qNMR Literature

The body of the scientific literature on qNMR technologies and applications continues to grow rapidly. This page is intended to provide a port of entry to the qNMR literature. Given its rapid growth, the emphasis of this list has been shifted to provide links to reviews rather than original research papers. The UIC qNMR team has published two comprehensive reviews on qHNMR and its applications in natural products research. While the 2005 qNMR review provides 193 references covering the literaure since the 1950s until 2004, the 2012 qNMR review provides an additional 170 references for the period since 2005 until end of 2011. A further follow-up review is in preparation. In the meantime, select review publications will be linked on this page.

qNMR Literature since 2005

Giraudeau P. Quantitative 2D liquid-state NMR. Magnetic Resonance in Chemistry: 62 (6), 259–272 (2014) doi: 10.1002/mrc.4068
Abstract: Two-dimensional (2D) liquid-state NMR has a very high potential to simultaneously determine the absolute concentration of small molecules in complex mixtures, thanks to its capacity to separate overlapping resonances. However, it suffers from two main drawbacks that probably explain its relatively late development. First, the 2D NMR signal is strongly molecule-dependent and site-dependent; second, the long duration of 2D NMR experiments prevents its general use for high-throughput quantitative applications and affects its quantitative performance. Fortunately, the last 10 years has witnessed an increasing number of contributions where quantitative approaches based on 2D NMR were developed and applied to solve real analytical issues. This review aims at presenting these recent efforts to reach a high trueness and precision in quantitative measurements by 2D NMR. After highlighting the interest of 2D NMR for quantitative analysis, the different strategies to determine the absolute concentrations from 2D NMR spectra are described and illustrated by recent applications. The last part of the manuscript concerns the recent development of fast quantitative 2D NMR approaches, aiming at reducing the experiment duration while preserving – or even increasing – the analytical performance. We hope that this comprehensive review will help.

Davies SR, Jones K, Goldys A, Alamgir M, Chan BK, Elgindy C, Mitchell PS, Tarrant GJ, Krishnaswami MR, Luo Y, Moawad M, Lawes D, Hook JM. Purity assessment of organic calibration standards using a combination of quantitative NMR and mass balance. Analytical and Bioanalytical Chemistry: in press (DOI 10.1007/s00216-014-7893-6) (2014) doi: 10.1007/s00216-014-7893-6
Abstract: Quantitative NMR spectroscopy (qNMR) has been examined for purity assessment using a range of organic calibration standards of varying structural complexities, certified using the traditional mass balance approach. Demonstrated equivalence between the two independent purity values confirmed the accuracy of qNMR and highlighted the benefit of using both methods in tandem to minimise the potential for hidden bias, thereby conferring greater confidence in the overall purity assessment. A comprehensive approach to purity assessment is detailed, utilising, where appropriate, multiple peaks in the qNMR spectrum, chosen on the basis of scientific reason and statistical analysis. Two examples are presented in which differences between the purity assignment by qNMR and mass balance are addressed in different ways depending on the requirement of the end user, affording fit-for-purpose calibration standards in a cost-effective manner.

REVIEW 2012 Comprehensive Overview of qNMR methodology and Review of qHNMR Applications in Natural Products Chemistry - An update

Pauli GF, Jaki BU, Gödecke T, 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); doi: 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.

Routine qHNMR Experiment and Standard Parameter Set for Bruker, Jeol, and Varian Spectrometers

Pauli G.F.; Jaki B.; Lankin D. A Routine Experimental Protocol for qHNMR Illustrated with Taxol. Journal of Natural Products 2007, 70(4), 589-595.

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.

qNMR Literature until 2004

REVIEW 2005 Comprehensive Overview of qNMR methodology and Review of qHNMR Applications in Natural Products Chemistry

Pauli G.F.; Jaki B.; Lankin D. Quantitative 1H NMR: Development and Potential of a Method for Natural Products Analysis. Journal of Natural Products 2005, 68(1), 133-149.

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.

Pauli G.F.; Jaki B.; Lankin D.; Burton, I.; Walter, J. Quantitative NMR (qNMR) of Bioactive Natural Products in Bioactive Natural Products: Detection, Isolation and Structural Determination, 2nd Edition, Colegate, S; Molyneux, R (Eds.), CRC Press (2008)

Abstract: The benefits and long-term implications of quantitative NMR (qNMR) outweigh the investment in effort that is required when qNMR is incorporated into routine characterization protocols for biologically active natural products. Informally, qNMR can be considered “the new kid on the block” in a neighborhood inhabited by a myriad of routine 1D and 2D NMR experiments, apart from other spectroscopic methods (MS, CD/ORD, IR etc). This book chapter deals with quantitative 1H NMR (qHNMR), explains qNMR concepts, and outlines qNMR technology. By providing examples of applications, it develops a perspective of how the new “fellow citizen” can be advantageously integrated into the neighborhood of advanced analytical tools and foster the characterization of bioactive natural products.

