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

  • Dominik Bergs
  • Bernhard Burghoff
  • Matthias Joehnck
  • Georg Martin
  • Gerhard Schembecker


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® STAR RP-18 endcapped 3 μm Hibar® 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.


HPLC Steviol glycosides Rebaudioside A Stevioside Validation Isocractic analysis 


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Copyright information

© Bundesamt für Verbraucherschutz und Lebensmittelsicherheit (BVL) 2012

Authors and Affiliations

  • Dominik Bergs
    • 1
  • Bernhard Burghoff
    • 1
  • Matthias Joehnck
    • 2
  • Georg Martin
    • 3
  • Gerhard Schembecker
    • 1
  1. 1.Laboratory of Plant and Process Design, Department of Biochemical and Chemical EngineeringTechnische Universität DortmundDortmundGermany
  2. 2.Merck KgaADarmstadtGermany
  3. 3.LANXESS Deutschland GmbHLeverkusenGermany

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