Process Patent Protection: Characterizing Synthetic Pathways by Stable-Isotopic Measurements - Pharmaceutical Technology

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Process Patent Protection: Characterizing Synthetic Pathways by Stable-Isotopic Measurements
The authors describe the methods by which precise analyses of stable-isotopic abundances can be used in security and forensic applications for pharmaceutical materials. These methods include product and process authentication of raw materials, pharmaceutical intermediates, drug substances, formulated drug products, and synthetic pathways. Collectively, these methods can be used to investigate and mitigate patent infringement. In the future, more complete examples will be presented containing full isotopic..


Pharmaceutical Technology


The precision of isotopic analyses is typically calculated by two methods. Pooled standard deviations of raw data are typically computed from sets of duplicate or triplicate measurements (12). From those pooled standard deviations, standard deviations of mean values pertaining to specific substances are calculated. More specifically, the standard deviation of a mean value is the pooled standard deviation divided by n , in which n is the number of measurements performed on a given sample (13). For carbon, nitrogen, oxygen, and sulfur, the resulting 95% confidence intervals for a result are typically in the range of 0.1 to 0.4‰. For hydrogen, the 95% confidence interval is typically 3‰.

Discussion

The precise quantitation of stable-isotopic compositions in pharmaceutical intermediates and products requires both mass balance and isotopic fractionation equations that are applicable to both single and multistep reaction sequences. We start from the most basic requirement of mass balance and then consider isotopic fractionations in a single reaction. Finally, we discuss applications to the protection of process patents.

Mass balance. For A + B → C, in which reactants A and B are quantitatively converted to product C, two mass balances can be written:










in which, m A, m B, and m C are molar amounts of carbon (or any other element) in A, B, and C and the isotopic compositions of that carbon (or any other element) in A, B, and C are given by δA, δB, and δC. Equation 1 is a mass balance (i.e., carbon in = carbon out) whereas Equation 2 is an isotopic mass balance (13C in = 13C out). Under the conditions postulated (quantitative conversion), the isotopic composition of C can be computed from those of A and B (10, 11).

Isotopic fractionation. For isotopic fractionations, calculations must take into account factors such as reaction completeness and isotope effects. These effects will cause the isotopic composition of C to differ from that computed using the mass balance equation and assuming quantitative conversion of reactants to products.

To provide a concrete example, assume that A is present in excess whereas B, the limiting reactant, is quantitatively converted to product. In that case:










in which n A, n B, and n C represent the numbers of carbon atoms (or any other element of interest) in A, B, and C. Because A is not quantitatively converted to product, the isotopic compositions of the A-derived positions in C can differ from those in the initial reactant. In this case, the isotopic offset is expressed as ΔA. As Figure 1 shows, its value depends on the isotope effects and on the fraction of A that remains unconsumed. If the reaction conditions, particularly the magnitude of the excess of A, are consistent, ΔA will be constant. Because the n values are known exactly, ΔA can be determined from Equation 3 after isotopic analysis of the reactants and product (i.e., determination of δA, δB, and δC).

Values of δA, δB, and δC do not affect the values of ΔA. Accordingly, once ΔA is known for a given reaction and a set of conditions, it is necessary only to know two of the δ values to compute the third. Thus, for example, when ΔA, δA, and δB, are known, the isotopic value of the product (δC) can be calculated.

If neither A nor B is consumed completely during the course of the reaction, and if the rate of the chemical reaction (or position of the chemical equilibrium) is sensitive to isotopic substitution on both reactants, it is necessary to consider values of ΔA and ΔB:










If reaction conditions cannot be manipulated so that f A and f B (and thus ΔA and ΔB) can be independently driven to completion (i.e., zero), it will be possible to determine only the sum, n AΔA + n BΔB. From theoretical considerations (14), ΔA and ΔB can be evaluated separately for all values of f A and f B if the isotope effects are known.

Of course, isotopic fractionations such as those previously described accumulate during the different steps of a multi-step synthesis scheme. They can, however, be individually and systematically differentiated, not only for multiple reactants but also for multiple isotopes. To provide an example, consider carbon-isotopic fractionations in a hypothetical four-step sequence:











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