Research Articles on quantitative NMR
(in chronologic order)

Pauli G.F. qNMR - A versatile concept for the validation of natural product reference compounds. Phytochem. Anal. 2001, 12(1), 28-42.

Jaki B.; Sticher O.; Veit M.; Fröhlich R.; Pauli G.F. Evaluation of Glucoiberin Reference Material from Iberis amara by Spectroscopic Fingerprinting. J. Nat. Prod. 2002, 65(4), 517-522.

Wittig J.; Leipolz I.; Graefe E.U.; Jaki B.; Treutter D.; Veit M. Quantification of procyanidins in oral herbal medicinal products containing extracts of Crataegus species. Arzneimittelforschung 2002, 52(2), 89-96.

Saito, T.; Nakaie, S.; Kinoshita, M.; Ihara, T.; Kinugasa, S.; Nomura, A.; Maeda, T. Practical guide for accurate quantitative solution state NMR analysis. Metrologia 2004, 41, 213-218.

Saed Al Deen T.; Brynn Hibbert D.; Hook J.M.; Wells R.J. Quantitative nuclear magnetic resonance spectrometry - II. Purity of phosphorus-based agrochemicals glyphosate (N-(phosphonomethyl)-glycine) and profenofos (O-(4-bromo-2-chlorophenyl) O-ethyl S-propyl phosphorothioate) measured by 1H and 31P QNMR spectrometry. Analytica Chimica Acta 2002, 474(1), 125-135.

Wells, R.; Cheung, J.; Hook, J. M. Dimethylsulfone as a universal standard for analysis of organics by QNMR. Accred. Qual. Assur. 2004, 9, 450-456.

Pinciroli, V.; Biancardi, R.; Visentin, G.; Rizzo, V. The Well-Characterized Synthetic Molecule: A Role for Quantitative 1H NMR. Org. Proc. Res. Devel. 2004, 8, 381-384.

Burton, I. W.; Quilliam, M. A.; Walter, J. A. Quantitative 1H NMR with external standards: use in preparation of calibration solutions for algal toxins and other natural products. Anal. Chem. 2005, 77, 3123-3131.

Hays, P. A. Proton nuclear magnetic resonance spectroscopy (NMR) methods for determining the purity of reference drug standards and illicit forensic drug seizures. J. Forensic Sci. 2005, 50, 1342-1360.

Rizzo, V.; Pinciroli, V. Quantitative NMR in synthetic and combinatorial chemistry. J. Pharm. Biomed. Anal. 2005, 38, 851-857.

Letot, E.; Koch, G.; Falchetto, R.; Bovermann, G.; Oberer, L.; Roth, H. J. Quality control in combinatorial chemistry: Determinations of amounts and comparison of the "purity" of LC-MS-purified samples by NMR, LC-UV and CLND. J. Comb. Chem. 2005, 7, 364-371.

Dumas, M.-E.; Maibaum, E. C.; Teague, C.; Ueshima, H.; Zhou, B.; Lindon, J. C.; Nicholson, J. K.; Stamler, J.; Elliott, P.; Chan, Q.; Holmes, E. Assessment of Analytical Reproducibility of 1H NMR Spectroscopy Based Metabonomics for Large-Scale Epidemiological Research: the INTERMAP Study. Anal. Chem. 2006, 78, 2199-2208.

Shao, G.; Kautz, R.; Peng, S.; Cui, G.; Giese, R. W. Calibration by NMR for quantitative analysis: p-Toluenesulfonic acid as a reference substance. Journal of Chromatography, A 2007, 1138, 305-308.

Ala-Korpela, M.; Lankinen, N.; Salminen, A.; Suna, T.; Soininen, P.; Laatikainen, R.; Ingman, P.; Jauhiainen, M.; Taskinen, M.-R.; Heberger, K.; Kaski, K. The inherent accuracy of 1H NMR spectroscopy to quantify plasma lipoproteins is subclass dependent. Atherosclerosis (Amsterdam, Netherlands) 2007, 190, 352-358.

Zulak, K. G.; Weljie, A. M.; Vogel, H. J.; Facchini, P. J. Quantitative H-1 NMR metabolomics reveals extensive metabolic reprogramming of primary and secondary metabolism in elicitor-treated opium poppy cell cultures. BMC Plant Biol 2008, 8, 5.

Craigie, J. S.; MacKinnon, S. L.; Walter, J. A. Liquid seaweed extracts identified using H-1 NMR profiles. J Appl Phycol 2008, 20, 665-671.

