13 C NMR Spectroscopy for the Differentiation of Enantiomers Using Chiral Solvating Agents Míriam Pe ́ rez-Trujillo,* ,†,‡ Eva Monteagudo, † and Teodor Parella †,‡ † Servei de Ressona ̀ ncia Magnè tica Nuclear, Facultat de Cie ̀ ncies i Biocie ̀ ncies, Universitat Autò noma de Barcelona, Bellaterra 08193, Catalonia, Spain ‡ Departament de Química, Facultat de Cie ̀ ncies i Biocie ̀ ncies, Universitat Autò noma de Barcelona, Bellaterra 08193, Catalonia, Spain * S Supporting Information ABSTRACT: The utility of 13 C NMR spectroscopy for the differentiation of enantiomers using chiral solvating agents (CSA) is stated. Three examples involving the enantiodiffer- entiation of a drug, a metabolite and a reactant in aqueous and organic solutions have been chosen to show it. The intrinsic high dispersion of 13 C nucleus, as well as the singlet nature of the signals in standard experiments makes 13 C NMR experiments a powerful alternative or complement to 1 H NMR experiments; specially, when studying pure compounds with complex proton spectra or mixtures of compounds, as in chiral metabonomics, where severe overlapping exists in proton spectrum. To evaluate and compare the quality of the enantioresolution of a NMR signal we introduce the enantiodifferentiation quotient, E, that considers the complexity of 1 H multiplets (and in general the width) of the original signal. It has been observed that the error in the measurement of the enantiomeric molar ratio can be related to the E value. The sensitivity and experimental time of a wide range of chiral analyte concentrations were also assessed. M ost of the endogenous metabolites are chiral com- pounds, as well as many drugs and reactants. The different character of two enantiomeric molecules is manifested when they are within a chiral environment, which is intrinsic in nature: endogenous metabolites can show different biological activities or functionalities (e.g., D- and L-serine 1 ) and drugs and exogenous metabolites can manifest different pharmaco- logical activities or toxicities (e.g., thalidomide, ethambutol, naproxen, and their degradation products). 2 Though the different behavior of enantiomeric molecules has been known for many decades, differentiating enantiomers and identifying them is still a tough task for scientists. Much attention is paid in distinguishing enantiomers in di fferent areas, such as pharmacology and the pharmaceutical industry, and in metabonomics, chiral metabonomics. In this work, we center on NMR spectroscopy and in the use of chiral solvating agents (CSA) 4-7 to induce enantiospecificity to the analysis. One of the main attractiveness of this methodology resides in its simplicity and easiness, which makes it compatible with chiral metabonomic studies. This approach is rapid, robust, involves minimal sample manipu- lation, and does not require derivatization or purification of the sample. The induction of enantiodifferentiation is the result of the formation of diastereoisomeric complexes between the CSA (a chiral single enantiopure molecule) and the two enantiomers of the analyte, via noncovalent interactions. The resulting complexes can be differentiated by NMR via their different complexation-induced chemical shift (δ) changes. Because the two diastereoisomeric complexes are in fast exchange with the individual components on the NMR time scale, a population- weighted average set of chemical shifts results. Typically, these differences are denoted as ΔΔδ and reported as the difference in the observed change in the δ of one enantiomer (e.g., Δδ R ) with respect to the other (e.g., Δδ S ), where these changes are relative to the chemical shift of the enantiomers in the absence of a chiral auxiliary. 1 H is by further the most used nuclei for observing the enantiodifferentiation by 1D 1 H NMR spectroscopy, 7 since protons are present in almost all molecules (together with carbon atoms) and because of its high sensitivity (other studies have been done using 19 F 4 and 31 P 8 NMR spectroscopy). However, it has two main drawbacks: the intrinsic low dispersion of the spectrum (chemical shift range from 0 to 12 ppm) and the signal multiplicities due to 1 H- 1 H scalar coupling, which turns out into typical situations of severe overlapping that hampers the observation of enantiodiffer- entiated peaks and/or situations of partial enantioresolution that can lead to considerable errors in the accurate R/S molar ratio measurement. The overlapping problem becomes critical when studying systems with complex 1 H NMR spectra. Typical cases are: the enantiomeric mixture of a pure compound with a complex proton spectrum and a (complex) mixture of different Received: July 25, 2013 Accepted: October 14, 2013 Published: October 14, 2013 Article pubs.acs.org/ac © 2013 American Chemical Society 10887 dx.doi.org/10.1021/ac402580j | Anal. Chem. 2013, 85, 10887-10894