Clearing the undergrowth: detection and quantification of low level impurities using ¹⁹F NMR

Abstract

A new method for the analysis of low level impurities in sparsely fluorinated species allows measurement of clean high dynamic range ¹⁹F spectra, fully decoupled and free of interfering signals from ¹³C isotopomers.

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The high sensitivity and wide chemical shift range of ¹⁹F NMR^1–4 make it potentially very attractive for characterising fluorine-containing impurities. In pharmaceutical chemistry, for example, a quarter of current drugs contain one or more fluorines,⁵ and regulatory authorities require all impurities above 0.1% of a main active pharmaceutical ingredient to be identified and quantified.⁶ Both 1D ¹⁹F NMR and ¹⁹F DOSY have been used for the detection of minor fluorinated impurities.⁷ One major technical problem is the difficulty of exciting quantitatively the very wide chemical shift range of ¹⁹F, but solutions now exist for both 1D⁸ and DOSY⁹ experiments. However, there remains the problem of ¹³C isotopomer signals. At around 0.54% of the intensity of ¹²C isotopomer signals, these are in the same range as impurity signals of interest and often have similar chemical shifts, and therefore complicate their identification and quantitation. The obvious solution is to use broadband ¹³C decoupling to collapse the heteronuclear J-couplings. This can work well for ¹H spectra, albeit at the expense of some sample heating.^10–15 However, ¹⁹F is exquisitely sensitive to chemical environment and its large secondary isotope shift means that the decoupled (¹⁹F–¹³C) signals have slightly different chemical shifts from the parent (¹⁹F–¹²C) signals, so decoupling just halves the number of ¹⁹F–¹³C signals, rather than hiding them all under the parent. Here we show how to acquire clean ¹⁹F spectra without interference from ¹³C isotopomers and with no heteronuclear (¹H or ¹³C) splittings. The new method does not use ¹³C decoupling, minimising sample heating, and should greatly facilitate the detection and quantification of low-level impurities by ¹⁹F NMR.

Fig. 1 shows ¹⁹F spectra of a slightly degraded sample of rosuvastatin (1, Scheme 1), used for treating dyslipidaemia, spiked with small amounts of precursors 2 and 3. The proton-decoupled spectrum of Fig. 1a (multiplet structure renders the proton coupled spectrum, shown in Fig. S4 of the ESI,† uninformative) is complicated by the presence of both one-bond and long-range ¹³C satellites; one of the two satellite signals due to the presence of ¹³C at the ortho position with respect to fluorine is almost degenerate with (8 ppb from) the signal of 2.

Fig. 1 (a) ¹H decoupled ¹⁹F spectrum; (b) ¹H decoupled ¹⁹F spectrum acquired with the pulse sequence of Fig. S1a of the ESI,† with one-bond satellites filtered out and long-range couplings decoupled; (c) ¹H decoupled, ¹³C isotopomer-suppressed ¹⁹F spectrum acquired with the pulse sequence of Fig. 2. Assignments are shown for rosuvastatin (1), its ipso, ortho and meta ¹³C isotopomers (¹J_CF, 1* and 1**), BEM (2), DPPO (3), a diastereomeric impurity of 1 (1a), and a degradation product (1b). All spectra used the same acquisition time of 13.5 h.

Scheme 1 Rosuvastatin (1), two of its precursors, BEM (2) and DPPO (3), and fluconazole (4).

Acquiring a spectrum with this resolution with full broadband decoupling is uncomfortably close to the limits of many instruments, because of the long high-power irradiation required, but if the one-bond ¹³C satellite signals are suppressed (see Section S1 of the ESI†), low power irradiation can be used to decouple the remaining longer-range (≥two-bond) couplings. This gives the spectrum of Fig. 1b, in which a singlet signal is seen for the 2.2% of ortho-¹³C 1. Had full ¹³C decoupling been used, the ipso-¹³C signal of 1, midway between the one-bond satellites in Fig. 1a, would have been degenerate with that of impurity 1a (a diastereomer). In the spectrum of Fig. 1c, in contrast, which was obtained with the new method, no resolvable signals at all are seen from ¹³C isotopomers, and there is no interference with the signals of the minor components of the sample.

