Isotope Ratio Determination: A Closer Look

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Isotope Ratio Determination A Closer Look

• Kevin Roberson
• Analytical Seminar
• November 23, 2009

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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
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On a Molecular Level

• A molecules isotopic composition heavily depends
  on
• Starting material
• Manner in which its prepared

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Whats the Big Deal?

• Operation Cyber Chase

• The Fight Against Counterfeiting

• Worlds largest counterfeit prescription drug
  ring
• Shutdown 200 illegal e-pharmacies

• Focus on the presence of impurities
• Chromatographic techniques
• Pattern recognition method
• Near-Infrared
• Raman
• Methods do not focus on the active pharmaceutical
  ingredient (API)

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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)

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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

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Isotope Ratio Mass Spectrometry

• Pros

• Cons

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

• Only obtain global isotope distribution

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Goal

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

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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

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Instrumentation

• Elemental Analyzer

• Oxidative combustion
• Converts analyte to H2O and CO2
• Oxidative furnace temp. 1020 oC
• Reductive furnace temp. 650 oC
• Column temp. 60 oC

• Thermal 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)
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Instrumentation (cont.)
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Results
Fig. 3. (a) Naproxen d13C vs. dD (b) naproxen
d13C vs. d18O (c) naproxen d18O vs. dD.
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Results (cont.)
Fig. 4. Naproxen isotope ratios. Plot made with
raw isotope ration data normalize using Pt
(value-min)/range.
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Results (cont.)
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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

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Multiple Synthetic Routes
Org. Process Res. Dev., 1997, 1 (1), pp 7276
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Multiple Synthetic Routes
Org. Process Res. Dev., 1997, 1 (1), pp 7276
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Isotopic Composition of multistep-synthesized
products

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

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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.

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13C NMR Spectroscopy

• Pros

• Cons

• Site-specific isotope ratio determination
• Separation of the signal of each molecular site
• Quantification of the nuclei under each peak
• Produces multiple parameters

• Large amounts of product
• Long analysis time
• Restricted to molecules with low molecular weight
  lt300

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Goal

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

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Experimental
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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

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IRMS measurements

• EA-Delta-V Advantage

• Sample size 1 2.5 mg

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NMR Spectroscopy Experiments

• Acetaminophen

• 250 mg dissolved in DMSO-d6 (600 µL)
• Solution was then filtered into 5 mm o.d. tube

Bruker DRX 500

• Acetylsalicylic acid

• 300 mg dissolved in DMSO-d6 (400 µL) CHCl3 (200
  µL )
• Solution was then filtered into 5 mm o.d. tube

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Results
Scheme 1. Molecular structures of (A)
acetylsalicylic acid and (B) acetaminophen with
carbon positions numbered in order of decreasing
13C chemical shift.
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Results (cont.)
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Results (cont.)
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Results (cont.)
Fig. 1. Principal component analysis (PCA) on the
acetylsalicylic acid data set using the 13C of
the aromatic carbons C3C8 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 C2C5 as variables component 1
(C1) vs. component 2 (C2).
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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

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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

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Critique

• Wokovich Paper

• Silvestre Paper

• Able to screen for manufacturer?
• Need a more controlled experiment to distinguish
  geographic provenance
• Quality of manufacturer should be considered

• Technique was shown to have potential to make
  significant contribution
• Can 13C SNIF-NMR stand alone?
• Expanded on PCA data

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Acknowledgements

• Dr. Megan Macnaughtan
• Macnaughtan Research Group
• Audience