1a*
1b,c
b* a
Beijing Key Laboratory of Active Substances Discovery and Druggability Evaluation,
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union
Medical College, Beijing 100050, China. b
State Key Laboratory of Bioactive Substance and Function of Natural Medicines,
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union
Medical College, Beijing 100050, P. R. China. c
Institute of Agro-products Processing Science and Technology, Chinese Academy of
Agricultural Sciences, Beijing 100193, People’s Republic of China.
*Corresponding authors. Email: zhangleilei@imm.ac.cn; dqyu@imm.ac.cn
1. These authors contributed equally to this work.

Abstract
A simple and accurate analytical method was developed for simultaneous and quantitative analysis of 6 triterpenoid saponins in Schefflera kwangsiensis via high-performance liquid chromatography (HPLC) with mass spectrometry (MS) in this study. Separation was performed on a Thermo hypersil GOLD C
18 column (150 mm×2.1 mm, 5 µm). A mobile phase consisted of methanol/acetonitrile/8mM ammonium acetate in water was used with a flow rate of 0.3 mL/min. The analytes were detected by mass spectrometry with the electrospray ionization (ESI) source combined with negative monitoring and full scan mode, and analysed by extracted ion chromatography (EIC). This established HPLC-ESI-MS analysis demonstrated good linearity, sensitivity, stability, precision, accuracy and recovery.
Therefore, this analytical method has great potential to be a novel tool to qualify Schefflera kwangsiensis.
Keywords: Schefflera kwangsiensis ; triterpenoid saponins; quantitative analysis ；
HPLC-ESI-MS

Figure S1 .The optimization of extraction conditions.
1) The extractedl ion chromatography of different extract solvent (A) Water (B) 45% methaonol (C) 70% methaonol (D) 100% methaonol
Compound 1
A
B
C
D
Compound 2
A
B
C
D
Compound 5

A
B
C
D
LC/MS analyses were performed on ThermoFisher Accela liquid chromatography system coupled with Exactive Plus Orbitrap mass spectrometer (Thermo-Fisher,
Bremen, Germany) via an ESI interface. The compounds were chromatographically resolved on a Thermo Hypersil GOLD C
18
column (150 mm×2.1 mm, 5 µm) connected with an Agilent Zorbax SB-C
18
guard column (12.5 mm×4.6 mm, 5 µm) at room temperature was applied for all analyses. A three-component mobile phase system was used, which contained acetonitrile (A), methanol (B), and water containing 8 mM ammonium acetate (C). A gradient program was used as follows: 0 min,20:20:60; 40 min, 30:30:40; 50 min, 45:45:10; 55 min, 45:45:10 (A:B:C, V/V/V).
The flow rate was 0.3 mL/min. All the data were processed by using Thermo Xcalibur
2.2 ChemStation software. A 5-min post-run time was set to fully equilibrate the column. The sample injection volume was 10 µL. For MS analysis, high-purity nitrogen (N
2
) (>99.99%) was used as the nebulizing gas. Ions were generated using electrospray ionization on the standard Thermo IonMax ESI source. The instrument worked in a full-scan acquisition mode in the range 300-2000 Da at a mass resolution of 35000 FWHM, allowing a maximum C-Trap fill time of 200ms. The optimized parameters in the negative ion mode were as follows: Capillary temperature and heater temperature 350 °C and 300 °C, respectively, spray voltage 2.5 kV, sheath gas
40 psi, auxiliary nitrogen pressure 12 L/min, Automatic gain control (AGC) target valve was set at 3×10
6
charges.

2) The extractedl ion chromatography of different extract time (A) 30 min (B) 60 min (C) 120min
Compound 1
A
B
C
Compound 2
A
B
C
Compound 5

A
B
C
The detection method for optimization of extraction time was same as above applied in optimization of extraction solvent, except that positive ion mode was used in optimization of extraction time .
3) The effect of cold soak (A) with cold soak (B) without cold soak
Compound 1
A
B
Compound 2

A
B
Compound 5
A
B
The detection method for investigation of the effect on cold soak was same as above applied in optimization of extraction solvent, except that elution condition was different. A two-component mobile phase system was used, which contained methanol
(A), and water containing 8 mM ammonium acetate (B). A gradient program was used as follows: 0 min,10% B; 10 min, 25% B; 45 min, 50% B; 50 min, 92% B, 55 min, 92%
B (A:B, v/v). A 5-min post-run time was set to fully equilibrate the column. The flow rate was 0.3 mL/min.

Figure S2.
TIC of six investigated compounds and internal standard compound.
Figure S3 . Purity test of refence compounds by HPLC at 210 nm.
4
5
6
1
2
3
HPLC detection conditions
ThermoFisher Accela instrument was equipped with a quaternary solvent deliver system, an autosample, PDA detector and a column compartment.
The compounds were chromatographically resolved on a Thermo Hypersil GOLD C
18 column (150 mm X 2.1 mm, 5 µm) connected with an Agilent Zorbax SB-C18 guard column(12.5 mm X 4.6 mm, 5 µm) at room temperature. Methanol-H
2
O mobile phase

