The story of the pharmaceutical spin doctors

Nobel prizes, the ability to ‘see’ the 3-D structure of compounds and a big headache for the drug counterfeiters – we trace how NMR has given rich pickings to the pharmaceutical industry

IN 1952 the Nobel Prize for Physics was awarded jointly to Felix Bloch and Edward Purcell for their independent but complementary work on nuclear magnetic resonance (NMR). Exactly fifty years on, in 2002, this time it was the Nobel Prize for Chemistry that was shared, with Kurt Wüthrich achieving his award for the application of nuclear magnetic resonance spectroscopy to the elucidation of three-dimensional structure and dynamics of protein molecules.

The progress made in the fifty years between the two Nobel Prizes is a stunning illustration of the way that NMR spectroscopy has progressed to become one of the most powerful analytical and research tools for the modern chemist. Elegant solutions have been found which largely overcome the problem of NMR being the least sensitive of the commonly used spectroscopic techniques. Nowadays, many research papers cite th

However it is not just in the field of pharmaceutical research that NMR is important. As a routine analytical tool, it has several applications that set it apart from other chemical techniques and allow chemists to get results of commercial importance more quickly and more simply than other techniques.
NMR excels in the identification of unknown compounds such as metabolites or drug degradation products. It is also used for impurity profiling or determination of the drug's optical purity, and increasingly, has a crucial role to play in the detection and identification of the growing problem of counterfeit drug products.

It’s worth remembering that in one sense, NMR is extremely limited. At a very basic level of understanding, it relies on the fact that all subatomic particles (electrons, protons and neutrons) have the property of spin. In many atoms (such as 12C) these spins are paired against each other, so that the nucleus of the atom has no overall spin. NMR is “blind” to such atoms. However, in some atoms (such as 1H and 13C) the nucleus does possess an overall spin, and it is the nuclei with spin, whether half-integer (i.e. 1/2, 3/2, 5/2) or integer (i.e. 1, 2, 3) that can be detected by NMR.
Fortunately, pharmaceuticals are composed of relatively few chemical elements (largely hydrogen, carbon, oxygen, nitrogen, and phosphorus, as well as the halogens and sometimes metals), and most of these have one or more isotopes that may be detected by NMR experiments. However, due to low natural abundance and/or low relative sensitivity, the focus is usually on hydrogen (1H), carbon (13C), fluorine (19F) and phosphorus (31P).

The most important role NMR plays in pharmaceutical analysis is its use in elucidating and/or confirming the structures of drug-related substances. However, NMR is also used to study drug impurities and contaminants including solvents, synthetic precursors, synthetic intermediates, and decomposition products. In the case of natural products NMR may be used to determine the identity of co-extractives. It also has a role to play in the study of drug metabolism where it has been used for identification and quantification of many metabolites.

Crucially, the interactions that occur between nuclei within molecules does not apply between molecules. This opens up the potential to use NMR on mixtures as well as on pure compounds meaning NMR can be used as a rapid screening method, and a useful tool in the fight against counterfeit pharmaceuticals.

According to the European Commission, seizures of counterfeit pharmaceuticals in Europe rose five-fold in 2006 compared with 2005, with a huge proportion of the counterfeits emerging from the rapidly expanding, but less regulated, economies of India and China. Indeed, the EC claims that India, the United Arab Emirates and China account for over 80% of the fake medicines entering Europe.
Counterfeit products can take many forms. The drug product itself may be copied, with the most sophisticated counterfeits actually containing the correct active ingredients, though not always in the correct amounts. The crudest copies contain no active ingredients of any kind, and are often made up of the simplest fillers. The most dangerous might contain toxic chemicals, though by definition, any counterfeit is dangerous since it will not provide the medical benefit it purports to provide.
Similarly, packaging might be copied, and it is not unheard of for a genuine product to be withdrawn, supposedly for disposal, only to be re-packaged in fake packaging and sold elsewhere. Likewise, documentation can be counterfeit, and it is well within the capabilities of the determined counterfeiter to attempt to copy or fake any part of the product or its associated paperwork.

NMR can be very useful in profiling a suspect product, and comparing it against the genuine item. For the manufacturer whose products have been targeted, it is always worthwhile to understand the nature of the counterfeit product, even if it is already very easy to tell that it is fake. Whilst the poorest copies can be detected by eye, sometimes microscopic techniques are needed, perhaps to study parameters such as the quality of intagliation on a tablet, the thickness of the card used for packaging, the printing on the packaging, or the type of material used as a filler. More powerful analytical techniques can be allied to the microscopes if necessary, perhaps to look at the elemental composition of coatings, or the microstructure of powders and these techniques can be relevant to more detailed investigation of the counterfeit, as well as confirming that counterfeiting has taken place.

Where NMR comes into its own is as a relatively simple, and non-destructive way of revealing where no active is present, or where a different active has been used. In the latter case, perhaps following separation, the NMR spectrum gives the experienced chemist an immediate handle on the structure of the ‘rogue’ active. Indeed, NMR is one of the most powerful techniques for the identification of unknown compounds, such as the rogue actives used in counterfeits.

Identification of an unknown contaminant usually begins with creating a high-resolution H-NMR spectrum. The chemical shifts and integration reveal the relative number of protons (aliphatic, olefinic, aromatic) and the coupling patterns may suggest their relative proximity. Next, C-NMR and DEPT (distortionless enhancement by polarization transfer) spectra are used to obtain the number of different carbons and the numbers of protons attached to each. Often, the combination of proton and carbon data is enough to solve the structure of the unknown. If not, further NMR experiments can be used to establish the connectivity and proximity of atoms in the molecule, and hence define structure. Should NMR “invisible” nuclei (such as chlorine or oxygen) be present, MS and/or IR data may be needed in order to complete the structure, though the locations of such heteroatoms can not infrequently be deduced from the NMR chemical shifts of the carbons (and associated hydrogens) to which they are attached.

Of course, unknown chemicals can also arise in genuine products as impurities. When a 1H-NMR spectrum is taken for a drug, additional peaks will indicate the presence of impurities and careful examination will often permit identification. The percentage of each impurity present can be deduced by integration, or comparison of relative peak heights. Provided each impurity gives unique resonance peaks, large numbers of impurities may be quantified from a single spectrum.

Where the impurity(ies) cannot be identified immediately, it is often possible to separate it (them) from the drug and then perform the required NMR techniques to identify the material. Alternatively, if an HPLC method exists for separating the impurity, the eluted peak may be collected and analysed by NMR.

For a number of drugs the presence or absence of optical isomers can be of critical importance. NMR provides a means for determining the optical purity for many of these products. Many of the early studies were done on lower-field-strength instruments, using chiral lanthanide shift reagents CLSRs, and the limit of quantification was typically 1–5%. Later studies, using higher-field-strength instruments and chiral shift reagents provided much greater sensitivity, with detection limits in some cases of less than 0.1%. These kinds of results make NMR a very attractive method for determining if pharmaceutical products meet the requirements set by pharmacopoeias of many countries.

NMR already plays a major role in pharmaceutical analysis and as with many other technologies, as sensitivities increase and new methods are developed, the possibilities seem set to increase. There is no doubt, for example, that NMR will be of considerable benefit to the newer biotech pharmaceuticals in exploring the structure and interactions of new drug products. In analysing traditional drug products, NMR, especially in conjunction with other analytical techniques such as MS and HPLC, will continue to play its part in ensuring the safety and purity of existing supplies, and in investigating cases of suspected counterfeit.
 

By Dr John Sheridan & Dr Ellen Norman of Reading Scientific Services.