Original Article revista.iq.unesp.br | Vol. 47 | n. 3 | 2022 | 32 Eclética Química Journal, vol. 47, n. 3, 2022, 32-38 ISSN: 1678-4618 DOI: 10.26850/1678-4618eqj.v47.3.2022.p32-38 Voltammetric glassy carbon sensor approach for the extended stability studies of doxorubicin in lyophilized dosage form Carlos Eduardo Peixoto Cunha1+ , Edson Silvio Batista Rodrigues1 , Jerônimo Raimundo de Oliveira Neto1 , Vernon Somerset2 , Stephânia Taveira1 , Lívia Sgobbi3 Eric de Souza Gil1 1. Federal University of Goiás, Pharmacy, Goiânia, Brazil. 2. Cape Peninsula University of Technology, Faculty of Applied Sciences, Cape Town, South Africa. 3. Federal University of Goiás, Chemistry, Goiânia, Brazil. +Corresponding author: Carlos Eduardo Peixoto Cunha, Phone: +55 62 981120522 Email address: cedcunhap35@hotmail.com ARTICLE INFO Article history: Received: November 03, 2021 Accepted: May 05, 2022 Published: July 01, 2022 Section Editor: Assis Vicente Benedetti Keywords 1. electrochemistry 2. quality control of medicines 3. analytical validation 4. voltammetry 5. anticancer drugs ABSTRACT: Doxorubicin (DOX) is an anthracycline antibiotic that is widely used in the clinical treatment of cancer patients. DOX has a high market value. Electroanalytical methods for DOX analysis are an alternative and promising approach compared to chromatographic techniques. In this context, electroanalysis provides a low-cost method for determining drugs such as DOX lyophilized powder for the injection. Differential pulse voltammetry with a glassy carbon electrode was used. DOX stability after reconstitution was performed, and the correct time for safe administration to patients in hospitals was determined. The electroanalytical method showed a limit of detection of 0.54 µmol L-1 and limit of quantification of 1.83 µmol L–1, which is enough for the application in quality control of DOX. The high- performance liquid chromatography analysis was also applied in pharmaceutical samples containing DOX to compare with the proposed method, showing that the obtained results are relatively similar for both methods. Therefore, the electroanalytical approach shows the viability of an attractive alternative technique for applying this sensor for drug quality control. https://revista.iq.unesp.br/index.php/ecletica https://doi.org/10.26850/1678-4618eqj.v47.3.2022.p32-38 mailto:cedcunhap35@hotmail.com https://orcid.org/0000-0002-6740-7185 https://orcid.org/0000-0003-2965-0421 https://orcid.org/0000-0002-0261-4554 https://orcid.org/0000-0001-8643-1853 https://orcid.org/0000-0003-3844-6334 https://orcid.org/0000-0002-0372-9396 https://orcid.org/0000-0001-9161-0127 Original Article revista.iq.unesp.br 33 Eclética Química Journal, vol. 47, n. 3, 2022, 32-38 ISSN: 1678-4618 DOI: 10.26850/1678-4618eqj.v47.3.2022.p32-38 1. Introduction Doxorubicin hydrochloride (DOX) (Fig. 1) is an anthracycline antibiotic which starting material is Streptomyces percetius var. cesium, which is widely used in the clinical treatment of patients with leukemias and tumors in the lung or breast. Doxorubicin hydrochloride is composed of an amino sugar linked to anthraquinone aglycone, as shown in in its chemical structure (Fig. 1). Its mechanism of action is elucidated in four different ways, i.e., a) inhibition of deoxyribonucleic acid (DNA) synthesis in tumor cells; b) creation of free radicals, which can damage DNA; c) induction of DNA damage due to DOX interference with topoisomerase II; and d) induction of apoptosis (Alhareth et al., 2012; Li et al., 2020; Skalová et al., 2020). Figure 1. Doxorubicin chemical structure. Doxorubicin