Pressurized capillary electrochromatographic assay of trimethoprim impurities using 1 μm particle-based columns

One micrometre silica particles, derivatized with C18, were electrokinetically packed into a 75-μm-i.d. capillary. The resulting column was evaluated for the separation of trimethoprim (TMP) and its impurities using pressurized capillary electrochromatography (pCEC), starting from a capillary liquid chromatographic (CLC) separation. These samples require gradient elution when separated by high performance liquid chromatography (HPLC), but with the new columns isocratic elution suffices for their separation by CLC or pCEC. Only 70,000 theoretical plates/m for impurity C were achieved using CLC mode at relative low pressure (78 bar) although very small particles were utilized. When a voltage above 2 kV (50 V/cm) was applied, unknown peaks appeared, which was assumed due to an electrophoretic effect with the unknown peaks resolving as a result of the applied voltage. In order to minimize these unfavorable contributions, only a low voltage was applied, still leading to higher separation performances and shorter separation times than in CLC. The optimal analyzing conditions in pCEC included a pressure of 78 bar, an applied voltage of 1 kV, and a mobile phase consisting of 80 mM sodium perchlorate (pH 3.1)/methanol (60/40, v/v). These conditions were used to separate and quantify four major impurities in TMP within 22 min. The obtained calibration curves were linear (r > 0.9980) in concentration ranges between 0.005 and 0.1 mg/mL for impurities A and C; 0.02 and 0.10 mg/mL for impurity F; and 0.01 and 0.10 mg/mL for impurity H. The detection limits (S/N = 3) for impurities A, C, F, and H were 0.52, 0.84, 3.18, and 2.41 μg/mL, respectively. The calibration curves were successfully applied to analyze spiked bulk samples, with mean recoveries ranging from 92% to 110%. The developed method can therefore be considered simple, rapid, and repeatable.