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13C satellite-free 1H NMR spectra Pinelopi Moutzouri, Peter Kiraly, Andrew Richard Phillips, Steven R. Coombes, Mathias Nilsson, and Gareth A. Morris Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03787 • Publication Date (Web): 30 Oct 2017 Downloaded from http://pubs.acs.org on October 31, 2017
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Analytical Chemistry
13
C satellite-free 1H NMR spectra
Pinelopi Moutzouri,† Peter Kiraly, † Andrew R. Phillips,‡ Steven R. Coombes,§ Mathias Nilsson† and Gareth A. Morris*† †
School of Chemistry, University of Manchester, Oxford Road, Manchester M13 9PL, UK.
‡
Pharmaceutical Sciences, AstraZeneca, Silk Road Business Park, Macclesfield, SK10 2NA, UK.
§
Pharmaceutical Technology and Development, AstraZeneca, Silk Road Business Park, Macclesfield, SK10 2NA, UK.
ABSTRACT: A new NMR experiment (Destruction of Interfering Satellites by Perfect Echo Low-pass filtration, DISPEL) is introduced that facilitates the analysis of low-level components in high dynamic range mixtures by suppressing one-bond 13 C satellite signals in 1H spectra. Since the natural abundance of 13C is around 1.1%, these satellites appear at 0.55% of the intensity of a parent peak, mimicking and often masking impurity signals. The new experiment suppresses one-bond 13C satellite signals, with high efficiency, at negligible cost in signal-to-noise ratio, and over a wide range of one-bond coupling constants, without the need for broadband 13C decoupling.
The identification and quantification of minor components in mixtures pose challenges in many areas of chemistry. In pharmaceutical manufacturing, for example, it is a requirement that all impurities above 0.1 % of a main active pharmaceutical ingredient should be identified and quantified.1 The presence of isotopomers containing 13C (natural abundance 1.1 %) is a major complication, as it gives rise to multiple 13C satellite signals at around 0.54 % of the parent peak intensity. 19F NMR is a powerful emerging tool in the analysis of low level impurities, giving highly sensitive and well-resolved spectra2-6. Recent experiments have made the use of 19F NMR even more attractive by allowing uniform quantitative excitation of its wide chemical shift range7,8, and suppressing 13C satellite signals9. However, the great majority of experiments use 1 H NMR, where the approach used in the ODYSSEUS experiment9 is not directly applicable. The much narrower chemical shift range and extensive multiplet structure greatly increase the potential for confusion and/or overlap between signals of low-level species and 13C satellites. Moreover the extensive coupling complicates the design of spectral editing pulse sequences. However the much smaller secondary isotope effects on the chemical shift mean that long-range 1H-13C couplings cause few if any problems. A new technique is proposed that gives excellent suppression of one-bond 13C satellite signals, over a wide range of one-bond coupling constants, at a very small cost in sensitivity. A variety of different methods have been used to circumvent problems of overlap between 13C satellites and signals of interest, including changing the solvent, concentration, temperature or pH of the sample to move the signals relative to one another,10,11 and using broadband 13 C decoupling to collapse the heteronuclear couplings12-17. The first class of methods is tedious and time-consuming,
relying on trial and error. Broadband 13C decoupling is more attractive, and in some cases works well, but its performance is very dependent on the sample, solvent and instrumentation used. As shown in Figure S10 of the Supplementary Information, even with the most recent instrumentation and a sample that is only moderately lossy, the high radiofrequency power required causes sample heating which broadens, distorts and shifts signals. The sidebands introduced by in the spectrum can also complicate spectral analysis. As shown in Figure S10 and S11, even with bilevel adiabatic decoupling18 careful optimisation is required to balance the conflicting demands of minimizing heating and avoiding decoupling sidebands. A subtler problem is that the secondary isotope effect on the proton chemical shift means that the decoupled signals from 13C isotopomers have slightly different (typically 1 – 2 ppb) chemical shifts from those of 12C isotopomers which slightly broadens the bases of the decoupled resonances. The new method described here avoids all these problems by editing the 1H spectrum to remove 13C satellite signals. Figure 1a shows the 1H spectrum of a sample of the proton pump inhibitor omeprazole (1, Scheme 1), used to treat excess stomach acid, spiked with a small amount (0.15 %) of its precursor omeprazole sulfide (2). The signals of 2, of which the 1H spectrum can be seen in Figure 1c, are approximately 4 times less intense compared to the 13 C satellites of omeprazole. The 13C satellites of 1, being dominant over the weaker impurity signals, complicate analysis; for example as seen in Figure 1f a signal of 2 close to 2.3 ppm is almost degenerate with a satellite signal of 1. However in the 1H spectrum of Figure 1b, obtained with the new one-bond 13C satellite suppression method, the absence of the 13C satellites makes it straightforward to
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identify those signals that do not originate from omeprazole. Here in addition to the signals of 2, identifiable by comparison with the omeprazole sulfide spectrum of Figure 1c, a small number of very weak signals, marked with asterisks, of an unknown impurity can be seen. Less obvious but evident in Figure 1d and 1g, the spectral region around 6.8 ppm shows that the 13C satellite signal of omeprazole at 6.76 ppm completely masks one of the omeprazole sulfide signals. The new method, shown in Figure 2, is an extension of the 1JCH low-pass filter,19-23 used for example in the HMBC20 and ODYSSEUS9 experiments, in which a 90° 13C pulse is used to
Scheme 1. Omeprazole (1), its precursor omeprazole sulfide (2), quinine (3), and cinchonidine (4). convert 1H coherence that is antiphase with respect to 13C into unobservable multiplet quantum coherence. The presence of 1H-1H scalar coupling means that this basic low-pass filter causes the phases of multiplet components to diverge, distorting and complicating phase-sensitive spectra. This J modulation can be refocused (for short times) by using a perfect echo24-26 instead of a simple spin echo.27 The perfect echo uses an orthogonal 90° pulse between two spin echoes to reverse the sense of J modulation. Provided the duration 2 τ of each echo is short compared to the inverse of the couplings JHH (2 τ JHH