The new method, using the pulse sequence of Fig. 2, is compatible with several different hardware configurations; the results shown here used a single high band radiofrequency (RF) amplifier and a (¹H/¹⁹F),¹³C triple-resonance probe with a double-tuned high band coil. The experiment consists of three parts: a low-pass filter to suppress one-bond ¹³C satellite signals; a J_CF – modulated spin echo; and time-shared acquisition during which the ¹⁹F signal is recorded under ¹H decoupling.

Fig. 2 ODYSSEUS (optimal decoupling yielding satellite suppression-edited ultraclean spectra) pulse sequence for the acquisition of ¹H decoupled, ¹³C isotopomer-suppressed ¹⁹F spectra. Closed narrow rectangles represent 90° hard RF pulses, and open wide rectangles 180° hard RF pulses. The delay Δ is set to 1/(2 ¹J_FC). Adiabatic bilevel ¹H decoupling during time-shared acquisition uses two types of WURST pulse with different durations and amplitudes. In systems with ¹⁹F–¹⁹F coupling, both 180° ¹⁹F pulses should be selective. Further experimental details are given in the Experimental section of the ESI.†

The low-pass J filter,^16–20 which converts ¹⁹F antiphase signals into unobservable heteronuclear multiple quantum coherences when Δ = 1/(2 ¹J_CF), suppresses the one-bond ¹³C satellite signals. Since a ¹⁹F spin echo is needed to refocus the fluorine chemical shift, there is time to use two ¹³C 90° pulses in a two-stage filter; if a wide range of ¹J_CF values is present, further stages can be added.

The modulated spin echo, which is analogous to a heteronuclear 2D J resolved experiment,^21–23 makes the phases of the remaining ¹³C satellite signals depend on the evolution time t₁, while the desired signals from the ¹²C isotopomers are unaffected. Weighted averaging of experiments with different t₁ cancels the modulated signals, leaving a clean spectrum. In practice the most effective way to perform this averaging is by double Fourier transformation and integral projection onto F₂ of the F₁ range spanned by the lineshape of the parent signal. This suppresses all satellite signals that would be resolvable in the 1D spectrum, while preserving the quantitative character of the spectrum. The final ¹³C 90° pulse deals with the problem of the phasetwist lineshape^24–26 of a 2D J spectrum by suppressing the sine-modulated dispersive part of the signal. The remaining cosine-modulated signal can then be selected by zeroing the imaginary component after the first Fourier transformation, leading to signals that are doubled in F₁ but have 2D absorption mode lineshapes. The choice of increment 1/sw1 in t₁ is determined by the range of couplings to be suppressed (sw1 > ⁿJ_CH), and the number of increments ni by the T₂ of the parent signal (ni > sw1 T₂). Relaxation losses during t₁ lead to a small sensitivity penalty for the new method, about a factor of 2 here (apparent on comparing Fig. 1a and c).

The data acquisition section of the pulse sequence uses time-shared decoupling because the ¹H and ¹⁹F channels share the same coil in the probe used. In normal circumstances, a simple WALTZ^27,28 or similar decoupling waveform would suffice to decouple ¹H from ¹⁹F, but the very high dynamic range of the sample means that the weak systematic signal modulations such methods induce would here give rise to significant decoupling sidebands (see Fig. S3, ESI†). These are suppressed very effectively here by the use of bilevel adiabatic decoupling.²⁹

As well as decoupling ¹H from ¹⁹F during acquisition, it can be helpful to decouple in the earlier parts of the sequence, to suppress any echo modulation caused by strong ¹H–¹H coupling. This is common in aromatic spin systems (as for example in Fig. S2 of the ESI†).^21,30,31 Here the quality of decoupling is less critical, so bilevel decoupling is not needed.