hydrochloride is highly prescribed for therapeutic use in the form of a lyophilized powder for injection, so it is necessary to ensure the quality of the drug. Doxorubicin hydrochloride has a high market value, so it is a drug with less accessibility to patients with less purchasing power However, hospitals allow the use of the DOX injection form to grant access to more patients. Nonetheless, it is necessary to carry out stability control after reconstitution of the lyophilized powder for DOX injection to ensure the drug’s efficacy and safety (Navas et al., 2013; Radi, 2003; Rodrigues et al., 2018). Quality control tests involve checking the content and possible impurities and stability of the drug. Techniques with good sensitivity are needed to perform the quality control tests for DOX, with the official methods used to determine the content consisting of high-performance liquid chromatography (HPLC) tests with UV detection and UV-VIS spectrometry (Felix and Angnes, 2018; Navas et al., 2013). These techniques require expensive instrumentation, centralized laboratory and well- qualified personnel, time-consuming sample pretreatment steps in addition to making use of many organic reagents, also not environment-friendly (ACS, 2019; Hahn and Lee, 2004; Shellaiah and Sun, 2020). As an alternative to those above conventional analytical methods, electrochemical sensors offer several remarkable attributes, such as low-cost instrumentation, elimination or reduction of sample pretreatment steps, fast response, and the possibility of online and in situ detection. Furthermore, electroanalytical methods provide good sensitivity, versatility, and cleaner and more sustainable analysis. The glassy carbon electrode (GCE) and the carbon paste electrode (CPE) have been used frequently among many electrodes employed in electroanalysis. Modifications of these electrodes, mostly with catalysts and/or nanoparticles, have displayed a significant increase in sensitivity for electrochemical detection (Hajian et al., 2017; Shah et al., 2018; Skalová et al., 2020). This work aims to perform the electroanalytical determination of DOX in lyophilized powder form, by differential pulse voltammetry (DPV) with a GCE sensor. We also aimed to verify the stability of DOX after reconstitution, establishing the correct time for safe administration to patients in hospitals. For comparison, DOX determination was also performed by HPLC-UV. 2. Materials and Methods Electrolyte solutions were prepared using high analytical grade salts, which were solved in Milli-Q water (conductivity ≤ 0.1 µS cm–1) (Millipore S. A., Molsheim, France). Analytical grade DOX was obtained from the United States Pharmacopeia (USP) (≥ 99%). Pharmaceutical formulations of DOX (10 mg mL–1) were kindly provided by a private oncology health unit (Rio de Janeiro/RJ – Brazil). The stock standard solution was prepared from a test dose of 27.18 mg in a 50 mL volumetric flask, then 25 mL of Milli-Q water was added and taken to ultrasound until complete solubilization, then the volume of the flask obtaining a concentration of 1.0 mmol L–1 DOX, the solution was prepared immediately before the experiments. The samples of the reconstituted drug destined for the evaluation of the stability against the ambient temperatures and at 2 to 8 °C by voltammetric tests were prepared from an initial solution of DOX reconstituted in water for injection in its original packaging and divided into four 10-mL amber glass volumetric flasks. In contrast, those for monitoring by https://revista.iq.unesp.br/index.php/ecletica https://doi.org/10.26850/1678-4618eqj.v47.3.2022.p32-38 Original Article revista.iq.unesp.br 34 Eclética Química Journal, vol. 47, n. 3, 2022, 32-38 ISSN: 1678-4618 DOI: 10.26850/1678-4618eqj.v47.3.2022.p32-38 chromatographic tests were packaged in four 100-mL amber glass volumetric flasks. The tests were performed at zero time and repeated in 2, 4, 6, 12, 24, 48, and 96 h in the samples exposed to the environmental conditions proposed by the study. 