Analytical Methods A reliable and rapid method for soluble sugars and RFO analysis in chickpea using HPAEC–PAD and its comparison with HPLC–RI Manu P. Gangola, Sarita Jaiswal, Yogendra P. Khedikar, Ravindra N. Chibbar ⇑ Department of Plant Sciences, College of Agriculture and Bioresources, University of Saskatchewan, 51 Campus Drive, Saskatoon, Saskatchewan S7N 5A8, Canada article info Article history: Received 15 November 2012 Received in revised form 1 November 2013 Accepted 19 December 2013 Available online 3 January 2014 Keywords: Myo-inositol Galactinol Raffinose family oligosaccharides (RFO) Raffinose Stachyose Verbascose High performance anion exchange chromatography (HPAEC) Pulsed amperometric detection (PAD) abstract A high performance anion exchange chromatography (HPAEC) coupled with pulsed amperometric detec- tion (PAD) was optimised to separate with precision, accuracy and high reproducibility soluble sugars including oligosaccharides present in pulse meal samples. The optimised method within 20 min sepa- rated myo-inositol, galactinol, glucose, fructose, sucrose, raffinose, stachyose and verbascose in chickpea seed meal extracts. Gradient method of eluting solvent (sodium hydroxide) resulted in higher sensitivity and rapid detection compared to similar analytical methods. Peaks asymmetry equivalent to one and res- olution value P1.5 support column’s precision and accuracy for quantitative determinations of soluble sugars in complex mixtures. Intermediate precision determined as relative standard deviation (1.8– 3.5%) for different soluble sugars confirms reproducibility of the optimised method. The developed method has superior sensitivity to detect even scarcely present verbascose in chickpea. It also quantifies myo-inositol and galactinol making it suitable both for RFO related genotype screening and biosynthetic studies. Ó 2014 Elsevier Ltd. All rights reserved. 1. Introduction Carbon storage and translocation is an important phenomenon in plants to sustain their growth and development. Raffinose fam- ily oligosaccharides (RFO) or a-galactosides constitute a group of soluble, non-reducing carbohydrates used to transport and store carbon in plant families like Cucurbitaceae, Leguminosae, Lamia- ceae, Oleaceae and Scrophulariaceae (Sprenger & Keller, 2000). RFO are non-structural carbohydrates characterised by the pres- ence of a (1 ? 6) linkage between the galactosyl and sucrose res- idues (Tapernoux-Luthi, Bohm, & Keller, 2004). RFO are ubiquitous in plant kingdom and only second to sucrose in concen- tration among soluble sugars (Frias et al., 1999). Raffinose is the first member of this family followed by stachyose and verbascose. RFO mainly accumulate in seeds during their later stages of development (Peterbauer et al., 2001) and play important physio- logical roles in plants, such as inducing desiccation tolerance, seed longevity (Koster, 1991), detoxification of reactive oxygen species (Bolouri-Moghaddam, Roy, Xiang, Rolland, & Van, 2010) and toler- ance against biotic and abiotic stresses (Cho et al., 2010; Liu, Dai, Xu, & Chong, 2007). However, human and mono-gastric animals cannot digest RFO and escape small intestinal digestion and absorp- tion due to lack of a-galactosidase enzyme required for the hydroly- sis of a (1 ? 6) glycosidic linkages (Saunders & Wiggins, 1981). However, microflora of large intestine metabolizes RFO and produce substantial amounts of carbon dioxide, hydrogen, and small quanti- ties of methane. Therefore, consumption of food with high RFO in humans causes stomach discomfort, flatulence and diarrhea (Veld- man, Veen, Barug, & Van Paridon, 1993). It also leads to nutrient defi- ciency in animal feed due to decreased intestinal absorption (Wiggins, 1984) and hence reduced availability of metabolizable en- ergy (Coon, Leske, Akavanichan, & Cheng, 1990). In human diet RFO stimulate growth of some therapeutic microorganisms such as Bifidobacterium spp. (a lactic acid bacteria) in large intestine and are considered as prebiotics (Peterbauer & Richter, 2001; Trojanová, Vlková, Rada, & Marounek, 2006). In pulse crops, a-galactosides con- tribute up to 9.5% of total dry matter that reduces their acceptability in human diet, particularly in western countries (Alonso, Rovir, Vegas, & Pedrosa, 2010; Martinez-Villaluenga, Frias, & Vidal- Valverde, 2008). In chickpea, stachyose (0.18–2.38 g/100 g) is re- ported as one of the major soluble sugars (Gangola, Khedikar, Gaur, Båga, & Chibbar, 2013). Therefore, to reduce the negative effects of pulses in human diet and increase its consumption, seed RFO con- centration needs to be reduced without affecting their beneficial attributes in humans and plants. To achieve this objective a rapid, precise and accurate method is needed to determine the concentra- tion of three RFO members, raffinose, stachyose and verbascose in small samples of pulse seeds. Chromatographic separation followed by visual detection [thin layer chromatography (TLC), Jones, DuPont, Ambrose, Frias, & 0308-8146/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.12.085 ⇑ Corresponding author. Tel.: +1 306 966 4969; fax: +1 306 966 5015. E-mail address: ravi.chibbar@usask.ca (R.N. Chibbar). Food Chemistry 154 (2014) 127–133 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem