With a combined 100+ years of practical NMR experience, our senior qNMR investigator team brings together expertise from academic and industrial environments.
This team is supported by postdoctoral fellows and PhD students who actively use qNMR methods in their research.
We have expertise in the following areas of quantitative NMR applications:
Feel free to contact us! Guido F. Pauli, PhD PharmD, College of Pharmacy, 833 S. Wood St., Chicago, IL 60612 (USA), gfp@uic.edu
Simmler C, Nikolic D, Lankin DC, Yu Y, Friesen JB, van Breemen RB, Lecomte A, Le Quemener C, Audo G, Pauli GF
Orthogonal Analysis Underscores the Relevance of Primary and Secondary Metabolites in Licorice
Journal of Natural Products 77: 1806-1816 (2014)
dx.doi.org/10.1021/np5001945
Abstract: Licorice botanicals are produced from the roots of Glycyrrhiza species (Fabaceae), encompassing metabolites of both plant and rhizobial origin. The composition in both primary and secondary metabolites (1°/2°Ms) reflects the physiologic state of the plant at harvest. Interestingly, the relative abundance of 1°Ms vs 2°Ms in licorice extracts remains undetermined. A centrifugal partition chromatography (CPC) method was developed to purify liquiritin derivatives that represent major bioactive 2°Ms and to concentrate the polar 1°Ms from the crude extract of Glycyrrhiza uralensis. One objective was to determine the purity of the generated reference materials by orthogonal UHPLC-UV/LC-MS and qHNMR analyses. The other objectives were to evaluate the presence of 1°Ms in purified 2°Ms and define their mass balance in a crude botanical extract. Whereas most impurities could be assigned to well-known 1°Ms, p-hydroxybenzylmalonic acid, a new natural tyrosine analogue, was also identified. Additionally, in the most polar fraction, sucrose and proline represented 93% (w/w) of all qHNMR-quantified 1°Ms. Compared to the 2°Ms, accounting for 11.9% by UHPLC-UV, 1°Ms quantified by qHNMR defined an additional 74.8% of G. uralensis extract. The combined orthogonal methods enable the mass balance characterization of licorice extracts and highlight the relevance of 1°Ms, and accompanying metabolites, for botanical quality control.
Liu Y, Chen SN, McAlpine JB, Klein LL, Friesen JB, Lankin DC,
Pauli G
Quantification of a Botanical Negative Marker without an Identical
Standard: Ginkgotoxin in Ginkgo biloba
Journal of Natural Products 77: 611-617 (2014)
dx.doi.org/10.1021/np400874z
Abstract: A new strategy for the analysis of natural products uses a combination of quantitative 1H NMR (qHNMR) and adsorbent-free countercurrent separation (CS) methodology to establish a quantification method for ginkgotoxin (4′-O-methylpyridoxine) in Ginkgo biloba preparations. The target analyte was concentrated in a one-step CS process using the ChMWat +2 solvent system (CHCl3–MeOH–H2O, 10:5:5) and subsequently assayed by qHNMR. While commercial G. biloba seeds contained 59 μg of ginkgotoxin per seed, the compound was below the limit of detection (9 ppm) in a typical leaf extract. Due to the enrichment potential and loss-free operation of CS, the combination of CS and qHNMR is a generally suitable approach for threshold assays aimed at quantifying target compounds such as botanical negative markers at the low ppm level. As the proof of principle is demonstrated for relatively small CS capacities (20 mL, 1:40 loading) and modest NMR sensitivity (n = 16, 400 MHz, 5 mm RT probe), the approach can be adapted to quantification at the ppb level. The procedure enables the quantification of a botanical negative marker in the absence of identical reference material, which otherwise is a prerequisite for LC-based assays.
Simmler C, Napolitano JG, McAlpine JB, Chen SN, Pauli GF
Universal and Quantitative Analysis of Complex Analytes by NMR
Current Opinion in Biotechnology 25: 51-59 (2014)
dx.doi.org/10.1016/j.copbio.2013.08.004
Abstract: Nuclear Magnetic Resonance (NMR) is a universal and quantitative analytical technique. Being a unique structural tool, NMR also competes with metrological techniques for purity determination and reference material analysis. In pharmaceutical research, applications of quantitative NMR (qNMR) cover mostly the identification and quantification of drug and biological metabolites. Offering an unbiased view of the sample composition, and the possibility to simultaneously quantify multiple compounds, qNMR has become the method of choice for metabolomic studies and quality control of complex natural samples such as foods, plants or herbal remedies, and biofluids. In this regard, NMR-based metabolomic studies, dedicated to both the characterization of herbal remedies and clinical diagnosis, have increased considerably.
