NMR Spectroscopy Used for Quality Assurance by Pharmaceutical and Biotechnology Manufacturers

Billy Cook

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Introduction Nuclear Magnetic Resonance spectroscopy, abbreviated as NMR, is an instrumentation method which measures the absorption of electromagnetic radiation between 4 and 900 MHz, which corresponds to the radio-frequency range. It is one of the most powerful tools in modern science a chemist can use to determine the structure of a molecule. While spectroscopies that make use of the ultraviolet, visible, and infrared frequencies measure the absorption by the outer electrons, NMR measures the absorption by the nuclei. Nuclear magnetic resonance spectroscopy also requires a strong magnetic field to be present around the analyte in order for the nuclei to obtain the energy states needed for absorption. In 1924, the theory that certain nuclei may possess properties of spin and of a magnetic moment was postulated by Wolfgang Pauli. Because of these properties, a magnetic field would cause a splitting of the nucleis energy levels. Through experimentation, these theories were proven over the subsequent decade but it was not until 1946 that two independently working researchers, Felix Bloch of Stanford and Edward Purcell of Harvard, showed that the splitting of energy levels allows the nuclei to absorb electromagnetic radiation (1).

Instrumentation

Billy Cook, 3 Nuclear magnetic resonance makes use of the magnetic properties of certain nuclei within the sample, most often the 13C and 1H nuclei. In general, an NMR spectrometer creates an intense magnetic field which causes the nuclei to align either with or against the magnetic field. The nuclei are then exposed to radio waves which cause a shift in alignment of the nuclei. This shift gives off energy which is then detected by the instrument and can be represented by a graph commonly using either absorption or intensity on the vertical axis and magnetic field, frequency, or chemical shift on the horizontal axis. Other than the ability to easily determine the structure of the sample, a major benefit of NMR spectroscopy is the fact that the sample is not destroyed in the process. Two main types of nuclear magnetic resonance spectrometers are marketed by manufacturers. The first type, wide-line spectrometers, uses magnets with relatively low strengths of a few tenths of a tesla and is much simpler, and thus cheaper, than the second type, high-resolution spectrometers, which utilizes much stronger magnets with a strength range of 1.4-23 teslas. Originally all high-resolution NMR spectrometers used a permanent magnet or electromagnet to create the magnetic field and thus were considered continuous wave. The experiments performed independently by Bloch and Purcell, for instance, were carried out using continuous wave spectrometers. Continuous wave spectrometers have, however, mostly been replaced by Fourier transform spectrometers since their debut in the 70s. Instead of using a permanent magnet or electromagnet, Fourier transform spectrometers use superconducting magnets in order to create the required magnetic field. A major benefit of using Fourier transform spectrometers lies in their ability to provide efficient signal averaging which gives much greater sensitivity.

Billy Cook, 4 A Fourier transform spectrometer, or FT-NMR spectrometer, is composed of a central, highly-stable magnet within which a sample surrounded by a transmitter-receiver coil is placed. Radiation of a frequency in the radio region is emitted by a crystal-controlled frequency synthesizer. The emission reaches a pulse-switch and power amplifier, which intensifies the emission and transforms it into a reproducible pulse of radio frequency current in the transmitterreceiver coil. The radiation then reaches and interacts with the sample. The pulses attributes, which include the length, amplitude, shape, and phase, are able to be selected by the operator who then inputs them into the console which is controlled by the computer. A free induction decay, or FID, signal is then received by the transmitter-receiver coil. The signal then undergoes amplification and is transmitted to a phase-sensitive detector, which produces a difference between the nuclear signals and the crystal oscillator output. A low-frequency, time-domain signal is then produced which is digitized by and stored in the computer. This signal then is analyzed by data analysis programs including one that performs a Fourier transform. The output data from the Fourier transform program is then represented on some display as a frequencydomain spectrum. Computers also have largely become very important to modern NMR spectroscopy. Their ability to take in the signal, compute the Fourier transformation of the FID, and perform other treatments of the obtained data has proved to be extremely useful (1).

