Validated LC–MS/MS method for simultaneous determination of doxorubicin and curcumin in polymeric micelles in subcellular compartments of MCF‐7/Adr cells by protein precipitation–ultrasonic breaking method

Abstract

A specific and sensitive LC-MS/MS with protein precipitation- ultrasonic breaking method has been developed and validated for simultaneous determination of doxorubicin (DOX) and curcumin (Cur) in DOX and Cur co-loaded hyaluronic acid-vitamin E succinatemicelles [(DOX + Cur)-polymeric micelles (PMs)] in subcellular compartments of resistant MCF-7/Adr cells. Sequential extraction of four subcellular protein fractions (cytosolic, membrane/organelle, nucleic and cytoskeleton) was performed directly from MCF-7/Adr cells after incubation with (DOX + Cur)-PMs. An ultrasonic breaking-methanol precipitation method was used for extraction of the fractions, and the micelle breaking efficiency with methanol was 98.1 and 97.6% for DOX and Cur, respectively. The analytes were analyzed using positive electrospray ionization coupled with multiple reaction monitoring. The calibration curves were linear over a concentration range of 0.5-400 ng/mL for DOX and 2-2000 ng/mL for Cur, and the recovery for the two analytes were >85% with negligible matrix effect. The intra-day and inter-day precision was <10.80% and relative error was within ±7.70%. The developed method was successfully applied for subcellular determination of DOX and Cur in MCF-7/Adr cells. Moreover, Cur and (DOX + Cur)-PMs had a marked promoting effect on the distribution of DOX in the nucleic protein fraction.

UHPLC-MS/MS Assay for Quantification of Legubicin, a Novel Doxorubicin-Based Legumain-Activated Prodrug, and Its Application to Pharmacokinetic and Tissue Distribution Studies

“…Various published extraction methods, including protein precipitation using methanol [ 10 , 11 , 12 ], acetonitrile precipitation combined with sonication [ 13 ], liquid–liquid extraction [ 14 , 15 , 16 ], solid-phase extraction [ 17 , 18 , 19 ], and electromembrane extraction [ 20 ] have been employed for the extraction of Leu-DOX and its metabolized products or DOX and its metabolites from diverse matrices such as plasma, urine, and tissues. In this specific study, we examined the viability of a direct protein precipitation technique for the concurrent identification of Leu-DOX and DOX.…”

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

“…The matrix effects for doxorubicin, doxorubicinol, doxorubicinone, doxorubicinolone, 7-deoxydoxorubicinone, and daunorubicin (IS) were 112.9-119.7%, 94.8-109.6%, 105.1-117.9%, 99.0-111.0%, 98.3-108.3%, and 88.0%, respectively, with RSD ≤ 14.2% at low, medium, and high QC levels (Table 2), indicating the presence of a small matrix effect. Liquid−liquid extraction using chloroform:methanol (4:1, v/v) resulted in higher sensitivity and smaller matrix effects for doxorubicin and its four metabolites compared with the protein precipitation method using methanol, acetonitrile, or acetone [13][14][15]18,19,21,22,[25][26][27]31,32,34,35]. Our method yielded LLOQ levels of doxorubicin (0.5 ng/mL) and its major four metabolites (0.1-0.01 ng/mL) that were better than those reported using the least plasma volume of 10 µL.…”

“…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,…”