High-Throughput Method for the Simultaneous Determination of Doxorubicin Metabolites in Rat Urine after Treatment with Different Drug Nanoformulations.

Lara Zorić,Leo Frkanec,Ruža Frkanec,Nikša Drinković,Akif Emre Türeli,Ivana Vinković Vrček,Nazende Günday-Türeli,Vedran Micek

doi:10.3390/molecules27041177

Abstract

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).

Highlights

The treatment of malignant diseases poses many challenges that need to be overcome, due to their wide prevalence and high mortality rates [1]
Anthracyclines are a group of antitumor drugs that were isolated from the genus Streptomyces in the 1960s, and their main representatives are doxorubicin (DOX, depicted in Figure 1) and daunorubicin [2]
The developed and validated method, which had high sensitivity, selectivity, and accuracy, has been successfully applied for the determination of DOX and its metabolites in rat urine treated with different DOX formulations

Summary

Introduction

The treatment of malignant diseases poses many challenges that need to be overcome, due to their wide prevalence and high mortality rates [1]. Anthracyclines are a group of antitumor drugs that were isolated from the genus Streptomyces in the 1960s, and their main representatives are doxorubicin (DOX, depicted in Figure 1) and daunorubicin [2]. They are used in the treatment of numerous solid tumors and hematological malignancies, including leukemia, lymphoma, breast, and ovarian tumors [3,4]. DOX enters cells by free diffusion, and achieves its action. Molecules 2022, 27, 1177 malignancies, including leukemia, lymphoma, breast, and ovarian tumors [3,4]. DOX enters cells by free diffusion, and achieves its action in the nucleus and mitochondria.

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