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 M S method has been developed to determine tissue distribution of betulinic a cid 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 th e 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 effl uent was analyzed by selected ion monitoring for the negative pseudo-molecu lar ion of betulinic acid [M-H](-) at m/z 455. The limit of detection for b etulinic 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. N o interference was noted due to endogenous substances. These methods of ana lysis should be of value in future studies related to the development and c haracterization of betulinic acid. (C) 1999 Published by Elsevier Science B .V. All rights reserved.