Theory Nuclear magnetic resonance spectroscopys usefulness as an analytically tool is only rivaled by its complexity. While the IR, Vis, and UV spectroscopies examine the interactions

Billy Cook, 5 between electrons and radiated energy, NMR spectroscopy examines the interactions between the nuclei and radiated energy. In many atoms the spins of the subatomic particles pair against each other to cause the nucleus to have no overall spin. However, some atoms have nuclei that do have an overall spin due to the different spins of their subatomic particles. As a rule, if the number of neutrons and protons both are even, the nucleus will have no spin. Conversely, if the number of neutrons and protons added together is odd, the nucleus has a half spin. If the number of neutrons and protons added together is even, the nucleus has a full spin. With the spin being denoted as I, quantum mechanics shows that the nucleus will have 2I+1 possible orientations, thus a nucleus with a spin of would have 2 possible orientations. In the absence of a magnetic field the energies of the orientations are equal but if a magnetic field has been applied, the energy levels of the orientations split (2). The Boltzmann distribution states that the population of nuclei in each energy level is determined by thermodynamics. This means that the lower energy level will contain slightly more nuclei than the higher level. With electromagnetic radiation, the nuclei in the lower energy state can be excited into the higher energy state. The difference in energy of the two states dictates what frequency of radiation is needed for this excitation to occur. Since the nucleus possesses a positive charge and has a spin, a small magnetic field is generated by the nucleus. This property causes the nucleus to possess a magnetic moment which is directly proportional to its spin. In comparison, the energy of an energy level is directly proportional to the nucleus magnetic moment and the strength of the magnetic field generated by the nucleus. This means that if the magnetic field is increased, the energy of the energy state is as well.

Billy Cook, 6 When a nucleus is in a lower level of energy, its magnetic moment is not in opposition with the applied field. This means that as the nucleus spins on its axis in the presence of an applied magnetic field, the nucleus axis of rotation will precess around the magnetic field. The frequency of this precession is known as the Larmor frequency and is equal to the transition frequency. The potential energy of the precessing nucleus is directly proportional to the cosine of the angle between the applied magnetic field and the axis of nuclear rotation, i.e. the angle of precession. If the nucleus absorbs energy, the angle of precession will change. For example, for a nucleus with a spin of , absorbed radiation would flip the nucleus magnetic moment so that it would oppose the applied magnetic field and excite it into the higher energy state. If a sample has a small proportion of nuclei in the lower energy state and thus can absorb emitted radiation, there is a possibility that the populations of nuclei in the higher and lower energy levels will become equal. If the populations become equal, no further absorption of radiation will occur and the spin system will become saturated. Relaxation processes help a system out of saturation by allowing nuclei to return to the lower energy levels. Although thermodynamics states that it would be more stable for the nuclei in the higher energy state to undergo relaxation into the lower energy state, the probability of re-emission of the electromagnetic radiation varies and other ways of relaxation need to be explored. Two relaxation processes, spin-lattice relaxation and spin-spin relaxation, are the most significant concerning nuclei returning to a lower state. Spin-lattice relaxation involves the interaction between the lattice field of the sample and the nuclei in the higher energy states. The sample which holds the nuclei to be observed in an NMR spectroscopy experiment is called the lattice. A magnetic field is generated through the vibrational and rotational motions of the nuclei in the lattice, which is known as the lattice field.

Billy Cook, 7 The field lattice has many components, some of which are equal in frequency and phase to the Larmor frequency of the particular nuclei. Said components of the lattice field have the ability to interact with the nuclei in the higher energy level whereby they can cause them to lose energy and relax to the lower energy level. As the nuclei loses energy, the amount of rotation and vibration in the lattice increases. The relaxation times of the nuclei are dependent on the mobility of the lattice and as the mobility increases, the rotational and vibrational frequencies increase as well. This increases the probability for a component of the lattice field to interact with excited nuclei, thus increases the probability for the relaxation of the nuclei. Spin-spin relaxation involves the interaction between adjacent nuclei with agreeing precessional frequencies and differing magnetic quantum states. During this form of relaxation, the nuclei with differing quantum states exchange energies. This causes the nucleus in the lower energy state to become excited into the higher energy state and the nucleus in the higher energy state to be relaxed into the lower energy state. Although no net change in the populations of the energy states, the average time the nuclei will stay in an excited state is decreased. This may cause the relaxation rate to become too quick which could result in line broadening. Differences may occur in the absorption frequency of the nucleus being examined. This effect is called chemical shift, measured in parts-per-million (ppm), and is dependent upon the group to which the nucleus is bonded. Chemical shifts occur due to a secondary magnetic field caused by the circulation of electrons. These secondary fields may decrease or increase the field that a nucleus may respond. In the presence of an applied magnetic field, electrons that are bonded to the nucleus may precess around the nucleus in a plane that is perpendicular to the applied magnetic field. This