Napolitano JG, Gödecke T, Lankin DC, Jaki BU, McAlpine JB, Chen SN, Pauli GF
Orthogonal Analytical Methods for Botanical Standardization: Determination of Green Tea Catechins by qNMR and LC-MS/MS
Journal of Pharmaceutical and Biomedical Analysis 93: 59-67 (2014)
dx.doi.org/10.1016/j.jpba.2013.06.017
Abstract: The development of analytical methods for parallel characterization of multiple phytoconstituents is essential to advance the quality control of herbal products. While chemical standardization is commonly carried out by targeted analysis using gas or liquid chromatography-based methods, more universal approaches based on quantitative 1H NMR (qHNMR) measurements are being used increasingly in the multi-targeted assessment of these complex mixtures. The present study describes the development of a 1D qHNMR-based method for simultaneous identification and quantification of green tea constituents. This approach utilizes computer-assisted 1H iterative Full Spin Analysis (HiFSA) and enables rapid profiling of seven catechins in commercial green tea extracts. The qHNMR results were cross-validated against quantitative profiles obtained with an orthogonal LC–MS/MS method. The relative strengths and weaknesses of both approaches are discussed, with special emphasis on the role of identical reference standards in qualitative and quantitative analyses.
Napolitano JG, Lankin D, McAlpine JB, M. Niemitz, SP Korhonen, Chen SN, Pauli GF
Proton Fingerprints Portray Molecular Structures: Enhanced Description of the 1D 1H NMR Spectra of Small Molecules
Journal of Organic Chemistry 78: 2827-2839 (2013)
dx.doi.org/10.1021/jo4011624
Abstract: The characteristic signals observed in NMR spectra encode essential information on the structure of small molecules. However, extracting all of this information from complex signal patterns is not trivial. This report demonstrates how computer-aided spectral analysis enables the complete interpretation of 1D 1H NMR data. The effectiveness of this approach is illustrated with a set of organic molecules, for which replicas of their 1H NMR spectra were generated. The potential impact of this methodology on organic chemistry research is discussed.
Napolitano JG, Lankin D, Graf T, Friesen JB, Chen SN, McAlpine JB, Oberlies, NH, Pauli GF
HiFSA Fingerprinting of Isomers with Near Identical NMR Spectra:
The Silybin/Isosilybin Case
Journal of Organic Chemistry 78: 2827-2839 (2013)
dx.doi.org/10.1021/jo302720h
Abstract: This study demonstrates how regio- and diastereo-isomers with near-identical NMR spectra can be distinguished and unambiguously assigned using quantum mechanical driven 1H iterative Full Spin Analysis (HiFSA). The method is illustrated with four natural products, the flavonolignans silybin A, silybin B, isosilybin A, and isosilybin B, which exhibit extremely similar coupling patterns and chemical shift differences well below the commonly reported level of accuracy of 0.01 ppm. The HiFSA approach generated highly reproducible 1H NMR fingerprints that enable distinction of all four isomers at 1H frequencies from 300 to 900 MHz. Furthermore, it is demonstrated that the underlying numeric 1H NMR profiles, combined with iterative computational analysis, allow parallel quantification of all four isomers, even in difficult to characterize reference materials and mixtures. The results shed new light on the historical challenges to the qualitative and quantitative analysis of these therapeutically relevant flavonolignans and open new opportunities to explore hidden diversity in the chemical space of organic molecules.
