A Novel Derivatization Ultraviolet Spectrophotometric Method for the Determination of Dihydroartemisinin using p-Nitroaniline

Purpose: To develop a novel ultraviolet (UV)–spectrophotometric method for the determination of dihydroartemisinin (DHA) in tablets using p-nitroaniline (PNA) as a derivatizing agent.
Methods: Derivatization was based on the reaction between methanol solutions of dihydroartemisinin (DHA) and p-nitroaniline (PNA) in acid medium (1M HCI) at elevated temperature and for a short reaction time. Optimal detector response was obtained within 15 min when the reaction was carried out at 90^o C in a molar ratio of 2:1 (DHA:PNA). The method used for analysis was validated and a linear calibration curve constructed in the range of 30 – 100 µg/mL for the reaction mixture at an absorbance of 290 nm.
Results: Separation of adduct from PNA was better achieved on reversed phase thin layer chromatography (TLC) using acetonitrile : water (60:50) or on high performance liquid chromatography (HPLC) with retention times of 2.8 min for PNA and 5.8 min for the adduct. The limit of detection was 6 µg/mL. The method was precise and accurate in the range 100.70 - 100.96 %, with intraday and interday precisions of less than 2 % at concentrations of 40 and 80 µg/mL, respectively. The new method was applied to the assay of two brands of dihydroartemisinin tablets with accuracy similar to that of the International Pharmacopoeia (IP) UV-spectrophotometric method (p > 0.05).
Conclusion: The derivatization method is simple, direct, devoid of dilutions and inexpensive in terms of reagent requirements and analyte volume, and has a shorter reaction time, cpmpared with IP method. Based on the foregoing, the method can be adopted as an alternative to the official assay method for routine quality control of dihydroartemisinin tablets.

Keywords
Derivatization; Ultraviolet spectrophotometry; Dihydroartemisinin; p-Nitroaniline; Analysis; Assay; Quality control