Stevia Sweetener Analyses Using an Agilent ZORBAX Eclipse Plus Phenyl-Hexyl Column

John W. Henderson Jr. and Judy Berry, Agilent Technologies, Inc.

An Agilent ZORBAX Rapid Resolution HT Eclipse Plus Phenyl-Hexyl column resolved many compounds from Stevia rebaudiana Bertoni plant extracts using an isocratic reversed phase method with UV and MS detection instead of a typical approach employing an amino column with a refractive index or other special detector. The Eclipse Plus Phenyl-Hexyl method's advantages include high peak capacity, isocratic mobile phase, low acetonitrile consumption, and MS compatibility.

The FDA has no objection to Rebaudioside A having GRAS (generally recognized as safe) status as a sweetener for food and drink (1). Stevia sweeteners may contain other steviol glycosides as well, mainly Stevioside, Rebaudioside C and Dulcoside A. Because the FDA GRAS confirmation is only for the use of Rebaudioside A at 95% purity or above in food and beverages, stevia extracts must be highly purified and characterized prior to use. Due to the lack of chromophores, many methods for sugars and similar compounds like steviol glycosides use refractive index, evaporative light scattering, or other special detectors. These methods often use an amino bonded-silica based column too. Hydrolysis of the amino-silica bond in a high aqueous environment, and special detectors, are some limitations to this approach. This method, however, takes advantage of the RRHT Eclipse Plus Phenyl-Hexyl column for enhanced resolution and the more prevalent UV /MS detectors.

Figure 1

Experimental

Instrumentation and chromatographic parameters are found in Figure 1. Dietary supplements, all containing "stevia extract" were diluted to 0.1g/mL (powders), or 1:10 (v:v) (liquid) in H₂O.

Results and Discussion

Two stevia dietary supplements were analyzed on an Eclipse Plus Phenyl Hexyl column. The ratio of the two major diterpenoid glycosides was quite different with Rebaudioside A being more prevalent in the powdered sweetener, and Stevioside being dominant in the liquid (Figure 1a, b). Rebaudioside A is considered the sweeter and better tasting of the two, although other minor glycosides may play a role in the overall taste quality. Rebaudioside A and Stevioside were identified with commercial standards and calibration curves were constructed, demonstrating linearity from about 100 to 3000 ppm (2).

A third stevia extract, was found to have many more components compared to the previous two samples. A G1956B Single Quad MSD was connected to the G1316C DAD detector outlet to also identify Dulcoside A and Rebaudioside C (Figure 1c, d) using the [M-1]^- ion for identification. The narrow bore column dimensions and formic acid in the mobile phase enhance electrospray ionization, making this well suited for ESI-MS. The isocractic mobile phase makes it easy to transfer from one LC to another because delay volume associated with gradients is not a factor.

The RRHT Eclipse Phenyl-Hexyl column contains 1.8-µm particles for high efficiency, and operated at a pressure of 400 bar under these conditions, well within the operating limits of the RRHT column (600 bar) and the 1200SL RRLC (600 bar).

Conclusion

The Eclipse Plus Phenyl-Hexyl method is useful for pure and complex stevia extracts. Benefits include high efficiency and peak capacity, rugged column life, popular UV and/or ESI-MS detection, and isocratic mobile phase.

References

(1) CFSAN/Office of Food Additive Safety December 17, 2008 U.S. FDA response letter GRAS notice no. GRN 000253.

(2) Isocratic Stevia Sweetener Analysis using Selective ZORBAX Columns, Agilent Pub. 5990-3933EN April 2009.

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