A validated assay for measuring doxorubicin in biological fluids and tissues in an isolated lung perfusion model: matrix effect and heparin interference strongly influence doxorubicin measurements

Abstract Doxorubicin is an antineoplasic agent active against sarcoma pulmonary metastasis, but its clinical use is hampered by its myelotoxicity and its cumulative cardiotoxicity, when administered systemically. This limitation may be circumvented using the isolated lung perfusion (ILP) approach, wherein a therapeutic agent is infused locoregionally after vascular isolation of the lung. The influence of the mode of infusion (anterograde (AG): through the pulmonary artery (PA); retrograde (RG): through the pulmonary vein (PV)) on doxorubicin pharmacokinetics and lung distribution was unknown. Therefore, a simple, rapid and sensitive high-performance liquid chromatography method has been developed to quantify doxorubicin in four different biological matrices (infusion effluent, serum, tissues with low or high levels of doxorubicin). The related compound daunorubicin was used as internal standard (I.S.). Following a single-step protein precipitation of 500 μl samples with 250 μl acetone and 50 μl zinc sulfate 70% aqueous solution, the obtained supernatant was evaporated to dryness at 60 °C for exactly 45 min under a stream of nitrogen and the solid residue was solubilized in 200 μl of purified water. A 100 μl-volume was subjected to HPLC analysis onto a Nucleosil 100–5 μm C18 AB column equipped with a guard column (Nucleosil 100–5 μm C 6 H 5 (phenyl) end-capped) using a gradient elution of acetonitrile and 1-heptanesulfonic acid 0.2% pH 4: 15/85 at 0 min→50/50 at 20 min→100/0 at 22 min→15/85 at 24 min→15/85 at 26 min, delivered at 1 ml/min. The analytes were detected by fluorescence detection with excitation and emission wavelength set at 480 and 550 nm, respectively. The calibration curves were linear over the range of 2–1000 ng/ml for effluent and plasma matrices, and 0.1 μg/g–750 μg/g for tissues matrices. The method is precise with inter-day and intra-day relative standard deviation within 0.5 and 6.7% and accurate with inter-day and intra-day deviations between −5.4 and +7.7%. The in vitro stability in all matrices and in processed samples has been studied at −80 °C for 1 month, and at 4 °C for 48 h, respectively. During initial studies, heparin used as anticoagulant was found to profoundly influence the measurements of doxorubicin in effluents collected from animals under ILP. Moreover, the strong matrix effect observed with tissues samples indicate that it is mandatory to prepare doxorubicin calibration standard samples in biological matrices which would reflect at best the composition of samples to be analyzed. This method was successfully applied in animal studies for the analysis of effluent, serum and tissue samples collected from pigs and rats undergoing ILP.