Synthesis of an IS and Steviol Glycoside Analysis by a Validated Internal Standard Method
| Autor: | Tom Struyf, Stijn Ceunen, Wim M. De Borggraeve, Gert Steurs, Jan M.C. Geuns |
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| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
Internal standard
Chromatography 010405 organic chemistry Calibration curve Chemistry Fraction (chemistry) 010402 general chemistry 01 natural sciences High-performance liquid chromatography 0104 chemical sciences Psychiatry and Mental health chemistry.chemical_compound Column chromatography Steviol Glycosides Standard addition Quantification Validation Internal Standard Method Rebaudioside A Steviol glycoside Purification |
| Popis: | The internal standard (IS) method is the best method for the analysis of samples, as it is independent of errors in injection volume, changes in sample volumes, and changes in sensitivity of the detector, etc. Use of an internal standard allows for the correction of losses due to sample clean-up of complex samples. An ideal IS is a compound that has properties very similar to, and that behaves as the compounds to be analysed. Ideally, only in the last step of analysis (HPLC), the IS should be well separated from the compounds of the mixture to be analysed. After testing several existing compounds with negative results, we decided to synthesise the 19-O-β-D-galactopyranosyl-13-O-β-D-glucopyranosyl-steviol as IS. This is the 19-galactosyl ester of steviolmonoside (13-O-β-D-glucopyranosyl-steviol). The IS was made according to published methods. Steviolmonoside (SM) was made from purified commercial rubusoside (Rub) by refluxing it in 10% KOH for 2 h. SM was precipitated and crystallized from MeOH. The hydroxyls of the glucose unit of SM were protected by acetylation. The acetylated SM was crystallized from acetone and dissolved in 1,2-dichloroethane. Then Ag2CO3 on Celite and tetra-acetylated galactopyranosyl bromide were added and the mixture was refluxed for 2 h. After cooling, BaO in MeOH was added to remove the acetyl groups. The 1,2-dichloroethane fraction was then extracted three times with equal volumes of water and the water fraction containing the IS was further purified on a C18 flash chromatography column. Traces of unreacted SM were removed by preparative HPLC on an Alltima C18 column (250 mm × 22 mm, particle size 10 µm) with AcCN:water (35:65, 20 ml/min). Detection was at 210 nm (KNAUER, “Smartline” UV detector 2500). The collected IS fraction from the HPLC was completely dried. Mixtures of steviol glycosides (SVglys) containing IS could be purified over SPE cartridges without change of the SVgly over IS ratio. The calibration curves for rebaudioside A (RebA) and stevioside (ST) were linear between 0.012 and 0.95 and between 0.013 and 1.13 mM for RebA and ST, respectively. The accuracy was checked by the standard addition method. It was concluded that the IS method gives an excellent precision and accuracy. ispartof: American Journal of Analytical Chemistry vol:9 issue:11 pages:547-559 status: published |
| Databáze: | OpenAIRE |
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