Chemistry: analytical and immunological testing – Nuclear magnetic resonance – electron spin resonance or other...
Reexamination Certificate
2002-04-22
2004-11-09
Gakh, Yelena G. (Department: 1743)
Chemistry: analytical and immunological testing
Nuclear magnetic resonance, electron spin resonance or other...
C436S174000, C436S002000, C436S086000, C436S094000, C436S095000
Reexamination Certificate
active
06815213
ABSTRACT:
The present invention relates to a process for the analysis of a sample of a complex molecule relative to a reference batch of the same complex molecule, so as particularly to determine their degree of similarity and/or the characterization of their process of production.
Counterfeiting complex products has become a veritable scourge, in particular in the fine chemical, cosmetic and pharmaceutical industries. The detection of counterfeiting of complex products by physico-chemical analysis is often based on the analysis of traces of secondary products of the synthesis, of catalysts or impurities. For example, it has been determined by chromatographic analysis in liquid phase (Asakawa, Shuichi; Kato, Koichi; Inoma, SusumuHakodate Customs Laboratory, Hakodate-shi, 040, Japan, Kanzei Chuo Bunsekishoho (1997), 36, 37-43) that certain glyphosphate base herbicides, produced in the United States and imported into Japan, infringe Japanese patents. By also using a gas phase chromatographic technique coupled with mass spectrometry, E. Charton, M. Wierer, J. M. Spieser, A. Van Dorsselaer, and G. Rautmann (European Department for the Quality of Medicines, Council of Europe, Strasbourg, F-67029, Pharm. Pharmacol. Commun. (1999), 5(1), 61-66) have been able to detect a counterfeit of a medication, somatropine, described in the European pharmacopea, which was in fact a product derived from somatropine of human origin. Conventional chemical methods have permitted proving that tablets of a narcotic substance, fenethylline, were prepared by copying a German patent (N. Al-Gharably and A. R. Al-Obaid, College of Pharmacy, Kind Saud University, Riyadh, 11451, Saudi Arabia, J. Forensic Sci. Soc. (1994), 34(3), 165-7). Similarly, counterfeiting of antibiotics of the &bgr;-lactam series have been studied by capillary electrophoresis, at the “National Forensic Chemistry Center” of the “U.S. Food and Drug Administration”, 1141 Central Parkway, Cincinnati, Ohio, 45202, USA and described in the Journal of Chromatography, A (1994), 674(1-2), 153-63.
These compositional methods are not always effective and they can lead to false positives. Moreover, they cannot be used in all cases because of the absence of characteristic tracers.
Another process for the authentication of the origin of a product constituted by a mixture of organic compounds, is described in French patent 2.673.291. This process comprises a separative analysis step for the product by gas phase chromatography, a step of transformation to CO
2
by combustion of the compounds of the product, followed by a step of analysis by isotopic mass spectrometry so as to measure the enrichment in C
13
of each compound of the mixture before choosing a compound to mark, particularly by modifying the enrichment in C
13
of this compound or by adding similar molecules whose richness in C
13
has first been increased or decreased. Enrichment by isotopic marking necessary for the authentication of the origin of a product is a major drawback of this process. Thus, this step requires the manufacturer to modify his industrial process to be able to mark and authenticate his products. This requirement is connected to the series of steps used in the analysis process, these steps being unable to obtain sufficiently detailed information as to the origin of the products to avoid a marking step of the product by enrichment.
There is also known, as is described in British Patent 2120007, an analysis process consisting in fragmenting a molecule by means of an electron beam in a mass spectrometer chamber with double focusing to obtain metastable ions analyzable by means of said mass spectrometer. However, in this process, the step of fragmentation does not permit obtaining molecular sub-entities, products perfectly stable and isolable, but rather metastable ions of a lifetime of the order of several fractions of a second. Moreover, the nature of the fragments as well as the molecular site where the cleavage is carried out by the electron beam of the mass spectrometer, are conditioned by the presence of the isotope to be determined. These two characteristics of this process distinguish it fundamentally from a process in which a mass spectrometer is used for the isotopic ratios.
Correspondingly, more powerful analytic techniques have been developed. Such is the case of the mass spectrometry of isotopic ratios (MSIR). Thus, it is possible to characterize the natural specific isotopic fractionation by Nuclear Magnetic Resonance Specific Natural Isotope Fractionation (NMR-SNIF method) by measuring the isotopic contents at several molecular sites (or even al the sites) of a molecule. However, this technique is at present used only for simple molecules that can be directly analyzed.
An object of the present invention is to provide a process for the analysis of complex molecules based on an original methodology using isotopic techniques in natural abundance.
Another object of the present invention is to provide a process for the analysis of complex molecules permitting differentiating a batch of complex molecules relative to another batch and establishing a posteriori the history of the process of production of such a complex molecule without having first modified the process for production of such a complex molecule.
To this end, the invention has for its object a process for the analysis of a sample of a complex molecule relative to a reference batch of the same complex molecule so as particularly to determine their degree of similarity and/or the characterization of their process of production, characterized in that the complex molecule is cleaved into at least two molecular sub-entities, in that, if necessary, at least one of the cleavage products is cleaved into at least two new molecular sub-entities and in that this cleavage operation is repeated on at least a portion of the cleavage products until there are obtained analyzable and isolable molecular sub-entities, and in that there is determined, as a function of the atomic sites of the cleavage products in question, by generally chemical cleavage reactions, the isotope or isotopes to be studied, in that there is established, for at least one portion of the cleavage products, their isotopic profile and in that the isotopic profile of the cleavage products is compared to the isotopic profile of the first materials already cataloged and taking part in the synthesis process of the reference complex molecule and/or in the isotopic profile of the cleavage products of the reference complex molecule subjected to the same cleavage reactions.
The performance of the above steps permits applying such a process to no matter what complex molecule, without having proceeded to marking, particularly by enrichment in isotopes of the complex molecule to be analyzed.
REFERENCES:
patent: 0 567 276 (1993-11-01), None
patent: 2 673 291 (1992-08-01), None
patent: 2 120 007 (1983-11-01), None
Martin et al. “A new method for the identification of the origin of natural products. Quantitative deuterium NMR at the natural abundance level applied to the characterization of anetholes”, J. Am. Chem. Soc., (1982), 104(9), 2658-9.*
Caer et al. “Determination of site-specific carbon isotope ratios at natural abundance by carbon-13 nuclear magnetic resonance spectroscopy”, Analytical Chemistry (1991), 63(20), 2306-13.*
Vallet et al. “Combination of mass spectrometry and site-specific NMR isotope analyses in the characterization of amino acids” Journal of Agricultural and Food Chemistry (1992), 40(1), 81-7.*
Hanneguelle et al. “Authentication of essential oils containing linalool and linalyl acetate by isotopic methods”, Journal of Agricultural and Food Chemistry (1992), 40(1), 81-7.*
Martin et al. “Application of 2H SNIF-NMR and 13C SIRA-MS Analyses to Maple Syrup: Detection of Added Sugars” J. Agric. Food Chem. (1996), 44(10), 3206-3213.*
Jamin et al. “Multi-element and multi-site isotopic analysis of nicotine from tobacco leaves”, Plant, Cell and Environment (1997 20, pp. 589-599.
Martin Gerard
Martin Gilles
Eurofins Scientific
Gakh Yelena G.
Young & Thompson
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