet.jpg

26. Results Scheme 1. Molecular structures of (A) acetylsalicylic acid and (B) acetaminophen with carbon positions numbered in order of decreasing 13C chemical shift.

27. Results (cont.)

28. Results (cont.)

29. Results (cont.) Fig. 1. Principal component analysis (PCA) on the acetylsalicylic acid data set using the 13C of the aromatic carbons C3–C8 as variables: component 1 (C1) vs. component 2 (C2). Note that sample N◦ 15 is very atypical. Fig. 2. Principal component analysis (PCA) on the acetaminophen data set using the 13C of the aromatic carbons C2–C5 as variables: component 1 (C1) vs. component 2 (C2).

30. Conclusions and Future Work • Effectively demonstrated the feasibility of the approach • Each batch was shown to have an individual isotopic fingerprint • Same batch showed same isotopic profile

31. Future Work • Reduction of the NMR analysis time by using relaxation agents or establishing new pulse sequences • Differentiation of batches produced by different synthetic pathways • Application of a variety of chemometric tools to link isotope data to the processes

32. Critique Wokovich Paper Silvestre Paper Technique was shown to have potential to make significant contribution Can 13C SNIF-NMR stand alone? Expanded on PCA data • Able to screen for manufacturer? • Need a more controlled experiment to distinguish geographic provenance • Quality of manufacturer should be considered

33. Acknowledgements • Dr. Megan Macnaughtan • Macnaughtan Research Group • Audience