Objective: To establish a liquid chromatography-mass spectrometry (LC-MS/MS) method for content determination of ten compounds including kaempferol-3-O-β-D-glucoside

diphyllin-4-O-β-D-glucoside

diphyllin-4-O-β-D-glucopyranosyl-(1→2)-β-D-arabinopyranoside

podophyllotoxin-4-O-β-D-glucoside

4'-demethylpodophyllotoxin

kaempferol

podophyllotoxone

diphyllin

quercetin

and podophyllotoxin from Diphylleia sinensis in different batches

analyze the similarities and differences in the chemical constituents between different batches of samples

and provide a new scientific basis for establishing a comprehensive evaluation system on the quality of D. sinensis. Method: The separation was performed on COSMOSIL-C18 column (4.6 mm×250 mm

5 μm) at 30℃ with a flow rate of 0.3 mL·min-1

with acetonitrile-0.1% formic acid solution as the mobile phase for gradient elution (0-30 min

30% acetonitrile;30-40 min

30%-32% acetonitrile.40.0-40.5 min

32%-50% acetonitrile;40.5-50.0 min

50%-60% acetonitrile;50-85 min

60% acetonitrile;85.0-85.5 min

60%-30% acetonitrile;85.5-95.0 min

30% acetonitrile)

and the detection wavelength was set at 300 nm. The analyte was monitored by positive electrospray ionization(ESI) in multiple reaction monitoring mode to determine the content of ten compounds in different batches of D. sinensis. Result: Ten components showed good linear relationships within the investigated concentration ranges(r>0.999 1) as follows:0.07-2.23

0.39-12.40

0.05-1.68

0.12-3.88

0.05-1.73

0.08-2.70

0.05-1.00

0.04-1.10

0.04-0.58

and 0.18-5.67 mg·L-1

with RSDs of the precision

repeatability and stability less than 2%

and the average recovery rate was in the range of 98.61%-99.93% respectively. Significant differences were found in the chemical constituents of the D. sinensis between different batches. Conclusion: The method is rapid and sensitive with high precision

and it can be used to detect the content of components in D. sinensis from different batches. This method would provide important data for establishing and completing the medicinal quality comprehensive evaluation system ofD. sinensis.