School of Pharmacy and Biomedical Sciences, Faculty of Clinical and Biomedical Sciences, University of Central Lancashire, Preston PR1 2HE, United Kingdom; Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard University, New Delhi 110062, India.
Department of Pharmaceutical Analysis and Quality Assurance, Roland Institute of Pharmaceutical Sciences, Berhampur, Ganjam, Odisha 760010, India.
J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Jun 1;1200:123261. doi: 10.1016/j.jchromb.2022.123261. Epub 2022 Apr 25.
The described work entails the development of a simple, sensitive, green, and robust high-performance liquid chromatographic (HPLC) method for simultaneous estimation of temozolomide (TMZ) and γ-linolenic acid (GLA). The chemometric factor screening study helped identify the critical method parameters optimized using Box-Behnken design for improved understanding and enhancing the method performance. Chromatographic separation was performed on a Kinetex® C column (150 × 4.6 mm, 5 µm particle size) using methanol: water (pH adjusted to 3.5 using 0.5% v/v O-phosphoric acid) as the mobile phase at 0.5 mL/min flow rate and diode array detection between 210 and 360 nm. The linearity of the method was observed for concentrations of TMZ and GLA ranging between 1 and 100 µg.mL (R = 0.999, p < 0.05). Accuracy evaluation showed good percent recovery within 97.9-100%, while intra- and inter-day precision showed RSD values within 0.37%-1.01%. The limit of detection and quantification for TMZ was found to be 0.75 and 1.0 μg.mL, respectively, while the values 0.55 and 1.0 μg.mL, respectively, were observed for GLA. System suitability (96.9-102.8%), its limits, and robustness evaluation indicated good percent recovery within, while RSD values were found to be within the acceptable limit of less than 2%. The method was specific for its ability to detect the analytes and their degradation products during forced degradation studies, which also indicated that TMZ was highly prone to alkaline conditions while GLA showed mild degradation in all the studied conditions. The estimation of both the analytes from lipid nanoparticles formulation showed good values for total drug content (82.6-85.3%), entrapment efficiency (95.4 to 98.7%), and drug loading (25.2 to 38.4%). Overall, the results indicated that the developed method was reliable for its accuracy, precision, sensitivity, and specificity for simultaneous estimation of the analytes. The method was found to be stability-indicating in nature and suitable for simultaneous estimation of TMZ and GLA from the developed nanoparticles formulation. Further, employing a greenness assessment approach established the method greenness.
本工作旨在开发一种简单、灵敏、绿色且稳健的高效液相色谱（HPLC）法，用于同时测定替莫唑胺（TMZ）和 γ-亚麻酸（GLA）。化学计量因素筛选研究有助于确定使用 Box-Behnken 设计优化的关键方法参数，以提高对方法性能的理解和增强方法性能。在 Kinetex® C 柱（150×4.6mm，5μm粒径）上进行色谱分离，以甲醇：水（使用 0.5%v/v 磷酸调至 pH3.5）为流动相，流速为 0.5mL/min，二极管阵列检测波长在 210 至 360nm 之间。TMZ 和 GLA 的浓度在 1 至 100μg.mL 范围内呈线性（R=0.999，p<0.05）。准确度评价显示，97.9-100%的回收率良好，而日内和日间精密度显示 RSD 值在 0.37%-1.01%之间。TMZ 的检测限和定量限分别为 0.75 和 1.0μg.mL，而 GLA 的相应值分别为 0.55 和 1.0μg.mL。系统适用性（96.9-102.8%）、其限度和稳健性评价表明，回收率良好，RSD 值在可接受的 2%以内。该方法具有特异性，能够在强制降解研究中检测到分析物及其降解产物。这也表明，TMZ 对碱性条件高度敏感，而 GLA 在所有研究条件下均有轻度降解。从脂质纳米粒制剂中估算两种分析物的总药物含量（82.6-85.3%）、包封效率（95.4 至 98.7%）和药物载量（25.2 至 38.4%）均表现良好。总的来说，结果表明，所开发的方法准确、精密、灵敏且专属性强，可用于同时估算分析物。该方法具有稳定性指示性质，适用于从开发的纳米粒制剂中同时估算 TMZ 和 GLA。此外，采用绿色评估方法确定了该方法的绿色性。