Billy Cook, 8 creates a secondary field that opposes the applied magnetic field which then causes the nucleus to experience an overall smaller field, thus shielding the nucleus from the influence of the applied field (3). Thus the strength of the external field must be increased in order for nuclear resonance to occur. Shielding of the nucleus is directly related to the surrounding electron density, therefore it would be expected that the shielding of the nucleus would increase with increased electronegativity of its neighboring groups. The level of shielding can therefore be used to determine the structure of a compound as less-shielded nuclei appear in the lower field and the higher-shielded nuclei appear in the higher field of an NMR spectrum (4).
1

H-NMR has the benefit of providing further evidence towards the structure of a

compound (6). On a typical 1H-NMR spectrum, the different absorption bands tend to be wide and have a bell curve-like shape to them. However, if the same spectrum is obtained by using a higher resolution the different absorption bands may show to actually be multiplet separated into any integer of sub-bands, i.e. component peaks of the multiplet. Component peaks of a multiplet will have the same spacing, in hertz, in between them no matter the number of peaks or the location of the multiplet on the spectral field. This spacing is known as the coupling constant. The ratio of the areas under the peaks can be described as the nth row of Pascals triangle, e.g. a quartet would have the ratio of 1:3:3:1. Since nuclei possess small magnetic fields, they tend to have an effect on each other and change the energy, and therefore frequency, of nearby nuclei as they resonate. This phenomenon is known as spin-spin coupling which, in NMR, the most important type is scalar coupling. Scalar coupling is the interaction between two nuclei which occurs through the chemical bonds and can be felt up to a distance of three bonds (4). As an example, if a proton has a singlet signal at a chemical shift of 1 ppm and is in a molecule with another proton that is a distance of three

Billy Cook, 9 bonds away, the adjacent group will cause the signal at 1 ppm to split into two. One peak of the newly formed doublet would be a number of hertz higher than the original signal of 1 ppm while the other peak would be the same number of hertz lower than the original signal. Each peak would then inherit half the area of the original singlet peak. The amount of splitting, i.e. the difference in frequency between the peaks, would have a constant magnitude across the spectrum which is known as a coupling constant. Coupling constants are independent of the strength of an external magnetic field being that they are the result of the presence of magnetic fields belonging to other nuclei instead of the external magnetic field itself. The units of the coupling constant, therefore, are hertz instead of ppm.

Applications The applications of nuclear magnetic resonance are vast, some of which include the detection of impurities for the quality control of drugs and uncovering counterfeits (5). Potential sources for impurities may be leftover starting materials or intermediates, by-products, degradation products, and many others (2). Some impurities may be deleterious to the drugs therapeutic efficacy while others may have a similar effect or no effect at all. Either way, a standard should be maintained and impurities must be controlled regardless of their effect on the therapeutic effect. The limit allowed for small-molecule drugs is 0.1% impurity and drugs often contain a percentage higher than this (3). All impurities in excess of this level ought to be detected in order to help avoid their formation or develop a more sufficient purification method in order to decrease their quantity to a tolerable level. Once an unavoidable impuritys structural

Billy Cook, 10 information has been determined through NMR, the impurity can be synthesized in order to obtain an impurity standard for use in toxicology studies. The counterfeiting of drugs is also a major problem which nuclear magnetic resonance spectroscopy can alleviate. Although counterfeiting of other goods such as clothes, jewelry, and others may pose no danger, the counterfeiting of drugs can be seriously dangerous and even lifethreatening. Even though methods to ensuring quality have been employed in order to detect them, sub-standard active pharmaceutical ingredients are found more often than not in the last few years. In 2009 the World Health Organization estimated doses of glibenclamide in China contained six times the normal dose, and in Tanzania an antimalarial drug was discovered to have less than the sufficient amount of active ingredients. In one study, NMR was compared to attenuated total reflection-infrared spectroscopy (ATR-IR) and Raman spectroscopy in its ability to assess the levels of impurity in heparin samples. This study was brought on by a widespread occurrence in 2008 of pharmaceutical grade heparin that was contaminated with oversulfated chrondroitin sulfate (OSCS). In the study a number of samples of heparin from the 2008 market, along with pure heparin sodium intentionally contaminated with known amounts of OSCS and dermatan sulfate (DS), were analyzed using the previously listed techniques in order to evaluate them. The study showed that each technique was very able to detect the levels of impurities, but the detection of them would most likely depend on the experience and expertise of the potential investigator. However, evaluation of the different recorded spectra by statistical classification techniques worked best with the NMR spectra. While this does not make NMR the superior technique, it does show it has some usefulness beyond other techniques in the detection of impurities (10).

