Separation of furostanol saponins by supercritical fluid chromatography

Highlights

• SFC was for the first time applied for separation of hydrophilic furostanol saponins.

• The methoxylation of the hydroxyl at C-22 position of furostanol saponins in the eluted process by using methanol as co-solvent was suppressed.

• SFC was effective in separating the similar furostanol saponins which share the same aglycone and vary in sugar chain.

• SFC could be applied for profiling the Chinese materia medicas which mainly contain steroidal saponins.

Abstract

Supercritical fluid chromatography (SFC) has good separation efficiency and is suitable for separating weakly polar compounds. Furostanol saponins, as an important kind of steroidal saponins, generally have two sugar chains, which are polar and hydrophilic. The hydroxyl group at the C-22 position of furostanol saponins is active and easily reacts with lower alcohols under appropriate conditions. The separation of hydrophilic furostanol saponins was tested by SFC in this study. The effects of chromatographic conditions on the separation of the mixed furostanol saponins and their hydroxyl derivatives at the C-22 position were studied. The conditions for SFC, which included different column polarity, modifier, additive, and column temperature, were tested. After optimization, the mixed 10 similar structures of furostanol saponins were separated in 22 min on the Diol column at a temperature of 40 °C. The mobile phase was CO₂ (mobile phase A) and methanol (containing 0.2% NH₃∙H₂O and 3% H₂O) (mobile phase B). The backpressure was maintained isobarically at 11.03 MPa. SFC was found to be effective in separating the furostanol saponins that shared the same aglycone but varied in sugar chains. SFC was sensitive to the number and type of sugars. The resolution of furostanol saponin isomers was not ideal. The extract of Dioscorea zingiberensis C. H. Wright was profiled by SFC–quadrupole time-of-flight mass spectrometry. The main saponins of the extract were well separated. Therefore, SFC could be used for separating hydrophilic furostanol saponins and analyzing traditional Chinese medicines that mainly contained steroidal saponins.

Introduction

Supercritical fluid chromatography (SFC) is a chromatographic separation technique that uses a supercritical fluid as the mobile phase. In the past, a solid adsorbent bonded with a polymer was used as a stationary phase [1], [2], [3]. Nowadays, stationary phases for SFC are based on silica particles, which are either bare silica or silica bonded with different ligands. Metal porphyrin compounds were separated successfully by the supercritical fluid (dichlorodifluoromethane and chlorodifluoromethane) for the first time by Klesper in 1962 [4]. The supercritical fluid has stronger dissolving ability than gases, and faster diffusion speed and lower viscosity than liquids [5], [6], [7]. Currently, supercritical CO₂ is mainly used as the principal mobile phase component in SFC. Because supercritical CO₂ is nontoxic and nonpolar, the less polar compounds, such as carotenoids [8], [9], tocopherols [10], [11], and lipid compounds [12], [13], are well separated by SFC. A few reports were available about the separation of polar compounds by SFC. The isoflavones and their glycosides were well separated by SFC using a polar organic modifier [14]. The six sesquiterpenoid alkaloids were separated completely on the BEH 2-EP column in 10 min using SFC–DAD–mass spectrometry (MS). The mobile phase was supercritical CO₂–methanol. The flow rate, column temperature, and backpressure were 1.0 mL/min, 45 °C, and 13.8 MPa, respectively. Simultaneously, the characterization and rapid identification of Tripterygium wilfordii Hook F were carried out [15].

Steroidal saponins are found in the plant families Dioscoreaceae, Agavaceae, Alliaceae, Liliaceae, and so on [16], which show a wide range of pharmacological activities such as antifertility, antiplatelet aggregation, antidiabetes, cytotoxicity, and antitumor [17], [18]. Furostanol saponins are an important kind of steroidal saponins. They are a class of compounds in which the F ring of spirostanol saponins cracks and the hydroxyl group at the C-26 position mostly reacts with glucose to form the glucoside. They have two sugar chains, which provide polar and hydrophilic property. Furostanol saponins are unstable when dissolved in methanol, ethanol, or other lower alcohols. When they are dissolved in these solvents, the hydroxyl group at the C-22 position is converted into methoxyl or ethoxyl [19]. A previous study demonstrated that SFC was advantageous in separating the hydrophobic spirostanol saponins and complementary to the reversed-phase liquid chromatography [20]. The separation of furostanol saponins using SFC has not been reported yet. In this study, three furostanol saponins (timosaponin BII, protodioscin, and protogracillin) were chosen for optimizing SFC conditions. Different column polarities, modifiers, additives, and column temperatures were tested to show the influence of the retention behavior on the hydroxyl group at the C-22 position derivatized for furostanol saponins. Ten similar furostanol saponins were analyzed in 22 min under optimized conditions by SFC. Simultaneously, the extract of D. zingiberensis C. H. Wright was analyzed to study the chromatographic behavior of furostanol saponins and their application in traditional Chinese medicines (TCMs) containing steroidal saponins using SFC.