Abstract: Mupirocin, a broad-spectrum antibiotic produced by the fermentation of Pseudomonas fluorescens, plays an important role in the clinical treatment of bacterial skin infections. Mupirocin production relies on microbial fermentation, a highly complex bioprocess. Critical parameters such as fermentation temperature, pH, and cultivation duration directly influence metabolic pathways. The final product is not a single compound but a multi-component mixture that contains both the active pharmaceutical ingredient and associated fermentation by-products. Additionally, it includes degradation products formed during downstream processing or storage. This study established a multi-step purification method utilizing preparative high performance liquid chromatography (prep-HPLC) for the isolation of mupirocin-related impurities B, D, and E. The HPLC analysis was performed on a Unitary C8 column (250 mm×4.6 mm, 5 μm) using a mobile phase consisting of A (7.7 g/L ammonium acetate solution adjusted to pH 5.7) and B (tetrahydrofuran). The gradient conditions were applied: 0-6 min, 25%B; 6-35 min, 25%B-30%B; 35-55 min, 30%B; 55-55.01 min, 30%B-25%B; 55. 01-60 min, 25%B. The flow rate was set at 1.0 mL/min and the column temperature was maintained at 35 ℃. First, the post-crystallization mother liquor was analyzed to confirm the presence of the target impurities. The mobile phase employed for purification consisted of A (0.1% formic acid aqueous solution) and B (acetonitrile). To reduce sample complexity, the post-crystallization mother liquor was diluted with 50% acetonitrile aqueous solution to a final mass concentration of approximately 400 mg/mL. A preparative separation was performed using a C18 column (250 mm×100 mm, 10 μm). The following gradient conditions were applied: 0-40 min, 25%B-50%B; 40-50 min, 50%B, at a flow rate of 280 mL/min and an injection volume of 90 mL. Fractions 1 to 8 (Fr.1-8) were collected, desalted, and rotary-evaporated. The resulting residues were then redissolved in 50% acetonitrile aqueous solution to a final mass concentration of approximately 100 mg/mL. Subsequent analysis revealed that Fr.8, which contained impurity B, exhibited high purity with minimal interference from other impurities. Further purification was performed using a C18 column (250 mm×50 mm, 10 μm). The following gradient conditions were applied: 0-25 min, 35%B-50% B; 25-35 min, 50%B, at a flow rate of 70 mL/min and an injection volume of 10 mL. This process yielded highly purified fractions of impurity B. Impurities D and E were detected in Fr.5. To remove interfering components eluting before and after the target impurity peaks, initial purification was performed using a C18 column (250 mm×50 mm, 10 μm). The gradient conditions were applied: 0-35 min, 35%B-50%B; 35-60 min, 50%B, at a flow rate of 70 mL/min and an injection volume of 10 mL. Fr.5-1 and Fr.5-2 were collected. Subsequent analysis revealed that the X4 stationary phase provided better resolution of impurities D and E compared with the C18 phase (R_s=1.63 vs. 1.12). Therefore, Fr.5-1 and Fr.5-2 were further purified using an X4 column (250 mm×50 mm, 10 μm). The gradient conditions were applied: 0-50 min, 25%B-35%B, at a flow rate of 70 mL/min and an injection volume of 5 mL, yielding purified fractions of impurities D and E. Desalting was performed on a C18 column using mobile phase A (H₂O) and B (acetonitrile). Prior to loading, the fractions were diluted twofold with water. After sample loading, desalting was carried out with 10%B for 20 min, followed by 100%B until complete elution of the sample. The detection wavelength was set at 240 nm. The collected fractions were concentrated to dryness by rotary evaporation at 25 ℃, and the purified impurities were further lyophilized. Based on the orthogonality of the separation mechanisms of the C18 and X4 stationary phases, impurities B, D, and E were successfully isolated with chromatographic purities of 98.7%, 96.64% and 99.6%. Their weights of them were 963, 629.2 and 1096.4 mg, respectively. These impurities meet the purity requirements for drug impurity reference standards and can serve as standard substances for mupirocin quality control. The developed method exhibits high sample loading capacity, separation efficiency, and stability, providing a valuable methodological reference for the preparation of mupirocin-related substances and facilitating the establishment of its impurity profile.

Key words: Mupirocin, Impurities, Preparative high-performance liquid chromatography, Purification, Structural characterization