Tromethamine (TMM), often encountered in a final drug product, exhibits interesting chemical properties as a counter ion, buffer, or active ingredient. European and US pharmacopeias propose titration against hydrogen chloride for TMM assays. However, this method can be a hindrance when using drugs containing low concentrations of TMM in complex buffered formulations. Due to the lack of chromophores and the high hydrophilicity of TMM, we performed a simple and reliable hydrophilic interaction chromatography coupled with a charged aerosol detector (HILIC-CAD) separation approach as an alternative for TMM analysis. An amide stationary phase and a mobile phase consisting of a binary mixture of acetonitrile and 10 mM ammonium formate, pH 3 (80/20, V/V) were used. As the CAD response deeply depends on parameters such as stationary phases and pH buffer, we investigated their impact and explored the optimal signal conditions. Including TMM analogs such as tris(hydroxymethyl) nitromethane and 2-amino-2-ethyl-1,3-propanediol allowed us to select these parameters appropriately. The effects of the evaporation temperature, flow rate, and power function value (PFV) on the CAD signal response were also studied and optimized. The method was validated according to the ICH Q2 R1 guidelines. A linear response (mean R2 > 0.997) covering the range for low TMM concentrations (170-520 μg/mL) was achieved. Satisfactory intra-day and inter-day precisions were obtained with RSDs lower than 1.9% and 2.8%, respectively. The trueness ranged from 99.6% to 101.2%, and the LOD was found to be 1.1 μg/mL. The HILIC-CAD method has been applied to a sterile TMM solution for injection.
Keywords: Charged aerosol detector; FIA; HILIC stationary Phases; Tromethamine; Validation.
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