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Scientists have used stable isotope analysis for decades, employing isotope ratio mass spectrometry to trace the isotopic fingerprints of natural materials. Within the past few years, pharmaceutical manufacturers and regulators have also begun to test the technique, which is relatively inexpensive and extremely precise, to authenticate pharmaceutical ingredients and products (Figures 1-3; Refs. 1-5).
In addition, isotopic analysis can also provide information about the manufacturing process, during which raw materials are manufactured into intermediates, active pharmaceutical ingredients (APIs) and, finally, into drug products. Stable-isotopic values are affected by the fractionation that occurs during the manufacturing process (Figure 4). The measurement of variables such as reaction type and rate, and separation processes such as recrystallization can be used to monitor reactions in various steps of Process Analytical Chemistry (PAC).
Recently, Molecular Isotope Technologies LLC (Niantic, Conn.) conducted joint studies with FDAs Division of Pharmaceutical Analysis using isotopic analysis to study the API, Naproxen [6,7]. Now, Molecular Isotope Technologies and Johnson & Johnson (J&J) are exploring the techniques potential in helping identify counterfeit products based on their processes, or to provide information in process patent infringement cases .
In this article, we discuss how isotopic analysis can be used to track the isotopic fractionation that occurs in synthetic pharmaceutical manufacturing processes. We also present and discuss preliminary results of J&J studies using isotopic analysis to evaluate the antiepileptic drug, Topiramate. So far, these results suggest that this analytical tool could be a practical, cost-effective way to analyze and control pharmaceutical manufacturing processes.
Fundamentals of isotopic analysis
To very briefly review the basics, stable isotopes exist naturally as mass variants of chemical elements. Carbon-13, for instance, consisting of seven neutrons and six protons, accounts for approximately 1.1 percent of all carbon in nature; carbon-12 accounts for approximately 98.9 percent. The variation in the 13C/12C ratiosand other isotopic ratios of pharmaceutical componentscan be used to trace the isotopic provenance or fingerprint of APIs or drug products via isotope-ratio mass spectrometry. Figure 1 is a schematic diagram of an Elemental Analyzer/Mass Spectrometer. In an EAMS, samples are dropped from a carousel to a high-temperature oven where they are combusted to small molecules such as CO2 or N2, whose isotope ratios are subsequently measured on an isotope ratio mass spectrometer.
When both the sources of starting materials and of the manufacturing process are fixed, stable-isotopic values for the products will be predictably constant and provide an identifiable and reproducible fingerprint. Over the last six years, authentication of pharmaceutical APIs and drug products, based on their isotopic compositions, has been shown to be a highly specific means of identifying the manufacturers and individual batches of products [1-5]. Both pharmaceutical manufacturers and regulatory agencies are interested in the technology as a forensic layer of product security.
However, FDA-DPAs Naproxen studies show that natural stable-isotopic ratios can also be measured to identify pharmaceutical material sources and determine the processes by which APIs are made (Figure 2). In 2002, J&J decided to test the techniques effectiveness in distinguishing between 53 samples of Topiramate that were made by three different synthetic pathways [6,7]. The initial goal was to determine whether the technique could be used as a tool in process patent infringement cases.
The observed isotopic results for the Topiramate samples can be grouped into a relatively small number of clusters consistent with synthetic fractionation. Three major isotopic-fractionating processes include:
- Synthetic reaction pathways (reaction mechanisms determining batch-to-batch isotopic variations via synthetic fractionation)
- Fractional crystallization
- Size fractionation (sieving, particle sorting).
Isotopic fractionation between light and heavy isotopes occurs when chemical reactions do not proceed to completion, or when multiple products are formed, and those isotopes are unevenly distributed among the reactants and products. In principle, the isotopic compositions of chemical products can be predicted from the isotopic compositions of the starting materials together with knowledge of the fractionations. However, fractionations can be quantitatively predicted only when complete mass balances are available and when the kinetic and equilibrium isotope effects associated with all relevant chemical reactions are accurately known .
Synthetic isotope fractionations are potential quantitative process monitors that could be used to integrate specific reaction variables that contribute to the isotopic composition of the synthetic product. In a given process for which the isotopic compositions of the reactants are known and the synthetic-isotopic fractionation has previously been determined, the isotopic composition of the product has a predictable isotopic value. If the observed value is not as predicted, then something in the synthetic process has varied. That may have been the reaction rate as modulated by factors such as pressure, temperature, reagent abundance, etc. Isotopic composition integrates such reaction variables and therefore can be used to monitor them.