Quantitative <sup>1</sup>H NMR:  Development and Potential of a Method for Natural Products Analysis

Pauli, Guido F.; Jaki, Birgit U.; Lankin, David C.

doi:10.1021/np0497301

Cited by 473 publications

(480 citation statements)

References 164 publications

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“…Samples were dissolved in acetone-D6 and analyzed by standard proton NMR spectrometry for identification and qNMR for quantitative analysis (Pauli et al 2005).…”

Section: Qnmr Purity Assessmentmentioning

confidence: 99%

Curcuminoids in Turmeric Roots and Supplements: Method Optimization and Validation

et al. 2015

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Curcuma longa L. rhizomes are used extensively as a spice in food preparations and dietary supplements for their anti-inflammatory and antioxidant properties. An expert review panel (ERP) evaluated analytical methods for the quantitation of individual curcuminoids for the purpose of identifying a method for official method status. It was requested that several modifications be undertaken to improve method performance prior to subjecting the chosen method to a singlelaboratory validation. Two separate Plackett-Burman factorial studies were used to identify factors that contributed to the chromatographic separation and extraction of curcuminoids. Significant factors were further optimized to produce the improved HPLC method for curcuminoid separation. This method was then subjected to a single-laboratory validation according to the AOAC International guidelines for linearity, detection limits, precision, and accuracy. The two most significant factors impacting the quantitation of curcuminoids were column temperature and extraction solvent, which were optimized to 55°C and 100 % methanol, respectively. The validation was performed on 12 raw materials and finished products containing turmeric roots. The method precision was reported using HorRat values which were within recommended ranges of the AOAC guidelines. Overall accuracy of the method was accessed at three separate levels for each analyte and ranged from 99.3-100.9 %. The validated method is suitable for quantitation of individual curcuminoids in turmeric raw materials and finished products and is recommended for consideration as an official method by the AOAC International.

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“…Samples were dissolved in acetone-D6 and analyzed by standard proton NMR spectrometry for identification and qNMR for quantitative analysis (Pauli et al 2005).…”

Section: Qnmr Purity Assessmentmentioning

confidence: 99%

Curcuminoids in Turmeric Roots and Supplements: Method Optimization and Validation

et al. 2015

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“…One-dimensional (1D) 1 H NMR spectroscopy has been used for decades as an analytical tool for identifying small molecules and measuring their concentrations. 1, 2 Traditionally, quantitative analysis by NMR has been restricted to relatively simple mixtures with minimal peak overlap.…”

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confidence: 99%

Method for Determining Molar Concentrations of Metabolites in Complex Solutions from Two-Dimensional ¹H−¹³C NMR Spectra

et al. 2007

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One-dimensional (1D) 1 H nuclear magnetic resonance (NMR) spectroscopy is used extensively for high-throughput analysis of metabolites in biological fluids and tissue extracts. Typically, such spectra are treated as multivariate statistical objects rather than as collections of quantifiable metabolites. We report here a two-dimensional (2D) 1 H-13 C NMR strategy (Fast Metabolite Quantification, FMQ by NMR) for identifying and quantifying the ∼40 most abundant metabolites in biological samples. To validate this technique, we prepared mixtures of synthetic compounds and extracts from Arabidopsis thaliana, Saccharomyces cerevisiae and Medicago sativa. We show that accurate (technical error 2.7%) molar concentrations can be determined in 12 minutes using our quantitative 2D 1 H-13 C NMR strategy. In contrast, traditional 1D 1 H NMR analysis resulted in 16.2% technical error under nearly ideal conditions. We propose FMQ by NMR as a practical alternative to 1D 1 H NMR for metabolomics studies in which 200-400 mg (preextraction dry weight) samples can be obtained.One-dimensional (1D) 1 H NMR spectroscopy has been used for decades as an analytical tool for identifying small molecules and measuring their concentrations. 1, 2 Traditionally, quantitative analysis by NMR has been restricted to relatively simple mixtures with minimal peak overlap. In these applications, 1D 1 H NMR is a natural choice, because its peaks scale linearly with concentration and its analytical precision is usually independent of the chemical properties of target molecules. Recently, interest has surged in using NMR for high-throughput analysis of complex biological processes at the metabolic level. 3, 4 These studies, defined as "metabolomics" or "metabonomics", place an emphasis on biomarker discovery or disease classification and are typically centered on unfractionated biological fluids and tissue extracts. 1D 1 H NMR spectra of these samples typically contain hundreds of overlapping resonances (Figure 1) that make traditional NMR-based analytical practices, such as resonance assignment and accurate peak integration, a challenging prospect. As a result, sophisticated statistical tools have been developed to translate spectral data into biologically meaningful information. 4, 5All statistical tools used for analyzing complex spectra face the same fundamental barrier: overlapped peaks do not scale in the discrete linear fashion that typifies well-isolated peaks. They scale as the sum of the total overlapped resonance. Consequently, multivariate and correlation statistics are reporters of overlapped spectral density, not concentrations of specific compounds. Although peak overlap does not interfere with the reproducibility of traditional analyses, 6 it does prevent accurate quantification. Two approaches can be used to overcome this barrier, one mathematical the other experimental. The mathematical approach is to fit overlapped 1 D NMR spectra with modeled peaks. This approach has been successfully applied by Weljie and co-workers. 7 The e...

