title: Steviol quantification at the picomole level by high-performance liquid chromatography.
abstract: A simple and highly sensitive reversed-phase high-performance liquid chromatographic method (RP-HPLC) has been developed for the determination of steviol (SV) using dihydroisosteviol (DHISV) as an internal standard (IS). SV and DHISV were derivatized by reaction of the acids with 4-(bromomethyl)-7-methoxycoumarin in an aprotic solvent (DMF or acetone). The resulting ester derivatives were separated on an ODS column (250 x 4.6 mm i.d., 5 microm particle size) using fluorescence detection with excitation at 321 nm and emission at 391 nm. The mobile phase consisted of acetonitrile/water (80:20 v/v) with a flow rate of 1 mL min(-)(1). A linear relationship was observed for concentrations between 0.5 and 50 microg/mL of SV, and the detection limit was 100 pg. For application of this method to samples of beer fortified with stevioside, a simple procedure for extraction of the beer with diethyl ether and derivatization in DMF was applied. Whereas beer samples spiked with SV gave a linear response over the range 0.1-15 microg/mL beer, no SV could be detected in beer samples enriched in stevioside that had been stored for over 3 years. The application of the method to plant samples involved preparation of an acid fraction containing the SV analyte, derivatization, and sample cleanup using small silica columns and thin-layer chromatography. A sensitive determination of 594 ng of steviol present in 100 mg of dry plant material was performed with high precision and accuracy.
authors.affiliation: Laboratory of Plant Physiology, Katholieke Universiteit Leuven, Kasteelpark Arenberg 31, B-3001 Leuven, Belgium.
authors.forename: Veerle J Y
authors.initials: VJ
authors.lastname: Minne
authors.forename: Frans
authors.initials: F
authors.lastname: Compernolle
authors.forename: Suzanne
authors.initials: S
authors.lastname: Toppet
authors.forename: Jan M C
authors.initials: JM
authors.lastname: Geuns
mesh_terms: D001515:Beer / Q000032:analysis; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004224:Diterpenes / Q000032:analysis* / Q000378:metabolism; D045786:Diterpenes, Kaurane; D005960:Glucosides / Q000032:analysis / Q000378:metabolism; D008832:Microchemistry / Q000379:methods*; D018515:Plant Leaves / Q000737:chemistry; D011753:Pyrones; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D036625:Stevia / Q000737:chemistry; D013997:Time Factors
chemical_list: D004224:Diterpenes; D045786:Diterpenes, Kaurane; D005960:Glucosides; D011753:Pyrones; C012043:stevioside; C012042:steviol; C113859:dihydrokavain