Quantitative analysis of 23-hydroxybetulinic acid in mouse plasma using electrospray liquid chromatography/mass spectrometry.
23-Hydroxybetulinic acid is a newly isolated derivative of betulinic acid. The agent exhibits potential anti-tumor activity and functions in this regard via apoptosis. In support of pharmacokinetic and toxicological evaluations, a new assay based on liquid chromatography/mass spectrometry (LC/MS) was developed for the quantitative analysis of 23-hydroxybetulinic acid. Sample preparation consisted of extraction of the plasma by the addition of methylene chloride 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. Molecules of 23-hydroxybetulinic acid and the internal standard limonin were detected using selected ion monitoring at m/z 471 and 469, respectively. The limit of detection of 23-hydroxybetulinic acid was 0.05 pg (0.11 fmol) injected on-column (10 pg/mL, 5 microL injection volume), and the limit of quantitation was 10 pg (21.19 fmol, 2 ng/mL, 5 muL injection volume). 23-Hydroxybetulinic 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 3.0 and 4.8%, respectively. The utility of the assay was demonstrated by measuring 23-hydroxybetulinicacid in mouse plasma following intragastric administration (IG) in vivo. Pharmacokinetic parameters were calculated using the 3P97 pharmacokinetic software package. A two-compartment, first-order model was selected for pharmacokinetic modeling. The result showed that after IG of 200 mg/kg 23-hydroxybetulinic acid, the plasma concentrations reached peaks at 2 h with C(max) of 3.1 microg/mL. The 200 mg/kg 23-hydroxybetulinic acid suspension IG doses were found to have long elimination half-lives of 25.6 h and low bioavailability of 2.3%. No interference was noted due to endogenous substances. These analytical methods should be of value in future studies related to the development and characterization of 23-hydroxybetulinic acid.
Yang
Min
M
Research Center of Pharmacokinetics, China Pharmaceutical University, Nanjing 210009, P.R. China. ymzfk@163.com
Wang
Guang Ji
GJ
Wang
Su Jun
SJ
Li
Xiao Tian
XT
Xu
Yu Ping
YP
Wang
Song Pei
SP
Xiang
Jing De
JD
Pan
Shang Ren
SR
Cao
Guo Xian
GX
Ye
Wen Cai
WC
D000818:Animals; D000970:Antineoplastic Agents / Q000097:blood* / Q000493:pharmacokinetics; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004365:Drugs, Chinese Herbal; D051379:Mice; D008813:Mice, Inbred ICR; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014315:Triterpenes / Q000097:blood* / Q000493:pharmacokinetics
C468651:23-hydroxybetulinic acid; D000970:Antineoplastic Agents; D004365:Drugs, Chinese Herbal; D014315:Triterpenes