Determination of esculentoside A in dog plasma by LC-MS/MS method: application to pre-clinical pharmacokinetics.

A simple and rapid high performance liquid chromatography electrospray ionization ion-trap tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the quantitative determination of esculentoside A (EsA) in dog plasma using ginsenoside Rg1 as the internal standard (IS). After liquid-liquid extraction (LLE) with n-butanol, the analyte and IS were separated on a Diamonsil C(18) (2.1 mm × 50 mm, 3 μm) column with the mobile phase of methanol-water containing 0.1% acetic acid (70:30, v/v) at a flow rate of 0.2 ml/min. An ion trap mass spectrometer equipped with an electrospray ionization source performed in selected reaction monitoring (SRM) mode was used as the detector. The precursor-product ion transitions were m/z 849.3 M+Na→m/z 805.3 for EsA and m/z 823.3 M+Na→m/z 643.3 for IS. The total chromatographic run time was 5 min. The method was sensitive enough with a lower limit of quantitation (LLOQ) of 5 ng/ml and had a good linearity (r(2)>0.997) over the linear range of 5-500 ng/ml. The mean extraction recovery of EsA from spiked plasma samples was over 75%. The intra- and inter-precisions were no more than 8.8% and accuracies were within the range of -4.6 to 8.7%. All the validated data were within the accepted criteria as stated in the FDA bioanalytical method validation guideline. The developed method was suitable for the quantification of EsA and successfully applied to the pharmacokinetic study of EsA after an oral administration to beagle dogs.