Determination of doxorubicin and metabolites in murine specimens by high-performance liquid chromatography

doi:10.1016/S0378-4347(98)00165-0

Journal of Chromatography B: Biomedical Sciences and Applications

Volume 712, Issues 1–2, 7 August 1998, Pages 129-143

https://doi.org/10.1016/S0378-4347(98)00165-0 Get rights and content

Abstract

A sensitive and selective reversed-phase high-performance liquid chromatographic method for the quantification of doxorubicin and its metabolites doxorubicinol, 7-deoxydoxorubicinone and 7-deoxydoxorubicinolone was developed and validated for a variety of murine specimens. Daunorubicin was used as internal standard. Sample pretreatment involved liquid–liquid extraction of 200 μl sample with 1 ml of chloroform–1-propanol (4:1, v/v). Chromatographic separation was achieved isocratically on a LiChrosorb RP-8 analytical column at ambient temperature. The mobile phase consisted of acidified water (pH 2.05)–acetonitrile–tetrahydrofuran (80:30:1, v/v/v). The column effluent was monitored fluorimetrically at an excitation wavelength of 460 nm and an emission wavelength of 550 nm. The lower limits of quantitation were in the range 1.8–2.4 nM. Spiked murine specimens and samples from treated mice were subjected to stability studies. The results demonstrated the importance of validation in all relevant specimens, since the accuracy and precision were highly matrix-dependent. Accuracies and precisions of measured drug concentrations in liver, spleen, muscle, gastrointestinal tissues, diluted bile, feces and urine were lower than in the other matrices. Doxorubicin was unstable in diluted bile, but not in the other specimens. The method is suitable for studying the pharmacokinetics of doxorubicin and its metabolites in mice.

Introduction

Doxorubicin is an anthracycline antibiotic which is widely used in the treatment of human malignancies such as leukemia, lymphoma and a number of solid tumors, particularly breast cancer. It is produced by the fungus Streptomyces peucetius (var. caesius). Since its discovery in 1969 by Arcamone et al. [1]several biochemical effects of doxorubicin have been described which may contribute to its therapeutic action. The compound can intercalate between DNA bases or generate free radicals, but also interaction with cellular membranes and inhibition of the nuclear enzyme topoisomerase II have been reported (reviewed in [2]). Drug resistance, however, is a major impediment to successful chemotherapy. The presence or upregulation of a large transmembrane protein, called P-glycoprotein, is one of the factors which has been associated with resistance to anthracyclines [3]. P-glycoprotein is not only found in various malignant tissues but also in many normal tissues. To get more insight into the physiological and pharmacological role of P-glycoprotein we study the pharmacokinetics of anticancer agents, including doxorubicin, in mice with a genetic disruption of this protein 4, 5, 6.

Two important routes of metabolic transformation of doxorubicin in animals and man are the reduction of the side chain carbonyl group to the secondary alcohol doxorubicinol (I) and the reductive deglycosidation with the formation of 7-deoxydoxorubicinone (II) and 7-deoxydoxorubicinolone (III) (Fig. 1, reviewed in [7]).

To study the pharmacokinetics of doxorubicin in small laboratory animals such as mice a sensitive analytical assay is a prerequisite. Although numerous analytical methods for the determination of doxorubicin and its metabolites have been described, including radiolabeled assays [8], fluorescence assays 9, 10, 11, and methods based on thin-layer chromatography 8, 11or high-performance liquid chromatography (HPLC) 12, 13, 14, 15, 16, an assay suited for the individual quantification of doxorubicin and metabolites in many different murine specimens has not been reported to date. We have previously described a sensitive and selective analytical reversed-phase HPLC method for doxorubicin in plasma samples of HIV-infected patients [16]. However, it is important to investigate the accuracy and precision of an analytical assay for each relevant specimen, since the results may be strongly dependent on the matrix. Therefore, we have now designed and thoroughly validated an HPLC assay for doxorubicin and metabolites in all relevant murine specimens. The validation program followed the guidelines as given in [17].

Section snippets

Chemicals

Doxorubicin·HCl and daunorubicin·HCl (Fig. 1) were purchased as powder for injection from Pharmacia-Farmitalia-Carlo Erba (Milan, Italy) and Rhône-Poulenc Rorer (Cedex, France), respectively. The metabolites doxorubicinol (I), 7-deoxydoxorubicinone (II) and 7-deoxydoxorubicinolone (III) were kindly provided by Pharmacia-Farmitalia-Carlo Erba. Bovine serum albumin (BSA) was obtained from Organon (Boxtel, The Netherlands). Hypnorm originated from Janssen (Tilburg, The Netherlands) and Dormicum

Results and discussion

The metabolism of doxorubicin to the secondary alcohol doxorubicinol (I) and the formation of 7-deoxydoxorubicinone (II) and 7-deoxydoxorubicinolone (III) is shown in Fig. 1. However, two additional metabolites of doxorubicin have been found in man, namely the aglycones doxorubicinone and doxorubicinolone [14]. To separate doxorubicin and all its metabolites, including these 7-hydroxy-aglycones, chromatography was initially performed using two coupled LiChrosorb RP-8 analytical columns. A

