Determination of betulinic acid in mouse blood, tumor and tissue homogenates by liquid chromatography-electrospray mass spectrometry

A rapid and sensitive high-performance liquid chromatography-electrospray MS method has been developed to determine tissue distribution of betulinic acid in mice. The method involved deproteinization of these samples with 2.5 volumes (v/w) of acetonitrile-ethanol (1:1) and then 5 mu l aliquots of the supernatant were injected onto a C-18 reversed-phase column coupled with an electrospray MS system. The mobile phase employed isocratic elution with 80% acetonitrile for 10 min; the flow-rate was 0.7 ml/min. The column effluent was analyzed by selected ion monitoring for the negative pseudo-molecular ion of betulinic acid [M-H](-) at m/z 455. The limit of detection for betulinic acid in biological samples by this method was approximately 1.4 pg and the coefficients of variation of the assay (intra- and inter-day) were generally low (below 9.1%). When athymic mice bearing human melanoma were treated with betulinic acid (500 mg/kg, i.p.), distribution was as follows: tumor, 452.2 +/- 261.2 mu g/g; liver, 233.9 +/- 80.3 mu g/g; lung, 74.8 +/- 63.7 mu g/g; kidney, 95.8 +/- 122.8 mu g/g; blood, 1.8 +/- 0.5 mu g/ml. No interference was noted due to endogenous substances. These methods of analysis should be of value in future studies related to the development and characterization of betulinic acid. (C) 1999 Published by Elsevier Science B.V. All rights reserved.