qNMR--a versatile concept for the validation of natural product reference compounds

The validation of reference compounds for natural products is a domain of the same physico-chemical methods that are used for their isolation, especially those techniques involving coupled high-resolution chromatography. Acknowledging the great problem of co-eluting impurities contained in the 'biogenetic cocktail' of a plant extract, there is a strong demand for non-chromatographic alternatives in the quality assessment of reference compounds. Because of this, the concept of qNMR is introduced as a versatile tool based on qualitative and quantitative 1H-NMR allowing the precise and simultaneous determination of both the compound content as well as the amount and nature of the impurities. As opposed to measuring carbons, 1H-NMR benefits from much higher sensitivity and is far more versatile for routine analysis with respect to time and cost. Since quantification of impurities is reliant upon their identification and, therefore, limited by knowledge about their structure, the concept emphasizes the high demand for qualitative reference dossiers including quality NMR data for profiling potential impurities which may be analogues, isomers, or degradation or oxidation products of the reference compounds. The qNMR concept is developed with focus on its potential in the certification and quality control of reference compounds. Taking into account published work in the field of quantitative NMR, selected natural products are analysed in order to elaborate suitable experimental parameters and to obtain preliminary validation data. The method is discussed with respect to sensitivity, precision and selectivity. Typical relative errors are found to be below 2% for the quantification of both the major analyte and the minor impurities even when the latter are contained at the 1% level only. Documentation of the conformity of signal integration and precision is based on measurements of a certified reference standard. Determination of the natural 13C isotope is suggested as an elegant method of validation because the content values could be reproduced with errors below 1%. The qNMR concept offers a rapid and efficient way to assess the purity of natural products in a single analytical step without the need of performing multiple analyses, while still offering the option to retain the substance. Thus, qNMR pays tribute to the increasing demands in reference compound certification, but also holds out the prospect of easy access to the valid characterisation of natural products tested in vitro or in vivo for their biological and pharmacological effects.