Department of Drug Analysis, Faculty of Pharmacy, University of Belgrade, 450 Vojvode Stepe, 11000 Belgrade, Serbia.
Department of Drug Control and Examination, Sector for Pharmacy, Military Medical Academy, Crnotravska 17, 11000 Belgrade, Serbia.
J Chromatogr Sci. 2021 Jun 21;59(7):650-658. doi: 10.1093/chromsci/bmab004.
Vancomycin and teicoplanin are glycopeptide antibacterials that inhibit the bacteria cell wall synthesis showing activity against gram-positive bacteria. Development of the sensitive method is of great importance for quality control of these drugs that are fermentation products. Modification of the fermentation conditions could cause the differences in the relative amount of the total substance or component, as it is the case with teicoplanin. The main objective of this study was development of the sensitive and effective ultra high performance liquid chromatography - tandem mass sprectrometry (UHPLC-MS/MS) method for simultaneous quantification of vancomycin, all six subcomponents of teicoplanin, and its pharmacopoeial impurity A in pharmaceutical forms. The scientific-based Quality by Design approach was implemented in the MS and UHPLC method development. Detection and quantification of analytes were carried out in positive electrospray ion mode by multiple reaction monitoring. Capillary voltage, cone voltage and collision energy were optimized by implementing experimental design methodology and optimal values for each fragment ion were obtained by performing experiments according to 'Rechtschaffen' design matrix. An ACQUITY CSH Phenyl-hexyl (2.1 × 50 mm, particle size 1.7 μm) column was chosen for the separation under the gradient elution mode with the mobile phase consisted of 0.1% formic acid in water (mobile phase A) and acetonitrile (mobile phase B). Optimal gradient elution parameters were achieved by applying 'Rechtschaffen' design too. Method operable design regions were constructed for investigated MS and chromatographic parameters. The method was fully validated, and its applicability was confirmed throughout the ability to follow the behavior of vancomycin and teicoplanin under stress conditions.
万古霉素和替考拉宁是糖肽类抗菌药物，通过抑制细菌细胞壁的合成发挥作用，对革兰氏阳性菌具有活性。开发敏感的方法对于这些发酵产品的质量控制非常重要。发酵条件的改变可能会导致总物质或成分的相对量发生变化，就像替考拉宁一样。本研究的主要目的是开发一种灵敏有效的超高效液相色谱-串联质谱法（UHPLC-MS/MS），用于同时定量测定药物制剂中的万古霉素、替考拉宁的全部六种组分及其药典杂质 A。在 MS 和 UHPLC 方法开发中采用了基于科学的质量源于设计方法。通过多反应监测，在正电喷雾离子模式下进行检测和定量分析。通过实施实验设计方法优化了毛细管电压、锥孔电压和碰撞能量，并根据“Rechtschaffen”设计矩阵进行实验，获得了每个片段离子的最佳值。采用 ACQUITY CSH Phenyl-hexyl（2.1×50mm，粒径 1.7μm）柱，在梯度洗脱模式下进行分离，流动相由 0.1%甲酸水（流动相 A）和乙腈（流动相 B）组成。通过应用“Rechtschaffen”设计也实现了最佳的梯度洗脱参数。为研究的 MS 和色谱参数构建了操作设计区域。该方法经过充分验证，并通过在应激条件下能够跟踪万古霉素和替考拉宁的行为，确认了其适用性。