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Critical Evaluation Nuclear magnetic resonance spectroscopy can be used for a wide array of purposes. It can provide a great amount of detailed information on selected molecules. Perhaps the most useful purpose is in a research lab setting where a chemist can verify the structure of a product or test the quality of the product. The latter may be of more important however, as it can be applied to pharmaceutical substances. If a substance intended for medical use is contaminated with impurities, the results could be hazardous, if not life-threatening. While some impurities may add to the drugs effectiveness or cause no change at all, others may decrease the drugs effectiveness or worse, cause harm to the individual. Either way NMR spectroscopy is a technique very capable of detecting impurities within a product and is being used as such. Over the past few years, there has been much development on the technique and the level of experience has grown significantly. With this, the technique improves every day in its ability to provide a broad range of information on a sample NMR spectroscopy can be an extremely powerful tool by which a chemist is able to easily determine the structure of a sample or detect impurities within a product. With experience the spectra obtained through NMR spectroscopy is fairly easy to read and with experimentally proven spectra of known compounds for comparison at a chemists disposal, reading spectra can become nearly effortless. Results from this form of spectroscopy provide a broad range of information and results can be obtained on solutions as well as solids (9). Although the technique is not as sensitive as other forms of spectroscopy and thus a larger amount of sample is required for proper analysis, NMR spectroscopy does not destroy the sample in the process. The only

Billy Cook, 12 limitations of the technique are in the probability of line broadening making the results hard to interpret and no information is given on the molecular weight (although the number of different nuclei is provided). Overall, nuclear magnetic resonance spectroscopy is very useful in providing information on the structure along with other aspects of molecules as well as detecting the presence of other compounds within what should be a pure sample. Development of the technique increases with each day, with which comes the ability to further regulate the quality of drugs and a fundamental understanding of the nature of molecules.

References 1. Skoog, D. A.; Holler, F. J.; Crouch, S. R.; Principle of Instrumental Analysis, 6th Edition, David Harris: Belmont, CA, 2007; 498-522 2. Rodrigues, J.E.A.; Erny, G.L.; Barros, A.S.; Quantification of organic acids in beer by NMR. Analytica Chimica Acta [Online] 2010, 674, 166-175 3. Kubicki, D; Gryff-Keller, A; Szczecinski, P; A combined DFT NMR study of cyclic 1,2diones and methyl ethers of their enols. Journal of Molecular Structure [Online] 2012, 1021, 95-101

Billy Cook, 13 4. Holzgrabe, U.; Malet-Martino, M.; Analytical challenges in drug counterfeiting and falsification- The NMR approach. Journal of Pharmaceutical and Biomedical Analysis [Online] 2011, 55, 679-687 5. Winter, W.; Deubner, R; Holzgrabe, U.; Multivariate analysis of nuclear magnetic resonance data characterization of critical drug substance quality of gentamicin sulfate. Journal of Pharmaceutical and Biomedical Analysis [Online] 2005, 38 ,833-839 6. Podgorskii, V. V.; Mikhalev, A. S.; Kalabin, G. A.; Structure of chemical compounds, methods of analysis and process control. Pharmaceutical Chemistry Journal [Online] 2011, 45, 52-55 7. Parmar, K. D.; Patel, N. M.; Patel, P. M.; A Review: Impurity Profile Study. Journal of Pharmacy Research [Online] 2011, 2566-2569 8. Sherikar, O.D.; Mehta, P.J.; Khatri, D. M.; Various approaches for impurity profiling of pharmaceuticals. Journal of Pharmacy Research [Online] 2011, 1937-1942 9. Heyman, H.M.; Meyer, J.J.M.; NMR-based metabolomics as a quality control tool for herbal products. South African Journal of Botany [Online] 2012, 82, 21-32 10. Keire, D. A.; Ye, H.; Trehy, M.L.; Characterization of currently marketed heparin products. Anal Bioanal Chem [Online] 2011, 399, 581-591