Development and validation of a stability-indicating HPLC-UV method for the determination of Thiocolchicoside and its degradation products

A stability indicating high performance liquid chromatography method has been developed for the determination of thiocolchicoside (TCC) and its main degradation products thiocolchicoside S-oxide (D1SO) and 3-O-demethylthiocolchicine (D3) in liquid and solid formulations. The method was developed based on a previous forced degradation study showing that TCC underwent chemical degradation by acid/base catalyzed hydrolysis and oxidation being the main degradation products D3 and D1SO respectively. The analytes separation and quantification were achieved on a Synergi™ 4 μm Polar-RP 80 Å, column 150 × 4.6 mm (Phenomenex) using the mobile phase constituted (flow rate 1 mL min⁻¹) of eluant A: 20 mM sodium acetate buffer (pH 5.0) and eluant B: MeOH:CH₃CN (20:80); the elution was performed in gradient mode detecting the analytes at 254 nm. The method showed linearity for TCC assay in the 5–15 μg mL⁻¹, range and for unknown (TCC_fu) and known (D1SO and D3) degradation products assay, in the 0.5–10 μg mL⁻¹ range: all the square of the correlation coefficients were greater than 0.999. The precision, determined in terms of intra-day and inter-day were expressed as RSDs and resulted to be 1.19, 1.10, 1.37 and 1.04% and 0.95, 0.83, 1.30 and 0.72 for TCC, TCC_fu, D1SO and D3, respectively. The method demonstrated also to be accurate; indeed, the average recoveries were 102.1/102.0% for TCC (ampoules and hard capsules respectively), 101.3/100.3% for TCC_fu, 101.7/100.2% for D1SO, and 101.4/101.4% for D3. The robustness was also evaluated by variations of mobile phase composition and pH. Finally, the applicability of the method was evaluated by analysis of commercial liquid and solid dosage forms.