Relaxation time estimation for qNMR of APIs Anna Lamieri, Katrina Badiola and Joshua Freem Pharmaron, United Kingdom
Solution-phase Quantitative Nuclear Magnetic Resonance (qNMR) spectroscopy is currently adopted by the Pharmaceutical Industry for sample quantitation and purity determination, most commonly using a certified internal standard of known purity. The accuracy of qNMR results strongly rely on the time allowed between the pulse sequence and signal acquisition, the relaxation delay parameter (D 1 ); the magnitude of the D 1 must allow for complete recovery of the spin magnetisation to thermal equilibrium after pulse excitation. 1,2 The time constant for longitudinal (spin-lattice) relaxation (T 1 ) is the slowest spin relaxation phenomenon of nuclei, and thus the magnitude of T 1 is the principal factor that influences the magnitude of the D 1 value for qNMR. The longest T 1 amongst the nuclei of a sample must be estimated to determine the optimal D 1 . 3 Practically, the D 1 value is set such that D 1 ≥ 5T 1 which provides at least 99.3% magnetization recovery and ~0.7% error due to incomplete relaxation, in common 90° pulse sequence qNMR experiments. 4,5 Inversion Recovery (IR) represents the ‘golden standard’ for the measurement of T 1 of nuclei, despite resulting in very long experimental times. 6 In this investigation, the T 1 of proton nuclei of common qNMR standards and Active Pharmaceutical Ingredients (APIs) is estimated by IR and compared to data acquired with the more recently developed, faster T 1 measurement methods: the ‘null-time experiment’ (d null ) and ‘Faster Longitudinal relaxation Investigated by Progressive Saturation’ (FLIPS). 7,8 The d null method is determined to be fit-for-purpose but is prohibitively time-consuming when T 1 relaxation times are measured for multiple environments. The FLIPS method is accurate with advantageously rapid T 1 determinations that also require minimal user input during data acquisition. Acquisition parameters of the FLIPS method are evaluated, and the 30° FLIPS pulse sequence typically yields conservative T 1 values compared to IR, thus being better suited for D 1 parameter determination, and allowing for improved robustness in qNMR method development for APIs. By incorporating FLIPS experiments prior to qNMR analysis, this study concludes that the D 1 parameters currently adopted in qNMR methods are likely disproportionate and may be significantly shortened whilst maintaining high levels of accuracy. References 1. N. Amin and T. Claridge, Quantitative NMR Spectroscopy , University of Oxford, Oxford, 2017. 2. A. A. Crook and R. Powers, Molecules , 2020, 25 , 5128-5085. 3. B. V. N. Phani Kumar and R. R. Reddy, Annual Reports on NMR Spectroscopy , 2020, 99 , 1-56. 4. T. Schoenberger, Guideline for qNMR Analysis , Drugs Working Group DWG-NMR-001, ENFSI, Bundeskriminalamt, 2019. 5. A. O. Mattes, D. Russell, E. Tishchenko, Y. Liu, R. H. Cichewicz and S. J. Robinson, Concepts in Magnetic Resonance , 2018, 45A , 21422-21430. 6. J. Kowalewski, Nuclear Magnetic Resonance , 1 st edn., 2021, vol. 46, ch. 3, 76-138. 7. University of Wisconsin-Madison , Zero(Null)-Crossing Estimates of 1 H T1 Values, Madison, 2015. 8. R. Wei, C. L. Dickson, D. Uhrín and G. C. Lloyd-Jones, The Journal of Organic Chemistry , 2021, 86 , 9023-9029.
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