Validated HPLC-DAD and Spectrophotometric Methods for Simultaneous Determination of Ternary Mixture of Analgin, Caffeine, and Ergotamine

Background: Amigrain (R), the market formulation of the ternary mixture of analgin, caffeine, and ergotamine, is used for the symptomatic treatment of migraine. Objective: The aim of the work is to develop and validate an high-performance liquid chromatography-diode array-fluorescence (HPLC-DAD) method and a novel spectrophotometric method for simultaneous determination of analgin, caffeine, and ergotamine in their pharmaceutical formulation. Method: The HPLC separation of the ternary mixture was carried out using an Inertsil-C8 column and a gradient elution of mobile phase composed of acetonitrile and ammonium format buffer (pH 4.2), the ultraviolet detection for analgin and caffeine was carried out at lambda = 280, 254 nm, and fluorometric detection for ergotamine was carried out at lambda exc =310 nm, lambda emm =360 nm. The two spectrophotometric methods were double divisor ratio spectra derivative (DDRD) and ratio dual wavelength (RDW) methods. The first method was used for determination of ergotamine at 355 nm and caffeine at 268 nm by the third and first derivative. The second one was based on using amplitude difference for the determination of caffeine and analgin. Results: HPLC and spectrophotometric methods were applied over the concentration ranges of 50-400, 25-200, and 0.5-10 mu g/mL and 10-35, 2-30, and 10-70 mu g/mL for analgin, caffeine, and ergotamine for the two methods. Conclusions: The proposed methods were successfully applied for the determination of the cited drugs in their pharmaceutical formulation, and the obtained results were statistically compared with those of the reported methods without any significant difference. Highlights: The developed HPLC-DAD method has a high sensitivity for ergotamine. The spectrophotometric methods offer novelty, green solvent usage, and economic cost.