Gödecke T, Napolitano JG, Rodriguez Brasco MF, Chen SN, Jaki BU, Lankin D, Pauli GF
Validation of a Generic qHNMR Method for Natural Products Analysis
Phytochemical Analysis 24: 581-597 (2013)
dx.doi.org/10.1002/pca.2436
Abstract: Introduction - Nuclear magnetic resonance (NMR) spectroscopy is increasingly employed in the quantitative analysis and quality control (QC) of natural products (NP) including botanical dietary supplements (BDS). The establishment of QC protocols based on quantitative 1H NMR (qHNMR) requires method validation. Objective - Develop and validate a generic qHNMR method. Optimize acquisition and processing parameters, with specific attention to the requirements for the analysis of complex NP samples, including botanicals and purity assessment of NP isolates. Methods - In order to establish the validated qHNMR method, samples containing two highly pure reference materials were used. The influence of acquisition and processing parameters on the method validation was examined, and general aspects of method validation of qHNMR methods discussed. Subsequently, the method established was applied to the analysis of two NP samples: a purified reference compound and a crude mixture. Results - The accuracy and precision of qHNMR using internal or external calibration were compared, using a validated method suitable for complex samples. The impact of post-acquisition processing on method validation was examined using three software packages: TopSpin, Mnova and NUTS. The dynamic range of the qHNMR method developed was 5000:1 with a limit of detection (LOD) of better than 10 µm. The limit of quantification (LOQ) depends on the desired level of accuracy and experiment time spent. Conclusion - This study revealed that acquisition parameters, processing parameters and processing software all contribute to qHNMR method validation. A validated method with a high dynamic range and general workflow for qHNMR analysis of NP is proposed.
Simmler C, Hajirahimkhan A; Lankin DC, Bolton J, Jones T, Soejarto DD; Chen SN, Pauli GF
Dynamic Residual Complexity of the Isoliquiritigenin-Liquiritigenin Interconversion During
Bioassays
Journal of Agricultural and Food Chemistry 61: 2146-2157 (2013)
dx.doi.org/10.1021/jf304445p
Abstract: Bioactive components in food plants can undergo dynamic processes that involve multiple chemical species. For example, 2′-hydroxychalcones can readily isomerize into flavanones. Although chemically well documented, this reaction has barely been explored in the context of cell-based assays. The present time-resolved study fills this gap by investigating the isomerization of isoliquiritigenin (a 2′-hydroxychalcone) and liquiritigenin (a flavanone) in two culture media (Dulbecco’s modified eagle medium and Roswell Park Memorial Institute medium) with and without MCF-7 cells, using high-performance liquid chromatography–diode array detector–electrospray ionization/atmospheric pressure chemical ionization–mass spectrometry for analysis. Both compounds were isomerized and epimerized under all investigated biological conditions, leading to mixtures of isoliquiritigenin and R/S-liquiritigenin, with 19.6% R enantiomeric excess. Consequently, all three species can potentially modulate the biological responses. This exemplifies dynamic residual complexity and demonstrates how both nonchiral reactions and enantiomeric discrimination can occur in bioassay media, with or without cells. The findings highlight the importance of controlling in situ chemical reactivity, influenced by biological systems when evaluating the mode of action of bioactives.
Qiu F, Cai G, Jaki BU, Lankin DC, Franzblau SG, Pauli GF
Quantitative Purity-Activity Relationships of Natural Products: The Case of Anti-Tuberculosis Active Triterpenes from Oplopanax horridus
Journal of Natural Products 76: 413-419 (2013)
dx.doi.org/10.1021/np3007809
Abstract: The present study provides an extension of the previously developed concept of purity–activity relationships (PARs) and enables the quantitative evaluation of the effects of multiple minor components on the bioactivity of residually complex natural products. The anti-tuberculosis active triterpenes from the Alaskan ethnobotanical Oplopanax horridus were selected as a case for the development of the quantitative PAR (QPAR) concept. The residual complexity of the purified triterpenes was initially evaluated by 1D- and 2D-NMR and identified as a combination of structurally related and unrelated impurities. Using a biochemometric approach, the qHNMR purity and anti-TB activity of successive chromatographic fractions of O. horridus triterpenes were correlated by linear regression analysis to generate a mathematical QPAR model. The results demonstrate that impurities, such as widely occurring monoglycerides, can have a profound impact on the observed antimycobacterial activity of triterpene-enriched fractions. The QPAR concept is shown to be capable of providing a quantitative assessment in situations where residually complex constitution contributes toward the biological activity of natural products.
