Continuation of Reversed-Phase HPLC Analysis Studies of Steviol Glycosides Isolated From Stevia rebaudiana Bertoni

Additional High Performance Liquid Chromatography (HPLC) studies were performed on the nine sweet steviol glycosides reported in Joint Expert Committee on Food Additives (JECFA) namely rebaudioside A, steviolbioside, stevioside, rubusoside, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside F, and dulcoside A isolated from the leaves of Stevia rebaudiana. Using Reversed-Phase (RP) HPLC method, individual retention times and area for nine naturally occurring ent-kaurane diterpene glycosides of S. rebaudiana have been determined at five different temperatures: 40, 45, 50, 55, and 60 oC at three different pH 2.4, 2.6, and 2.8. HPLC results suggested that temperatures 50 and 55 oC at pH 2.4 would be ideal condition for better separation of steviol glycosides.


Mobile Phase Preparation
All solvents utilized for reversed-phase HPLC mobile phase preparation were degassed at least fifteen minutes before starting experimentation.The method employed for separation of steviol glycosides 1-9 was an isocratic binary solvent mobile phase system with a 32:68 mixture of acetonitrile and 10 mmol/L sodium phosphate buffers.Buffer solutions were prepared for each pH 2.4, 2.6, and 2.8 using the method described earlier (Chaturvedula & Zamora, 2014).

Standard Preparation
Each standard steviol glycoside 1-9 was prepared separately at a concentration of 10 mg/ml.The dilutions for all steviol glycosides (1-7) were made using the phosphate buffer, but the two compounds steviolbioside (8) and rebaudioside B (9) were prepared dissolving in methanol.The mixture of standard steviol glycosides 1-9 was prepared in such way that final dilutions were made to set the concentration at 1.0 mg/mL of each compound in the mixture.

Instrumentation and Conditions
An Agilent (Wilmington, DE) 1100 HPLC System, including a quaternary pump, a temperature controlled column compartment with additional 6 port switching valve, an auto sampler and VWD absorbance detector, was used for separation of steviol glycosides 1-9.The detector was set-up at UV 210 nm.The data acquisition was done using a Chemastation A 10.02 software.The column used for HPLC analysis was a reversed-phase C18 (2) 100 A Phenomenex (Torrance CA) (Length: 250 mm, inner diameter 4.6 mm, particle size: 5 µm); pH was measured using meter Metler Toledo seven compact pH/ion S220 (Switzerland).Branson Ultrasonic Cleaner Model 2510 (Maplewood, NJ) was used for degassing HPLC solvents.

Analysis Procedure
For the RP-HPLC method, the column was flushed with 50 mL of 90% ACN to waste before use and the samples were bracketed with standards by injecting them at the beginning and at the end of a run for accuracy of their retention times. 1 µL of steviol glycoside mixture (1-9) has been injected at temperatures 40, 45, 50, 55, and 60˚C also three different pH values 2.4, 2.6 and 2.8.All the nine steviol glycosides were detected under UV at 210 nm.The two compounds rebaudioside A (2) and stevioside (3) were injected once at the beginning and once at the end of the sequence for consistency and reproducibility of HPLC chromatograms.

Results and Discussion
In this study, we have used RP-HPLC method under isocratic binary solvent mobile phase system (32:68; acetonitrile and phosphate buffer) at temperatures 40, 45, 50, 55, and 60 ºC.Further, the pH of the buffer used in the HPLC method was tested between 2.0 to 4.0 and results found that pH 2.4, 2.6 and 2.8 gave good separation compared to others.Hence, we were describing the HPLC analysis of compounds 1-9 at temperatures 40, 45, 50, 55, and 60 ºC under three pH conditions 2.4, 2.6 and 2.8.Although the steviol glycosides 1-9 are weak UV absorbers, they give an adequate signal at 210 nm to meet the required quantitation limit (LOQ) of 0.5 mg/L.Duplicate runs were performed at temperatures 40, 45, 50, 55, and 60˚C at each pH 2.4, 2.6 and 2.8; results found almost identical.

Conclusion
We are herewith reporting a qualitative HPLC method for the identification of nine steviol glycosides rebaudioside D (1), rebaudioside A (2), stevioside (3), rebaudioside F (4), rebaudioside C (5), dulcoside A (6), rubusoside (7), rebaudioside B (8), and steviolbioside (9) based on the RP-HPLC method using UV detection system.From the above experiments it has been concluded that temperatures 50 and 55 ºC would be ideal under pH 2.4 for separation of steviol glycosides having identical number of sugar units attached to the C-19 acid and C-13 hydroxyl groups.