Historical Background of qNMR. Quantitative NMR (qNMR) is almost as old as NMR itself. Early reports regarding the achievable precision of quantitation are inconsistent, and some of them even tended to deny NMR a role as a precision method by estimating the error to be in the 10% range. Interestingly, and with notable exceptions,textbook literature often does not emphasize the quantitative aspects of NMR and, thus, does not motivate educators and researchers to consider qNMR as an analytical tool. This stands in contrast to the authors' recent personal discussions with experienced NMR spectroscopists, as well as to the tenor of the publications cited in this review, according to which the quantitative power of 1H NMR and its broad applications are greatly underestimated. Moreover, recent developments in the field have provided evidence that NMR can be developed as a precise quantitative tool and, in time, can even be a primary analytical method.
As can be seen from Figure 1, there is a steadily increasing interest in qNMR over the past 40 years, as measured by the number of publications in the field (Chemical Abstracts). However, taking into account the overall rapid increase of publications in science, and especially when considering the statistics for natural products related qNMR (solid bars in Figure 1), there seems to be almost no gain in interest in the past 15 years, a period that has been exceptionally productive in terms of NMR hardware development. It must be noted, however, that the metabolomic studies mentioned below, which often involve (semi-) quantitative NMR analysis, are not included in this statistical picture, because the necessary qNMR keywords cannot be searched successfully since they are not included in the database entries of the corresponding publications. The importance of the qNMR methodology in this recently emerging area of research, however, indicates the rising impact of qNMR methodology on natural products research in general.
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Literature Background of qNMR. Because qNMR has been living in the shadow of the multifaceted and multidimensional qualitative NMR used in structure analysis, neither has it been used as widely and routinely nor is a recent and comprehensive overview of the literature available. However, Szantay and Evilia have reviewed systematically the general experimental factors known to interfere with quantitative determinations in NMR. Their articles cover relaxation, digitization, and instrumental parameters and provide valuable sources of information independent from the observed nuclei. Certainly noteworthy, while exclusively dealing with analyses of drugs and pharmaceuticals, is the extensive qHNMR work by Turczan and co-workers at the FDA, which to our best knowledge has not been summarized in a review format. Their experience shows that typical errors fall in the 0.5-2% range, and their reports serve as a valuable resource when it comes to the selection of qNMR reference standards (see below). The essential lack of reports describing the application of qHNMR in natural product research is confirmed in a 1989 1H/13C NMR review by Pieters and Vlietinck, who concluded that, despite the great potential of qNMR, suitability has to be established for each individual case. The excellent review series focused on 1H NMR by Rackham that begun in 1975, unfortunately, has been discontinued, leaving almost all of high-field qHNMR uncovered. The present review seeks to fill this gap and to provide a comprehensive survey of the qHNMR literature by discussing recent and forthcoming technological innovations, while concentrating on the applications of qHNMR to complex samples (mixtures) such as materials that are obtained from natural sources. Because the second most studied organic NMR nucleus (13C) is considerably less sensitive (1.6% of 1H sensitivity for an equal number of nuclei, augmented by a sensitivity loss due to the 1.1% natural abundance of 13C) and affords quantitative information significantly more difficult to obtain, in particular for small natural product samples, this review will focus on the 1H variant of qNMR (qHNMR). Unless NMR technology achieves another quantum leap in sensitivity, it is reasonable to hypothesize that proton NMR will remain the most suitable nucleus for quantitative studies, especially for natural products, and is preferred over the much more dispersive, but inherently less sensitive heteronuclei. One exception is 19F, with 88% of 1H sensitivity (100% 19F natural abundance), which due to its negligible background interference is evolving into a preferred quantitative tool in drug metabolism studies. The application of 19F NMR for studying natural products is very limited at this time,and while not presently enjoying practical widespread utility, it could become an important qualitative and quantitative screening tool in the search for new naturally occurring organofluorine compounds. Another relatively sensitive nucleus, which has been used for extensive qualitative and quantitative applications, is 31P NMR (7% of 1H sensitivity, 100% 31P natural abundance), but this is also beyond the scope of this review.
The present review (see organizational Figure 2) covers the scientific literature from 1982 to July 2004. A thorough manual screening has been performed of ca. 8000 primary hits obtained from the Chemical Abstracts database through the use of SciFinder, when searching the concept "quantitative NMR" (see Figure 1). It has been the experience of the authors in their own research involving qNMR, and from extensive communications with colleagues, that qNMR is much more frequently applied in the industrial sector and in the regulatory environment than is reflected in the published scientific literature. Therefore, the proceedings of two conferences have been included in the literature survey: first, beginning with 1988, the abstracts of the Experimental NMR Conference (ENC),which represents a foremost platform for information exchange on NMR topics, and second the recent "Small Molecules Are Still Hot" (SMASH) NMR conferences,albeit providing few of the documented qNMR applications. The review is organized in two major sections. The first section deals with a discussion of the experimental aspects of the qNMR experiment, and the second section is devoted to a compilation of relevant applications of the qNMR technique for natural product analysis.