Acta Pharm. 55 (2005) 81-91

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Original research paper  

Rapid and sensitive HPLC method for the estimation of doxorubicin in dog blood - The silver nitrate artifact


1Drug Delivery Research Laboratory, Center of Relevance and Excellence in NDDS, Pharmacy Department, G.H. Patel Building, M.S. University of Baroda, Vadodara-390002, Gujarat, India
2Bioanalytical division, Sun Pharma Advanced Research Center, Akota Road, Baroda-390020, India
Received April 16, 2004      Accepted February 9, 2005

A rapid and sensitive high performance liquid chromatographic (HPLC) assay utilizing fluorimetric detection (excitation at 480 nm, emission at 560 nm) for the determination of doxorubicin in dog blood was developed and validated. Treatment of blood samples containing doxorubicin with AgNO3 (as protein precipitant) resulted in appearance of an additional peak in the chromatogram of doxorubicin at 11.5 min along with the parent peak (tR = 5.5 min). The latter peak was not found when treated with other protein precipitants such as trichloroacetic acid and methanol. Construction of calibration curve based on area of both peaks alone did not result in linearity of the curve. However, summation of areas of both peaks resulted in a curve with good linearity and correlation coefficient (R2 = 0.9985). Appearance of second peak may be due to the interaction of doxorubicin with cellular components of blood in the presence of AgNO3 leading to the formation of complex with reduced polarity. Analysis of the quality control samples showed good accuracy (96.7-100.42) and precision (RSD = 2,6-5,7%). The proposed method could be advantageous in estimation of doxorubicin incorporated into targeted delivery systems that concentrate in blood cells and quantify the absolute blood concentration of doxorubicin.

Keywords: doxorubicin, HPLC estimation, silver nitrate, dog blood, dog plasma, fluorescence detection