Development and Validation of HPLC Method for the Determination and Quantification of Colchicine in Gloriosa superba

Aims: In the present study our aim is to develop and validate a reverse phase HPLC method for both determination and quantification of cochicine in different parts of Gloriosa superba. The developed HPLC method is validated for parameters as mentioned in ICH guidelines.

Place and Duration of Study: Study was undertaken in Department of Forest Products, University of Horticulture and Forestry, Nauni, Solan, Himachal Pradesh, India and in the period between June 2015 and July 2016. The plant material used for testing of developed and validated method was collected in October, 2015 and is identified in the Herbarium section of the above said Department with reference number 13900.

Methodology: The system used is of Waters binary HPLC unit with Waters HPLC pump 515, dual λ absorbance detector 2487 and Empower II software. Standard of colchicine was purchased from Sigma Aldrich, (USA) and was used for HPLC method development and validation. The developed HPLC method was validated for parameters as mentioned in ICH guidelines.

Results: The analytical column, Sunfire C18 (4.6×250mm, 5µm) was operated at ambient temperature. Isocratic elution with A acetonitrile and B 3% glacial acetic acid was at a flow rate of 1 ml/min. UV detection was done at 245nm and run time was given ten minutes for standards and fifteen minutes for samples. LOD and and LOQ of the developed method was found as 0.002 µg/ml and 0.011 µg/ml respectively.

Conclusion: Method was found to be satisfactory in terms of high accuracy, precision and robustness. The method was successfully applied to the extracts made of different plant parts of Gloriosa superba.