Fast and isocratic HPLC-method for steviol glycosides analysis from Stevia rebaudiana leaves

Abstract

A fast isocratic HPLC method for analysis of steviol glycosides from Stevia rebaudiana leaves was developed with a high selectivity for nine known steviol glycosides and low eluent consumption. The analytical method was performed on a Purospher Ò STAR RP-18 endcapped 3 lm Hibar Ò RT 250-4.6 column at 50°C with an eluent composition of water (65 vol. %) and acetonitrile (35 vol. %). With a flow rate of 1 mL min -1 , nine known steviol glycosides were detected selectively after 15 min. Method validation for rebaudioside A showed a LOD of 0.0004 mg mL -1 and a LOQ of 0.0038 mg mL -1 . Particularly with regard to moderate solubility, the method is linear up to a concentration of rebaudioside A of 4.8 mg mL -1 . The linear calibration curve was obtained with a coefficient of determination of 0.9997 ± 0.0002 and a total error of 2.01 % RSD (n = 12). The accuracy of the method was determined by the percentage mean recovery rate to 100.99 ± 2.01 %. The intra-day precision was in a range of 0.12 to 1.96 % RSD and the inter-day precision varies from 0.02 to 1.89 % RSD. Small changes in operating conditions like eluent composition (65 ± 2 vol. %), temperature (50 ± 10°C) or flow rate (1 ± 0.2 mL min -1 ) do not affect the performance of the analytical method. The reliable and robust proposed HPLC method can be applied for quantification of steviol glycosides in Stevia rebaudiana Bertoni leaves in laboratories and quality control in industry.

Improvement method of fast and isocratic RP-HPLC analysis of major diterpene glycoside from Stevia rebaudiana leaves

“…The developed method still has complete resolution profile in complex matrices of S. rebaudiana leaves extract. The resolution obtained was greater than the separation profile of rebaudioside A and stevioside chromatogram produced by the method developed by Berg et al (2012) [7]. Chromatogram separation of stevioside and rebaudioside A from standard mixture solution and S. rebaudiana leaves extract can be seen in Fig.…”

“…The results obtained from this study provided a better value than study who received LOD of 1.07 mg/L and LOQ of 3.56 mg/L [2]. The method developed by Bergs et a.l (2012) has a lower detection limit, 0.40 µg/mL compared to this study [7]. Further, this study established better LOQ compared to Berg et al(2012) which has LOQ at 3.8 µg/mL [7].…”

“…The third solution (S3), 1 mL extract was added with 1 mL of a standard solution (stevioside and rebaudioside A solutions). Each solution was analyzed in three replicates [7,24]. Percent recovery was determined by equation 1 as follows:…”

“…Various methods of analysis stevioside and rebaudioside A have been developed, including the method of analysis Reversed-Phase High Performmance Liquid Chromatography (RP-HPLC) and Normal-Phase High Performance Liquid Chromatography (NP-HPLC) and Ultra High Performance Liquid Chromatography (UHPLC) [1,[3][4][5][6][7][8][9]. Liquid Chromatography-Mass Spectrometry (LC-MS) was also developed for diterpene glycoside determination [10][11][12][13][14][15].…”

“…This method has developed to determine diterpene glycoside in S. rebaudiana [2][3]7,22]. The method giving better results was developed by Bergs et al (2012).…”