Napolitano J, Lankin D, Chen SN, Pauli GF
Complete 1H NMR Spectral Analysis of Ten Chemical Markers of Ginkgo biloba
Magnetic Resonances in Chemistry 50: 569-575 (2012)
dx.doi.org/10.1002/mrc.3829
Abstract: The complete and unambiguous 1H NMR assignments of ten marker constituents of Ginkgo biloba are described. The comprehensive 1H NMR profiles (fingerprints) of ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, bilobalide, quercetin, kaempferol, isorhamnetin, isoquercetin, and rutin in DMSO-d6 were obtained through the examination of 1D 1H NMR and 2D 1H,1H-COSY data, in combination with 1H iterative full spin analysis (HiFSA). The computational analysis of discrete spin systems allowed a detailed characterization of all the 1H NMR signals in terms of chemical shifts (δH) and spin-spin coupling constants (JHH), regardless of signal overlap and higher order coupling effects. The capability of the HiFSA-generated 1H fingerprints to reproduce experimental 1H NMR spectra at different field strengths was also evaluated. As a result of this analysis, a revised set of 1H NMR parameters for all ten phytoconstituents was assembled. Furthermore, precise 1H NMR assignments of the sugar moieties of isoquercetin and rutin are reported for the first time.
Pauli GF, Gödecke T, Jaki BU, Lankin DC
Quantitative 1H NMR: development and potential of a method for natural products analysis - An Update
Journal of Natural Products 75: 834-851 (2012)
dx.doi.org/10.1021/np200993k
Abstract: Covering the literature from mid-2004 until the end of 2011, this review continues a previous literature overview on quantitative 1H NMR (qHNMR) methodology and its applications in the analysis of natural products. Among the foremost advantages of qHNMR is its accurate function with external calibration, the lack of any requirement for identical reference materials, a high precision and accuracy when properly validated, and an ability to quantitate multiple analytes simultaneously. As a result of the inclusion of over 170 new references, this updated review summarizes a wealth of detailed experiential evidence and newly developed methodology that supports qHNMR as a valuable and unbiased analytical tool for natural product and other areas of research.
Napolitano J, Gödecke T, Rodriguez Brasco MF, Jaki BU, Chen SN, Lankin DC, Pauli GF
The Tandem of Full Spin Analysis and qHNMR for the Quality Control of Botanicals Exemplified with Ginkgo biloba
Journal of Natural Products 75: 238-248 (2012)
dx.doi.org/10.1021/np200949v
Abstract: Botanical dietary supplements and herbal remedies are widely used for health promotion and disease prevention. Due to the high chemical complexity of these natural products, it is essential to develop new analytical strategies to guarantee their quality and consistency. In particular, the precise characterization of multiple botanical markers remains a challenge. This study demonstrates how a combination of computer-aided spectral analysis and 1D quantitative 1H NMR spectroscopy (qHNMR) generates the analytical foundation for innovative means of simultaneously identifying and quantifying botanical markers in complex mixtures. First, comprehensive 1H NMR profiles (fingerprints) of selected botanical markers were generated via 1H iterative full spin analysis (HiFSA) with PERCH. Next, the 1H fingerprints were used to assign specific 1H resonances in the NMR spectra of reference materials, enriched fractions, and crude extracts of Ginkgo biloba leaves. These 1H fingerprints were then used to verify the assignments by 2D NMR. Subsequently, a complete purity and composition assessment by means of 1D qHNMR was conducted. As its major strengths, this tandem approach enables the simultaneous quantification of multiple constituents without the need for identical reference materials, the semiquantitative determination of particular subclasses of components, and the detection of impurities and adulterants.
Goedecke T, Napolitano J, Yao P, Nikolic D, Dietz B, Bolton J, van Breemen R, Chen SN, Lankin D, Farnsworth N, Pauli GF
Integrated standardization concept for Angelica botanicals using quantitative NMR
Fitoterapia 83: 18-32 (2012)
dx.doi.org/10.1016/j.fitote.2011.08.017
Abstract: Despite numerous in vitro/vivo and phytochemical studies, the active constituents of Angelica sinensis (AS) have not been conclusively identified for the standardization to bioactive markers. Phytochemical analyses of AS extracts and fractions that demonstrate activity in a panel of in vitro bioassays, have repeatedly pointed to ligustilide as being (associated with) the active principle(s). Due to the chemical instability of ligustilide and related issues in GC/LC analyses, new methods capable of quantifying ligustilide in mixtures that do not rely on an identical reference standard are in high demand. This study demonstrates how NMR can satisfy the requirement for simultaneous, multi-target quantification and qualitative identification. First, the AS activity was concentrated into a single fraction by RP-solid-phase extraction, as confirmed by an alkaline phosphatase, (anti-)estrogenicity and cytotoxicity assay. Next, a quantitative 1H NMR (qHNMR) method was established and validated using standard compounds and comparing processing methods. Subsequent 1D/2D NMR and qHNMR analysis led to the identification and quantification of ligustilide and other minor components in the active fraction, and to the development of quality criteria for authentic AS preparations. The absolute and relative quantities of ligustilide, six minor alkyl phthalides, and groups of phenylpropanoids, polyynes, and poly-unsaturated fatty acids were measured by a combination of qHNMR and 2D COSY. The qNMR approach enables multi-target quality control of the bioactive fraction, and enables the integrated biological and chemical standardization of AS botanicals. This methodology can potentially be transferred to other botanicals with active principles that act synergistically, or that contain closely related and/or constituents, which have not been conclusively identified as the active principles.
