New NMR Tools for Impurity Analysis was written by Jane Power and submitted for the degree of Doctor of Philosophy in the Faculty of Engineering and Physical Sciences at the University of Manchester, on 31st March 2016.NMR spectroscopy is rich in structural information and is a widely used technique for structure elucidation and characterization of organic molecules; however, for impurity analysis it is not generally the tool of choice. While 1H NMR is quite sensitive, due to its narrow chemical shift range (0 - 10 ppm) and the high abundance of hydrogen atoms in most drugs, its resolution is often poor, with much signal overlap. Therefore, impurity signals, especially for chemically cognate species, are frequently obscured. 19F NMR on the other hand offers extremely high resolution for pharmaceutical applications. It exhibits far wider chemical shift ranges (± 300 ppm) than 1H NMR, and typical fluorinated drugs, of which there are many on the market, have only one or two fluorine atoms. In view of this, 19F NMR is being considered as an alternative for low-level impurity analysis and quantification, using a chosen example drug, rosuvastatin. Before 19F NMR can be effectively used for such analysis, the significant technical problem of pulse imperfections, such as sensitivity to B1 inhomogeneity and resonance-offset effects, has to be overcome. At present, due to the limited power of the radiofrequency amplifiers, only a fraction of the very wide frequency ranges encountered with nuclei such as fluorine can be excited uniformly at any one time. In this thesis, some of the limitations imposed by pulse imperfections are addressed and overcome. Two new pulse sequences are developed and presented, CHORUS and CHORUS Oneshot, which use tailored, ultra-broadband swept-frequency chirp pulses to achieve uniform constant amplitude and constant phase excitation and refocusing over very wide bandwidths (approximately 250 kHz), with no undue B1 sensitivity and no significant loss in sensitivity. CHORUS, for use in quantitative NMR, is demonstrated to give accuracies better than 0.1%. CHORUS Oneshot, a diffusion-ordered spectroscopic technique, exploits the exquisite sensitivity of the 19F chemical shift to its local environment, giving excellent resolution, which allows for accurate discrimination between diffusion coefficients with high dynamic range and over very wide bandwidths. Sulfur hexafluoride (SF6) is investigated and shown to be a suitable reference material for use in 19F NMR. The bandshape of the fluorine signal and its satellites is simple, without complex splitting patterns, and therefore good for reference deconvolution; in addition, it is sufficiently soluble in the solvent of choice, DMSO-d6.To demonstrate the functionality of the CHORUS sequences for low-level impurity analysis, 470 MHz 1H decoupled 19F spectra were acquired on a 500 MHz Bruker system, using a degraded sample of rosuvastatin, to reveal two low-level impurities. Using a standard Varian probe with a single high frequency channel, simultaneous 1H irradiation and 19F acquisition was made possible by time-sharing. Simultaneous 19F{1H} and 19F{13C} double decoupling was then performed using degraded and fresh samples of rosuvastatin, to reveal three low-level impurities (in the degraded sample) and low-level 1H and 13C modulation artefacts.

This thesis is focussed on the investigation of matrices for matrix-assisted diffusion-ordered spectroscopy (MAD). Diffusion-ordered spectroscopy (DOSY) is a family of experiments where the resonances in the chemical shift dimension are further dispersed in an extra dimension according to diffusion coefficient. A typical DOSY spectrum shows one single diffusion coefficient for all the resonances coming from one single species. However, If two or more resonances overlap, the diffusion resolution of the DOSY spectrum is compromised and a spurious diffusion coefficient results, intermediate between the species. In case of signal overlap, the use of more advanced processing methods aids to separate two analytes that differ by at least 30% in diffusion coefficient. In practice, many mixtures contain species of similar diffusion coefficients whose resonances overlap in the chemical shift dimension. The addition of co-solutes can modify the chemical environment (matrix), with which different analytes interact to different extents, and enhance the diffusion resolution of DOSY. However, the addition of co-solutes can risk the benefits of DOSY by increasing the probability of signal overlap. Signal overlap in MAD is avoided by using a 1H NMR-invisible surfactant such as sodium perfluorooctanoate (NaPFO), which has replaced each proton by a fluorine atom. PFO micelles are a tunable matrix which allows the separation of analytes via coulombic interactions by adjusting the pH. Differences in diffusion coefficient in NaPFO solution can be analysed using a modified Lindman's law to model the diffusion coefficient as a function of pH. The model rationalises the binding constants of analytes to PFO micelles with good accuracy, subject to the spectral data quality. Another alternative to resolve diffusion coefficients using the invisible MAD approach is by means of a commercially available alkyl surfactant like cetyltrimethylammonium bromide (CTAB). CTAB in high ionic strength solution forms worm-like micelles whose resonances can be filtered out from the final DOSY spectrum. CTAB worm-like micelles have short transverse relaxation times compared to all of the analytes in the mixture. If a transverse relaxation filter is positioned at the beginning of a standard DOSY pulse sequence, as in PROJECT-Oneshot, the strong CTAB signals vanish and leave behind only the analyte resonances and hence avoid signal overlap. Finally, the use of bovine serum albumin (BSA) as a potential invisible matrix, using a similar approach to CTAB worm-like micelles is investigated, using a relaxation-weighted DOSY pulse sequence to suppress most of the BSA background signal (at a cost in analyte signal to noise ratio). An alternative to suppress most of the BSA background and preserve most of the analyte signal is by means of mild transverse relaxation filtration and spectral editing to obtain an edited DOSY spectrum that shows only the analyte signals. Nonetheless, it is a shame that useful MAD results can only be obtained under a narrow set of conditions: i) different mole ratios BSA: analyte to aid diffusion resolution, ii) mild T2 filtration to improve analyte signal to noise ratio and iii) spectral editing to remove residual BSA background.