RENJIE ZHAN

Purpose(s)

• Ursolic acid (UA) is a natural compound found abundantly in fruits and plants. Preclinical evidence shows that UA has hepatoprotection, muscle buildup, anti-inflammatory effects. It is being used as a dietary supplement in the U.S. market.
• Sulfation of UA is reported to be a major pathway according to our lab data. However, limited information is available regarding UA sulfation due to the lack of authentic UA sulfation product (UAS).
• The purpose of this study is to synthesize UAS and develop HPLC-ESI-MS analytical method to examine and quantify UA in vitro sulfation pathway by using our chemically synthesized UAS standard.

Method(s)

• UAS chemical synthesis: Concentrated sulfuric acid and acetic anhydride were mixed in dry pyridine and heated to 55 °C. UA was dissolved in dry pyridine and added to the mixture, then stirred at 55 °C for 30 minutes. The reaction was cooled on ice, then 28% ammonium hydroxide was added with further stirring, followed by filtration and drying under reduced pressure. Elemental analysis was performed by Atlantic Microlab, Inc.
• UAS biosynthesis: 10 μM UA was incubated in human liver cytosol (0.5 mg/mL) for up to 2 hours with a PAPS regenerating system.
• UAS analytical method development: Waters 2695 HPLC module and Waters QDa mass detector
were utilized. Chromatographic conditions are listed
below:
Column: Agilent Zorbax SB-CN (4.5 x 150 mm, 5 μm) Gradient elution: acetonitrile (from 20 to 90%) and aqueous acetic acid, pH~3.0 (q.s. to 100%)
Mass detector SIR mode: 455 (-), UA; 535 (-), UAS Injection volume: 10 uL
Run time: 12 min

Result(s)

• UAS chemical synthesis and verification: UAS exhibited an m/z of 535.39 [M-H]- (calculated mass: 535.35). Elemental analysis was consistent with the monoammonium salt monohydrate, C30H53NO7S (expected: C 63.02%, H 9.34%, S 5.61%, N 2.45%; found C 63.10%, H 9.35%, S 5.32%, N 2.43%), FW 571.81. Yield was 91%. UAS was resistant to hydrolysis, as expected for an aliphatic sulfate.
• UAS determination and quantification by HPLC-ESI-MS: UAS peak eluted at 5.3 min, UA peak eluted at 6.1 min in chromatograms. Specifically, LLOD and LLOQ for UAS in enzymatic matrix were 0.007 μM and 0.02 μM respectively (LLOD = 3Sb/slope; LLOQ=10Sb/slope); linearity for UAS calibration curve was good (r2 > 0.99) within the determination range (0.1-5 μM).
• UAS biosynthesis: Biologically and chemically synthesized UAS had identical elution time on both C18 column (Cogent bidentate, 4.6 x 75 mm, 4 μm; mobile phase: aq. Ammonium formate/methanol) and CN column (described above): 4.4 min and 5.3 min, respectively.