A fully validated microbiological assay for daptomycin injection and comparison to HPLC method

abstract Daptomycin (DPT) was the first lipopeptide antibiotic available for commercialization. It is active against gram-positive bacteria, including resistant strains. This work aimed to develop and validate a turbidimetric microbiologic assay to determine daptomycin in an injectable form. A 3x3 design was employed, at concentrations of 1, 2 and 4.0 µg/mL. The microorganism test used was Staphylococcus aureus ATCC 6538p, and Antibiotic Medium 3 was used as the culture medium. Method validation demonstrated that the bioassay was linear (r=0.9995), precise (RSD=2.58%), accurate (recovery 100.48± 2.11%), and robust. Degradation kinetics was also performed in an alkaline medium, indicating that daptomycin degradation follows first order kinetics under these conditions. The analyses of degraded solutions showed that daptomycin degradation products do not possess bactericidal activity. The bioassay was compared to HPLC method that was previously developed and no significant difference was found between them (p>0.05). The method proved to be appropriate for daptomycin injection quality control.

摘要：达托霉素（Daptomycin，DPT）是首款实现商业化的脂肽类抗生素，对包括耐药菌株在内的革兰氏阳性菌均具有抗菌活性。本研究旨在开发并验证一种比浊微生物测定法，用于注射用达托霉素的含量测定。实验采用3×3设计，设置1、2与4.0 µg/mL三个浓度梯度。试验菌株选用金黄色葡萄球菌ATCC 6538p，培养基采用抗生素培养基3号（Antibiotic Medium 3）。方法学验证结果表明，该生物测定法线性关系良好（r=0.9995）、精密度优异（相对标准偏差（Relative Standard Deviation，RSD）=2.58%）、准确度可靠（回收率为100.48±2.11%）且稳健性良好。此外，本研究还在碱性介质中开展了降解动力学实验，结果显示达托霉素在该条件下的降解符合一级动力学规律。对降解溶液的分析表明，达托霉素的降解产物不具备杀菌活性。将该生物测定法与此前开发的高效液相色谱（HPLC）法进行对比，二者无显著性差异（p>0.05）。该方法适用于注射用达托霉素的质量控制。