LC–MS/MS method development for quantification of doxorubicin and its metabolite 13‐hydroxy doxorubicin in mice biological matrices: Application to a pharmaco‐delivery study

Abstract

This study describes the development of simple, rapid and sensitive liquid chromatography tandem mass spectrometry method for the simultaneous analysis of doxorubicin and its major metabolite, doxorubicinol, in mouse plasma, urine and tissues. The calibration curves were linear over the range 5-250 ng/mL for doxorubicin and 1.25-25 ng/mL for doxorubicinol in plasma and tumor, over the range 25-500 ng/mL for doxorubicin and 1.25-25 ng/mL for doxorubicinol in liver and kidney, and over the range 25-1000 ng/mL for doxorubicin and doxorubicinol in urine. The study was validated, using quality control samples prepared in all different matrices, for accuracy, precision, linearity, selectivity, lower limit of quantification and recovery in accordance with the US Food & Drug Administration guidelines. The method was successfully applied in determining the pharmaco-distribution of doxorubicin and doxorubicinol after intravenously administration in tumor-bearing mice of drug, free or nano-formulated in ferritin nanoparticles or in liposomes. Obtained results demonstrate an effective different distribution and doxorubicin protection against metabolism linked to nano-formulation. This method, thanks to its validation in plasma and urine, could be a powerful tool for pharmaceutical research and therapeutic drug monitoring, which is a clinical approach currently used in the optimization of oncologic treatments.

Insights into Doxorubicin-induced Cardiotoxicity: Molecular Mechanisms, Preventive Strategies, and Early Monitoring

“…DOX delivery via nano-formulations is one of the preventative techniques to successfully reduce this metabolism-dependent cardiotoxicity. Besides diminishing the cardiovascular side effect of DOX through the slow release of free drug from the nanocarrier, nano-formulations are meant to improve free drug availability, delivery, and accumulation at the tumor site (Mazzucchelli et al, 2017). Other metabolites of DOX such as DOX deoxyaglycone, DOX hydroxyaglycone, and doxorubicinol hydroxyaglycone are also suspected of increasing cardiac dysfunction by HPLC with fluorescence detection (Fogli et al, 1999) Daunorubicin, idarubicin, DOX, epirubicin, and their 13-dihydro metabolites in human plasma samples…”

“…Liquid chromatography with tandem mass spectrometry (Mazzucchelli et al, 2017) Concentration of DOX and DOXL in mouse plasma, liver, kidney, tumor, urine Plasma: 0.04 (DOX); 0.24 (DOXL) Liver: 0.12 (DOX); 0.3 (DOXL) Kidney: 0.43 (DOX); 0.32 (DOXL) Tumor: 0.52 (DOX); 0.35 (DOXL) Urine: 0.025 (DOX); 0.09 (DOXL) Ultra-HPLC fluorescence (Pérez-Blanco et al, 2014) Concentrations of DOX and DOXL in human plasma DOX = 8 ng/ml DOXL = 3 ng/ml Radionuclide angiocardiography (Lu, 2005;Panjrath and Jain, 2006) Measurement of LVEF Not applicable…”

Liquid Chromatography–Tandem Mass Spectrometry for the Simultaneous Determination of Doxorubicin and its Metabolites Doxorubicinol, Doxorubicinone, Doxorubicinolone, and 7-Deoxydoxorubicinone in Mouse Plasma

“…The Luna Omega C18 column exhibited better separation, an excellent peak shape, and good sensitivity for the analytes using a gradient elution of 0.1% formic acid in 95% methanol and 0.1% formic acid in 0.1% formic acid in 5% methanol compared with the Halo C18 and Acquity BEH C18 columns. Using methanol instead of acetonitrile [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33] as the strong eluent of the mobile phase resulted in increased ionization efficiency and linearity range for the five analytes. Daunorubicin, an analog of doxorubicin, was selected as and the internal standard (IS).…”

“…Doxorubicinol, a major alcohol metabolite of doxorubicin formed by carbonyl reductases 1 and 3, is implicated in off-target cardiotoxicity of doxorubicin-treated patients [3,[7][8][9][10]. High-performance liquid chromatography (HPLC) with fluorescence [11][12][13][14][15][16][17][18][19] or ultraviolet (UV) [20,21] detection, LC with mass spectrometry (LC-MS) [22,23] or tandem mass spectrometry (LC-MS/MS) [24][25][26][27][28][29][30][31][32][33][34][35][36], and capillary electrophoresis [37][38][39] methods have been used to analyze doxorubicin alone or with its metabolite doxorubicinol in various biological matrices, such as blood, serum, plasma, cells, and tissues. Protein precipitation with methanol, acetonitrile, or acetone [13][14][15]18,19,21,22,[25][26][27]31,…”

“…Protein precipitation with methanol, acetonitrile, or acetone [13][14][15]18,19,21,22,[25][26][27]31,32,34,35]; liquid-liquid extraction with ethyl acetate or a mixture of chloroform and methanol [11,16,17,20,29,33]; and solid-phase extraction [23,24,28,30,36] methods have been used as sample clean-up procedures for a variety of biological samples. These methods resulted in lower limit of quantification (LLOQ) levels of 0.2-50 ng/mL for doxorubicin ; 0.5-1.25 ng/mL for doxorubicinol [11,23,25,27,28,30,33]; and 1-5 ng/mL for doxorubicinone, doxorubicinolone, 7-deoxydoxorubicinone, and 7-deoxydoxorubicinolone [11,12] using large volumes of blood, plasma, or serum (50-1000 µL). To date, no studies have used an LC-MS/MS method for the simultaneous determination of doxorubicin and its major four metabolites in plasma samples, but some have used HPLC with fluorescence detection [11,12].…”