Two isocratic reversed-phase methods that have distinct selectivity, and use popular UV/MS or UV detection, are presented for analyzing Rebaudioside A and other steviol glycosides. A Rapid Resolution High Throughput (RRHT) Eclipse Plus Phenyl-Hexyl method using UV and MS detection resolved many compounds from a Stevia rebaudi- ana Bertoni plant extract instead of the other approach employing an amino column with a refractive index (RI) or other special detector. The RRHT Eclipse Plus Phenyl- Hexyl method’s advantages include high peak capacity, isocratic mobile phase, low acetonitrile consumption, and unique selectivity compared to a ZORBAX Carbohydrate Analysis column method. Three commercially available Stevia sweeteners were ana- lyzed for Rebaudioside A and Stevioside, the two major diterpenoid glycosides present in stevia leaves.
The document describes two isocratic reversed-phase liquid chromatography methods for analyzing Rebaudioside A and other steviol glycosides in Stevia sweeteners. The primary method utilizes an Agilent ZORBAX Eclipse Plus Phenyl-Hexyl column coupled with UV/MS detection, offering high peak capacity and low acetonitrile consumption. An alternative method using a ZORBAX Carbohydrate Analysis column is also presented as a secondary confirmatory technique due to its distinct selectivity.
| Mobile Phase (Primary) | Water (0.1% formic acid): ACN (0.1% formic acid) (82:18) |
|---|---|
| Flow Rate (Primary) | 0.42 mL/min |
| Detection Wavelength (Primary) | 204 nm |
| Temperature (Primary) | 35 °C |
| Mobile Phase (Secondary) | Water: ACN (22:78) |
| Flow Rate (Secondary) | 1.5 mL/min |
| Temperature (Secondary) | 30 °C |
The primary method uses UV/MS or UV detection, specifically a DAD and a single quad MSD with electrospray ionization.
It is recommended as a secondary or confirmatory method due to its significantly different selectivity compared to the Eclipse Plus Phenyl-Hexyl column.
The isocratic mobile phase offers no reequilibration time and eliminates baseline drift common with gradients.