Vishwanathan, K.; Babalola, K.; Wang, J.; Espina, R.; Yu, L.; Adedoyin, A.; Talaat, R.; Mutlib, A.; Scatina, J. Obtaining exposures of metabolites in preclinical species through plasma pooling and quantitative NMR: addressing metabolites in safety testing (MIST) guidance without using radiolabeled compounds and chemically synthesized metabolite standards. Chem. Res. Toxicol. 2009, 22, 311-322.

Kontogianni, V. G.; Exarchou, V.; Troganis, A.; Gerothanassis, I. P. Rapid and novel discrimination and quantification of oleanolic and ursolic acids in complex plant extracts using two-dimensional nuclear magnetic resonance spectroscopy-Comparison with HPLC methods. Anal. Chim. Acta 2009, 635, 188-195.

Albers, M. J.; Butler, T. N.; Rahwa, I.; Bao, N.; Keshari, K. R.; Swanson, M. G.; Kurhanewicz, J. Evaluation of the ERETIC method as an improved quantitative reference for 1H HR-MAS spectroscopy of prostate tissue. Magn. Res. Med. 2009, 61, 525-532.

Recent Publications Employing qNMR Methodology

Manetti C.; Bianchetti C.; Bizzarri M.; Casciani L.; Castro C.; D'Ascenzo G.; Delfini M.; Di Cocco M.E.; Lagana A.; Miccheli A.; Motto M.; Conti F. NMR-based metabonomic study of transgenic maize. Phytochemistry 2004, 65(24), 3187-3198.

Fernandez-Ramirez A.; de la Luz Salazar-Cavazos M.; Rivas-Galindo V.; Ceniceros-Almaguer L.; de Torres N.W. Determination of isoperoxisomicine A1 content in peroxisomicine A1 batches by 1H NMR. Anal. Lett. 2004, 37(12), 2433-2444.

Wells R.; Cheung J.; Hook J.M. Dimethylsulfone as a universal standard for analysis of organics by QNMR. Accred. Qual. Assur. 2004, 9(8), 450-456.

Jaki, B.; Franzblau, S. G.; Pauli, G. F. Phytochem. Anal. 2004, 15, 213-219.


Ph.D. Dissertations on quantitative NMR
(in chronologic order)

Al-Deen, Tareq. Validation of quantitative nuclear magnetic resonance (QNMR) spectroscopy as a primary ratio analytical method for assessing the purity of organic compounds: a metrological approach; Ph.D. Dissertation, School of Chemical Sciences, University of New South Wales: Sydney (Australia), 2002

Abstract: Recently, there has been a strong demand for non-chromatographic alternatives for the purity assessment of organic compounds. The potential offered by quantitative NMR spectrometry (QNMR) as a viable alternative to chromatographic methods for quantifying major component and impurity is considerable. The NMR spectrum of a solution shows all soluble organic substances present, and the signal intensity is directly proportional to the quantity of the nucleus being measured. QNMR is a primary ratio analytical method (i.e. one that does not depend on calibration with a pure sample of the analyte) as just a single internal standard, unrelated to the target analyte, can be used to determine the percentage purity of the analyte providing the NMR signals of the internal standard do not overlap with those of the target analyte. A method validation study for QNMR methodology (Chapter 2) was performed and statistical designs were applied to examine the following validation aspects: accuracy, precision, sensitivity, selectivity, repeatability, reproducibility, robustness and ruggedness, limits of detection and limits of quantification. The QNMR method and validation was documented so that it can be clearly and unambiguously implemented. The amount of substance (as % active ingredient) was measured for a range of commercial agricultural and pharmaceutical organic compounds by 1H, 31P and 19F NMR spectrometry. The measurement uncertainty associated with these quantitative analyses is described in compliance with ISO guide 17025, and a detailed description of the preparation of the related uncertainty budgets is reported (Chapter 3). The traceability of the measurements to the SI is demonstrated by the use of certified reference standards. QNMR is shown to be one of the most precise and accurate quantitative methods that gives highly repeatable and reproducible measurements provided that certain requirements are met. Such influence factors and requirements are discussed in depth from the experimental and theoretical point of view (Chapter 4). Results show that the accuracy and precision are dependent on the selected acquisition and processing parameters. Results for 1H QNMR of the purity of the chosen chemical were compared with HPLC data and found to be of better precision (Chapter 5). Measurements obtained by 31P and 19F NMR (Chapter 6 & 7) were also of high quality and agreed with the 1H QNMR measurements.

Malz, Frank. Quantitative NMR-Spektroskopie als Referenzverfahren in der analytischen Chemie; Ph.D. Dissertation, Humboldt-Universität Berlin: Berlin, 2003, 147 pp.

