Simultaneous determination of andrographolide, dehydroandrographolide and neoandrographolide in dog plasma by LC–MS/MS and its application to a dog pharmacokinetic study of Andrographis paniculata tablet

• Simultaneous determination of AP, DP and NP in dog plasma by LC–MS/MS.
• MRM mode was applied to achieve high specificity and sensitivity.
• Application to a dog pharmacokinetic study of A. paniculata tablet.
• The LLOQ is as low as 0.50 ng/mL for AP, 1.00 ng/mL for DP and 0.20 ng/mL for NP.

In this study, a sensitive and rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated to simultaneously determinate andrographolide (AP), dehydroandrographolide (DP), and neoandrographolide (NP) in plasma of beagle dogs after oral administration of Andrographis paniculata tablet (A. paniculata). The analytes and bilobalide (internal standard) were separated on an Agilent ZORBAX XDB-C18 column (50 mm × 2.1 mm, 3.5 μm) by using gradient elution consisting of methanol and water at a flow rate of 0.50 mL/min in 7 min. Multiple reaction monitoring (MRM) mode was performed to quantify data under monitoring precursor-product ion transitions of m/z 348.8 → 286.9, 330.9 → 107.9, 479.1 → 160.8 and 325.0 → 163.0 for AP, DP, NP and internal standard (IS) at negative ion mode, respectively. This method was developed at linearity ranging from 0.50 to 250 ng/mL for AP, 1.00 to 500 ng/mL for DP and 0.20 to 100 ng/mL for NP. The accuracy of each analyte ranged between 94.8% and 107.1% and the precision was within 14.6%. No significant matrix effect was observed. AP, DP and NP were stable during sample storage, preparation and analytic procedures. Furthermore, this method was successfully applied in the investigation of the pharmacokinetic profile of AP, DP and NP in beagle dogs after oral administration of A. paniculata tablet (49.5 mg for AP, 7.0 mg for DP, 22.0 mg for NP). Biological half-life (t1/2) was 2.08 ± 0.99, 3.13 ± 1.19 and 1.07 ± 0.38 h for AP, DP and NP, respectively. The areas under curves (AUC0–t) of AP, DP and NP was 494.50 ± 150.64, 26.01 ± 8.72 and 78.78 ± 18.29 ng h/mL, respectively.