Determination of six components of Andrographis paniculata extract and one major metabolite of andrographolide in rat plasma by liquid chromatography-tandem mass spectrometry.

Andrographis paniculata (AP) has been widely used in Asian countries to treat many kinds of diseases for several decades. Hutchison Medipharma Ltd. developed an aqueous ethanol extract of A. paniculata (APE) named as HMPL-004 to treat inflammatory bowel diseases. The representative chemical components of HMPL-004 include andrographolide (AND), neoandrographolide (NAND), 14-deoxyandrographolide (DAND), 14-deoxy-11,12-didehydro-andrographolide (DDAND), apigenin-7-O-β-d-glucuronopyranoside (AODG) and chlorogenic acid (CLA). HM5013620 is the major circulating metabolite of AND. The purpose of this study was to develop a bioanalytical method to determine all seven compounds in rat plasma using liquid chromatography coupled to electrospray tandem mass spectrometry (LC-MS/MS). The assay was fully validated according to FDA guidelines. The LC-MS/MS detection was operated in the negative mode, and the multiple reaction monitoring (MRM) mode was used for the quantification. The analyte extraction was performed by protein precipitation with acetonitrile after adding a small volume (2% of the total volume) of 10% formic acid into plasma to stabilize AND under bench-top condition (ice-bath). The linear ranges of the analytes were 8-2000ng/mL for DDAND and 4-2000ng/mL for others. Validation results demonstrate that AND, NAND, DAND, DDAND, CLA, AODG and HM5013620 can be rapidly, accurately, precisely and robustly quantified in rat plasma. Furthermore, the method was successfully applied to characterize the pharmacokinetic profiles of all seven compounds in Sprague-Dawley rats after a single oral administration of 750mg of HMPL-004.