Development and validation of a stability-indicating RP-HPLC method for estimation of Lefamulin in pure and pharmaceutical drug products

Chemicals

We used water and acetonitrile of Honeywell HPLC grade. Hydrogen peroxide (30% w/w), sodium hydroxide, potassium phosphate, orthophosphoric acid, hydrochloric acid of analytical-grade from Sigma-Aldrich were employed.

Standards and samples

Lefamulin acetate, which was procured from Kaifeng Pharmaceutical (Group) Company Limited China, was gratefully donated by Horizon Healthcare (Pvt.) Limited in Pakistan. The injection of 150 mg/15 mL and the 600 mg tablets of XENLETA® were bought from open market. Horizon Healthcare Pakistan granted all the commercial-grade excipients used throughout the validation study.

Instrumentation

Shimadzu Corporation of Japan quaternary gradient HPLC LC-10AD is outfitted with a degasser, column temperature oven, and a multi-channel photodiode array detector was used for the analytical method development and validation studies. The data curation was carried out by using Lab Solution software. To obtain a chromatographic separation, a Welchrom Ultisil® XB-C18 column with a 4.6 mm internal diameter, 25 cm length, and 5 µm particle size was employed. Analytical balance (ATX-224; Shimadzu Japan) was used to measure the weights of the standards and samples. The sonication process was carried out using an S15H ultra sonicator Elma Germany, and the pH of the mobile phase was maintained using a pH meter S210 from Mettler Toledo Switzerland. The Sartorius Germany filtration assembly was used to filter the Mobile phase. The glassware used during the whole study was calibrated. The climatic chambers HPP1060 Memmert Germany were used for the temperature and humidity stress experiments, while the xenon lamp 50W CEL-HXB F300 from China was used for the photolytic stress studies.

Chromatographic conditions

10 µL volumes of standards and samples were injected while the mobile phase flow rate was held at 1 mL/min, the column temperature was kept at 30 °C, and 210 nm was the detection wavelength. The HPLC needle rinsing solution was acetonitrile and water (50:50 v/v).

Preparation of mobile phase and diluting solution

The chromatographic separation used an isocratic mobile phase elution consisting of 0.05 M potassium phosphate buffer with a pH adjustment of 2.5 from diluted orthophosphoric acid. Then the buffer was combined with the acetonitrile as the organic modifier in equal volumes. The 45% v/v solution of acetonitrile in distilled water was used as a diluting solution.

Preparation of standard solution and calibration curves solutions

Lefamulin acetate, which is equivalent to 150 mg of Lefamulin base, was dissolved by diluent in a 100 mL volumetric flask to obtain the standard stock solution. Then, 3–7.5 mL were transferred to separate 50 mL volumetric flasks and diluted with a diluting solution to generate 90–225 ppm solutions in order to develop the calibration curve. The target concentration of standard solution was 150 ppm.

Preparation of tablet sample solution

The stock tablet sample solution was prepared by transferring the weight equivalent to one tablet (nominal amount of Lefamulin 600 mg) from powdered tablets to a 200 mL volumetric flask, diluted with diluent, and dissolved with the aid of ultra Sonicator for 15 min, finally made the volume to mark with diluent. The 5 mL of stock solution was diluted with diluent to 100 mL for preparation of the final sample solution.

Preparation of injection sample solution

The stock injection sample solution was prepared by diluting the 15 mL portion of injection (nominal amount of Lefamulin 150 mg) from cumulative volume of injections to 100 mL volumetric flask, diluted with diluent, and dissolved with the aid of ultra Sonicator for 15 min, finally made the volume to mark with diluent. The 5 mL of stock solution was diluted with diluent to 50 mL for preparation of the final sample solution.

Preparation of laboratory formulated tablet and injection sample solutions

The laboratory-formulated tablet sample solution was prepared by mixing the excipients (Croscarmellose sodium; 5 mg, colloidal silicon dioxide; 1 mg, magnesium stearate; 1 mg, mannitol; 45 mg, microcrystalline cellulose; 26 mg, polyethylene glycol; 1 mg, polyvinyl pyrrolidone K30; 5 mg, talc; 1 mg, and titanium dioxide; 2 mg) with 671 mg of Lefamulin acetate drug substance, and transferred in 200 mL volumetric flask, diluted with diluent, and dissolved with the aid of ultra Sonicator for 15 min, finally made the volume to mark with diluent. The 5 mL of stock solution was diluted with diluent to 100 mL for preparation of the final sample solution. Similarly, the Lefamulin laboratory injection sample solution was prepared by mixing the Lefamulin acetate with sodium chloride, citric acid, and trisodium citrate, diluted as per the above injection sample solution. Three replicates of these solutions were injected, measured in the % assay, and compared with commercial products.

Force degradation studies

Force degradation studies were employed to evaluate the stability characteristics of the Lefamulin analytical method [10]. Various stress conditions, oxidative, photolytic, thermal, and hydrolysis (acid and basic), were applied. The 150 ppm pure drug substance solution was exposed to acidic stress (10 mL of 0.1 N hydrochloric acid), basic stress (10 mL of 0.1 N sodium hydroxide solution), and oxidative stress (3% v/v hydrogen peroxide solution) and left in the dark for 5 days before injecting. The drug substance solution was subjected to thermal-humidity stress by being kept at 60 °C temperature and 75% relative humidity for 5 days. The standard solution was exposed to an illuminance of nearly 1500 lx for 5 days to apply the photolytic stress. The temperature control for acidic and basic stress samples along with their control blank solutions was 40 °C, while for oxidative stress along with its control blank was 25 °C.

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