Development of HPLC Analytical Techniques for Diterpene Glycosides fromStevia rebaudiana(Bertoni) Bertoni: Strategies to Scale-Up

Abstract

Stevia rebaudiana is a plant well-known as a source of dozens of high potency non-caloric natural sweeteners, many of them still uncharacterized. Herein, we describe the development of several routine HPLC (high performance liquid chromatography) analytical techniques to better separate steviol glycosides. Different stationary phases/columns were used: reverse phase C-18, amino, HILIC (hydrophilic interaction chromatography), Sepaxdiol and Synergi. The advantages and disadvantages of each method for resolving the very polar and less polar elution regions are demonstrated. Strategies for scale up of the purification process of steviol glycosides to make it more economic are also summarized, emphasizing the advantages of using high efficiency normal phase chromatography in relation with reverse phase. Additionally, we also developed an easy and useful way for predicting yields and composition of the pools resulting from the preparative chromatography. The development of these analytical and preparative liquid chromatography techniques allowed the isolation of several steviol glycosides in gram quantities at relatively low cost.

Development of a high‐performance liquid chromatography procedure to identify known and detect novel C‐13 oligosaccharide moieties in diterpene glycosides from Stevia rebaudiana (Bertoni) Bertoni (Asteraceae): Structure elucidation of rebaudiosides V and W

“…The crystalline products were rebaudioside A and stevioside, which accounted for approximately 50% of the starting mass. Pools rich in rebaudioside N from several large scale chromatographies for isolation of rebaudioside C in quantity were combined to obtain 140 g. All this material was fractionated on a high efficiency reverse‐phase chromatography column (7.5 id x 50 cm, 10 μm spherical C18 gel) using a three‐step gradient with H 2 O/AcOH (0.5%; 4.5 L) and CH 3 CN: H 2 O: AcOH (15:85:0.1 from fractions 1–7; 25:75:0.1 fractions 8–16; 40:60:0.1 fractions 17–25). One liter forerun was collected when mobile phase was switched to CH 3 CN/H 2 O/AcOH (15:85:0.1, v/v/%) and 25 fractions × 500 mL.…”

“…One liter forerun was collected when mobile phase was switched to CH 3 CN/H 2 O/AcOH (15:85:0.1, v/v/%) and 25 fractions × 500 mL. Fractions were analyzed by HPLC and ten main pools were prepared based on results from column analysis . A flow chart about the purification process of compounds 1 – 4 is shown in Supporting Information Fig.…”

“…Probably, isomers of these polar glycosides will coelute and if the aglycone-C-13 portion is the same, they will not be distinguishable from each other. Thus, we propose to use in case of coelution in this very polar region, an alternative Sepax-Diol or HILIC method that shows a well resolved region for steviol highly polar glycosides [17]. Rebaudiosides T and U were the most recent DGs discovered by our group [14], previously detected as new compounds by using this HPLC technique.…”

Assignment of sugar arrangement in branched steviol glycosides using electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

“…Therefore, many of the minor components of Stevia rebaudiana are better known today but many more of them remain uncharacterized. Chromatographic techniques, both analytical and preparative, have been developed to monitor and purify steviol glycosides . High‐performance liquid chromatography (HPLC) techniques with a wide array of chromatographic columns have been scouted .…”

“…Chromatographic techniques, both analytical and preparative, have been developed to monitor and purify steviol glycosides . High‐performance liquid chromatography (HPLC) techniques with a wide array of chromatographic columns have been scouted . As a result of the structural similarity of these compounds one to another, co‐elution of some components are commonly observed.…”