LC and MS condition optimization
SEPARATION
Liquid chromatography-tandem mass spectrometry (LC–MS/MS) was performed using a Nexera X2 LCMS-8060NX triple quadrupole mass spectrometer (Shimadzu, Japan) equipped with a SIL-40C XR autosampler, CTO-40C column oven, CBM-40lite system controller, FCV-20AH2 switching valve, DGU-403 degasser, and LC-40D XR solvent delivery (pump unit). The LC separation was conducted on a VertiSep AQS C18 column (100 mm × 3.0 mm, 3.0 µm) using a gradient mobile phase. The different solvents (including acetonitrile, methanol, and water, with or without formic acid) were tested to optimize sample elution systems.Mass spectrometry (MS) and ionization was optimized and operated in Multiple Reaction Monitoring (MRM) mode. MS detection was fully scanned by directly injecting each standard (10.00–100.00 ng/mL) into the MS instrument without separation on a column. MS optimization was performed under the positive and negative conditions of electrospray ionization (ESI) mode. The MRM transitions and the fragmentations of parent and daughter ions for each analyte, with the corresponding collision energy, are shown in Table MRM transitions and fragmentations of parent ions and daughter ions of andrographolide (AP1); 14-deoxy-11, 12-didehydroandrographolide (AP3); neoandrographolide (AP4); 14-deoxyandrographolide (AP6); and digoxin (IS).*Quantitative ion.