Fig. S5 (ESI†) shows the intermediate stage in the production of Fig. 1c at which the F₂ projection of the 2D is calculated. Each ¹³C isotopomer gives four symmetrically-disposed signals, with frequency coordinates (±J_CF/2, δ ± J_CF/2); in Fig. S5 (ESI†) both of the less shielded satellites overlap in F₂ with t₁-noise from the parent peak. Integration between the dotted lines produces the spectrum of Fig. 1c.

To test the quantification performance of the new method, the relative percentages of the impurities compared to the main drug substance were measured using the spectrum (Fig. S6 of the ESI†) of a fresh, undegraded, sample. Since the dynamic range of the spectrum is very high, lineshape fitting^32–36 was used instead of conventional integration. As shown in Table 1, the relative percentages measured agree well with those expected.

Table 1 Expected and measured relative percentages of components 2, 3 and impurity 1a as percentages of 1, for a fresh sample lacking degradation product 1b

	Expected (%)	Measured (%)
2	0.33	0.37 ± 0.03
3	0.17	0.18 ± 0.03
1a	0.28	0.26 ± 0.03

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In systems with mutually coupled fluorines, homonuclear J modulation interferes with ¹³C satellite suppression if hard 180° ¹⁹F pulses are used in Fig. 2. Selective 180° pulses avoid this problem, as shown in Fig. 3 for the antifungal drug fluconazole, which has J_FF = 8.1 Hz. Fig. 3b and c were acquired separately using the selective analogue of Fig. 2 to excite the regions around −107 and −111 ppm respectively, revealing the degradation products 4a, 4b and 4c.

Fig. 3 (a) ¹H decoupled ¹⁹F spectrum of a degraded sample of the antifungal drug fluconazole (4); (b and c) ¹H decoupled, ¹³C isotopomer-suppressed ¹⁹F spectra acquired separately for each parent signal using the pulse sequence of Fig. 2 with selective ¹⁹F 180° pulses.

¹³C isotopomer signals can pose significant challenges in identifying and quantifying impurities down to the 0.1% level. The novel approach introduced here of filtering out, rather than decoupling, these signals offers the possibility of acquiring clean, high dynamic range ¹⁹F spectra without interference from species containing ¹³C. A slightly simpler approach can be used in proton spectra.

This work was supported by AstraZeneca and by the Engineering and Physical Sciences Research Council (grant number EP/N033949/1). All raw experimental data, and the pulse sequence code, can be downloaded from DOI: http://10.15127/1.304823.

