Development and validation of a dosing method for colchicine capsules for a stability study

Context
The control laboratory of our health establishment was asked to participate in a clinical trial requiring colchicine capsules at 0.5 mg.

Objective
To develop and validate a stability-indicating assay method for colchicine using reverse-phase high-performance liquid chromatography (HPLC) coupled with a diode-array ultraviolet detector and a mass spectrometer to study the stability of these capsules.

Materials and methods
The stationary phase chosen was a C18 column (100 x 3 mm, 2.6 µM) thermostated at 40°C and the mobile phase was a mixture 40/60 (v/v) acetonitrile and ammonium formate buffer (5 mM, pH 3.5) Detection of the analyte was performed by 2D UV (λ = 254 nm and λ = 350 nm), 3D UV (λ = 210 to 400 nm) and mass spectrometry. Injection volume was 20 µL with a flow rate of 0.25 ml/min.
Analytical validation (linearity, precision, accuracy and specificity) of the assay method was performed according to current ICH. Forced degradation (FD) tests [1] were performed to validate the stability indicating properties of the method. The contents of the capsules were exposed to alkaline (1 M NaOH, 1h, +/- 80°C), acid (1 M HCl, 1h, +/- 80°C), thermal (80°C, 1h), oxidative (H2O2 at 30%) and photolysis (UV) treatment in order to reveal possible degradation products. An analysis of the data obtained associated with the study of the literature data allowed to identify some of them.

Results
The retention time of colchicine is approximately 2.5 min. The analytical method was found to be linear for concentrations between 15 and 35 µg/ml (r2 > 0.99). The intra- and inter-day precision (CV < 5%) as well as the accuracy (CV < 5%) are in conformity. Concerning the specificity of the method, no interference was observed between colchicine and the excipients tested (saccharose, magnesium stearate, polyvidone, aluminium lacquer, erythrosine (E127), lactose). Only the FD tests under alkaline conditions show degradation products (1.9 min). The analysis of the mass spectrum under alkaline conditions allows to find Colchicine (400 Da) and various fragments corresponding to the degradation products (64 Da, 81 Da, 227 Da and 386 Da).

Conclusion
The analytical assay method is validated and the FD tests have demonstrated the stability-indicating characteristics. Our control laboratory will therefore be able to carry out the stability study of colchicine capsules at 0.5 mg in the context of this clinical trial.

References
[1] Coord. V. Sautou. 1st ed. SFPC/GERPAC, 2013. Methodological guidelines for stability studies of hospital pharmaceutical preparations.

See the poster