2.1. Electroanalytical tests Voltammetric measurements were performed using a potentiostat/galvanostat PGSTAT model 204 with a FRA32M module (Metrohm Autolab, Eco Chemie, Netherlands) integrated with NOVA 2.1 software. All measurements were carried out in a 1-mL one- compartment electrochemical cell coupled to a three- electrode system consisting of a GCE, a Pt wire counter electrode and an Ag/AgCl/KClsat reference electrode (both purchased from Lab Solutions, São Paulo, Brazil). The experimental conditions used for the DPV were pulse amplitude of 50 mV, pulse width of 0.5 s, and scan rate of 10 mV s−1. All voltammetric assays were performed in 0.1 mol L−1 phosphate buffer solution (PBS), pH 7.0. The DP voltammograms were background-subtracted and baseline-corrected. All experiments were conducted in triplicate, and data were analyzed using Origin Pro 9 software (Northampton, MA, USA). Between each voltammogram reading, the GCE was sanded with 0.3 m alumina suspension and subsequently rinsed with Milli-Q water, in order to renew the electrode surface, thus ensuring the reproducibility of the tests. 2.2 Chromatography tests For the chromatographic assay, a stainless-steel column (250 × 4.6 mm) was used, packed with octadecylsilyl silica gel for chromatography (5 µm) (Hypersil C18). The elution was isocratic with the mobile comprised of acetonitrile and acid surfactant solution (containing 0.288% w/v dodecyl sulfate and 0.225% w/v orthophosphoric acid) in a 50:50 (v/v) proportion. The analyses were performed at room temperature (25 °C). The flow was 1 mL min–1, the wavelength was 254 nm, and the injection volume was10 µL. The samples were prepared with 0.01% w/v of the reconstituted solution for injection diluted in the mobile phase, compared to the USP standard’s declared value (USP, 2020; Zhao and Dash, 1999). 2.3 Method validation The method was developed and validated for linearity, accuracy, precision, quantification limit, detection limit, and selectivity in accordance with ICH Q2 (R1). The linearity of the method was performed through three analytical curves using DOX standard solutions. The results were statistically analyzed by linear regression analysis using the least squares method (ICH, 2014). 3. Results and discussion 3.1 Electroanalytical tests The DP voltammogram obtained with GCE showed one main oxidation peak for DOX at Epa = 0.33 V corresponding to oxidation in the quinonic portion of the anthracyclines drug class, followed by a second and third oxidation peaks, at Epa = 0.60 V and Epa = 0.75 V that are explained by the adsorption of DOX, attributed to the formation of a hydrogen bond between the hydroxyl groups of the phenolic compound and the carbonyl group (Piovesan and Spinelli, 2014). Figure 2. Calibration curve of DOX with DPV with GCE in PBS pH 7.0. A calibration curve (Fig. 2) was constructed to determine the ideal concentration for DOX recovery in the stability study. Linearity was found on the calibration curve for the anodic peak Epa1 a.a. 0.33 V. As Epa1 presented a good linearity coefficient (r² = 0.9993), it was used to calculate the regression equation: y = (2.81± 