Nuclear Magnetic Resonance Spectroscopy as a Quantitative Tool To Determine the Concentrations of Biologically Produced Metabolites: Implications in Metabolites in Safety Testing

Abstract

Nuclear magnetic resonance (NMR) spectroscopy has traditionally been considered as an indispensable tool in elucidating structures of metabolites. With the advent of Fourier transform (FT) spectrometers, along with improvements in software and hardware (such as high-field magnets, cryoprobes, versatile pulse sequences, and solvent suppression techniques), NMR is increasingly being considered as a critical quantitative tool, despite its lower sensitivity as compared to mass spectrometry. A specific quantitative application of NMR is in determining the concentrations of biologically isolated metabolites, which could potentially be used as reference standards for further quantitative work by liquid chromatography/mass spectrometry. With the recent demands from regulatory agencies on quantitative information on metabolites, it is proposed that NMR will play a significant role in strategies aimed at addressing metabolite coverage in toxicological species. Traditionally, biologically isolated metabolites have not been considered as a way of generating "reference standards" for further quantitative work. However, because of the recent FDA guidance on safety testing of metabolites, one has to consider means of authenticating and quantitating biologically or nonbiologically generated metabolites. 1H NMR is being proposed as the method of choice, as it is able to be used as both a qualitative and a quantitative tool, hence allowing structure determination, purity check, and quantitative measurement of the isolated metabolite. In this publication, the application of NMR as a powerful and robust analytical technique in determining the concentrations of in vitro or in vivo isolated metabolites is discussed. Furthermore, to demonstrate the reliability and accuracy of metabolite concentrations determined by NMR, validation and cross-validation with gravimetric and mass spectrometric methods were conducted.

Application of Quantitative NMR in Pharmacological Evaluation of Biologically Generated Metabolites: Implications in Drug Discovery

“…The results are presented in Table 1. It was shown by Espina et al (2009) during the validation of the quantitative NMR method that the concentrations of metabolites measured using this approach were within 10% of the gravimetrically determined nominal values. Hence, the measured concentration of the isolated metabolite was used to obtain the absolute amount of metabolite that was isolated from the biological source.…”

“…With recent improvements in NMR technology, NMR is now considered to be an important quantitative tool despite its lower sensitivity compared with those of the more widely used mass spectrometers. In response to the demands from regulatory agencies to better define the exposures of metabolites in humans and in preclinical species (U.S. Food and Drug Administration, 2008), strategies based on the application of NMR as a quantitative tool for metabolite quantitation have been proposed (Dear et al, 2008;Espina et al, 2009). NMR was used in the past as an analytical tool to determine concentrations of synthetic and biosynthetic products (Warren et al, 1976;Vinson and Kozak, 1978;Werner et al, 1997;Groen et al, 1998;Silvestre et al, 2001;Holzgrabe et al, 2005;Pauli et al, 2005Pauli et al, , 2007Pauli et al, , 2008Rizzo and Pinciroli, 2005;Diehl et al, 2007;Shao et al, 2007), metabolites, catabolites, and endogenous compounds in biological fluids (MaletMartino et al, 1986;Monté et al, 1994;Desmoulin et al, 2002;Orhan et al, 2004;Skordi et al, 2004;Moazzami et al, 2007), or impurities present in products (Hays, 2005;Malz and Jancke, 2006).…”

“…The exact concentration of the isolated metabolite was measured with NMR using the calibration curve constructed with compound 1 as described previously by Espina et al (2009). In brief, the NMR spectra of the parent compound were obtained at five different concentrations and a calibration curve was created on the basis of the integration of aromatic proton signals (average).…”

Meeting Report: Metabolites in Safety Testing (MIST) Symposium—Safety Assessment of Human Metabolites: What’s REALLY Necessary to Ascertain Exposure Coverage in Safety Tests?

“…The currently available techniques may be integrated to reliably find and structurally identify the metabolites from the plasma and urine samples that are already collected in typical phase I clinical trials. And semiquantitation of metabolites using liquid chromatography (LC)-UV, LC/MS/MS peak area ratio comparison (10,11,13), radiolabeled calibrant (5-7), and quantitative NMR standards (8,9,14) can be employed to compare the exposures to the metabolites in animals to humans. This allows a sponsor to comply with regulatory expectations for metabolite safety assessment without the need to wait for the conventional 14 C-ADME studies that are usually conducted in phases II or III.…”

Analytical Methods for Quantification of Drug Metabolites in Biological Samples

“…Such approach has become even more important for metabolite evaluation in the light of recently introduced FDA guidelines on metabolites in safety testing, which recommends that all metabolites greater than 10 percent of parent drug should be examined [1]. Some further examples of metabolite quantification using accelerator MS [47], inductively coupled plasma MS [43], chemiluminescene nitrogen detector [48], quantitative NMR [49] and evaporative light-scattering detector [50] are given.…”