High-performance liquid chromatographic analysis of idarubicin and fluorescent metabolites in biological fluids

Abstract

A specific, sensitive, and reliable high-performance liquid chromatographic (HPLC) method for the determination of idarubicin (IDA) and its known fluorescent metabolites idarubicinol (IDAol) and 4-demethoxy-daunomycinone (AG1) in biological fluids (human plasma and urine) was developed and tested. Plasma samples were solid-phase-extracted (C18 bonded silica cartridges). Complete separation of unchanged drugs and metabolites was achieved on a Cyanopropyl chromatographic column (25 cm x 4.6 mm inside diameter; particle size, 5 microns) using fluorescence detection (excitation wavelength, 470 nm; emission wavelength, 580 nm). Sensitivity was better than 0.2 ng/ml for all analytes; rates of recovery of unchanged drug and metabolites were better than 84.5% (IDA), 80.3% (IDAol), and 83.9% (AG1). The interassay coefficient of variation was 6.5% for IDA, 5.8% for IDAol, and 9.8% for AG1. Mean intra-assay precision was 4.6% for IDA, 5.9% for IDAol, and 5.0% for AG1 at sample concentrations of above 1 ng/ml and 12.1% for IDA, 10.8% for IDAol, and 14.1% for AG1 at sample concentrations of below 1 ng/ml.

Quantitative liquid chromatographic analysis of anthracyclines in biological fluids

“…From the 35 publications included in this overview, 12 quantify only one main compound [26,[42][43][44][45][46][47][48][49][50][51][52]. Others determine a main compound and its reduced metabolite [27,[53][54][55][56][57][58][59], sometimes together with additional 8 metabolites [24,[60][61][62][63][64][65][66][67]. One method could be applied to each of the four pairs (main compound and respective reduced metabolite) individually [68].…”

“…Besides silica based reversedphase C18 [46,47,56,60,66,67,70], C8 [72] and C2 [44,65] sorbents, also polymeric Oasis HLB [48,49,57,71] and MCX [50] sorbents have been employed. A Biotrap 500 MS online SPE column has also been used [69].…”

“…To avoid losses due to protein binding, samples have sometimes been diluted [50,65,66,71,72] or have been subjected to a protein precipitation step [57] prior to loading on the sorbent.…”

“…Occasionally, a stronger (additional) wash-step was included: 25 or 30% methanol at neutral pH [50,56,66], 40% methanol at alkaline conditions [48,49] or hexane [46]. Mild washing conditions improve recoveries, especially if the reduced metabolites or glucuronic acid conjugates have to be included in the assay.…”

“…Methanol (pure or in combination with an acid or tetrahydrofuran) [47][48][49]56,57,60,66,67], acetonitrile -acidic buffer mixtures [71,72] and chloroform -alcohol combinations [46,70] have been applied to elute the C18, C8 and Oasis HLB sorbents. The C2 sorbents and Biotrap 12 formate buffer (pH 3.55) : acetone : isopropanol (60:32:8, v/v/v) mixture [44], while the Oasis MCX sorbent was eluted with an alkaline methanol -acetonitrile mixture [50].…”