¹³C/¹²C isotope ratios of organic acids, glucose and fructose determined by HPLC-co-IRMS for lemon juices authenticity

Highlights

• Focus on the application of HPLC-co-IRMS to control lemon juice authenticity.

• Technical optimisation for simultaneous determination of δ¹³C values of organic acids, glucose, fructose.

• Lemon data bank elaboration based on carbon isotopic ratios of organic acids, glucose and fructose.

• Application to commercial lemon and lime juices authenticity.

Abstract

High performance liquid chromatography linked to isotope ratio mass spectrometry via an interface allowing the chemical oxidation of organic matter (HPLC-co-IRMS) was used to simultaneously determine carbon 13 isotope ratio (δ¹³C) of organic acids, glucose and fructose in lime and lemon juices. Because of the significant difference between organic acids and sugars concentrations, the experimental protocol was optimised by applying a “current jump” to the IRMS device. The filament current is increased of 300 μA during elution in order to enhance IRMS sensitivity. Then, analysis were performed on 35 lemon and lime fruits from various geographical origins and squeezed in the laboratory. An overall average δ¹³C values of −25.40 ± 1.62‰, −23.83 ± 1.82‰ and −25.67 ± 1.72‰ is found for organic acids mixture mainly made up of citric acid, glucose and fructose, respectively. These authentic samples allowed the definition of a confidence domain to which have been confronted 30 commercial juices (24 “pure juices” and 6 coming from concentrate). Among these 30 samples, 10 present δ¹³C values outside the defined range revealing an added “C4” type organic acids or sugars, addition not specified on the label that is not in agreement with EU regulation.

Introduction

Lemons and limes are commercialised as fresh fruits or as juices, oils can also be found on the market. Until now, geographical origin does not appear as a real commercial argument and few labels mentioned the lemon/lime juice origin. As a result, geographical origin studies on these matrix are scarce (Barnes, 1997, Pellerano et al., 2008). The price of lemon and concentrate juice is determined on the basis of titratable acidity, i.e. citric acid concentration essentially, and sugar content. Thus, adulterations could be performed by the addition of exogenous sugar and/or citric acid. Moreover, European legislation elaborated regulations fixing the fruit juices trade including protected geographical indication (PGI). The addition of various acidifying compounds is authorised as well as the addition of sugars according to European reglementation (Directive, 1995, Directive, 2001). The use of such authorised compounds must be mentioned in the ingredients list of the product label and the simultaneous addition of sugar and acidifying product is prohibited (Directive, 2001). Moreover, sugar addition in fruit juices will be no more authorised from 28 October 2013 (Directive, 2012). As a result, most of the research works are focus on the characterisation of the authenticity of main components of lemon juice and several pathways were envisaged to detect such additions.

Concentration ratio between citric and isocitric acid has been used to detect added citric acid. Unfortunately, this concentration ratio range is large leading to undetected citric acid addition (AIJN). Citric acid, industrially produced, results from the fermentation of various sources like beet and cane molasses, corn syrups i.e. from plants with either “C3” or “C4” type metabolisms. One of the main differences between these plants carbohydrates is their carbon 13 isotope ratio (δ¹³C or ¹³C/¹²C): “C3” carbohydrates δ¹³C values are in the range −34 < δ¹³C < −24‰ whereas for “C4” carbohydrates, the range is −17 < δ¹³C < −10‰ (Meier-Augenstein, 1999). As lemons have a “C3” metabolism, only important amount of “C3” type compound could influence isotopic ratios. Therefore, isotope ratio mass spectrometry (IRMS) determination became a choice method for “C4” type citric acid and sugars adulteration (Doner, 1985, Jamin et al., 1998a). In order to improve the authenticity control, isotope ratio of fruit proteins were used as an internal reference (Jamin et al., 1998b). Combination of multi-isotope analysis also allowed the detection of “C3” type citric acid adulteration (Gonzalez et al., 1998, Jamin et al., 2005). In those studies, a prerequisite to obtain isotopic information is the extraction and separation of each component from the fruit matrix, moreover for some experiments, citric acid needs to be derivated to be studied by ²H-NMR. These purification steps could limit the use of those techniques in authenticity control routine applications. Moreover, the determination of carbon 13 isotope ratio of sugars is performed on the overall mixture (sucrose + glucose + fructose) and this global determination of isotope ratios could hide some sugars additions.

Recently, the link between high performance liquid chromatography (HPLC) and isotope ratio mass spectrometry (IRMS) was achieved through an interface insuring a chemical oxidation (co). A recent review (Godin & McCullagh, 2011) details some applications of this technique that allows δ¹³C determination of single compounds coming from a complex mixture without any purification steps. Despite its potential uses in food authenticity, it has been scarcely applied for this type of application. To our knowledge, the link HPLC-co-IRMS has only been applied for honey, (Cabanero et al., 2006, Elflein and Raezke, 2008) caffeine authentication, (Zhang, Kujawinski, Federherr, Schmidt, & Jochmann, 2012) and wine (Cabanero et al., 2008, Cabanero et al., 2010, Guyon et al., 2011). As HPLC-co-IRMS allows δ¹³C ratio measurement of major wine components, (Guyon et al., 2011) this technique has been applied to the authentication of lemon matrix which is essentially made up of water, acids and sugars, (Souci, Fachmann, and Kraut, 2000).

This study presents the application of HPLC-co-IRMS to the determination of carbon isotope ratio of major lemon components. An original strategy was set up to answer to the important difference in concentration between acids and sugars. Organic acids, glucose and fructose δ¹³C values were determined on 35 authentic lemon or lime juices obtained from the fruits squeezed in the laboratory. Then 30 commercial lemon or lime juices were analysed and their δ¹³C values confronted to authentic databank values.