A Simple and Sensitive LC-MS/MS Method for Determination of Four Major Active Diterpenoids from Andrographis paniculata in Human Plasma and Its Application to a Pilot Study

Abstract

Andrographis paniculata contains four major active diterpenoids, including andrographolide (1), 14-deoxy-11, 12-didehydroandrographolide (2), neoandrographolide (3), and 14-deoxyandrographolide (4), which exhibit differences in types and/or degrees of their pharmacological activity. Previous pharmacokinetic studies in humans reported only the parameters of compound 1 and its analytical method in human plasma. The purpose of this study was to develop a simple, sensitive, and selective liquid chromatography tandem-mass spectrometry technique for the simultaneous determination of all four major active diterpenoids in the A. paniculata product in human plasma. These four diterpenoids in plasma samples were extracted by a simple protein precipitation method with methanol and separated on a Kinetex C18 column using a gradient system with a mobile phase of acetonitrile and water. The liquid chromatography tandem-mass spectrometry was performed in the negative mode, and the multiple reaction monitoring mode was used for the quantitation. The method showed a good linearity over a wide concentration range of 2.50-500 ng/mL for 1 and over the range of 1.00-500 ng/mL for the other diterpenoids with a correlation coefficient R(2) > 0.995. The lower limit of quantification of 1 was found to be 2.50 ng/mL, while those of the other diterpenoids were 1.00 ng/mL. The intraday and interday accuracy (relative error) ranged from 0.03 % to 10.03 %, and the intraday and interday precisions (relative standard deviation) were in the range of 2.05-9.67 %. The extraction recovery (86.54-111.56 %) with a relative standard deviation of 2.78-8.61 % and the matrix effect (85.15-112.36 %) were within the acceptance criteria. Moreover, these four major active diterpenoids were stable in plasma samples at the studied storage conditions with a relative error ≤-9.79 % and a relative standard deviation ≤ 9.26 %. Hence, this present method was successfully validated and used in the pilot study to determine the pharmacokinetic parameters of all four major active diterpenoids in human plasma after multiple oral doses of the A. paniculata product were administered to a healthy, Thai female volunteer.

Clinical Parameters following Multiple Oral Dose Administration of a Standardized Andrographis paniculata Capsule in Healthy Thai Subjects

“…Furthermore, in consideration of A. paniculata for the treatment of diseases, the key component is compound 1, which is the major diterpenoid found in the highest amount in the plant. Our previous pharmacokinetic study of major active diterpenoids after multiple oral doses of A. paniculata in healthy subjects, with a dose-normalized basis, showed that the amount of 2 that could reach blood circulation was approximately 14 times higher than that of 1 [23,24]. Furthermore, our previous in vitro and in vivo studies showed that the effects of A. paniculata and its major bioactive diterpenoids give some reasons for concern.…”

“…The simultaneous determination of major active diterpenoids of A. paniculata in human plasma was employed using a validated LC-MS/MS technique [23]. Briefly, 50.0 ng/mL of cortisone (internal standard) and 800 µL of methanol were added to a 200-µL aliquot of plasma samples.…”

A validated LC–MS/MS method for clinical pharmacokinetics and presumptive phase II metabolic pathways following oral administration of Andrographis paniculata extract

“…While the clinical benefits of the extract (60 mg of andrographolide/dose) have been evaluated, the data on the pharmacokinetics of this dose remain limited. Therefore, one of the objectives of this study was to investigate the clinical pharmacokinetics of andrographolide (AP1), a major phytoconstituent of this plant, and of other diterpenoid derivatives 6 – 8 including: 14-deoxy-11, 12-didehydroandrographolide (AP3), neoandrographolide (AP4), and 14-deoxyandrographolide (AP6) after a single oral administration of A. paniculata extract (equivalent to 60 mg/dose of andrographolide).…”

Inhibition of UGT2B7 Enzyme Activity in Human and Rat Liver Microsomes by Herbal Constituents

“…The value is lower than the IC 50 value of 6.18 µM obtained in this study. Another two pharmacokinetic studies of andrographolide showed that its maximum concentration in plasma were 58.6 ng/mL (0.17 µM) and 32.41 ng/mL (0.09 µM) after ingestion of 200 mg and 32.64 mg of andrographolide for three times a day, respectively [ 59 , 60 ]. As the andrographolide concentration in plasma reportedly ranged from 0.09 to 3.8 µM which is less than the obtained IC 50 value in HLM (6.18 ± 1.27 µM), inhibition of hepatic systemic clearance of UGT2B7 substrates in humans seems implausible.…”