Development and validation of a high-performance liquid chromatographic method with a fluorescence detector for the analysis of epirubicin in human urine and plasma, and its application in drug monitoring
Introduction
Anthracyclines are some of the most commonly used and most effective anti-cancer drugs. Although their multidirectional mechanism of action is not fully explained, the drug interaction with the DNA-topoisomerase II complex is considered to be the basis of activity responsible for cytostatic and cytotoxic effects. Blocking the complex prevents further transformations, which leads to the inhibition of DNA and RNA synthesis, resulting in cell death [1]. In addition, it has been proven that patients receiving anthracyclines are exposed to oxidative stress associated with increased production of free radicals, which may be one of the causes of serious side effects, such as cardiotoxicity [2]. The first drugs of this class used in the treatment of cancer were daunorubicin and doxorubicin. They had a wide spectrum of activity and high efficacy in treating various cancers. However, the life-threatening side effects became the reason for the search for drug analogs with the same efficacy but lower toxicity [3], [4]. As a result, a semi-synthetic analogue of doxorubicin – 4′-epidoxorubicin, called epirubicin, has been developed. Epirubicin, as a more lipophilic compound than doxorubicin, penetrates the cell membrane more easily and has a faster activity in cancer cells. In addition, epirubicin is much more metabolized via the glucuronic acid conjugation process than all other anthracyclines. The pathway leads to a reduction in drug accumulation and consequently to a reduced risk of side effects. Decreased negative influence on myocytes of epirubicin may result from the fact that it is not involved in the formation of free radicals as much as doxorubicin or daunorubicin [5], [6].
Epirubicin is a compound of great significance in clinical practice, as it can be used in both monotherapy and combination therapy. There are many chemotherapeutic regimes, which include epirubicin, most commonly it is supported by an alkylating antineoplastic agent. Epirubicin can be administered within adjuvant and neoadjuvant therapy of various cancers of children (rhabdomyosarcoma) and adults (cancer of breast, lung, stomach and ovary) [7], [8], [9], [10]. Although epirubicin causes fewer side effects than doxorubicin, they still pose serious problems for cancer patients and oncologists. Apart from vomiting, nausea, hair loss, fatigue, myelosupression, anemia and infections, the greatest risk for the patient’s life and its quality is associated with cardiotoxicity [4], [11].
Studies show that the risk of cardiotoxicity of epirubicin depends on its single and cumulative doses and some unmodified factors, such as gender, age, concomitant diseases and medications [12]. In clinical practice, the doses of epirubicin are calculated per body weight or surface of the patient and depend on the type of cancer. However, such dosing may not be optimal for some patients, since the action and metabolism of epirubicin may depend on inter-individual differences in the pharmacokinetics of the drug.
Better therapeutic results with lower toxic effects might be achieved with more personalized anti-cancer therapy based on monitoring the level of epirubicin in plasma or urine. However, for this purpose it would be necessary to develop new effective methods of detection. The first information on the technique of detection and determination of epirubicin in biological samples was reported in 1984 [13]. Since then, many papers have been published on various methods for the determination of this drug, mainly in human plasma [14], [16], [17], [18], [19], [20], [21], [22], [23], [24], and serum [15], [25], [26], [27]. In addition, analyzes were performed on samples of urine [14], [15], [28], [29], [30], bile [14], human saliva [19], [21], horse serum [31] and urine, plasma, bile and tissue of rats [32]. Most of them were performed by liquid chromatography using a fluorescence detector (FL) [14], [15], [19], [21], [22], [23], [24], [31], [32] and an ultraviolet (UV) detector [16], [27]. For FL detection, the wavelength of the excitation radiation was 470–480 nm and the emitted radiation 550–580 nm. Only in one study the excitation wavelength was 254 nm [31]. Epirubicin was also detected using liquid chromatography coupled to tandem mass spectrometry (MS) with electrospray ionization (LC-ESI-MS/MS) [17], [18], [26], [28], [29], [30] and atmospheric-pressure chemical ionization (LC-APCI-MS/MS) [25]. An electrochemical method was also applied for the quantification of epirubicin in human plasma [20].
The widespread use of FL and UV–Vis detectors for epirubicin determination is undoubtedly associated with their presence in all modern laboratories. Their low price and the possibility of developing relatively simple analytical methods make them a willingly used analytical tool. However, it should be highlighted that the FL detector allows for almost 30 times higher sensitivity than the UV detector, which is particularly important for the examination of biological samples, where analytes concentrations are low. In addition, naturally occurring epirubicin fluorescence eliminates the necessity of derivatization reactions, which reduces time and improves test results when using the FL detector. Currently, the methods of determination of epirubicin based on the FL detector allow achieving limits of detection (LOD) at the level of 0.255 ng/mL with 1 mL of biological fluid required [14].
