Development and validation of a liquid chromatographic method to quantify sucrose, glucose, and fructose in tubers of Solanum tuberosum Group Phureja

Abstract

A High Performance Liquid Chromatography (HPLC) method was developed and validated to quantify sucrose (non-reducing sugar), glucose, and fructose (reducing sugars) in raw tubers of Solanum tuberosum Group Phureja. Chromatographic analysis was performed using an AMINEX HPX 87H column, at 18 °C, linked to a refraction index detector, at 35 °C. The eluent was 10mM sulfuric acid. The conditions established for the method provided an optimum separation of sugars, citric acid, and malic acid, with resolution values higher or equal to one. Among the four sugar extraction methods tested, the double 50% (v/v) aqueous methanol extraction gave the highest level of analytes. Recovery of this extraction method ranged between 94.14 and 99.77%. The HPLC method was validated for repeatability, reproducibility, linearity, and limits of detection, and quantification. Relative standard deviation was found to be lower than five, when testing repeatability and reproducibility, which is suitable considering a range of acceptability from 5.3 to 7.3. Additionally, the regression analyses supported the method linearity in a range of quantification from 3 to 100 mg/L with regression coefficients values greater than 0.998 for the three analytes. Limits of detection were 3.0 mg/L for the three sugars and limits of quantification were 2.0 mg/L for sucrose and 3.0 mg/L for glucose and fructose. Four Colombian commercial cultivars (Criolla Guaneña, Criolla Paisa, Criolla Galeras, and Criolla Colombia) and five landrace accessions from the Colombian Core Collection of Group Phureja were grown in the district of Usme (Bogotá) fields to analyze their sugar contents. Sucrose, glucose, and fructose contents were found ranging from 0.93 to 3.11 g/100 g tuber dried weight (DW), from 0.25 to 4.53 g/100 g tuber DW, and from 0.10 to 1.49 g/100 g tuber DW, respectively. Therefore, a high range in the variability of sugar contents was found among genotypes. However, the variability was low among technical replicates of the same genotype, revealing an accurate quantification of sugars in Group Phureja. This method can be used to assess the amount of reducing and non-reducing sugars accumulation in potato germplasm.

Keywords: Group Phureja; HPLC; Non-reducing sugars; Reducing sugars.