Notes and references

1. S. Trefi, V. Gilard, S. Balayssac, M. Malet-Martino and R. Martino, J. Pharm. Biomed. Anal., 2008, 46, 707–722.
2. J. C. Lindon and I. D. Wilson, eMagRes, John Wiley & Sons, Ltd, 2007 DOI:10.1002/9780470034590.emrstm1479.
3. S. Trefi, V. Gilard, M. Malet-Martino and R. Martino, J. Pharm. Biomed. Anal., 2007, 44, 743–754.
4. W. He, F. Du, Y. Wu, Y. Wang, X. Liu, H. Liu and X. Zhao, J. Fluorine Chem., 2006, 127, 809–815.
5. J. Wang, M. Sánchez-Roselló, J. L. Aceña, C. del Pozo, A. E. Sorochinsky, S. Fustero, V. A. Soloshonok and H. Liu, Chem. Rev., 2014, 114, 2432–2506.
6. ICH, Impurities in new drug substances Q3A (R2) International Conference on Harmonisation, IFPMA, Geneva, Switzerland, 2006.
7. N. Mistry, I. M. Ismail, R. Duncan Farrant, M. Liu, J. K. Nicholson and J. C. Lindon, J. Pharm. Biomed. Anal., 1999, 19, 511–517.
8. J. E. Power, M. Foroozandeh, R. W. Adams, M. Nilsson, S. R. Coombes, A. R. Phillips and G. A. Morris, Chem. Commun., 2016, 52, 2916–2919.
9. J. E. Power, M. Foroozandeh, P. Moutzouri, R. W. Adams, M. Nilsson, S. R. Coombes, A. R. Phillips and G. A. Morris, Chem. Commun., 2016, 52, 6892–6894.
10. A. J. Shaka, P. B. Barker and R. Freeman, J. Magn. Reson., 1985, 64, 547–552.
11. T. Fujiwara, T. Anai, N. Kurihara and K. Nagayama, J. Magn. Reson., Ser. A, 1993, 104, 103–105.
12. R. Fu and G. Bodenhausen, Chem. Phys. Lett., 1995, 245, 415–420.
13. L. S. Simeral, Appl. Spectrosc., 1995, 49, 400–402.
14. Ē. Kupče and R. Freeman, Chem. Phys. Lett., 1996, 250, 523–527.
15. R. Freeman and Ē. Kupče, NMR Biomed., 1997, 10, 372–380.
16. A. Bax, R. H. Griffey and B. L. Hawkins, J. Magn. Reson., 1983, 55, 301–315.
17. A. Bax and M. F. Summers, J. Am. Chem. Soc., 1986, 108, 2093–2094.
18. H. Kogler, O. W. Sørensen, G. Bodenhausen and R. R. Ernst, J. Magn. Reson., 1983, 55, 157–163.
19. L. Müller, J. Am. Chem. Soc., 1979, 101, 4481–4484.
20. N. T. Nyberg and O. W. Sørensen, Magn. Reson. Chem., 2006, 44, 451–454.
21. G. Bodenhausen, R. Freeman, G. A. Morris and D. L. Turner, J. Magn. Reson., 1977, 28, 17–28.
22. G. Bodenhausen, R. Freeman, R. Niedermeyer and D. L. Turner, J. Magn. Reson., 1976, 24, 291–294.
23. L. Müller, A. Kumar and R. R. Ernst, J. Magn. Reson., 1977, 25, 383–390.
24. P. Bachmann, W. P. Aue, L. Müller and R. R. Ernst, J. Magn. Reson., 1977, 28, 29–39.
25. A. Bax, A. F. Mehlkopf and J. Smidt, J. Magn. Reson., 1979, 35, 373–377.
26. R. Freeman, S. P. Kempsell and M. H. Levitt, J. Magn. Reson., 1979, 34, 663–667.
27. A. J. Shaka, J. Keeler, T. Frenkiel and R. Freeman, J. Magn. Reson., 1983, 52, 335–338.
28. A. J. Shaka, J. Keeler and R. Freeman, J. Magn. Reson., 1983, 53, 313–340.
29. Ē. Kupče, R. Freeman, G. Wider and K. Wüthrich, J. Magn. Reson., Ser. A, 1996, 122, 81–84.
30. R. Freeman, G. A. Morris and D. L. Turner, J. Magn. Reson., 1977, 26, 373–378.
31. A. Kumar and R. R. Ernst, Chem. Phys. Lett., 1976, 37, 162–164.
32. M. Ala-Korpela, Y. Hiltunen, J. Jokisaari, S. Eskelinen, K. Kiviniitty, M. J. Savolainen and Y. A. Kesäniemi, NMR Biomed., 1993, 6, 225–233.
33. Y. Hiltunen, M. Ala-Korpela, J. Jokisaari, S. Eskelinen, K. Kiviniitty, M. Savolainen and Y. A. Kesäniemi, Magn. Reson. Med., 1991, 21, 222–232.
34. V. V. Mihaleva, S. P. Korhonen, J. van Duynhoven, M. Niemitz, J. Vervoort and D. M. Jacobs, Anal. Bioanal. Chem., 2014, 406, 3091–3102.
35. H. M. Parsons, C. Ludwig and M. R. Viant, Magn. Reson. Chem., 2009, 47, S86–S95.
36. P. Soininen, J. Haarala, J. Vepsäläinen, M. Niemitz and R. Laatikainen, Anal. Chim. Acta, 2005, 542, 178–185.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6cc08836h

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