0.09) + (0.48 ± 0.01 × [DOX] (mol L–1). As the p-value (0) found in the ANOVA F test is less than 0.05, it was rejected the null hypothesis (zero slope) at the significance level of 5%, whereas the P-value of 2755.7954 of the t-test is greater than 0.05, therefore, it was not rejected the null hypothesis (intercept equal to zero) at the significance level of 5%. https://revista.iq.unesp.br/index.php/ecletica https://doi.org/10.26850/1678-4618eqj.v47.3.2022.p32-38 Original Article revista.iq.unesp.br 35 Eclética Química Journal, vol. 47, n. 3, 2022, 32-38 ISSN: 1678-4618 DOI: 10.26850/1678-4618eqj.v47.3.2022.p32-38 The correlation coefficient found of 0.9993 is greater than 0.9900, so it was concluded that there is an adequate linear relationship, showing residual sum of squares of 3.94 × 10–9. A comparison of the analytical parameters obtained by the proposed method and HPLC for the determination of DOX in pharmaceutical samples is described in Tab. 1. Table 1. Precision and Repeatability assays values for voltammetric and chromatographic assays. Samples Sample amount (%) Methods DPV – GCE (% ± SD) (n = 6) HPLC (% ± SD) (n = 6) DOX lyophilized powder for injection Repeatability (intraday) 100.0 101.4 ± 0.8 100.9 ± 0.2 DOX lyophilized powder for injection tablets Intermediary precision (interday) 100.0 101.0 ± 0.5 100.6 ± 0.1 SD: standard deviation. For comparison reasons, HPLC recovery assays were performed. Repeatability assays were performed within a single day with intervals of 2 h. Intermediary precision assays were performed on three different days with three different analysts with intervals of 4 h for each day. It can be seen from Tab. 1 that both methods displayed acceptable precision. The voltammetric method displayed slightly higher deviations of precision in both assays in comparison with HPLC, as expected. However, due to the great difference between equipment and preprocessing of both approaches, the precision difference shows that the method developed in this work is within an appropriate range of precision in comparison with pharmacopoeia methods. The results are further in agreement with the study conducted by Macêdo et al. (2020) and Cunha et al. (2019). Accuracy assays were also performed for both methods and the results are shown in Tab. 2. Table 2. Accuracy assays validation data for voltammetric and chromatographic assays for DOX lyophilized powder for injection. Samples Sample amount (%) Methods DPV – GCE (% ± RSD) HPLC (% ± RSD) DOX lyophilized powder for injection 80.0 81.4 ± 0.7 80.7 ± 0.2 100.0 101.1 ± 0.5 101.5 ± 0.2 120.0 120.7 ± 0.8 120.1 ± 0.4 RSD: Relative standard deviation. As observed in Tab. 2, the accuracy of both methods was satisfactory, with results within 5% deviation range. The chromatography approach showed only slightly lesser deviation than the voltammetric method with the GCE, corroborating the considerations made previously. Table 3 presents the limit of detection (LOD) and limit of quantification (LOQ) values of this study compared to the results obtained from the literature data used to determine DOX. Table 3. Comparisons of the limits of detection and the limits of quantification in the determination of DOX with other methods. Method LOD LOQ Reference DPV 0.54 µmol L–1 1.83 µmol L–1 This work HPLC 0.5 ng mL–1 5.0 ng mL–1 Skalová et al., 2020 DPV-PGE 9.9 µmol L–1 33.31 µmol L–1 Cunha et al., 2019 DPCSV 0.44 µmol L–1 0.6 µmol L–1 Deepa et al., 2020 HPLC 0.2 ng mL–1 0.6 ng mL–1 Thomaz et al., 2018 Next, a summary and a comparison of our method depicted in Tab. 3 were provided, which shows the current