Development of a novel method based on liquid chromatography-evaporative light scattering detection for the direct determination of streptomycin and dihydrostreptomycin in raw materials, pharmaceutical formulations, culture media and plasma.

A novel method for the non-derivatization liquid chromatographic determination of streptomycin (STR) and dihydrostreptomycin (DHSTR) was developed and validated based on evaporative light scattering detection (ELSD). Utilizing a ThermoHypersil BetaBasic C18 analytical column, evaporation temperature of 50 degrees C and pressure of nebulizing gas (nitrogen) of 3.5 bar, the optimized mobile phase was 1.25 mL L(-1) TFA aqueous solution, in an isocratic mode at a rate of 1.0 mL min(-1). STR was eluted at 5.6 min and DHSTR at 7.8 min with a resolution of 4.4. Linear calibration curves were obtained from 2 to 120 microg mL(-1) (r > 0.9990) for STR and 2-75 microg mL(-1) (r > 0.9994) for DHSTR, with a LOD equal to 0.7 and 0.5 microg mL(-1), respectively. The developed method was applied for the assay of STR and DHSTR (sulfate) in pharmaceutical raw materials and formulations, while the simultaneous direct determination of sulfate was feasible (tR = 2.5 min, LOD = 1.4 microg mL(-1), double logarithmic calibration curve in the range of 4-50 microg mL(-1), r > 0.9998). Modified isocratic mobile phase (H2O-ACN, 90:10, v/v, containing 1.25 mL L(-1) TFA), was used for the determination of streptomycin B impurity in STR sulfate raw material and a gradient mobile phase (H2O-ACN containing TFA) was used for the determination of DHSTR in the presence of penicillinG procaine. The developed method was also applied for the assay of commercial formulations (STR powder and DHSTR injection solution and suspension) (%recovery 98-102, %RSD < 1.3, n = 3 x 3), for the determination of STR in bacteria culture medium (%recovery 99.6, %RSD = 0.8, n = 3 x 3), and for the determination of DHSTR in human plasma (2.0-23.0 microg mL(-1)) after solid phase extraction using carboxylate cartridges (%recovery 98.4-101.8, %RSD = 3.2, n = 3 x 3).

Development of a novel liquid chromatography--evaporative light scattering detection method for bacitracins and applications to quality control of pharmaceuticals.

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 x 150 mm), for which the optimum (using modified Simplex algorithm) mobile phase was H2O-ACN (73:27, v/v) containing 0.80 microL 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 degrees 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 microg mL(-1). Detection limits for the individual components were in the range 1.0-1.6 microg 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).