Profiling of Stevia rebaudiana Extract by Accurate Mass Using HILIC and Reversed-Phase Chromatography

By: Craig Aurand, Reporter US Volume 27.2

Craig Aurand


There is growing public interest in low-calorie alternatives to carbohydrate-based sweeteners. Synthetic sweeteners are often regarded as having an undesirable aftertaste. Recent publications have shown a dramatic increase in attention toward natural extracts including the Stevia rebaudiana plant, not only for its sweetening effect but also for additional health benefits attributed to the plant. The major sweetening components are stevioside, rebaudioside A, rebaudioside C, and dulcoside A, each of which is over 300 times sweeter than sucrose-based sweeteners. The concern with the human consumption of the stevia leaf had been attributed to the possible mutagenic properties of steviol, but more recent studies conducted by the World Health Organization have established the safety for steviol and its glycosides.

In this study, an evaluation of the Stevia rebaudiana plant extract was conducted using modern chromatographic and mass spectrometry techniques for the determination of extracted components. The purpose was to evaluate the utility of performing two different modes of chromatographic separation for component identification. An accurate mass time of flight (TOF) mass spectrometer was used in the detection and identification of components. A novel software package was then utilized for the determination of common components between the two chromatographic modes and to depict the impact of chromatographic selectivity.

The concept behind the study was to utilize both reversed-phase chromatography and HILIC chromatography for the determination of extract components. By using two different modes of selectivity, components that co-retain, do not retain, or do not elute under one chromatographic mode may be resolved under a separate mode. By resolving a component chromatographically, a more accurate assessment of the component can be made without relying specifically on accurate mass data.

With traditional reversed-phase chromatography, analytes are primarily retained on an alkyl based stationary phase by partitioning interaction between the non polar stationary phase and the analyte. Though this mode of chromatography is widely accepted for separation of moderately polar to non-polar compounds, highly polar analytes often have minimal or no retention on these phases. More popular polar embedded stationary phases address this issue with the addition of a polar functional group within the alkyl chain. Polar embedded phases can enhance retention of polar compounds, but it is not a solution for all applications. Often highly polar analytes require alternative modes of chromatographic retention. In particular, HILIC chromatography allows for alternative selectivity by utilizing a highly polar stationary phase with a relatively non polar mobile phase. Under HILIC conditions, the partitioning of analytes is achieved through a preferential solvation of an aqueous environment on the polar surface. More polar analytes will partition more into the surface solvent and thus be retained longer than a less polar analyte. In addition to the partitioning, the polar surface of the stationary phase allows for adsorptive interactions via hydrogen bonding, dipole, etc. When ionic samples are separated, the potential for ion-exchange interactions also exists and in many cases becomes the dominant retention mechanism. Using silica-based stationary phases, ionized surface silanol groups may interact via ion-exchange with positively charged analytes.

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In this study, both reversed-phase and HILIC separations were conducted using the Ascentis® Express RP-Amide and Ascentis Express HILIC. The polar embedded group of the Amide was chosen over traditional C18 phases to increase the retention of the polar analytes in the stevia extract. The Ascentis HILIC allowed for alternative selectivities for polar analytes. Because of the large amount of unknown components in the stevia extract, using both reversed-phase and HILIC modes enabled orthogonal selectivity to resolve co-retained components and enable better determination of components in the extract with confirmation between the two modes.

Stevia leaves were obtained from Sigma Aldrich (S5381). Sample extraction of the stevia leaves was performed by weighing 400 mg of crushed stevia leaves into a 7 mL amber vial. A total of 4 mL of 50:50 acetonitrile:water was added and the sample was vortexed and sonicated for 3 minutes. The sample was then centrifuged for 2 minutes at 15000 rpm. The supernatant was then collected and analyzed directly.

The sample extract was analyzed using a gradient elution profile for both HILIC and reversed-phase chromatographic modes. Analysis was conducted using an Agilent® 1200SL Rapid Resolution system in sequence with an Agilent 6210 TOF mass spectrometer. The TOF enabled the use of accurate mass for determination of components. The acquired data was processed using the Mass Hunter software package. The data was pushed to the Mass Profiler package for statistical comparison of the two chromatographic modes. This software package enabled the identification of common components between the two chromatographic separations of the stevia extract. By performing this type of statistical comparison, the components attributed to the stevia extract were differentiated from components attributed to chromatographic anomalies. From this comparison the major components of the stevia extract were determined. Available standards were then used to confirm the identification of several of the components.

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Results and Discussion

Figure 1 and Figure 2 represent the total ion chromatogram for the stevia extract under both HILIC and reversedphase conditions. Both of these chromatographic separations demonstrate the complexity of the stevia extract. Table 1 depicts the major components that were common in both the reversed-phase and HILIC separations of the stevia extract. More than 250 components were identified with this comparison, but only the major components were targeted in this study. The highlighted components in Table 1 depict co-retention of analytes under reversed-phase conditions. A good example of using this orthogonal approach is observed in the case of steviobioside and ducloside A. Under the reversed-phase separation, these components were co-retained. By performing the separation under HILIC conditions, steviobioside and ducloside A were well separated. Other unidentified major components that were unresolved under the reversed-phase conditions were also separated under the HILIC conditions. The data in Table 1 also depicts the selectivity difference between the two chromatographic modes. Polar components that were poorly retained in the reversed-phase conditions were strongly retained under HILIC conditions. In two cases, where known components were identified, it was necessary to use standards to confirm their retention. Figures 3 and 4 depict the extracted ion chromatogram for the accurate mass of steviol and stevioside. As can be seen in both chromatograms, multiple peaks are observed for each of the accurate masses. In the case of stevioside, it is isobaric with rebaudioside B making identification difficult. A stevioside standard (Sigma Aldrich) was used for positive identification. In addition, the reversed-phase separation of the extract resulted in multiple peaks observed for the accurate mass of steviol. This was due to fragments from additional glycosides that resulted in a steviol fragment ion, again it was necessary to confirm the steviol retention with a standard.

Figure 1. Component Chromatogram of Stevia Extract on Ascentis Express HILIC

Figure 2. Component Chromatogram of Stevia Extract on Ascentis Express RP-Amide

Table 1. Major Component Retention Comparison Between HILIC and Reversed-Phase Modes

Figure 3. Stevia Extract on Ascentis Express RP-Amide, Extracted Ion Chromatogram for Steviol

Figure 4. Stevia Extract on Ascentis Express RP-Amide, Extracted Ion Chromatogram for Stevioside

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The profiling of the Stevia rebaudiana extract demonstrates the utility of performing orthogonal chromatographic modes when handling complex samples. The two modes of chromatography were complimentary for the determination of major components from the stevia extract. In most cases where coelution occurred in one chromatographic mode, the components were separated under the orthogonal mode. Though component identification was made easier through the accurate mass of the TOF, it was still necessary to have good chromatographic resolution to confirm component identity. In both cases, the Fused-Core particle demonstrated the ability to perform complex matrix analysis in both HILIC and reversed-phase separations.

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