qNMR Session at PANIC 2016

2:30 Session | Chairs’ Remarks: Torsten Schoenberger, Bundeskriminalamt and John Marino, NIST

2:40 Guido Pauli, University of Illinois, Chicago: Between Metabolomics and Metrology: The Development Paths of Quantitative NMR

• Abstract. The basic concepts of NMR as a quantitative analytical tool (qNMR) date back to the days of continuous wave (CW) spectral acquisition. While NMR’s fundamental ability to quantify pharmaceutical substances was demonstrated some 50 years ago, the advent of FT, 2D/nD, and biomolecular NMR led to a qualitative concentration on structure and diversification of experimental capability. Nurtured by the widespread notion that NMR is a specialized method, its quantitative utility remained largely underexplored and/or under-documented in the literature. Starting with the millennial turn, two major impulses for 1D 1H NMR (qHNMR) resulted from independent progress achieved in different fields: metrological investigations established and refined the basic analytical boundaries (accuracy, precisions, parametrization, intra-/inter-laboratory reproducibility), whereas natural products research demonstrated that qHNMR can fill a unique gap in the assessment of nature-derived materials such as isolates and samples that challenge LC-based methods. Both scientific paths used reference materials as (initial) focal points, and the recognition of qNMR as a relative primary analytical method provided strong support for the conclusion that qHNMR is an orthogonal analytical method with unique utility and potential. In over 15 years of qNMR-related research, we have continuously applied and developed qNMR methods with the ultimate goal of characterizing bioactive natural products more comprehensively, as well as more adequately with respect to their metabolomic origin and nature. This presentation seeks to provide an overall perspective by covering the following points: (a) key aspects of qNMR methodology investigated during earlier studies in our laboratory, including calibration (100% vs. internal vs. external calibration), the simultaneously useful and disturbing nature of 13C satellites and the development of {13C}qHNMR; (b) the recognition of qHNMR analysis as a means of validating hits for drug leads and detecting causes for “erratic” behavior in biological assays, ultimately leading to the establishment of the concept of (quantitative) purity-activity relationships ([q]PARs); (c) the recent institution of quantum-mechanics based 1H iterative Full Spin Analysis (HiFSA), representing a matchless method of generating qualitative fingerprints of compounds that are both highly compound specific and fully quantitative. Moving into metabolomic applications, (d) our most recent applications utilize qHNMR to realize a more holistic standardization of botanical dietary supplements. One application is the analysis of NMR fingerprints of the sweet-tasting Stevia glycosides using a new building block approach for these complex oligoglycosides with overlapping spectra. This “sweet qNMR” approach exemplifies the power of the simplistic 1D (q)NMR spectrum in the realm of bioactive organic molecules.

3:10 Markus Obkircher, Sigma-Aldrich: Recent Research Developments in the Certification of Organic Reference Materials by Quantitative NMR at the Highest Metrological Level

• Abstract. Quantitative NMR (qNMR) has become one of the most important tools for content determination of organic substances and quantitative evaluation of impurities. Sigma­Aldrich R&D demonstrated the validity, robustness and precision of the qNMR technique through the optimization of High­Performance qNMR (HP-qNMR®) to its maximum level of accuracy using metrological weighing equipment and a specially designed experimental setup for the certification of organic compounds with combined, expanded uncertainties down to 0.1%. An important step towards the implementation of a multi nucleus CRM for qNMR was accomplished by a successful concept study for 31P qNMR measurements with direct traceability to NIST SRM. Further research activities include the certification of very limited sample quantities (“micro”­qNMR) as well as the preparation of a primary qNMR SRM in collaboration with NIST. Sigma­Aldrich's qNMR technology very recently has been awarded by CITAC (Cooperation for International Traceability in Analytical Chemistry) with the 2013 best paper award.

3:40 Purnima Khandelwal, Bristol Myers Squibb: Polyethylene Glycol Quantitation in Biofluids supporting Drug Discovery

• Abstract. Conjugation of biological drugs to PEG improves pharmacokinetic profile. However, due to its large and polymeric nature, other analytical methods have difficulty in measuring it. We have developed an NMR quantitation method and applied it to several biofluid matrices and animal species to demonstrate its utility. Results using the new NMR method compare very well with the established radioisotope labeling technique.