Center for Neuroscience and Cell Biology, University of Coimbra, Rua Larga, 3004-504 Coimbra, Portugal.
AAPS PharmSciTech. 2013 Jun;14(2):649-55. doi: 10.1208/s12249-013-9950-4. Epub 2013 Mar 7.
A new, simple, inexpensive, and rapid 96-well plate UV spectrophotometric method was developed and validated for the quantification of compound 48/80 (C48/80) associated with particles. C48/80 was quantified at 570 nm after reaction with acetaldehyde and sodium nitroprusside in an alkaline solution (pH 9.6). The method was validated according to the recommendations of the ICH Guidelines for specificity, linearity, range, accuracy, precision, and detection and quantification limits (DL and QL). All the validation parameters were assessed in three different solvents, i.e., deionized water, blank matrix of chitosan nanoparticles, and blank matrix of chitosan/alginate nanoparticles. The method was found to be linear in the concentration range of 5 to 160 μg/ml (R(2)>0.9994). Intraday and interday precision was adequate, with relative standard deviation lower than those given by the Horwitz equation. The mean recoveries of C48/80 from spiked samples ranged between 98.1% and 105.9% for calibration curves done with the blank matrices and between 89.3% and 103.3% for calibration curves done with water, respectively. The DL were lower than 1.01 μg/ml and the QL were lower than 3.30 μg/ml. The results showed that the developed method is sensitive, linear, precise, and accurate for its intended use, with the additional advantages of being cost-effective and time-effective, allowing the use of small-volume samples, and the simultaneous analysis of a large number of samples. The proposed method was already successfully applied to evaluate the loading efficacy of C48/80 chitosan-based nanoparticles and can be easily applied during the development of other C48/80-based formulations.
建立并验证了一种新的、简单的、经济的、快速的 96 孔板紫外分光光度法，用于定量与颗粒结合的化合物 48/80（C48/80）。C48/80 在碱性溶液（pH 9.6）中与乙醛和硝普酸钠反应后，在 570nm 处进行定量。该方法根据 ICH 指南的要求进行了验证，包括专属性、线性、范围、准确度、精密度以及检测限（DL）和定量限（QL）。所有验证参数均在三种不同溶剂中进行评估，即去离子水、壳聚糖纳米颗粒空白基质和壳聚糖/海藻酸钠纳米颗粒空白基质。该方法在 5 至 160μg/ml 的浓度范围内呈线性（R²>0.9994）。日内和日间精密度良好，相对标准偏差低于 Horwitz 方程给出的值。用空白基质制作的校准曲线，从加标样品中测得的 C48/80 的平均回收率在 98.1%至 105.9%之间，而用水制作的校准曲线，平均回收率在 89.3%至 103.3%之间。DL 低于 1.01μg/ml，QL 低于 3.30μg/ml。结果表明，该方法灵敏、线性、精确、准确，适用于其预期用途，具有成本效益高、时间效益高、允许使用小体积样品以及同时分析大量样品等额外优势。该方法已成功应用于评估 C48/80 壳聚糖纳米颗粒的载药效率，并可轻松应用于其他 C48/80 制剂的开发过程中。