analytical approaches for the detection of DOX, from which it is possible to infer that an adequate sensitivity was obtained for the simplified electroanalytical approach used. The results further demonstrate the availability of a faster analytical method and at a lower cost compared to other methods. https://revista.iq.unesp.br/index.php/ecletica https://doi.org/10.26850/1678-4618eqj.v47.3.2022.p32-38 Original Article revista.iq.unesp.br 36 Eclética Química Journal, vol. 47, n. 3, 2022, 32-38 ISSN: 1678-4618 DOI: 10.26850/1678-4618eqj.v47.3.2022.p32-38 The electroanalytical method proposed in this work showed a LOD of 0.54 µmol L–1 and LOQ of 1.83 µmol L–1, which is sensitive enough for the application in quality control of DOX in lyophilized powder for injection. 3.2 Extended stability study test on a DOX pharmaceutical sample The GCE sensor was also used to monitor the stability of DOX lyophilized powder for injection applications. In this context, injectable DOX solutions were evaluated after reconstitution at room temperature and at temperatures from 2 to 8 °C. Analytical assays were performed at different times monitoring the decay of the DOX content after reconstitution, where the recovery in percentage after each period was evaluated, determining the stability after its reconstitution through the assay found (Tab. 4). As expected, the recovery values for voltammetric and chromatographic determinations showed DOX degradation after reconstitution. However, at room temperature, it shows DOX degradation after 48 h, while from 2 to 8 °C it remained stable until 96 h. (Tab. 4). All concentrations found were less than 5% of the relative standard deviation. Both methods showed effective approaches to assess the stability of DOX lyophilized powder for injection after reconstitution. Table 4. Results obtained for the recovery of DOX in the evaluation of the extended stability study at different times. Time (h) % DOX (at room temperature) % DOX (2–8 °C) DPV HPLC DPV HPLC 0 92.74 92.33 92.74 92.33 2 92.18 93.36 92.22 93.34 4 91.13 92.92 93.62 93.84 6 92.04 92.61 91.61 91.50 12 91.38 90.20 92.55 92.33 24 92.07 88.47 91.12 90.74 48 83.36 84.23 91.78 90.89 96 79.51 81.52 91.06 91.41 4. Conclusions The GCE sensor, when compared to other sensors and evaluation methods, offers efficiency in its analytical performance for the determination of lyophilized powder for injection. This characteristic, when associated with low cost, easy access, quick and efficient cleaning of the electrode surface area, indicates that the GCE sensor can be a useful tool for DOX analysis. Also, the GCE sensor exhibited satisfactory detection and recovery, although the standard deviation values were slightly higher than most of the sensors and methods applied. However, the results are following the specifications for such an analysis. The general analytical performance and the low cost of the material associated with the immediate analysis provided by both electrodes, consistently justify the choice of these analytical devices as alternative approaches to quality control and extended drug stability studies. Authors’ contribution Conceptualization: Cunha, C. E. P.; Gil, E. S.; Rodrigues, E. S. B; Sgobbi, L. F.; Taveira, S. F Data curation: Cunha, C. E. P.; Rodrigues, E. S. B.; Oliveira Neto, J. R; Sgobbi, L. F.; Taveira, S. F Formal Analysis: Cunha, C. E. P.; Rodrigues, E. S. B.; Oliveira Neto, J. R; Sgobbi, L. F.; Taveira, S. F Funding acquisition: Not applicable. Investigation: Cunha, C. E. P.; Rodrigues, E. S. B.; Oliveira Neto, J. R; Sgobbi, L. F.; Taveira, S. F Methodology: