Determination of oleanolic and ursolic acid in Chinese herbs using HPLC and γ‐CD as mobile phase modifier

Abstract

To separate and determine oleanolic acid and ursolic acid, a rapid and accurate HPLC using γ-CD as the mobile phase additive was developed. The effect of CD nature and concentration, and the acidity of the mobile phase on the chromatographic behavior of two bioactive triterpenes were systematically studied. Two bioactive triterpenes were completely separated (R = 3.11) on a Kromasil(®) C(18) column (150×4.6 mm id, 5 μm) with the mobile phase consisting of acetonitrile/0.1% phosphoric acid with 2 mM γ-CD as the mobile phase modifier (60:40, v/v). The flow rate was set at 1.0 mL/min and the eluent was detected at 210 nm for two bioactive triterpenes. The linearity of the method was excellent (r=0.9999) over the studied range of 6-300 μg/mL for oleanolic acid, and 12-600 μg/mL for ursolic acid. The LOD and LOQ were 1.5 and 5.0, 1.0 and 3.0 μg/mL for oleanolic acid and ursolic acid, respectively. The optimized method was successfully applied to separate and determine two bioactive triterpenes in five Chinese herbs. It is concluded that this method could be used for rapid and accurate qualitative and quantitative analysis of the two bioactive triterpenes in Chinese herbs.

Simultaneous analysis of five triterpenes in Centella asiatica by high performance liquid chromatography with cyclodextrins as the mobile phase additives

“…On the other hand, the complexation phenomenon increases the hydrophilicity of them in the mobile phase and then significantly decreases their retention time in the column. This is consistent with our previous report that γ-CD can form the inclusion complexes with triterpenes (oleanolic acid and ursolic acid), and decrease sharply their retention time and improve their separation 22 .…”

“…So, CDs are able to host hydrophobic compounds, and increase water solubility of them or alter other properties. As the mobile phase additives in HPLC, CDs can form the inclusion complexes with analytes, change the retention behavior of analytes, and be available for separation enantiomers and geometric isomers 20 – 22 or simultaneous analysis of glycosides and aglycone with isocratic elution 23 , 24 . Therefore, the addition of CDs into the mobile phase was investigated to alter the retention behavior of five triterpenes allowing simultaneous analysis of five triterpenes in C. asiatica using HPLC with isocratic elution.…”

Application of Response Surface Methodology for Optimisation of Simultaneous UHPLC-PDA Determination of Oleanolic and Ursolic Acids and Standardisation of Ericaceae Medicinal Plants

“…Considering the earlier studies, C18 is the stationary phase that is most commonly used in the LC separation of OA and UA [8,[15][16][17][18][19][20][21]26,38]. This is hardly surprising, as RP-18 columns are also the most popular ones when it comes to plant material standardisation in general and, therefore, easily available in laboratories performing such studies.…”

“…According to the literature [16,[18][19][20][21]28,[39][40][41][42], isocratic binary elution systems containing acetonitrile or methanol are preferred in analysis of native triterpenoid acids as preventing baseline drifts often observed in gradient modes at detection wavelengths set below 220 nm, the region which the both organic modifiers exhibit also the highest absorption in. In the corresponding mobile phases, the concentrations of both solvents are similar, usually 80%-95% (v/v) for methanol and 85%-90% (v/v) for acetonitrile.…”

“…To address these problems various methods have been applied. Adding of separation facilitating substances, like cyclodextrins [20,21], might improve the resolution, as might pre-column derivation [17,[22][23][24], which is sometimes used as a solution for low sensitivity. Better limits of detection may be also achieved with detectors other than UV, e.g., evaporative light scattering (ELS) or mass selective (MS) detectors [23][24][25][26].…”