OPTIMIZATION OF LC-QqQ-MS/MS METHOD FOR QUANTIFICATION OF ANDROGRAPHOLIDES IN HUMAN PLASMA

Andrographolides are diterpenoids found in the plant Andrographis paniculata and are widely used in traditional medicine, exhibiting anti- inflammatory properties.1 In this research we developed and validated high-performance liquid chromatography coupled with electrospray ionization triple-quadrupole tandem mass spectrometry (LC-QqQ-MS/MS) method for quantification of andrographolides in human plasma. The chromatographic analysis of pharmacological active andrographolide (AND), neoandrographolide (NAND) and 14-deoxy 11, 12- didehydroandrographolide (DAND) was performed on a 15 cm HSS Cyano column with a gradient mobile phase of ultra-pure water and acetonitrile with the addition of formic acid (0.1%) as a modifier at a flow rate of 0.8 mL/min in 21 min. The validation results showed acceptable linearity of the method (r>0.999) for all analytes within the calibration range 0.7–10 µg/mL. The acceptable accuracy of the method was indicated by recovery of 90–107 %. The limit of quantification was found to be between 2 and 70 ng/mL while the limit of detection was found between 0.6 and 20 ng/mL. Multiple reaction monitoring (MRM) mode was optimized to quantify data under monitoring precursor-product ion transitions of m/z 351.1 → 105.0, 351.5 → 91.0, 351.1 → 79.0 and 351.1 → 77.0 for AND, m/z 481.6  319.0, 481.6  301.2, 481.6  289.2, 481.6  95.1 for NAND and m/z 333.1  314.9, 333.1  297.2, 333.1  257.3, 333.1  91.0 for DAND (Figure 1). This advanced method is simple, selective, accurate and cost-effective. As a result, this assay can be easily transferred in clinical settings for pharmacokinetic studies and therapeutic drug monitoring programs. ACKNOWLEDGEMENTS This work has been supported in part by the Croatian Science Foundation under the project numbers [UIP-2017-05-3949, DOK-2021- 02-7922] and project FarmInova (KK.01.1.1.02.0021) funded by the European Regional Development Fund.