Chen SN, Lankin D, Chadwick L, Jaki B, Pauli GF
Dynamic Residual Complexity of Natural Products by qHNMR: Solution Stability of Desmethylxanthohumol
Planta Medica 75: 757-762 (2009)
Abstract: The use of chromatographic assays to assess the residual complexity of materials that are purified from natural sources by chromatographic means is, in a sense, a case of the fox watching the henhouse. Beside their static residual complexity, which is intrinsic to their metabolic origin, biologically active natural materials can also be involved in chemical reactions that lead to dynamic residual complexity. The present study examines the dynamics of the hop prenylphenol, desmethylxanthohumol (DMX), by means of quantitative 1H-NMR (qHNMR) in a setting that mimics in vitro and physiological conditions. The experiments provide a comprehensive, time-resolved, and mechanistic picture of the spontaneous isomerization of DMX into congeneric flavanones, including their 1H/2D isotopomers. Formation of the potent phytoestrogen, 8-prenylnaringenin (8PN), suggests that measurable estrogenic activity even of high-purity DMX is an artifact. Together with previously established qHNMR assays including purity activity relationships (PARs), dynamic qHNMR assays complement important steps of the post-isolation evaluation of natural products. Thus, qHNMR allows assessment of several unexpected effects that potentially break the assumed linkage between a single chemical entity (SCE) and biological endpoints.
Jaki BU, Franzblau SG, Chadwick LR, Lankin DC, Zhang F, Wang Y, Pauli GF
Purity Bioactivity Relationships – The Case of Anti-TB Active Ursolic Acid
Journal of Natural Products 71: 1742-1748 (2008)
Abstract: The present study explores the variability of biological responses from the perspective of sample purity and introduces the concept of purity−activity relationships (PARs) in natural product research. The abundant plant triterpene ursolic acid (1) was selected as an exemplary natural product due to the overwhelming number yet inconsistent nature of its approximate 120 reported biological activities, which include anti-TB potential. Nine different samples of ursolic acid with purity certifications were obtained, and their purity was independently assessed by means of quantitative 1H NMR (qHNMR). Biological evaluation consisted of determining MICs against two strains of virulent Mycobacterium tuberculosis and IC50 values in Vero cells. Ab initio structure elucidation provided unequivocal structural confirmation and included an extensive 1H NMR spin system analysis, determination of nearly all J couplings and the complete NOE pattern, and led to the revision of earlier reports. As a net result, a sigmoid PAR profile of 1 was obtained, demonstrating the inverse correlation of purity and anti-TB bioactivity. The results imply that synergistic effects of 1 and its varying impurities are the likely cause of previously reported antimycobacterial potential. Generating PARs is a powerful extension of the routinely performed quantitative correlation of structure and activity ([Q]SAR). Advanced by the use of primary analytical methods such as qHNMR, PARs enable the elucidation of cases like 1 when increasing purity voids biological activity. This underlines the potential of PARs as a tool in drug discovery and synergy research and accentuates the need to routinely combine biological testing with purity assessment..
