Track: Bioanalytics - Chemical - Reagents and Reference Standards - Characterization and Quality Control
Category: Late Breaking Poster Abstract
Synthesis and Chromatography of the Sulfation Metabolite of A Natural Product- Ursolic Acid
Purpose: Ursolic acid (UA) is a naturally occurring compound found abundantly in numerous herbs, fruits and plants. Preclinical evidence shows that UA has extensive potential pharmacological activities, such as hepatoprotection, muscle buildup, anti inflammatory and others, and it has been used as a dietary supplement in the U.S. market.  However, regarding UA metabolism and disposition, it has only been reported that UA undergoes glucuronidation but there is limited information on its sulfation metabolism pathway. Our lab has conducted UA sulfation metabolism experiments in human subcellular fractions previously, and concluded that UA also undergoes extensive sulfation in vitro.  Therefore, for the purpose of examining and quantifying UA sulfation metabolism and due to the lack of commercially available authentic UA sulfation metabolite (UAS), we propose a method to chemically synthesize UAS and use that as the authentic standard for subsequent determination and quantification of UAS formation both in vitro and in vivo. In the meantime, we propose to develop and establish an analytical method to quantify UAS by HPLC-ESI-MS. Methods: Chemical synthesis of UAS: Concentrated sulfuric acid and acetic anhydride were mixed in dry pyridine and heated to 55 oC, as described by Teske et al. UA was dissolved in dry pyridine and added to the mixture, then stirred at 55 oC 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. Analytical method development for UAS: HPLC-ESI-MS was adopted to verify and characterize the synthesis product. Both C18 and cyano columns were applied for methodology development. Column temperature was set at 35 oC; initially, negative ion scan (100-600 m/z) was performed; later, SIM was set at m/z 535.39 (-) for UAS, m/z 455.50 (-) and m/z 439 (+) for UA. For C18 column (Cogent bidentate, 4.6 x 75 mm, 4 um), gradient elution (A: 10 mM ammonium formate, pH 3.8; B: methanol) was performed as the following: 0-1.6 min A 28%, B 72%; 1.6-5 min A decreased to 10%, B increased to 90% linearly; 5-8.5 min A 10%, B 90%; 8.6-10 min A 28%, B 72%; For cyano column (Agilent, 4.5 x 150 mm, 5 um), gradient elution (A: acetic acid, pH 3; B: acetonitrile) was programed as: 0-1.5 min A 80%, B 20%; 1.6-4 min A decreased to 10%, B increased to 90% linearly; 4-7 min to A 10%, B 90%, 7-10 min A 0%, B 100%, 10-15 min A 80% B 20%. Also, UAS was biologically synthesized using human liver cytosol in the presence of UA, PAPS, sodium sulfate, and ATP. Results: 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. Both UA and UAS peaks were observed in chromatograms using the two analytical columns. UAS retention times were 4.4 min and 5.3 min, UA retention times were 7.8 min and 6.1 min for C18 and cyano column analytical methods respectively. The retention times for biologically- and chemically-produced UAS coincided. Conclusion: UAS was successfully chemically synthesized and verified by HPLC-MS, elemental analysis, and by comparison with biologically-produced UAS. Chemical synthesis of UAS is more efficient than enzymatic synthesis. UAS can be used as the authentic analytical standard for the future UA sulfation metabolism studies in vitro as well as in vivo/clinical PK studies on UA disposition. References:
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P. Phansalkar et al, AAPS PharmSci 360, Washington D.C. 2018.