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...

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Bibliographic Details
Published in:Rapid communications in mass spectrometry 2003-01, Vol.17 (18), p.2089-2092
Main Authors: Cheng, Xun, Shin, Young Geun, Levine, Barry S., Smith, Adaline C., Tomaszewski, Joseph E., van Breemen, Richard B.
Format: Article
Language:English
Subjects:
Animals
Chromatography, High Pressure Liquid
Dogs
Mice
Molecular Structure
Protein Binding
Rats
Reproducibility of Results
Sensitivity and Specificity
Spectrometry, Mass, Electrospray Ionization - methods
Triterpenes - blood
Triterpenes - metabolism
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Summary: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 fmol) injected on‐column (50 pg/mL, 10 μL injection volume), and the limit of quantitation was 2 pg (4.4 fmol, 200 pg/mL, 10 μL injection volume). Betulinic acid was stable in plasma samples at −20°C for at least 3 weeks. The intra‐day and inter‐day coefficients of variation of the assay were ≤6.4 and ≤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 μg/mL in mouse, rat or dog plasma, betulinic acid was 99.99% bound to serum proteins, and, at 5 μg/mL, betulinic acid was ≥99.97% bound. Copyright © 2003 John Wiley & Sons, Ltd.
ISSN:0951-4198
1097-0231
DOI:10.1002/rcm.1155