Quantitative analysis of betulinic acid in mouse, rat and dog plasma using electrospray liquid chromatography/mass spectrometry.
Betulinic acid is under development as a therapeutic agent for the treatment of metastatic malignant melanoma. In support of pharmacokinetic and toxicological evaluations, a robust assay based on liquid chromatography/mass spectrometry (LC/MS) was developed for the quantitative analysis of betulinic acid. Sample preparation consisted of deproteinization of the plasma by the addition of three volumes of acetonitrile and one volume of methanol followed by centrifugation. Aliquots of the supernatant were analyzed using an isocratic reversed-phase high-performance liquid chromatography (HPLC) system coupled to a negative ion electrospray mass spectrometer. Deprotonated molecules of betulinic acid and the isomeric internal standard oleanolic acid were detected using selected ion monitoring at m/z 455. The limit of detection of betulinic acid was 0.5 pg (1.1 fM) injected on-column (50 pg/mL, 10 microL injection volume), and the limit of quantitation was 2 pg (4.4 fM, 200 pg/mL, 10 microL injection volume). Betulinic acid was stable in plasma samples at -20 degrees C for at least 3 weeks. The intra-day and inter-day coefficients of variation of the assay were < or =6.4 and < or =9.0%, respectively. The utility of the assay was demonstrated by analyzing betulinic acid spiked into mouse, rat and dog plasma, by determining the extent of binding of betulinic acid to plasma proteins, and by measuring betulinic acid in mouse and rat plasma following intraperitoneal or intravenous administration in vivo. At 15 and 25 microg/mL in mouse, rat or dog plasma, betulinic acid was 99.99% bound to serum proteins, and, at 5 microg/mL, betulinic acid was > or =99.97% bound.
Cheng
Xun
X
College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612, USA.
Shin
Young Geun
YG
Levine
Barry S
BS
Smith
Adaline C
AC
Tomaszewski
Joseph E
JE
van Breemen
Richard B
RB
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004285:Dogs; D051379:Mice; D015394:Molecular Structure; D053978:Pentacyclic Triterpenes; D011485:Protein Binding; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014315:Triterpenes / Q000097:blood* / Q000378:metabolism; D000094062:Betulinic Acid
D053978:Pentacyclic Triterpenes; D014315:Triterpenes; D000094062:Betulinic Acid