Journal of Chromatography B, 885–886 (2012) 43–49 Contents lists available at SciVerse ScienceDirect Journal of Chromatography B j ourna l ho me page: www.elsevier.com/locate/chromb Optimisation of an HPLC method for the simultaneous quantification of the major sugars and organic acids in grapevine berries Hans A. Eyéghé-Bickong, Erik O. Alexandersson 1 , Liezel M. Gouws, Philip R. Young, Melané A. Vivier ∗ Institute for Wine Biotechnology, Department of Viticulture and Oenology, Stellenbosch University, PO Box X1, Matieland, Stellenbosch 7602, South Africa a r t i c l e i n f o Article history: Received 31 August 2011 Accepted 9 December 2011 Available online 20 December 2011 Keywords: Sugars Organic acids Grapevine berries High performance liquid chromatography a b s t r a c t A high performance liquid chromatographic method was developed to profile major sugars and organic acids in grapevine berries. Sugars and organic acids in grapevine berries were extracted by chloro- form/polyvinylpolypyrrolidone purification. The extracts were chromatographed on an Aminex HPX-87H ion-exchange HPLC column with 5 mM sulphuric acid as mobile phase. Chromatography was visualised via a diode array detector combined with a refractive index detector. The analysis was calibrated using external standard calibration and a novel equation was used to calculate the concentrations of malic acid and fructose from unresolved separation. For the method to be utilised for analysing a large num- bers of berry samples, each sample was directly injected after sample extraction and the extraction step was downscaled to allow the use of small amounts of sample material. The concentrations of sugars and organic acids in grapevine berry samples were normalised to the internal standard concentrations obtained after extraction of an internal standard mixture. The analysis method exhibits a good precision and a high analyte recovery from samples spiked with the standard mixture and is suitable for the profil- ing of major sugars and organic acids in grapevine berry samples at different stages of berry development. This is the first report on the combined profiling of the major sugars and organic acids in grapevine berries using milligram amounts of plant material with direct injection after sample extraction. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Sugars and organic acids are important primary metabolites that contribute to grapevine growth and berry development. These compounds are also considered key factors in grape and wine qual- ity. The hexoses, glucose and fructose, as well as the organic acids malic and tartaric acid are the most abundant compounds con- tributing to the grape juice sweetness and acidity respectively [1]. Their concentrations and/or ratios vary during the berry devel- opment and maturation stages. Organic acids are produced in both the grape leaves and berries and start accumulating in the grapevine berry at early stages of berry development [1]. Hexoses are produced in leaves and are transported to the berry where a characteristic accumulation over time occurs in the vacuole of berry cells [1]. After véraison, the concentration of hexoses accu- mulate considerably in the berry while the acid concentrations start to decline with the exception of tartaric acid, which remains relatively constant throughout ripening [1,2]. The accurate quan- tification of these metabolites in grapevine berries at the different ∗ Corresponding author. Tel.: +27 21 808 3773; fax: +27 21 808 3771. E-mail address: mav@sun.ac.za (M.A. Vivier). 1 Current address: Department of Plant Protection Biology, Swedish University of Agricultural Sciences, SE-230 53 Alnarp, Sweden. stages of berry development is important to follow berry devel- opment and ripening, specifically when evaluating the impact of viticulture practice on berry characteristics and subsequent wine properties. Most methods for the analysis of sugars and organic acids in grapevine berries and wines that rely on high performance liquid chromatography (HPLC) have commonly used only the grape musts and/or juices with different sample extraction protocols [2–7]. Most of these extraction protocols are followed by clean-up steps prior to HPLC analysis to eliminate problems of co-elution of compounds of interest. These clean-up steps are often accomplished by fractiona- tion through resin cartridges to separate the sugars and organic acids into two fractions before analysis [3,6,8]. Hunter et al. [8] developed a pre-analysis fractionation method that separated sug- ars and organic acid in two fractions before analysis from (1 g) whole, freeze-dried berries. The downside of fractionation meth- ods is that it requires a significant amount of plant material and two HPLC runs per sample (increase time requirement). However, it has been shown that direct injection from grape juice and wine can provide an alternative for routine analysis with greater accuracy [9]. Here we describe an analytical HPLC method to profile the major organic acids and sugars by direct injection after sample extraction using small amounts (80–100 mg) of whole, frozen, deseeded and ground berries (pulp and skin). This method would be 1570-0232/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2011.12.011