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“…16 Due to the close structural similarities of the taxoids, the assumption was made that the molecular weight of the taxoid impurities is similar to taxol, and the identical mass (854 amu) was taken into account as a ficticious weight. This approach has empirically been proven to provide very reasonable quantitative results in purity analyses that are based on the 100% method 5,17 (and unpublished data using absolute quantitation; see also error discussion below).…”

Section: The Qhnmr Evaluation Of Taxol Reference Materials and Relatementioning

confidence: 99%

“…While nD qNMR is yet to be fully established, the 1D qNMR method already allows a powerful discrimination of multi-component mixtures or impurities in isolates. In particular the 1D 1 H variant of qNMR, qHNMR, adds a combined quantitative-qualitative dimension to the structural NMR portfolio of any natural product, and has the potential to make a fundamental contribution to the bigger picture of structure-activity correlations.Considering the above, and in the context of more than 100 reports on the use of quantitative NMR in natural products and related sciences, mainly pharmaceutical, food and drug analyses, 5,6 there is a significant potential for a standardized qHNMR method, i.e., a method that can be applied in routine purity analysis and quantitation of complex mixtures. 7…”

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confidence: 99%

A Routine Experimental Protocol for qHNMR Illustrated with Taxol

2007

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Quantitative 1 H NMR (qHNMR) provides a value-added dimension to the standard spectroscopic data set involved in structure analysis, especially when analyzing bioactive molecules and elucidating new natural products. The qHNMR method can be integrated into any routine qualitative workflow without much additional effort by simply establishing quantitative conditions for the standard solution 1 H NMR experiments. Moreover, examination of different chemical lots of taxol and a Taxus brevifolia extract as working examples led to a blueprint for a generic approach to performing a routinely practiced 13 C-decoupled qHNMR experiment, and for recognizing its potential and main limitations. The proposed protocol is based on a newly assembled 13 C GARP broadband decoupled proton acquisition sequence that reduces spectroscopic complexity by removal of carbon satellites. The method is capable of providing qualitative and quantitative NMR data simultaneously and covers various analytes from pure compounds to complex mixtures such as metabolomes. Due to a routinely achievable dynamic range of 300:1 (0.3%) or better, qHNMR qualifies for applications ranging from reference standards to biologically active compounds to metabolome analysis. Providing a "cookbook" approach to qHNMR, acquisition conditions are described that can be adapted for contemporary NMR spectrometers of all major manufacturers. The world's pool of natural products plays an important role as an (in)exhaustible resource for evolutionary-shaped molecules. Natural products are valuable research tools, which in part is due to their biological potency (see comprehensive reviews [1][2][3][4] ). When natural products are used as biomedical agents, and in consistency with the pharmacophore model, it is the combination of their specific chemical structure and/or reactivity that forms their relationship with a biological target and ultimately defines their essential structural features. Consequently, chemical constitution plays a key role in biological activity, and, therefore, all structure related information obtainable from a biologically active agent is by default relevant. Ultimately, any variation of structural parameters has the potential to introduce variations in biological activity. This relationship holds regardless of the magnitude of the biological perturbation, i.e., whether there is slight or a substantial change in potency, or even an alteration in the type of biological response. The well-documented subtleties of mammalian hormonal steroids can serve as a distinguished example in this regard.⊥ Dedicated to Dr. Norman R. Farnsworth on the occasion of his 77 th birthday.* To whom correspondence should be addressed. Tel (312) 355-1949355- . Fax (312)-355-2693. gfp@uic.edu. Supporting Information Available.This material is available free of charge via the Internet at http://pubs.acs.org. Further information on qNMR methodology, instrument parameter files for the NMR major manufacturer's spectrometers, and original FIDs of the taxol qHNMR experime...

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Quantitative ¹H NMR: Development and Potential of a Method for Natural Products Analysis

Cited by 473 publications

References 164 publications

Curcuminoids in Turmeric Roots and Supplements: Method Optimization and Validation

Curcuminoids in Turmeric Roots and Supplements: Method Optimization and Validation

Method for Determining Molar Concentrations of Metabolites in Complex Solutions from Two-Dimensional ¹H−¹³C NMR Spectra

A Routine Experimental Protocol for qHNMR Illustrated with Taxol

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Quantitative 1H NMR: Development and Potential of a Method for Natural Products Analysis

Cited by 473 publications

References 164 publications

Curcuminoids in Turmeric Roots and Supplements: Method Optimization and Validation

Curcuminoids in Turmeric Roots and Supplements: Method Optimization and Validation

Method for Determining Molar Concentrations of Metabolites in Complex Solutions from Two-Dimensional 1H−13C NMR Spectra

A Routine Experimental Protocol for qHNMR Illustrated with Taxol

Contact Info

Product

Resources

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Quantitative ¹H NMR: Development and Potential of a Method for Natural Products Analysis

Method for Determining Molar Concentrations of Metabolites in Complex Solutions from Two-Dimensional ¹H−¹³C NMR Spectra