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Steviol quantification at the picomole level by high-performance liquid chromatography.
Veerle J Y Minne1, Frans Compernolle, Suzanne Toppet
1Laboratory of Plant Physiology, Katholieke Universiteit Leuven, Kasteelpark Arenberg 31, B-3001 Leuven, Belgium.
Journal of Agricultural and Food Chemistry
|April 29, 2004
Summary
A new reversed-phase high-performance liquid chromatography (RP-HPLC) method accurately quantifies steviol (SV) in beer and plant samples. This sensitive technique uses fluorescence detection and derivatization for precise steviol determination.
Area of Science:
- Analytical Chemistry
- Chromatography
- Natural Product Analysis
Background:
- Steviol (SV) is a key metabolite of steviol glycosides, important for understanding stevia-based sweetener applications.
- Accurate quantification of SV is crucial for quality control and research in food and pharmaceutical industries.
- Existing methods for SV determination may lack the sensitivity or applicability required for complex matrices like beer and plant extracts.
Purpose of the Study:
- To develop and validate a simple, highly sensitive reversed-phase high-performance liquid chromatographic (RP-HPLC) method for steviol (SV) determination.
- To apply the developed RP-HPLC method for the analysis of SV in fortified beer and plant samples.
- To establish the limit of detection and linearity range for SV quantification using the new method.
Main Methods:
- Development of a reversed-phase high-performance liquid chromatographic (RP-HPLC) method using dihydroisosteviol (DHISV) as an internal standard.
- Derivatization of SV and DHISV using 4-(bromomethyl)-7-methoxycoumarin for enhanced fluorescence detection.
- Separation on an ODS column with a mobile phase of acetonitrile/water (80:20 v/v) and fluorescence detection (Ex 321 nm, Em 391 nm).
- Sample preparation involving diethyl ether extraction for beer and acid fraction preparation/silica column cleanup for plant samples.
Main Results:
- The developed RP-HPLC method demonstrated high sensitivity with a detection limit of 100 pg for SV.
- A linear relationship was observed for SV concentrations between 0.5 and 50 µg/mL, and 0.1-15 µg/mL in spiked beer.
- SV was not detected in aged beer samples enriched in stevioside, suggesting potential degradation over time.
- Accurate determination of 594 ng of SV in 100 mg of dry plant material was achieved with high precision.
Conclusions:
- A robust and sensitive RP-HPLC method was successfully developed for the quantification of steviol (SV).
- The method is applicable to complex matrices such as beer and plant materials, offering valuable insights into SV stability and presence.
- The findings highlight the utility of fluorescence derivatization coupled with RP-HPLC for sensitive analysis of steviol in various samples.