Stability-indicating reversed-phase liquid chromatographic methods for the determination of aconitine and piperine in...

Simple and rapid stability-indicating HPLC methods were developed for the individual analysis of aconitine (ACN) and piperine (PIN) in Mahamrutynjaya rasa, an herbal dosage form containing Aconitum ferox, Piper nigrum, and Piper longum in combination. Separation of the ACN from its major and minor degradation products was successfully achieved on a reversed-phase C18 column (250 x 4.6 mm id, 5 microm particle size), with isocratic elution using a mixture of acetonitrile-KH2PO4 buffer (10 mM, pH 8 +/- 0.1; 50 + 50, v/v) at flow rate of 0.7 mL/min with UV detection at 227 nm. PIN separation was performed on a reversed-phase C18 column (250 x 4.6 mm id, 5 microm particle size), with isocratic elution in acetonitrile-KH2PO4 buffer (10 mM, pH 7 +/- 0.1; 35 + 65, v/v) at a flow rate of 1 mL/min with UV detection at 343 nm. The methods were validated with respect to linearity, precision, accuracy, specificity, system suitability, and robustness. The responses were linear in the drug concentration range of 10-100 microg/mL for both ACN and PIN. The percent recoveries of both the markers from a mixture of degradation products were in the range between 98-101%. The utility of the procedures was verified by their application to marketed formulations that were subjected to accelerated degradation studies. The methods could distinctly separate the drug and degradation products. The products formed in the marketed tablets were similar to those formed in the laboratory during stress studies.