| In vitro: |
| J Chromatogr A. 2005 Oct 14;1091(1-2):32-9. | | Simultaneous analysis of hyoscyamine, scopolamine, 6beta-hydroxyhyoscyamine and apoatropine in Solanaceous hairy roots by reversed-phase high-performance liquid chromatography.[Pubmed: 16395790] |
METHODS AND RESULTS:
A new high-performance liquid chromatographic method is described for tropane alkaloid analysis in genetically transformed root cultures of Datura innoxia Mill. and Atropa belladonna L. Sample preparation, tropane alkaloid extraction with chloroform-methanol-concentrated ammonia 15:5:1 (v/v/v), was followed by solid-phase extraction on Supelclean LC-18 cartridges. Optimized conditions and careful pH control resulted in high recovery and reproducibility. Simultaneous determination of Apoatropine, 6beta-hydroxyhyoscyamine, hyoscyamine and scopolamine was performed by HPLC on C18 (2) reversed-phase column. The application of Luna new-generation silica-based stationary phase resulted in excellent peak shapes using an ion-pair reagent and triethanolamine free mobile phase and allowed to exploit the full power of pH-dependent selectivity.
CONCLUSIONS:
Simplicity and improved selectivity make this method a preferred alternative of published ion-pair chromatographic methods. Validation studies proved that the global method has good repeatability and satisfactory recovery. Absolute limits of detection were 0.6, 0.6, and 0.8 ng for hyoscyamine, 6beta-hydroxyhyoscyamine, and scopolamine respectively. |
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