Exploration of the effects of chloride ions on the analysis of polar compounds at low concentrations by hydrophilic interaction liquid chromatography coupled to a charged aerosol detector: Application to tromethamine.

In this study, we discuss the origin of the slightly increased response of the charged aerosol detector when low-concentration polar drugs formulated with sodium chloride are analyzed by hydrophilic interaction liquid chromatography coupled to the charged aerosol detector. In the case of tromethamine mixed with saline solutions, we investigated several levels including the mobile phase, sample matrix, and detection. We show that the analysis of the rich-salted sample results in both interactions with the mobile phase modifiers and the stationary phase during the run time. With 150 mM NaCl as a compounding solution, a slight increase in the tromethamine peak area was observed (<5.5%). Our study suggests that chloride ions in excess sequentially interact firstly with the counterions from the organic modifiers and secondly with the analyte via the stationary phase and the contribution of hydrophilic interaction liquid chromatography retention mechanisms. Because of these effects, the hydrophilic interaction liquid chromatography-charged aerosol detector analysis of drugs in saline solutions requires particular attention, and a correction factor for quantitative purposes that accounts for formulation ions remains appropriate.

Complete 1 H, 13 C, and 15 N assignments of the minor isomer of codeine N-oxide.

Codeine N-oxide 2 is an active metabolite of codeine obtained by oxidation and observed as a degradant in codeine drug products such as syrups. Oxidation of codeine's N-methyl function can deliver two regio-isomers due to chirality of the tetra-substituted nitrogen. Hydrogen peroxide oxidation of codeine was performed and induced two different isomers in a 9:1 ratio; these isomers were isolated using preparative high performance liquid chromatography (HPLC) and fully characterized by nuclear magnetic resonance (NMR) techniques. We describe the complete assignment of the minor isomer of codeine N-oxide 3 and attribute a (S) configuration (N-methyl axial) of the tetra-substituted nitrogen. The effects of N-oxidation on the 15 N chemical shifts of the codeine are presented. The 15 N shifts were determined using the CIGAR-HMBC experiment at natural abundance, and the nitrogen resonance of codeine shifted downfield from 42.8 to 118.7 ppm for both N-oxide isomers.

Investigation of hydrophilic interaction liquid chromatography coupled with charged aerosol detector for the analysis of tromethamine.

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.