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.

Natural variation of sucrose, glucose and fructose contents in Colombian genotypes of Solanum tuberosum Group Phureja at harvest

“…Sucrose, glucose and fructose were extracted with 50% (v/v) aqueous methanol from 0.5 g freeze‐dried ground tissue and measured by HPLC. The HPLC method was developed and validated for Group Phureja potatoes using an AMINEX 87H column, 10 mmol L −1 sulfuric acid as eluent and refraction index detection …”

“…The HPLC method was developed and validated for Group Phureja potatoes using an AMINEX 87H column, 10 mmol L −1 sulfuric acid as eluent and refraction index detection. 29 Calibration curves were run every 48 samples. The lack of carryover in the long-term HPLC analysis was verified by injecting blanks every eight samples.…”

“…That study identified a wide genetic basis for the trait through the assessment of chip color using a visual scale in fried tubers from plots in nine environments . Recently, a method using high‐performance liquid chromatography (HPLC) for the analysis of sugars in Group Phureja tubers was developed and validated . The aim of this research was to study the natural variation of sucrose, glucose and fructose contents after harvest in Colombian Phureja genotypes using this HPLC method.…”

“…15 Recently, a method using high-performance liquid chromatography (HPLC) for the analysis of sugars in Group Phureja tubers was developed and validated. 29 The aim of this research was to study the natural variation of sucrose, glucose and fructose contents after harvest in Colombian Phureja genotypes using this HPLC method. This study will contribute to potato breeding programs and will generate phenotypic information with the purpose of advancing in the elucidation of the molecular basis of sugar content variation in tubers from this cultivated group.…”

Determination of Single Sugars, Including Inulin, in Plants and Feed Materials by High-Performance Liquid Chromatography and Refraction Index Detection

“…This method was found to be useful for the quantification of these three sugars in the enzymatic degradation of stachyose. Duarte-Delgado et al [33] published a method concerning the development and validation of a HPLC method with RI detection to quantify sucrose, glucose, and fructose in tubers of solanum, and reported an acceptable range of RSD from 5.3% to 7.3% and the limit of detection of 3.0 mg/L for the three sugars. Both parameters are less precise compared with the method presented here.…”