Cunha, C. E. P.; Rodrigues, E. S. B.; Oliveira Neto, J. R; Sgobbi, L. F.; Taveira, S. F Project administration: Gil, E. S. Resources: Gil, E. S. Software: Not applicable. Supervision: Gil, E. S. Validation: Somerset, V. Visualization: Cunha, C. E. P.; Rodrigues, E. S. B.; Oliveira Neto, J. R; Sgobbi, L. F.; Taveira, S. F Writing – original draft: Cunha, C. E. P.; Rodrigues, E. S. B.; Oliveira Neto, J. R Writing – review & editing: Gil, E. S.; Somerset, V. https://revista.iq.unesp.br/index.php/ecletica https://doi.org/10.26850/1678-4618eqj.v47.3.2022.p32-38 Original Article revista.iq.unesp.br 37 Eclética Química Journal, vol. 47, n. 3, 2022, 32-38 ISSN: 1678-4618 DOI: 10.26850/1678-4618eqj.v47.3.2022.p32-38 Data availability statement All dataset were generated or analyzed in the current study. Funding Not applicable. Acknowledgments We thank to AV Farma for their support and to the Federal University of Goiás (UFG) for supporting this work. References ACS Publications Home Page. 2019. https://pubs.acs.org/ (accessed 2019-02-21). Alhareth, K.; Vauthier, C.; Gueutin, C.; Ponchel, G.; Moussa, F. HPLC quantification of doxorubicin in plasma and tissues of rats treated with doxorubicin loaded poly(alkylcyanoacrylate) nanoparticles. J. Chromatogr. B Biomed. Appl. 2012, 887–888, 128–132. https://doi.org/10.1016/j.jchromb.2012.01.025 Cunha, C. E. P.; Rodrigues, E. S. B.; Alecrim, M. F.; Thomaz, D. V.; Macêdo, I. Y. L.; Garcia, L. F.; Oliveira Neto, J. R.; Moreno, E. K. G.; Ballaminut, N.; Gil, E. de S. Voltammetric Evaluation of Diclofenac Tablets Samples through Carbon Black-Based Electrodes. Pharmaceuticals. 2019, 12 (2), 83. https://doi.org/10.3390/ph12020083 Deepa, S.; Swamy, B. E. K.; Pai, K. P. Voltammetric detection of anticancer drug Doxorubicin at pencil graphite electrode: A voltammetric study. Sensors International 2020, 1, 100033. https://doi.org/10.1016/j.sintl.2020.100033 Felix, F. S.; Angnes, L. Electrochemical immunosensors – A powerful tool for analytical applications. Biosens. Bioelectron. 2018, 102, 470–478. https://doi.org/10.1016/j.bios.2017.11.029 Hahn, Y.; Lee, H. Y. Electrochemical behavior and square wave voltammetric determination of doxorubicin hydrochloride. Arch. Pharm. Res. 2004, 27 (1), 31–34. https://doi.org/10.1007/BF02980041 Hajian, R.; Tayebi, Z.; Shams, N. Fabrication of an electrochemical sensor for determination of doxorubicin in human plasma and its interaction with DNA. J. Pharm. Anal. 2017, 7 (1), 27–33. https://doi.org/10.1016/j.jpha.2016.07.005 ICH Harmonized Tripartite Guideline. Validation of analytical procedures: text and methodology (Q2) R1. Current Step 4. International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use. 2014. https://database.ich.org/sites/default/files/Q2%28R1%29%2 0Guideline.pdf (accessed 2021-02-28). Li, D.; Xu, Y.; Fan, L.; Shen, B.; Ding, X.; Yuan, R.; Li, X.; Chen, W. Target-Driven Rolling Walker Based Electrochemical Biosensor for Ultrasensitive Detection of Circulating Tumor DNA Using Doxorubicin@tetrahedron- Au Tags. Biosens. Bioelectron. 