References (20)

A.H Schinkel et al.
Cell
(1994)
H.S Schwartz
Biochem. Med.
(1973)
S Shinozawa et al.
J. Chromatogr.
(1980)
P.A Maessen et al.
J. Chromatogr.
(1988)
J.H Beijnen et al.
J. Pharm. Biomed. Anal.
(1991)
V.P Shah et al.
J. Pharm. Sci.
(1992)
J Cummings et al.
Eur. J. Cancer Clin. Oncol.
(1986)
F Arcamone et al.
Biotechnol. Bioeng.
(1969)
D.J Booser et al.
Drugs
(1994)
N Kartner et al.
Cancer Res.
(1983)

There are more references available in the full text version of this article.

Cited by (71)

Greenness estimation of chromatographic assay for the determination of anthracycline-based antitumor drug in bacterial ghost matrix of Salmonella typhimurium
2022, Sustainable Chemistry and Pharmacy
The determination of an anthracycline-based antitumor drug, doxorubicin (DOX), in the bacterial ghost (BG) matrix of Salmonella typhimurium and commercial injectable dosage form was performed using a rapid, sensitive, and environmentally greener “high-performance liquid chromatography (HPLC)” assay. DOX was determined chromatographically on a Nucleodur (150 mm × 4.6 mm) RP C18 column with a particle size of 5 μm. The greener solvent system consisted of ethanol, methanol, and acetic acid (50:49:1 percent v/v/v), which was delivered with a flow rate of 1.0 mL/min. DOX was detected at a wavelength of 480 nm. With a determination coefficient value of 0.9992, the suggested HPLC assay was linear in the range of 1–100 μg/mL. In addition, the suggested HPLC assay for DOX analysis was rapid (R_t = 2.19 min), accurate (percent recoveries = 98.12–101.55), precise (percent CV = 0.87–1.97), and sensitive. The applicability of suggested HPLC technique was demonstrated by measuring DOX in S. typhimurium BG matrix and commercial injectable dosage form. DOX recovery in commercial injectable dosage form was calculated to be 101.02 percent. The amount of DOX in BG matrix was determined to be 48.51 μg/mL. Using the AGREE metric technique, the overall analytical GREEnness (AGREE) score for the proposed HPLC assay was calculated to be 0.79, indicating an outstanding greenness profile. These findings suggested that the proposed HPLC assay might be used to analyze DOX in BG matrices and commercial dosage forms on a regular basis.
A review on various analytical methods for determination of anthracyclines and their metabolites as anti–cancer chemotherapy drugs in different matrices over the last four decades
2020, TrAC - Trends in Analytical Chemistry
Anthracyclines (ANT), belong to a group of antineoplastic drugs, are commonly used as chemotherapeutic agents in the treatment of various cancers. During the past decade, the number of cancer patients receiving ANT–based chemotherapy drugs has significantly increased. Generally, ANT are very cytotoxic agents and are known to be accompanied by several unpleasant adverse effects. Therefore, the development of a reliable, accurate and sensitive analytical method is necessary and important to analyze ANT in human biomonitoring. The objective of this review is to provide a summary of various analytical methods (spectroscopic, liquid chromatographic and electroanalytical) that have been reported during the last four decades for the analysis of the five most commonly used ANT, daunorubicin (DNR), doxorubicin (DOX), epirubicin (EPI), idarubicin (IDA), valrubicin (VAL) and their metabolites, alone or in combination with other drugs, in various matrices such as biological matrices, environmental samples and pharmaceutical formulations.
Pharmacokinetics of a liposomal formulation of doxorubicin in rats
2017, Saudi Pharmaceutical Journal
Background: Measuring free drug concentration following systemic administration of a liposomal drug is a crucial aspect of the assessment of its in vivo behavior. Therefore we require an efficient method to separate free drug in the plasma from encapsulated drug. Objectives: To study the pharmacokinetics of free doxorubicin (DOX) released from liposomal doxorubicin (L-DOX) in rats. Methods: L-DOX was prepared with encapsulation efficiency of 90% and was injected intravenously into rats. A solid-phase extraction (SPE) method coupled with UPLC–MS/MS was used to measure the concentration of F-DOX in rat plasma without disrupting the integrity of L-DOX. Results: This method exhibited a linear range of F-DOX from 0.2 to 200 ng/mL. Recovery, precision, linearity and accuracy of this technique appear satisfactory for pharmacokinetic study. The constituents of F-DOX ranged from 5.35% to 14.09% of total DOX in plasma at each time point measured after L-DOX administration. Conclusion: SPE method was suitable for studying the pharmacokinetics of F-DOX in rats receiving L-DOX.
A new kinetic-mechanistic approach to elucidate electrooxidation of doxorubicin hydrochloride in unprocessed human fluids using magnetic graphene based nanocomposite modified glassy carbon electrode
2016, Materials Science and Engineering C
