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 microl aliquots of the supernatant were injected onto a C18 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 microg/g; liver, 233.9 +/- 80.3 microg/g; lung, 74.8 +/- 63.7 microg/g; kidney, 95.8 +/- 122.8 microg/g; blood, 1.8 +/- 0.5 microg/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.