Evolution of Quantitative Measures in NMR: Quantum Mechanical qHNMR Advances Chemical Standardization of a Red Clover (Trifolium pratense) Extract

Abstract

Chemical standardization, along with morphological and DNA analysis ensures the authenticity and advances the integrity evaluation of botanical preparations. Achievement of a more comprehensive, metabolomic standardization requires simultaneous quantitation of multiple marker compounds. Employing quantitative 1H NMR (qHNMR), this study determined the total isoflavone content (TIfCo; 34.5–36.5% w/w) via multimarker standardization and assessed the stability of a 10-year-old isoflavone-enriched red clover extract (RCE). Eleven markers (nine isoflavones, two flavonols) were targeted simultaneously, and outcomes were compared with LC-based standardization. Two advanced quantitative measures in qHNMR were applied to derive quantities from complex and/or overlapping resonances: a quantum mechanical (QM) method (QM-qHNMR) that employs 1H iterative full spin analysis, and a non-QM method that uses linear peak fitting algorithms (PF-qHNMR). A 10 min UHPLC-UV method provided auxiliary orthogonal quantitation. This is the first systematic evaluation of QM and non-QM deconvolution as qHNMR quantitation measures. It demonstrates that QM-qHNMR can account successfully for the complexity of 1H NMR spectra of individual analytes and how QM-qHNMR can be built for mixtures such as botanical extracts. The contents of the main bioactive markers were in good agreement with earlier HPLC-UV results, demonstrating the chemical stability of the RCE. QM-qHNMR advances chemical standardization by its inherent QM accuracy and the use of universal calibrants, avoiding the impractical need for identical reference materials.

Screening and isolation of cyclooxygenase‐2 inhibitors from Trifolium pratense L. via ultrafiltration, enzyme‐immobilized magnetic beads, semi‐preparative high‐performance liquid chromatography and high‐speed counter‐current chromatography

“…The identification of the semi‐preparative HPLC and HSCCC peaks was based on the LC retention time and on the ESI‐MS, 1 H‐NMR, and 13 C‐NMR spectra. The 1 H‐NMR and 13 C‐NMR data are given in the Supporting Information and were found to be in good agreement with those of the corresponding compounds in the literature .…”

The value of universally available raw NMR data for transparency, reproducibility, and integrity in natural product research

“…This review identified a multitude of rationales, why and how raw NMR data can provide useful and/or unprecedented insights. Presented cases exemplify the importance of sharing raw NMR data in NP research and fall into seven areas of broader impact: (i) the enhancement of the integrity of structure elucidation, which has major implications on downstream activities; (ii) the ability to document the purity status of a given material and enable future meta-analysis and/or refinement of the evaluation; notably, the (semi-)quantitative evaluations are often feasible even if the more rigorous conditions of quantitative NMR (qNMR)29,389–391 were not satisfied during data acquisition, and even if no internal calibration was used (feasibility of the 100% qNMR method);29,390 (iii) the enhancement of the accuracy and general capabilities of dereplication methods, thereby addressing one of the major challenges in metabolomics; (iv) enhancement of the amount of information obtained from NMR spectra and the speed of the mining process, by employing newly emerging software tools that depend on the availability of larger data sets; (v) the catalysis of developments in the field of the less studied NMR-accessible nuclei, offering new opportunities for the study of NPs and their analogues, of these fields; (vi) the promotion of the proven capabilities of existing repositories; (vii) the prospects of extending NP and metabolomic knowledge into clinical applications, e.g. , by using magnetic resonance spectroscopy (MRS).…”

Quality Assessment and Quantification of Genistein in Dietary Supplements by High-Performance Liquid Chromatography, Quantitative Nuclear Magnetic Resonance, and Two-Dimensional Diffusion Ordered Spectroscopy 1h

“…The isoflavone content of four dietary supplements has been determined by HPLC, qNMR, and 2D 1 H DOSY NMR. The quantification of these compounds is generally carried out by HPLC or qNMR [29][30][31][32]. However, the use of 2D 1 H DOSY NMR allowed the detection of various components of these formulations in a single run.…”