2020, 148, 111826. https://doi.org/10.1016/j.bios.2019.111826 Macêdo, I. Y. L.; Alecrim, M. F.; Oliveira Neto, J. R.; Torres, I. M. S.; Thomaz, D. V.; Gil, E. S. Piroxicam voltammetric determination by ultra low cost pencil graphite electrode. Braz. J. Pharm. Sci. 2020, 56, e17344. https://doi.org/10.1590/s2175-97902019000317344 Navas, N.; Herrera, A.; Martínez-Ortega, A.; Salmerón- García, A.; Cabeza, J.; Cuadros-Rodríguez, L. Quantification of an intact monoclonal antibody, rituximab, by (RP)HPLC/DAD in compliance with ICH guidelines. Anal. Bioanal. Chem. 2013, 405 (29), 9351–9363. https://doi.org/10.1007/s00216-013-7368-1 Piovesan, J. V.; Spinelli, A. Determination of Quercetin in a Pharmaceutical Sample by Square-Wave Voltammetry Using a Poly(vinylpyrrolidone)-Modified Carbon-Paste Electrode. J. Braz. Chem. Soc. 2014, 25 (3), 517–525. https://doi.org/10.5935/0103-5053.20140019 Radi, A. Anodic voltammetric assay of lansoprazole and omeprazole on a carbon paste electrode. Pharm. Biomed. Anal. 2003, 31 (5), 1007–1012. https://doi.org/10.1016/S0731-7085(02)00707-0 Rodrigues, E. S. B.; Macêdo, I. Y. L.; Lima, L. L. S.; Thomaz, D. V.; Cunha, C. E. P.; Oliveira, M. T.; Ballaminut, N.; Alecrim, M. F.; Carvalho, M.F.; Isecke, B. G.; Leite, K. C. S.; Machado, F. B.; Guimarães, F. F.; Menegatti, R.; Somerset, V.; Gil, E. S. Electrochemical Characterization of Central Action Tricyclic Drugs by Voltammetric Techniques and Density Functional Theory Calculations. Pharmaceuticals 2018, 12 (3), 116 https://doi.org/10.3390/ph12030116 Shah, M.; Bourner, L.; Ali, S.; Al-Enazy, S.; Youssef, M. M.; Fisler, M.; Rytting, E. HPLC Method Development for Quantification of Doxorubicin in Cell Culture and Placental Perfusion Media. Separations. 2018, 5 (1), 9. https://doi.org/10.3390/separations5010009 Shellaiah, M.; Sun, K. W. Review on Sensing Applications of Perovskite Nanomaterials. Chemosensors. 2020, 8 (3), 55. https://doi.org/10.3390/chemosensors8030055 Skalová, Š.; Langmaier, J.; Barek, J.; Vyskočil, V.; Navrátil, T. Doxorubicin determination using two novel voltammetric https://revista.iq.unesp.br/index.php/ecletica https://doi.org/10.26850/1678-4618eqj.v47.3.2022.p32-38 https://pubs.acs.org/ https://pubs.acs.org/ https://doi.org/10.1016/j.jchromb.2012.01.025 https://doi.org/10.1016/j.jchromb.2012.01.025 https://doi.org/10.1016/j.jchromb.2012.01.025 https://doi.org/10.1016/j.jchromb.2012.01.025 https://doi.org/10.1016/j.jchromb.2012.01.025 https://doi.org/10.1016/j.jchromb.2012.01.025 https://doi.org/10.3390/ph12020083 https://doi.org/10.3390/ph12020083 https://doi.org/10.3390/ph12020083 https://doi.org/10.3390/ph12020083 https://doi.org/10.3390/ph12020083 https://doi.org/10.3390/ph12020083 https://doi.org/10.1016/j.sintl.2020.100033 https://doi.org/10.1016/j.sintl.2020.100033 https://doi.org/10.1016/j.sintl.2020.100033 https://doi.org/10.1016/j.sintl.2020.100033 https://doi.org/10.1016/j.bios.2017.11.029 https://doi.org/10.1016/j.bios.2017.11.029 https://doi.org/10.1016/j.bios.2017.11.029 https://doi.org/10.1016/j.bios.2017.11.029 https://doi.org/10.1007/BF02980041 https://doi.org/10.1007/BF02980041 https://doi.org/10.1007/BF02980041 https://doi.org/10.1007/BF02980041 https://doi.org/10.1016/j.jpha.2016.07.005 https://doi.org/10.1016/j.jpha.2016.07.005 https://doi.org/10.1016/j.jpha.2016.07.005 https://doi.org/10.1016/j.jpha.2016.07.005 https://doi.org/10.1016/j.jpha.2016.07.005 https://database.ich.org/sites/default/files/Q2%28R1%29%20Guideline.pdf https://database.ich.org/sites/default/files/Q2%28R1%29%20Guideline.pdf https://database.ich.org/sites/default/files/Q2%28R1%29%20Guideline.pdf https://database.ich.org/sites/default/files/Q2%28R1%29%20Guideline.pdf https://database.ich.org/sites/default/files/Q2%28R1%29%20Guideline.pdf https://database.ich.org/sites/default/files/Q2%28R1%29%20Guideline.pdf https://database.ich.org/sites/default/files/Q2%28R1%29%20Guideline.pdf https://doi.org/10.1016/j.bios.2019.111826 https://doi.org/10.1016/j.bios.2019.111826 https://doi.org/10.1016/j.bios.2019.111826 https://doi.org/10.1016/j.bios.2019.111826 https://doi.org/10.1016/j.bios.2019.111826 