Time Course of the Evolution of Malic and Lactic Acids in the Alcoholic and Malolactic Fermentation of Grape Must by Quantitative 1 H NMR (qHNMR) Spectroscopy ALBERTO AVENOZA,JESU Ä S H. BUSTO,* NOELIA CANAL, AND JESU Ä S M. PEREGRINA Departamento de Quı ´mica, Grupo de Sı ´ntesis Quı ´mica de La Rioja, Universidad de La Rioja, UA-CSIC, E-26006 Logron ˜o, Spain Quantitative NMR can be used to monitor several processes that take place in the transformation of the must of wine grapes. The study described here focused attention on monitoring of the malic and lactic acid levels during the alcoholic and malolactic fermentation processes. The method allows the simultaneous quantification of both acids through a range of 1-3.2 mmol/L. The effectiveness of each process was assessed and compared by carrying out precise analyses using enzymatic methods. KEYWORDS: Quantitative NMR; wine; grape must; fermentation; malic acid; lactic acid INTRODUCTION Nuclear magnetic resonance (NMR) spectroscopy is a power- ful analytical technique and has an outstanding position in the field of complex chemical analysis of agricultural and food products as a potent analytical procedure for chemical charac- terization (1, 2). The nondestructive nature of NMR is one of its most attractive features and allows rapid measurements, analysis of samples without laborious sample preparation, and the noninvasive study of samples. Some recent examples that have been reported include the analysis of coffee (3), olive oil (4), and tomatoes (5). Given that the main use of this technique is for structure elucidation, NMR method development has mainly focused on the enhancement of qualitative information, although the quantitative aspects have actually been addressed since the early days of NMR (6, 7). In a recent and excellent review (8) concerning this topic, Pauli and co-workers introduced the term qHNMR as an abbreviation for proton-specific quantitative NMR and highlighted the enormous potential of qHNMR in the identification, characterization, and discovery of bioactive natural products and its potential uses in the area of metabolome analysis. The simple integration method and chemometric analysis could be used to obtain appropriate results for quantification. Partial least squares (PLS) regression has been successfully used for the quantification of components with partially overlapped signals but needs a set of representative samples. An integration method could be correctly used with non-overlapped signals and with careful manual integration (9). Quantitative NMR analysis of other nuclei has also been developed to study gasoline (10), the purity of technical grade agrochemicals (11), and the analysis of lignins (12). In cases when an accurate quantitative determination can be performed, NMR techniques represent a very powerful analytical method not only for structural determination but also for quantitative analysis. Recent developments in this field have provided evidence that NMR can be developed as a precise quantitative tool and a primary ratio analytical method (13). Recently, Maniara et al. conducted a detailed metrological comparison between the use of traditional chromatographic techniques and NMR (14). It has also been demonstrated that NMR represents a robust method that does not suffer from any significant effect in terms of analyst, instrument, magnetic field strength, or experimental parameters (15). There are only a few examples in the literature of quantitative NMR studies on beverages, and these include the quantification of organic and amino acids in beer (9), the quantification of chlorogenic acid (16) and (-)- epicatechin (17) in apple cider, and the quantification of methanol in a traditional Cypriot spirit (18). Wines consist of several hundred components that are present at different concentrations, with the major components being water, ethanol, glycerol, sugars, and organic acids. The NMR spectroscopy of wine has proven to be useful for assessing wine quality, for example, in the verification of wine origin and age and the effects of adulteration (19). In recent years, the use of high-resolution NMR techniques in the study of wine has attracted the interest of several groups and, as a result, one- dimensional (1D) (20) and two-dimensional (2D) (21) NMR experiments have been explored. The use of 13 C NMR spec- troscopy was introduced in wine analysis by Rapp et al., who showed that 13 C NMR can successfully be used for the detection of sugars, alcohols, organic acids, and amino acids (22). In addition, Kos ˇir et al. used intensities from 1 H NMR spectra for the quantification of succinic and acetic acids in wine (23). Given this background, and bearing in mind the excellent versatility of NMR for the study of samples that exhibit a time * Author to whom correspondence should be addressed (fax +34 941 299655; e-mail hector.busto@dq.unirioja.es). J. Agric. Food Chem. 2006, 54, 4715-4720 4715 10.1021/jf060778p CCC: $33.50 © 2006 American Chemical Society Published on Web 06/07/2006