Verification of the authenticity of drugs by means of NMR relaxometry—Viagra® as an example
Graphical abstract
Introduction
One of the most commonly counterfeited drugs include phosphodiesterase type 5 (PDE-5) inhibitors. Viagra®, which contains sildenafil citrate as an active pharmaceutical ingredient, belongs to this group of drugs [1], [2]. It is estimated that approximately 6 million (that is about 60%) tablets containing derivatives of the PDE-5 inhibitors are purchased every year in Europe from spurious sources of distribution [3]. This carries serious health consequences due to interactions between PDE-5 inhibitors and other concomitant medication and dietary supplements, which can cause, among others, acute hypotension and hypoglycaemia and even lead to death [4], [5], [6], [7]. From this perspective it is of utmost importance to develop methods of identification and differential analysis of original and counterfeit drugs. This task is very difficult as counterfeit medicines often contain not only the known and legal active ingredients, but also their chemical derivatives. In the case of sildenafil the main difficulties are due the high number of possible isomers and their similar molar mass [8]. More than 50 chemical derivatives of sildenafil have been identified including dimethylosildenafil, homosildenafil, propoxyphenyl sildenafil, isobutyl sildenafil, thiodimethylsildenafil, propoxyphenyl sildenafil, etc. [9], [10]. These isomers have very similar UV spectra as well as almost the same retention times in chromatographic methods [9], [11], [12], [13], [14]. With the virtually infinite amount of derivatives of PDE-5 inhibitors, it is not possible to identify them using reference standards. Moreover there is an increasing number of modern forms of drugs containing derivatives of sildenafil: orodispersible tablets [15], orally soluble films [16], granules [17] or sublingual tablets [18] that may also be subject to counterfeiting. The new forms of drugs hinder the applicability of many methods traditionally used to identify counterfeit products. In consequence, at present there is no method allowing for reliable identification of counterfeit drugs containing PDE-5 inhibitors.
In this paper we explore the potential of 1H Nuclear Magnetic Resonance (NMR) relaxometry in this matter. NMR relaxometry is a unique experimental method probing mechanisms and characteristic time constants of dynamical processes in condensed matter, from simple liquids [19] via macromolecular systems [20] to solids [21], on the atomistic level. In contrary to NMR spectroscopy which is focused on molecular structure, NMR relaxometry probes molecular dynamics. This summarizes the concept of the work: as it has turned out that structure oriented methods do not prove to be sufficient for discriminating between original and counterfeit products with PDE-5 inhibitors, the next try is to attempt to reveal characteristic dynamical features of these materials. Standard NMR relaxation experiments are performed at a single frequency. Here the experiments are carried out in a frequency range encompassing four orders of magnitude, from about 5 kHz to 40 MHz (for 1H). This is possible by applying Fast Field Cycling (FFC) technique [21], [22] which has introduced a new dimension into relaxation studies − namely the possibility of varying the frequency (magnetic field). In consequence, by FFC NMR relaxometry one can detect motional processes across a huge range of time scales (from ms to ns) by single experiment. NMR relaxometry studies are non-destructive, which enables further analyses on the basis of the same material or it can be used by the relevant departments (police, court, custom). Moreover, there is no need either for special sample preparation (the whole form of the drug can be tested) or for any additional chemicals.
The paper is organized as follows. In Section 2 the principles of NMR relaxometry are presented and details of the experimental procedures and samples are given. Section 2.1 contains the basic mathematical formulae describing the relationship between the quantity measured in the NMR relaxation experiments (referred to as a spin-lattice relaxation rate) and dynamical parameters of the investigated material are provided. Then, in Section 3 the obtained 1H NMR relaxation results for original and counterfeit Viagra® are presented and quantitatively analyzed. Section 4 contains a discussion of the NMR results accompanied by FTIR (Fourier Transform Infra-red) spectra and AFM (Atomic Force Microscopy) pictures of the original and counterfeit products for comparison. Eventually in Section 5 the main conclusions of this work are summarized.
Section snippets
Materials and methods
The original Viagra® tablets produced by Pfizer, counterfeit drug and pure sildenafil citrate (active pharmaceutical ingredient of Viagra®) were compared. The original drug containing 100 mg of sildenafil citrate was purchased in a pharmacy in Poland. The counterfeit drug was purchased on the black market from a seller, who advertised it on the Internet. By pure sildenafil citrate we understand the compound, which is the European Pharmacopoeia (EP) Reference Standard. Sildenafil citrate was
Results
1H spin-lattice relaxation dispersion profiles for original and counterfeit Viagra® have been collected in the frequency range spanning four orders of magnitude: from 5 kHz up to 40 MHz (referring to 1H resonance frequency). The measurements were performed at room temperature, 330 K. Essential differences between relaxation properties of those compounds have been observed.
Fig. 1 shows time evolution of 1H magnetization (magnetization curves, ) recorded for original Viagra® at selected
Discussion
In this work 1H spin-lattice NMR relaxation studies have been performed in a frequency range from 4 kHz to 40 MHz to reveal differences in the relaxation, and hence dynamical, scenario of original and counterfeit Viagra®. The first observation is that for the counterfeit product the relaxation is clearly bi-exponential, while for original Viagra® the relaxation process is single-exponential. This effect is observed in the whole frequency range covering four orders of magnitude. This means that
Conclusions
1H spin-lattice relaxation studies have been performed in order to differentiate between original Viagra® and a counterfeit product. The relaxation experiments have been carried out in a frequency range from 4 kHz to 40 MHz. In the case of the counterfeit product the relaxation process is bi-exponential (has two components) in the whole frequency range, while for the original Viagra® the relaxation is always single exponential. Already this qualitative observation allows to distinguish between
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