Deubner, Ralph. Quantitative NMR-Spektroskopie zur Reinheitsbestimmung von Arzneistoffen; Ph.D. Dissertation, University of Wuerzburg: Wuerzburg, 2004 [pdf]

Abstract: Quantitative analysis It could be shown on the basis of different substances that the NMR spectroscopy is able to quantify impurities of pharmaceuticals. For the quantification of impurities of the antidepressive drug fluvoxamine the pharmacopoeia describes an ion-pair chromatographic method. Since the antidepressive activity resides on the E-isomer the content of the Z-isomer has to be lim-ited. Since ion-pair chromatography often lacks of robustness, qNMR is an alternative. The quantitative evaluation of 1H NMR spectra of a mixture of the two isomers is possible without extensive sample preparation. The signals of the hydrogens at position 2 of both isomers are well separated in the spectrum. If these are quantitative evaluated, under optimized conditions, e.g. with respect to T1-relaxation time, it is possible to limit the content of the Z-isomer to 0.2%. For analysis of degradation products of perphenazine enantate qNMR is a suitable method. Perphenazine enantate can be cleaved by ester hydrolysis. Using the integral area of the signal of the hydrogens at position 21 of per-phenazine in comparison to the integral area of the overlapping signals of the hydrogens at position 11 of both substances perphenazine and perphenazine enantate it was possible to quantify perphenazine as a degradation product of perphenazine as well perphenazine enantate in perphenazine. Additionally the area of the aromatic hydrogens can be used for the analysis of the oxidation. The oxidation of the sulfur of the the phenothiazine moiety to the sulfoxide and the sulfone changes the chemical shifts of the corresponding hydrogens. This enables a half-quantitative assessment. Finally it was possible to quantify the two epimers quinine and quinidine as an impurity in either drug. Again signals of both substances could be identified to be used for quantification. In both cases quinine as impurity of quinidine and vice versa the impurity can be limited to 2.5 per cent as required by the pharmacopeias. Gentamicin sulfate 1H-NMR spectroscopy was also used as an analytical method to characterize the composition of gentamicin. Gentamicin is produced from Micromonospora purpurea by fermentation and consists of different main and side components. The composition varies when applying different fermentation conditions. A number of deaths in connection with the application of the antibiotic drug gentamicin in the USA were reported. Different impurities were suspected to be responsible for these deaths. In the current pharmacopoeia monograph an HPLC method is described which is able to quantify all main components but does not separate all side components. In addition, the total elution time is long. Thus late eluting substances show very broad peaks. Furthermore the used pulsed amperometric detector is very sensitive and the method is not very robust over all. Using one- and two-dimensional routine NMR techniques and selective TOCSY experiments it was possible to assign all signals in the 1H- and 13C-NMR spectra of all main and side components. The area of the anomeric hydrogens is appropriate to evaluate the purity of gentamicin and the proportions between the main components. In the part of the 400 MHz 1H-NMR spectrum integration of the H20 signals of the main components is impossible due to the missing baseline separation. However, using 600 MHz spectra integration is possible. In this way the proportions between the main components can be determined. The results achieved in this way show good accordance to the results obtained with an MEKC separation. More than 40 gentamicin samples from different manufactures were studied, com-pared and divided into several groups, based on their impurity profile. As a lead impurity sisimocin has been identified. Besides that, the comparison of the impurity profiles enables to trace the trade ways. Some of the samples which led to the deaths were among the samples being classified in the impure groups.

Forshed, Jenny. Processing and analysis of NMR data: Impurity determination and metabolic profiling; Ph.D. Dissertation, Stockholm University, Department of Analytical Chemistry, 2005, [pdf]

Abstract: This thesis describes the use of nuclear magnetic resonance (NMR) spectrometry as an analytical tool. The theory of NMR spectroscopy in general and quantitative NMR spectrometry (qNMR) in particular is described and the instrumental properties and parameter setups for qNMR measurements are discussed. Examples of qNMR are presented by impurity determination of pharmaceutical compounds and analysis of urine samples from rats fed with either water or a drug (metabolic profiling). The instrumental parameter setup of qNMR and traditional data pre-treatments are examined. Spectral smoothing by convolution with a triangular function, which is an unusual application in this context, was shown to be successful regarding the sensitivity and robustness of the method in paper II. In addition, papers III and IV comprise the field of peak alignment, especially designed for 1H-NMR spectra of urine samples. This is an important preprocessing tool when multivariate analysis is to be applied. A novel peak alignment method was developed and compared to the traditional bucketing approach and a conceptually different alignment method. Univariate, multivariate, linear and nonlinear data analyses were applied to qNMR data. In papers I–II, calibration models were created to examine the potential of qNMR for these applications. The data analysis in papers III–VI was mainly explorative. The potential of data fusion and data correlation was examined in order to increase the possibilities of analysing the highly complex samples from metabolic profiling (papers V–VI). Data from LC/MS analysis of the same samples were used with the 1H-NMR data in different ways. Correlation analyses between the 1H-NMR data and the drug metabolites identified from the LC/MS data were also performed. In this process, data fusion proved to be a valuable tool.


Other Literature and Materials on quantitative NMR