Faculty of Pharmacy and Biochemistry, University of Zagreb, Ante Kovačića 1, 10000 Zagreb, Croatia.
Poliklinika Prof. Nikša Drinković, Boškovićeva Ul. 15, 10000 Zagreb, Croatia.
Molecules. 2022 Feb 9;27(4):1177. doi: 10.3390/molecules27041177.
Doxorubicin (DOX) is one of the most effective cytotoxic agents against malignant diseases. However, the clinical application of DOX is limited, due to dose-related toxicity. The development of DOX nanoformulations that significantly reduce its toxicity and affect the metabolic pathway of the drug requires improved methods for the quantitative determination of DOX metabolites with high specificity and sensitivity. This study aimed to develop a high-throughput method based on high-performance liquid chromatography with fluorescence detection (HPLC-FD) for the quantification of DOX and its metabolites in the urine of laboratory animals after treatment with different DOX nanoformulations. The developed method was validated by examining its specificity and selectivity, linearity, accuracy, precision, limit of detection, and limit of quantification. The DOX and its metabolites, doxorubicinol (DOXol) and doxorubicinone (DOXon), were successfully separated and quantified using idarubicin (IDA) as an internal standard (IS). The linearity was obtained over a concentration range of 0.05-1.6 μg/mL. The lowest limit of detection and limit of quantitation were obtained for DOXon at 5.0 ng/mL and 15.0 ng/mL, respectively. For each level of quality control (QC) samples, the inter- and intra-assay precision was less than 5%. The accuracy was in the range of 95.08-104.69%, indicating acceptable accuracy and precision of the developed method. The method was applied to the quantitative determination of DOX and its metabolites in the urine of rats treated by novel nanoformulated poly(lactic-co-glycolic acid) (DOX-PLGA), and compared with a commercially available DOX solution for injection (DOX-IN) and liposomal-DOX (DOX-MY).
多柔比星（DOX）是对抗恶性疾病最有效的细胞毒性药物之一。然而，由于剂量相关的毒性，DOX 的临床应用受到限制。开发显著降低其毒性并影响药物代谢途径的 DOX 纳米制剂需要改进方法，以高特异性和灵敏度定量测定 DOX 代谢物。本研究旨在开发一种基于高效液相色谱法与荧光检测（HPLC-FD）的高通量方法，用于定量测定不同 DOX 纳米制剂处理后的实验室动物尿液中的 DOX 及其代谢物。通过检查方法的特异性和选择性、线性、准确性、精密度、检测限和定量限来验证所开发的方法。使用盐酸伊达比星（IDA）作为内标（IS），成功分离和定量测定 DOX 及其代谢物多柔比星醇（DOXol）和多柔比星酮（DOXon）。在 0.05-1.6 μg/mL 的浓度范围内获得线性。DOXon 的最低检测限和定量限分别为 5.0 ng/mL 和 15.0 ng/mL。对于每个质控（QC）样品水平，日内和日间精密度均小于 5%。准确度在 95.08-104.69%范围内，表明所开发方法具有可接受的准确度和精密度。该方法应用于新型聚（乳酸-共-乙醇酸）（DOX-PLGA）纳米制剂处理的大鼠尿液中 DOX 和其代谢物的定量测定，并与市售多柔比星注射液（DOX-IN）和脂质体多柔比星（DOX-MY）进行比较。