The MS detection provides very good detection results. In the case of epirubicin quantification, it is over 140 times more sensitive than the FL detector and over 16,000 times than the UV detector. The main limitations of using MS detection include high costs, and the requirement of having qualified and experienced analysts. In addition, the application of mobile phases with the addition of non-volatile salts, which may improve the results, is not recommended due to the risk of damage of MS apparatus. To our best knowledge, the analysis of real samples obtained from cancer patients with the use of the MS was performed in plasma and saliva. However, the urine samples were never analyzed this way.
Preparation of samples with optimal choice of conditions is the most important part of the analysis, significantly influencing the results’ quality. In previous publications, samples were purified by deproteinization [15], [21], [32], concentrated during liquid-liquid extraction (LLE) [19], [21], [22], [25], solid phase extraction (SPE) [14], [16], [17], [18], [20], [24], [26], [28], [29], [30], [31], dispersive solid-phase extraction (d-SPE) [30] and online SPE [27]. The deproteinization was performed with acetonitrile (ACN) [15], methanol (MeOH) [32] and ethanol [21]. Chloroform/2-propanol [19], chloroform/1-heptanol [22], dichloromethane (DChM) [21] were used as the eluting solvent during LLE. The most frequently was used SPE method in which many modifications for improving epirubicin extraction have been proposed. Therefore, cartridges with different types of solid phases and various eluents for the extraction of the analytes were applied. For example, hydrophilic-lipophilic balance (HLB) [16], [17], [18], [20], C18 [14], [24], [26], [28], [29], [30], C2 [23], C8 [31], and Biotrap 500 MS [27] columns were used. Moreover, MeOH [14], [30], ACN [31] and mixtures of methylene chloride/2-propanol/MeOH [17], [24], [29], MeOH/formic acid [18], methylene chloride/2-propanol [28], chloroform/2-propanol [26], MeOH/ammonium hydroxide [16] and water/ACN [20] were used as eluting agents.
It should be emphasized that among all the developed methods for the determination of epirubicin in biological materials, only a few were used to analyze real samples [15], [17], [18], [19], [20], [21], [27]. All methods of sample preparation enabled to obtain acceptable validation parameters, but while analyzing real samples, it is important that the method is universal for various matrices. The development of one method which can be successfully applied to various biological materials is essential to determine the complete profile of the drug in the body. To the best of our knowledge, since 2013 there have been no publications reporting epirubicin measurement in the urine of cancer patients, despite the fact that the drug is eliminated mainly via bile and urine [32]. Also, a potential exposure to epirubicin present in urine for medical personnel has never been evaluated.
The main aim of the study was to develop a sensitive, precise and accurate method to quantify the epirubicin concentration in human urine and plasma, which can be included in routine clinical analysis. The next step of the study was to confirm the usefulness of the developed and validated method for epirubicin determination in clinical practice. Therefore, the profiles of epirubicin in urine and plasma samples of a 19-year-old patient with metastatic alveolar rhabdomyosarcoma (RMA) were calculated. The results were compared to those obtained in other studies to better understand the pharmacokinetics of epirubicin. In addition, attention was drawn to the requirement for medical personnel to comply with the safety rules in contact with biological fluids.
Section snippets
Reagents
Epirubicin hydrochloride (>98% purity) was purchased from Cayman Chemical Company (USA). Daunorubicin hydrochloride (DAU) (>98% purity) was obtained from Tocris Bioscience (Bristol, United Kingdom) whereas idarubicin hydrochloride (IDA) (>98% purity) and doxorubicin hydrochloride (>98% purity) were supplied by Cayman Chemical Company (USA). Chloroform, 2-propanol and DChM of analytical grade were obtained from Merck (Darmstadt, Germany). HPLC grade ACN and MeOH were provided by J.T. Baker
Results and discussions
The use of anthracyclines, including epirubicin, in systemic cancer therapy of adult and pediatric patients, requires conducting research that may improve the effects of these drugs while minimizing their complications. To our knowledge, in the last few years most LC methodologies have been developed for simultaneous determination of epirubicin and its metabolites or other analytes in plasma [14], [16], [17], [18], [19], [20], [21], [22], [23], [24], [32], serum [15], [25], [26], [27], [31],
Conclusion
In this study, a simple, accurate, precise, selective and sensitive method was developed for the quantitative determination of epirubicin in urine and plasma using the LC-FL technique. A new protocol of the sample pretreatment, chromatographic separation and detection conditions was proposed in comparison to the earlier published methods. Detection and quantification limits were the lowest among all previous results achieved on the fluorescence detector. Additionally, the volume of urine
CRediT authorship contribution statement
Natalia Treder: Conceptualization, Methodology, Software, Validation, Formal analysis, Writing - original draft, Writing - review & editing, Visualization, Project administration. Olga Maliszewska: Conceptualization, Methodology, Software, Validation, Writing - original draft, Data curation, Project administration, Visualization. Ilona Olędzka: Conceptualization, Methodology, Writing - original draft, Writing - review & editing. Piotr Kowalski: Conceptualization, Validation, Writing - review &
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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