Schinkovitz A, Pro S, Main M, Chen SN, Jaki BU, Lankin DC, Pauli GF
The Dynamic Nature of the Ligustilide Complex
Journal of Natural Products 71: 1606-1611 (2008)
Abstract: Monomeric phthalides such as Z-ligustilide (1) and Z-butylidenephthalide (2) are major constituents of medicinal plants of the Apiaceae family. While 1 has been associated with a variety of observed biological effects, it is also known for its instability and rapid chemical degradation. For the purpose of isolating pure 1 and 2, a gentle and rapid two-step countercurrent isolation procedure was developed. From a supercritical CO2 fluid extract of Angelica sinensis roots, the phthalides were isolated with high GC-MS purities of 99.4% for 1 and 98.9% for 2 and consistently lower qHNMR purities of 98.1% and 96.4%, respectively. Taking advantage of molarity-based qHNMR methodology, a time-resolved study of the dynamic changes and residual complexity of pure 1 was conducted. GC-MS and (qH)NMR analysis of artificially degraded 1 provided evidence for the phthalide degradation pathways and optimized storing conditions. Parallel qHNMR analysis led to the recognition of variations in time- and process-dependent sample purity and has impact on the overall assessment of time-dependent changes in complex natural products systems. The study underscores the importance of independent quantitative monitoring as a prerequisite for the biological evaluation of labile natural products such as monomeric phthalides..
Chen S, Turner A, Jaki B, Nikolic D, van Breemen R, Friesen B, Pauli GF
An Experimental Implementation of Chemical Subtraction
Journal of Pharmaceutical and Biomedical Analysis 46: 692-698 (2008)
Abstract: A preparative analytical method was developed to selectively remove (“chemically subtract”) a single compound from a complex mixture, such as a natural extract or fraction, in a single step. The proof of concept is demonstrated by the removal of pure benzoic acid (BA) from cranberry (Vaccinium macrocarpon Ait.) juice fractions that exhibit anti-adhesive effects versus uropathogenic Escherichia coli. Chemical subtraction of BA, representing a major constituent of the fractions, eliminates the potential in vitro interference of the bacteriostatic effect of BA on the E. coli anti-adherence action measured in bioassays. Upon BA removal, the anti-adherent activity of the fraction was fully retained, 36% inhibition of adherence in the parent fraction at 100 μg/mL increased to 58% in the BA-free active fraction. The method employs countercurrent chromatography (CCC) and operates loss-free for both the subtracted and the retained portions as only liquid–liquid partitioning is involved. While the high purity (97.47% by quantitative 1H NMR) of the subtracted BA confirms the selectivity of the method, one minor impurity was determined to be scopoletin by HR-ESI-MS and (q)HNMR and represents the first coumarin reported from cranberries. A general concept for the selective removal of phytoconstituents by CCC is presented, which has potential broad applicability in the biological evaluation of medicinal plant extracts and complex pharmaceutical preparations.
Pauli GF, Jaki BU, Lankin DC
A Routine Experimental Protocol for qHNMR Illustrated with Taxol
Journal of Natural Products 70: 589-595 (2007)
Abstract: Quantitative 1H 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 1H NMR experiments. Moreover, examination of different chemical lots of taxol (paclitaxel) and a Taxus brevifolia extract as working examples led to a blueprint for a generic approach to performing a routinely practiced 13C-decoupled qHNMR experiment and for recognizing its potential and main limitations. The proposed protocol is based on a newly assembled 13C 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.
Pauli
GF, Jaki BU, Lankin DC.
Quantitative 1H NMR: Development and Potential
of a Method for Natural Products Analysis. Journal of Natural Products 68: 133-149 (2005).
Abstract: Based on a brief revision of what constitutes state-of-the-art “quantitative experimental conditions” for 1H quantitative NMR (qHNMR), this comprehensive review contains almost 200 references and covers the literature since 1982 with emphasis on natural products. It provides an overview of the background and applications of qHNMR in natural products research, new methods such as decoupling and hyphenation, analytical potential and limitations, and compiles information on reference materials used for and studied by qHNMR. The dual status of natural products, being single chemical entities and valuable biologically active agents that need to be purified from complex matrices, results in an increased analytical demand when testing their deviation from the singleton composition ideal. The outcome and versatility of reported applications lead to the conclusion that qHNMR is currently the principal analytical method to meet this demand. Considering both 1D and 2D 1H NMR experiments, qHNMR has proved to be highly suitable for the simultaneous selective recognition and quantitative determination of metabolites in complex biological matrices. This is manifested by the prior publication of over 80 reports on applications involving the quantitation of single natural products in plant extracts, dietary materials, and materials representing different metabolic stages of (micro)organisms. In summary, qHNMR has great potential as an analytical tool in both the discovery of new bioactive natural products as well as the field of metabolome analysis.