Development of a novel liquid chromatography — Evaporative light scattering detection method for bacitracins and applications to quality control of pharmaceuticals Original Research Article

A novel liquid chromatography method for the direct determination of bacitracin main components (Bc-A, -B1, -B2 and -B3), a basic, cyclic polypeptide antibiotic, was developed and validated, based on ion pairs formation with trifluoroacetic acid (TFA) and evaporative light scattering detection (ELSD). The selected analytical column was the Waters Nova-pak C8 (3.9 × 150 mm), for which the optimum (using modified Simplex algorithm) mobile phase was H2O–ACN (73:27, v/v) containing 0.80 μL mL−1 of TFA, at a flow rate of 1.0 mL min−1. Optimized ELSD parameters were: nebulizing gas (nitrogen) pressure = 3.5 bar, evaporation temperature = 50 °C, detector gain = 12. Retention time of Bc-B1, -B2, -B3, -A and -F (oxidative degradation product of Bc-A) was 5.3, 5.8, 7.7, 8.7, 15.9 min, respectively, while zinc ions and related peptides were eluted at 1.3–1.9 min. A logarithmic calibration curve was obtained for each component (r > 0.998), while the concentration range of total bacitracin was 30–235 μg mL−1. Detection limits for the individual components were in the range 1.0–1.6 μg mL−1. The proposed method was applied for the direct determination of Bc components and related peptides in raw materials and pharmaceutical formulations (tablets, powder and aerosol) without tedious pretreatment (for tablets, a liquid–liquid extraction of magnesium with oxine was required). In the case of matrix interference, synthetic standards containing the same amounts of excipients or the standard addition technique were used. Recovery from spiked commercial formulations was ranged from 96.7% to 101.5% (in respect of total Bc).