Purification method for the isolation of monophosphate nucleotides from Champagne wine and their identification by mass spectrometry

Abstract

Monophosphate nucleotides are difficult to identify in Champagne wine because they are present in small concentrations in a complex mixture. A method for the isolation, separation and identification of reference compounds, which achieved on average 79% recovery (except for cytidine derivatives), was developed and applied to wine. Some monophosphate nucleotides were then isolated from a Champagne wine aged on lees for 8 years, by ultrafiltration followed by a semi-preparative HPLC step using a strong anion-exchange column. The fraction obtained was subjected to HPLC in a reversed-phase column to remove the salt previously introduced, before identification of compounds by HPLC coupled to a mass spectrometer. For the first time in wine, 5′-IMP, 5′-AMP, 5′-CMP, 5′-GMP, 5′-UMP and the 3′- and/or 2′-isomers of the four latter compounds were identified by comparing their HPLC and electrospray ionization mass spectrometry data with those of reference nucleotides.

Introduction

In the food industry, monophosphate nucleotides, particularly 5′-nucleotides such as 5′-guanosine monophosphate (5′-GMP) and 5′-inosine monophosphate (5′-IMP) [1] have commercial applications as flavoring ingredients and can be produced from enzymatic RNA degradation during yeast autolysis [2], [3], [4]. RNA represents more than 95% of the total content of nucleic acids within yeast cells [5] and is degraded more rapidly than DNA during autolysis [6]. One aspect of the traditional production of Champagne is the long aging on lees during which yeast autolysis occurs [7], releasing into the wine intracellular yeast constituents such as the degradation products of nucleic acids [8]. Formation of such nucleotides in Champagne wine can affect its sensory qualities [9], hence our interest in their identification. Some monophosphate nucleotides have already been detected and quantified by reversed-phase ion-pairing high-performance liquid chromatography (HPLC) with UV detection, in yeast extract [10], yeast autolysates [11] and beer [12]. On the other hand, unlike other nitrogenous compounds, nucleotides have received little attention in wine because their detection in this beverage is quite difficult. Wine monophosphate nucleotides are in an extremely complex mixture, together with organic acids, polysaccharides, phenolic compounds, proteins, peptides, amino acids, etc., which are present in higher concentrations and can therefore interfere with their measurement. Up to now, except for preliminary investigations [13], [14] performed with spectrophotometer observations, no study has led to the unequivocal identification of monophosphate nucleotides in Champagne wine. Generally, nucleic acids have been measured by methods based on HPLC with spectrometric detection. There are some difficulties using these methods due to low sensitivity and selectivity; indeed, complex mixtures contain many different types of products that interfere with the chromatographic detection. Although HPLC methods with electrochemical [15], [16], fluorometric [17], [18], [19], [20] and chemiluminescent nitrogen-specific detection [21] for the determination of some nucleotides have been reported, the method of choice for the identification of polar compounds such as nucleotides is mass spectrometry (MS), due to the development of soft ionization techniques such as electrospray ionization (ESI) [22].

The aim of our work was to develop a purification procedure for monophosphate nucleotides from Champagne wine to allow their identification by HPLC coupled to ESI-MS.