A novel magnetic nanocomposite was synthesized in one step using polymerization of magnetic graph oxide grafted with chlorosulfonic acid (Fe₃O₄–GO–SO₃H) in the presence of polystyrene. The prepared magnetic nanocomposite was characterized using transmission electron microscopy (TEM), dynamic differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), (Thermo-gravimetric/differential thermal analysis (DTA)), Fourier transform infrared (FTIR), and UV–Vis techniques. Magnetic nanocomposite was casted on the surface of the glassy carbon electrode (PS/Fe₃O₄–GO–SO₃H/GCE) and used for the detection and determination of doxorubicin hydrochloride (DOX) in human biological fluids. The cyclic voltammograms (CVs) of the modified electrode in aqueous solution displayed a pair of well-defined, stable and irreversible reductive/oxidation redox systems. CV study indicated that the oxidation process is irreversible and adsorption controlled. In addition, CV results indicated that DOX is oxidized via two electrons and three protons which is an unusual approach for the oxidation of DOX. A sensitive and time-saving procedure was developed for the analysis of DOX in plasma, cerebrospinal fluid, and urine with detection limit of 4.9 nM, 14 nM and 4.3 nM, respectively.
Internal standard method for the measurement of doxorubicin and daunorubicin by capillary electrophoresis with in-column double optical-fiber LED-induced fluorescence detection
2016, Journal of Pharmaceutical and Biomedical Analysis
Citation Excerpt :
These techniques include gas chromatography [3,4], liquid chromatography [5–7], UV spectrophotometry [8], fluorescence spectrophotometry [9,10] and so on. Although it has been widely used for the determination of anthracycline antibiotics [11–13], high-performance liquid chromatography (HPLC) suffers from poor selectivity and tedious procedures for sample preparation and chromatographic optimization. In recent years, capillary electrophoresis (CE) [14–16] has been applied in many areas due to its various advantages such as low consumption of chemicals, high resolution, short analysis time and the simplicity of operation.
An internal standard method has been developed for the simultaneous determination of anthracycline antibiotics, doxorubicin (DOX) and daunorubicin (DAN), in rabbit plasma using capillary electrophoresis (CE) with in-column double optical-fiber LED-induced fluorescence detection (CE-ICDOF-LED-FLD). Rhodamine B (RhB) was selected as an internal standard because its emission wavelength is similar to that of the anthracycline antibiotics. Parameters including buffer pH, buffer concentration, organic solvents and separation voltage have been investigated to explore the sensitivity and separation efficiency of DOX and DAN. The optimal electrophoretic separation conditions were a borate buffer (15 mM, pH 9.0) containing 50% acetonitrile (v/v), 10 s hydrodynamic injection at a height of 20 cm and a separation voltage of 15 kV. The developed CE-ICDOF-LED-FLD method provides limits of detection of 18 and 13 ng/mL for DOX and DAN in rabbit plasma samples, respectively. The recoveries ranging from 93.7 to 104.8% and the relative standard deviations at 1.1–1.7% were achieved for DOX and DAN in spiked rabbit plasma samples.
Sildenafil is not a useful modulator of ABCB1 and ABCG2 mediated drug resistance in vivo
2013, European Journal of Cancer
Recently, sildenafil was reported to be an inhibitor of P-glycoprotein (P-gp/ABCB1) and breast cancer resistance protein (BCRP/ABCG2) in vitro. We have now investigated the in vivo potency of sildenafil.
By using wild-type and Abcb1;Abcg2 knockout mice we have investigated the effect of sildenafil on the brain penetration of two substrate drugs (docetaxel and topotecan). Next we have investigated if sildenafil was able to improve the efficacy of doxorubicin against P-glycoprotein expressing CT26 colon cancer cells in syngeneic Balb/c mice.
Sildenafil administered orally at a dose of 50 mg/kg did not improve the brain penetration of docetaxel and topotecan, although the plasma level of sildenafil was already much higher than can be achieved in humans. On the other hand, sildenafil increased the plasma levels of the cytotoxic drugs, but not by inhibition of Abcb1 or Abcg2, since this effect was also seen in Abcb1;Abcg2 knockout mice. The brain penetration of sildenafil was more than 20-fold higher in Abcb1;Abcg2 mice versus wild-type mice, indicating that sildenafil is a good substrate of the two transporters. Sildenafil was also not able to improve the efficacy of doxorubicin against subcutaneous CT26 tumours. The doxorubicin level in tumour tissue did increase, but so did the concentration of doxorubicin in plasma and heart.
These results demonstrate that the potency and specificity of sildenafil as an inhibitor of ABCB1 and ABCG2 is not sufficient to warrant further clinical testing of this agent in combination with anticancer drugs.

View all citing articles on Scopus

View full text

Determination of doxorubicin and metabolites in murine specimens by high-performance liquid chromatography

Abstract

Introduction

Section snippets

Chemicals

Results and discussion

Cell

Biochem. Med.

J. Chromatogr.

J. Chromatogr.

J. Pharm. Biomed. Anal.

J. Pharm. Sci.

Eur. J. Cancer Clin. Oncol.

Biotechnol. Bioeng.

Drugs

Cancer Res.