system was used, which contained water with 0.05% formic acid (A) and acetonitrile with 0.05% formic acid (B). A gradient program was used as follows, 0 min-20%B,
10min-30%B, 45 min-70%B, 50 min-90%B, the flow rate was 0.3 mL/min, the detection wave was 210 nm, the sample injection volume was 10 µ L.
Table S1 . The linearity of calibration curves for 6 triterpenoid saponins (n=3)
Comp.
Molecular
Formula
1
2
Ions used for quantification
Rt
(min)
Equation
Linear range
(µg/mL)
R 2
C
60
H
94
O
27
622.3 ([M-2H] 2)
C
59
H
93
O
27 616.3 ([M-2H] 2)
29.21 Y = 3.1348 x-0.3331
30.30 Y= 0.8993 x + 0.0287
0.1025-6.5600 0.9983
0.0981 – 6.2800 0.9975
LOQ LOD
(ng/mL) (ng/ mL)
1.86
2.80
0.42
0.78
3 C
54
H
86
O
23
580.3
([M-CH
3
COO-H]
2-
)
31.37 Y= 2.8648 x + 0.3733 0.0981 – 6.2800 0.9957 1.05 0.39
4
5
6
C
60
H
95
O
27
C
36
H
56
O
9
580.3
631.4 ([M-H] )
31.37
C
48
H
75
O
18
([M-CH
3
COO-H] 2)
939.5 ([M-H] ) 38.94 Y=2.5322 x - 0.0679
45.77
Y = 2.0215 x + 0.1239
Y= 2.6652 x – 0.0207
0.0984 – 6.2960 0.9975
0.0986 – 6.3130
0.1016 – 3.2500
0.9989
0.9986
2.18
1.31
1.69
0.65
0.45
0.53
Table S2 . Intra- and Inter-day variability for the assay of six investigated compounds in Schefflera kwangsiensis (n=5)
Intra-day Inter-day
Compd Conc (ng/mL)
820.0
Found (ng/mL) RSD a (%)
852.42
± 11.33 1.3
Found (ng/mL)
842.41
± 37.33
1640.0 1709.72
± 49.32 2.9 1714.13
± 55.08
1
3280.0 3390.72
± 55.88 1.6 3260.98
± 119.64
RSD (%)
4.4
3.2
3.7
2
785.0
1570.0
3140.0
757.58
± 14.15
1491.71
± 50.84
2939.20
± 66.54
1.9
3.4
2.3
777.44
±
1509.35
2959.77
38.67
±
±
37.83
79.97
5.0
2.5
2.7

3
5
6
4
785.0
1570.0
3140.0
787.0
1574.0
3148.0
789.125
1578.25
3156.50
812.5
1625.0
3250.0
851.63
± 12.97
1694.94
± 56.35
3173.77
± 53.11
774.52
± 10.98
1617.26
± 47.95
3077.67
± 49.21
826.33
± 16.36
1664.36
± 56.51
2839.51
± 71.94
806.68
± 12.61
1630.36
± 50.10
3115.70
± 46.77
1.5
3.3
1.7
1.4
3.0
1.6
2.0
3.4
2.5
1.6
3.1
1.5
843.75
± 34.52
1707.78
± 46.29
3185.45
± 72.10
780.63
± 29.58
1619.16
± 36.63
3060.79
± 61.86
809.25
± 34.48
1650.52
± 38.96
2910.63
± 95.47
797.78
± 27.60
1624.67
± 40.30
3104.83
± 73.98
4.1
2.7
2.3
3.8
2.3
2.0
4.3
2.4
3.3
3.5
2.5
2.4 a ： RSD(%) = (SD/mean) × 100.
Table S3 . Recoveries of the six investigated compounds in Schefflera kwangsiensis
(n=3)
Compound
Spiked
(µg)
76.7
Found
(µg)
75.97
Recovery(%) a)
RSD(%) b)
1
2
153.4
230.1
50.85
101.7
138.93
206.38
49.44
91.18
1.13
0.91
0.90
12.7
6.7
4.3
3
152.55
39.55
79.10
118.65
136.53
41.76
82.58
127.85
0.98
0.90
0.90
1.06
1.04
1.08
4.4
6.3
3.3
1.8
8.5
2.3
4
33.90
67.80
34.31
67.64
1.01
0.99
7.0
6.4

5
6
101.70 109.04
62.48 71.44
124.96 114.86
187.44 163.27
46.80
93.60
49.89
98.77
140.40 146.69
1.07
1.14
0.92
0.87
1.07
1.06
1.04 a) Recovery (%) = (detected amount - original amount)/spiked amount × 100 b) RSD (%) = (standard derivation/mean) × 100
3.8
1.0
6.9
1.6
5.1
10.6
7.2
3
4
1
2
5
6
Table S4 . Stability of the six investigated compounds in Schefflera kwangsiensis (n=6
）
(ng/mL)
Compound 0h 5h 10h 15h 20h 25h Mean RSD(%)
2298.79 2193.30 2119.11 2180.79 2095.21 2239.47 2187.78
1425.43 1438.79 1439.83 1471.35 1449.49 1467.38 1448.711
1061.39 1119.41 1117.54 1105.76 1128.47 1142.49 1112.51
831.49 850.02 845.01 830.63 844.91 848.19 841.71
1780.02 1688.38 1615.59 1578.71 1622.36 1575.72 1643.47
1294.33 1231.52 1175.03 1168.23 1187.01 1152.39 1201.42
7.0
6.2
4.0
4.8
6.1
7.9