Ultrasonication Extraction and Solid Phase Extraction Clean-up for Quantification of Aristolochic Acid I in the Thai Herbal Antitussive 3 Formula by High Performance Liquid Chromatography with Photodiode Array Detection

Objective: To establish a reliable High Performance Liquid Chromatography method with photodiode array detection (HPLC-PDA) and a suitable extraction method for identification and quantification of Aristolochic acid I (AA-I) in 3 different sample types, i.e. Aristolochia tagala Cham.(ATC), Thai Herbal Antitussive 3 Formula (ATF3), and Thai Herbal Antitussive 3 Formula containing ATC (ATF3+ATC). Methods: Two extraction methods, ultrasonication extraction (UE) and solid phase extraction (SPE), were used in the present study. Reversed phase separations were performed with a SunFire C18 column and HPLC-PDA. Mobile phase was performed by 1 mL/min infusion of mixture of 0.1% o-phosphoric acid and methanol (35:65). Results: Calibration curve showed a good linearity with R2 equal to 0.9986. Every sample type has been analyzed. While the concentration of AA-I in ATF3 cannot be determined due to the absence of the AA-I peak, the AA-I concentrations in ATC and ATF3+ATC were 7.50-11.34 and 6.78-11.77 mg/mL respectively. In comparison with UE, SPE is more effective in eradication of the interfering compounds and improves the absorbance spectrum of the peak and increases extraction efficiency. Conclusion: A reliable HPLC-PDA and SPE methods for identification and quantification of AA-I in ATF3+ATC, ATF3, and ATC were established in the present study. The SPE was more effective than UE in the pretreatment step