A simple HPLC method for doxorubicin in plasma and tissues of nude mice

Abstract

Doxorubicin is a cytotoxic anthracycline that has been used for the treatment of several malignancies. Several HPLC methods have been reported for the quantification of doxorubicin in biological samples. Tissue matrix effect and sample size requirements, however, have been remaining issues for simple and easy-to-adapt analytical methods in small animal experiments. The present study established a simple HPLC method for doxorubicin in plasma and tissues (tumor, heart, spleen, liver, gastrointestinal tract, brain, lung, and kidney) of nude mice. Our method required a small sample volume (100 microL plasma and 10 mg tissue), which made it possible to use each blank tissue for calibration curves. The limit of quantification was 25 ng/mL in plasma and 0.1 to 0.4 microg/mg in other tissues with recovery rates ranging from 52.4 to 95.2%. The linearity, accuracy and precision in all tissues, except gastrointestinal tract (GIT), were found to be acceptable in the range of 25-2000 ng/mL plasma and 0.1-4 ng/mg tissue. This method was used successfully to determine the drug concentration in plasma and tissues of human tumor xenograft-bearing nude mice given intratumoral doxorubicin in a polymeric drug delivery system designed for sustained release. In conclusion, the present method may be useful as a simple and easy-to-adapt, yet, sensitive analytical method of doxorubicin for plasma and tissue pharmacokinetic studies in small animals such as nude mice.

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].…”

“…Zinc sulphate, always in the presence of methanol [24,45], acetone [26,52,55] or both [43] has also been applied for protein precipitation. Zinc, a positively charged metal ion, will interact with proteins, reducing a protein's solubility by altering its iso-electric point and by displacing protons, resulting in a lowering of the solution's pH.…”

“…As is common in SPE, the choice of the stringency of the washing solvent is a compromise; given the same retention of the main compound, more stringent washing steps may lead to cleaner extracts, but with less retention of several metabolites. Reversed-phase C18 stationary phases have been widely used [13,24,26,27,44,45,50,51,55,56,58,60,62,64,65,67,72], but also C8 [46,52,61,63], cyano [66,68], phenyl [47] and phenyl-hexyl [53] stationary phases have been chosen.…”

“…Isocratic water-acetonitrile mobile phases, containing a diluted acid [27,51,53,55,58,61,63] or an acidic buffer [52,56,60,64,65,67,68,72], sometimes in the presence of an additional organic modifier such as tetrahydrofuran [55,61,63,65] or triethylamine [27,51,56,58,64,72] have been employed frequently. Other isocratic mobile phases have been used occasionally [24,[44][45][46]62].…”

HPLC quantification of doxorubicin in plasma and tissues of rats treated with doxorubicin loaded poly(alkylcyanoacrylate) nanoparticles

“…Several RP-HPLC methods of quantification of Dox in rodent plasma were described during the last decade [10][11][12][13][14][15][16][17][18][19][20][21][22][23][24]. Sample preparation in general includes either a single step of protein precipitation [10][11][12] or solid phase extraction [13,14] or liquid-liquid extraction (LLE) [15] before injection into the chromatograph.…”