Isotope Ratio Determination: A Closer Look

Kevin RobersonAnalytical Seminar

November 23, 2009

Isotopic Composition

• Life story of a molecule, plant, and animal

• Effected by immediate environment

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Isotope ratios are heavily used as tracer in medical and biological studies for authenticity in traceability

On a Molecular Level

• A molecule’s isotopic composition heavily depends on:– Starting material– Manner in which it’s prepared

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What’s the Big Deal?

Operation Cyber Chase• World’s largest counterfeit

prescription drug ring• Shutdown 200 illegal e-

pharmacies

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)

http://149.101.225.20/dea/pubs/pressrel/pr042005b.jpg

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)

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

Isotope Ratio Mass Spectrometry

Pros

• Requires little compound• Fast acquisition• Relatively cheap• Analyze several nuclei

simultaneously

Cons

• Only obtain global isotope distribution

Goal

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

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

Fig. 1. Structure of naproxen (C14H14O3).

O

OH

CH3

H3CO

Instrumentation

Elemental Analyzer• Oxidative combustion • Converts analyte to H2O and CO2

• Oxidative furnace temp. 1020 oC• Reductive furnace temp. 650 oC• Column temp. 60 oCThermal Conversion Elemental Analyzer• Pyrolysis• Converts analyte to H2 and CO• Furnace temp. 1350 oC• Column temp. 85 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)

Instrumentation (cont.)

http://www.sahra.arizona.edu/programs/isotopes/methods/images/diagram.gif

Results

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

Results (cont.)

Fig. 4. Naproxen isotope ratios. Plot made with raw isotope ration datanormalize using Pt = (value−min)/range.

Results (cont.)

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

Multiple Synthetic Routes

Org. Process Res. Dev., 1997, 1 (1), pp 72–76

Multiple Synthetic Routes

Org. Process Res. Dev., 1997, 1 (1), pp 72–76

Isotopic Composition of multistep-synthesized products

• Isotopic composition of raw materials• Type of chemical reaction involved• The yield of each reaction• Purification step

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.

13C NMR Spectroscopy

Pros• Site-specific isotope ratio

determination• Separation of the signal of

each molecular site• Quantification of the nuclei

under each peak• Produces multiple

parameters

Cons• Large amounts of product• Long analysis time• Restricted to molecules

with low molecular weight <300

Goal

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

Experimental

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

IRMS measurements

• EA-Delta-V Advantage

• Sample size: 1 – 2.5 mg

NMR Spectroscopy Experiments

Acetylsalicylic acid

• 300 mg dissolved in DMSO-d6 (400 µL) + CHCl3 (200 µL )

• Solution was then filtered into 5 mm o.d. tube

Acetaminophen

• 250 mg dissolved in DMSO-d6 (600 µL)

• Solution was then filtered into 5 mm o.d. tube

http://www.kjemi.uio.no/nmr/dekor/DRX500magnet.jpg

Bruker DRX 500

Results

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

Results (cont.)

Results (cont.)

Results (cont.)

Fig. 1. Principal component analysis (PCA) on the acetylsalicylic acid data set usingthe 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).

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

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

Critique

Wokovich Paper• Able to screen for

manufacturer?• Need a more controlled

experiment to distinguish geographic provenance

• Quality of manufacturer should be considered

Silvestre Paper• Technique was shown to

have potential to make significant contribution

• Can 13C SNIF-NMR stand alone?

• Expanded on PCA data

Acknowledgements

• Dr. Megan Macnaughtan• Macnaughtan Research Group• Audience