https://doi.org/10.1016/j.bios.2019.111826 https://doi.org/10.1590/s2175-97902019000317344 https://doi.org/10.1590/s2175-97902019000317344 https://doi.org/10.1590/s2175-97902019000317344 https://doi.org/10.1590/s2175-97902019000317344 https://doi.org/10.1590/s2175-97902019000317344 https://doi.org/10.1007/s00216-013-7368-1 https://doi.org/10.1007/s00216-013-7368-1 https://doi.org/10.1007/s00216-013-7368-1 https://doi.org/10.1007/s00216-013-7368-1 https://doi.org/10.1007/s00216-013-7368-1 https://doi.org/10.1007/s00216-013-7368-1 https://doi.org/10.5935/0103-5053.20140019 https://doi.org/10.5935/0103-5053.20140019 https://doi.org/10.5935/0103-5053.20140019 https://doi.org/10.5935/0103-5053.20140019 https://doi.org/10.5935/0103-5053.20140019 https://doi.org/10.1016/S0731-7085(02)00707-0 https://doi.org/10.1016/S0731-7085(02)00707-0 https://doi.org/10.1016/S0731-7085(02)00707-0 https://doi.org/10.1016/S0731-7085(02)00707-0 https://doi.org/10.3390/ph12030116 https://doi.org/10.3390/ph12030116 https://doi.org/10.3390/ph12030116 https://doi.org/10.3390/ph12030116 https://doi.org/10.3390/ph12030116 https://doi.org/10.3390/ph12030116 https://doi.org/10.3390/ph12030116 https://doi.org/10.3390/ph12030116 https://doi.org/10.3390/ph12030116 https://doi.org/10.3390/separations5010009 https://doi.org/10.3390/separations5010009 https://doi.org/10.3390/separations5010009 https://doi.org/10.3390/separations5010009 https://doi.org/10.3390/separations5010009 https://doi.org/10.3390/chemosensors8030055 https://doi.org/10.3390/chemosensors8030055 https://doi.org/10.3390/chemosensors8030055 https://doi.org/10.1016/j.electacta.2019.135180 https://doi.org/10.1016/j.electacta.2019.135180 Original Article revista.iq.unesp.br 38 Eclética Química Journal, vol. 47, n. 3, 2022, 32-38 ISSN: 1678-4618 DOI: 10.26850/1678-4618eqj.v47.3.2022.p32-38 approaches: A comparative study. Electrochim. Acta. 2020, 330, 135180. https://doi.org/10.1016/j.electacta.2019.135180 Thomaz, D. V.; Leite, K. C. de S.; Moreno, E. K. G.; Garcia, L. F.; Alecrim, M. F.; Macêdo, I. Y. L.; Caetano, M. P.; Carvalho, M. F.; Machado, F. B.; Gil, E. de S. Electrochemical Study of Commercial Black Tea Samples. Int. J. Electrochem. Sci. 2018, 13 (6), 5433–5439. https://doi.org/10.20964/2018.06.55 US Pharmacopeia (USP). Doxorubicin lyophilized powde. 2020. https://online.uspnf.com/uspnf/document/1_GUID- 41785247-DA22-407F-B060-7182FDA4FD3A_3_en-US (accessed 2021-02-28). Zhao, P.; Dash, A. K. A simple HPLC method using a microbore column for the analysis of doxorubicin. J. Pharm. Biomed. Anal. 1999, 20 (3), 543–548. https://doi.org/10.1016/S0731-7085(99)00070-9 https://revista.iq.unesp.br/index.php/ecletica https://doi.org/10.26850/1678-4618eqj.v47.3.2022.p32-38 https://doi.org/10.1016/j.electacta.2019.135180 https://doi.org/10.1016/j.electacta.2019.135180 https://doi.org/10.20964/2018.06.55 https://doi.org/10.20964/2018.06.55 https://doi.org/10.20964/2018.06.55 https://doi.org/10.20964/2018.06.55 https://doi.org/10.20964/2018.06.55 https://doi.org/10.20964/2018.06.55 https://online.uspnf.com/uspnf/document/1_GUID-41785247-DA22-407F-B060-7182FDA4FD3A_3_en-US https://online.uspnf.com/uspnf/document/1_GUID-41785247-DA22-407F-B060-7182FDA4FD3A_3_en-US https://online.uspnf.com/uspnf/document/1_GUID-41785247-DA22-407F-B060-7182FDA4FD3A_3_en-US https://online.uspnf.com/uspnf/document/1_GUID-41785247-DA22-407F-B060-7182FDA4FD3A_3_en-US https://doi.org/10.1016/S0731-7085(99)00070-9 https://doi.org/10.1016/S0731-7085(99)00070-9 https://doi.org/10.1016/S0731-7085(99)00070-9 https://doi.org/10.1016/S0731-7085(99)00070-9