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Development of a sensitive and specific liquid chromatography/mass spectrometry method for the quantification of cucurbitacin I (JSI-124) in rat plasma.

PURPOSE
To develop a liquid chromatography/mass spectrometry (LC-MS) method for the quantitative analysis of cucurbitacin I (JSI-124), an anti-cancer inhibitor of the janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway, in rat plasma samples.


METHODS
Standard samples of cucurbitacin I were prepared from a stock solution (1 mg/mL) in methanol. Internal standard (I.S.) was 4-hydroxybenzophenone. Extraction of cucurbitacin I and I.S. from rat plasma was performed using acetonitrile/dichloromethane. LC-MS analyses were performed using a Waters Micromass ZQ 4000 spectrometer, and chromatographic separation was achieved using a Waters XTerraMSC18 3.5 microm (2.1 x 50 mm) column as the stationary phase. The mobile phase consisting of a mixture of acetonitrile: water containing 1% formic acid with initial ratio of 20:80, employing a linear gradient to a final ratio of 40:60 v/v over 13 minutes, was delivered at a constant flow rate of 0.2 mL/min. The mass spectrometer was operated in negative ionization mode and analytes were quantified with single ion recording (SIR) at m/z 559 for cucurbitacin I and m/z 196.8 for I.S.


RESULTS
Calibration curves with r2 > 0.999 were constructed over the concentration range of 5-10000 ng/mL for the solution of cucurbitacin I in methanol and 10-1000 ng/mL for rat plasma samples. The extraction recoveries were 86 and 98% for 50 ng/mL and 1000 ng/mL plasma concentration of cucurbitacin I, respectively. The intra- and inter-day coefficients of variation were less than 15%, and mean intraday errors were less than 10% at plasma concentration extending from 10-1000 ng/mL.


CONCLUSION
The developed assay is sensitive, specific, reproducible and reliable for quantitative analysis of cucurbitacin I. Application in a pharmacokinetic assessment was proven in the rats given the drug.