药学学报

No.	Retention time/min	Adduction	Actual value of m/z	Theoretical value of m/z	Relative error (×10^-6)	Molecular formula	Molecular weight	MS/MS
1	0.86	[M-H]^-	133.014 9	133.014 2	5.3	C₄H₆O₅	134.02	115.005 2; 71.014 2
2	1.85	[M-H]^-	169.014 8	169.014 2	3.5	C₇H₆O₅	170.02	125.024 9; 81.034 2
3	2.00	[M+FA-H]^-	405.140 8	405.140 2	1.5	C₁₆H₂₄O₉	360.14	359.136 5; 197.085 8; 179.074 1
4	7.32	[M-H]^-	165.056 7	165.055 7	6.1	C₉H₁₀O₃	166.06	165.054 6; 93.036 6
5	9.08	[M-H]^-	353.090 9	353.087 8	8.8	C₁₆H₁₈O₉	354.10	191.056 6
6	11.81	[M+FA-H]^-	525.165 7	525.161 3	8.4	C₂₃H₂₈O₁₁	480.16	525.161 2; 479.157 2; 283.000 0; 121.029 9
7	12.20	[M-H]^-	593.153 7	593.151 2	4.2	C₂₇H₃₀O₁₅	594.16	593.152 8; 503.115 5; 383.075 0; 353.063 7
8	12.79	[M+FA-H]^-	525.165 5	525.161 3	8.0	C₂₃H₂₈O₁₁	480.16	428.159 1; 479.159 8; 449.148 5; 327.110 4; 121.029 9
9	13.87	[M-H]^-	563.142 3	563.140 6	3.0	C₂₆H₂₈O₁₄	564.15	563.139 9; 545.136 9; 443.101 5; 383.073 5
10	13.93	[M+H]⁺	469.168 6	469.170 4	-3.8	C₂₂H₂₈O₁₁	468.16	469.169 6; 397.109 7; 307.116 3; 235.057 4
11	14.40	[M-H]^-	415.104 9	415.103 4	3.6	C₂₁H₂₀O₉	416.11	415.107 0; 253.050 5; 224.055 7
12	15.26	[M-H]^-	417.121 5	417.119 1	5.8	C₂₁H₂₂O₉	418.13	417.117 1; 255.066 7; 135.009 5; 119.050 5
13	15.47	[M-H]^-	549.166 8	549.161 3	10.0	C₂₆H₃₀O₁₃	550.17	549.162 0; 255.066 4; 135.008 4
14	16.27	[M-H]^-	461.075 1	461.072 5	5.6	C₂₁H₁₈O₁₂	462.08	285.041 5; 267.027 7; 175.024 2
15	16.50	[M-H]^-	623.167 4	623.161 7	9.1	C₂₈H₃₂O₁₆	624.17	623.158 0; 314.042 1; 243.031 4
16	16.66	[M+H]⁺	307.116 9	307.117 6	-2.3	C₁₆H₁₈O₆	306.11	307.119 7; 259.060 4; 235.062 7; 221.045 1
17	17.17	[M-H]^-	623.201 9	623.198 1	6.1	C₂₉H₃₆O₁₅	624.21	623.197 8; 461.166 4; 161.024 7
18	18.44	[M-H]^-	623.202 3	623.198 1	6.7	C₂₉H₃₆O₁₅	624.21	623.196 4; 461.162 4; 161.022 5
19	18.63	[M-H]^-	433.114 7	433.114 0	1.6	C₂₁H₂₂O₁₀	434.12	433.113 3; 271.061 5; 151.004 4
20	18.74	[M+FA-H]^-	497.168 5	497.166 4	4.2	C₂₂H₂₈O₁₀	452.17	497.173 2; 451.161 6; 271.097 6
21	20.52	[M+H]⁺	447.127 1	447.128 6	-3.4	C₂₂H₂₂O₁₀	446.12	285.075 5; 269.041 1
22	21.16	[M-H]^-	549.164 3	549.161 3	5.5	C₂₆H₃₀O₁₃	550.17	549.163 9; 255.066 3; 135.010 7
23	21.33	[M+FA-H]^-	475.128 6	475.124 6	8.4	C₂₂H₂₂O₉	430.13	475.133 6; 267.068 2; 252.042 8
24	21.68	[M-H]^-	417.121 0	417.119 1	4.5	C₂₁H₂₂O₉	418.13	417.117 8; 255.067 5; 135.008 8
25	22.02	[M-H]^-	445.081 3	445.077 6	8.3	C₂₁H₁₈O₁₁	446.08	269.046 0; 113.025 4
26	23.54	[M-H]^-	165.057 2	165.055 7	9.1	C₉H₁₀O₃	166.06	136.037 4; 93.033 9
27	24.18	[M-H]^-	445.080 2	445.077 6	5.8	C₂₁H₁₈O₁₁	446.08	269.045 1; 241.052 0; 197.063 1
28	25.58	[M+H]⁺	439.158 2	439.159 9	-3.9	C₂₁H₂₆O₁₀	438.15	277.108 9; 259.097 4; 205.048 1
29	25.76	[M-H]^-	459.097 3	459.093 3	8.7	C₂₂H₂₀O₁₁	460.10	459.092 7; 283.061 8; 268.037 6
30	26.82	[M-H]^-	459.095 6	459.093 3	5.0	C₂₂H₂₀O₁₁	460.10	459.094 8; 283.060 9; 268.038 1
31	27.49	[M-H]^-	271.061 4	271.061 2	0.7	C₁₅H₁₂O₅	272.07	271.063 0; 177.018 0; 151.006 1
32	30.52	[M-H]^-	269.046 6	269.045 5	4.1	C₁₅H₁₀O₅	270.05	269.043 9; 223.043 4
33	30.55	[M+H]⁺	271.060 2	271.060 1	0.4	C₁₅H₁₀O₅	270.05	271.061 4; 169.011 9; 123.006 5
34	30.78	[M+FA-H]^-	845.494 1	845.490 4	4.4	C₄₂H₇₂O₁₄	800.49	845.498 3; 799.490 0; 637.431 1; 475.375 6
35	32.49	[M+H]⁺	269.079 9	269.080 8	-3.3	C₁₆H₁₂O₄	268.07	269.080 2; 254.054 8; 237.055 2; 197.056 6
36	32.99	[M+FA-H]^-	829.499 6	829.495 5	4.9	C₄₂H₇₂O₁₃	784.50	829.502 4; 783.493 5; 637.424 8; 475.375 0
37	35.00	[M-H]^-	955.497 4	955.490 8	6.9	C₄₈H₇₆O₁₉	956.50	955.492 3; 793.436 6
38	36.07	[M-H]^-	283.061 6	283.061 2	1.4	C₁₆H₁₂O₅	284.07	283.060 8; 268.038 6; 163.003 7
39	36.6	[M+H]⁺	255.064 2	255.065 2	-3.9	C₁₅H₁₀O₄	254.06	255.063 7; 153.018 0
40	37.14	[M-H]^-	373.094 7	373.092 9	4.8	C₁₉H₁₈O₈	374.10	373.092 0; 358.066 0; 343.044 2; 328.020 4
41	37.35	[M-H]^-	821.399 1	821.396 5	3.2	C₄₂H₆₂O₁₆	822.40	821.391 7; 351.054 7
42	39.53	[M-H]^-	821.403 2	821.396 5	8.2	C₄₂H₆₂O₁₆	822.40	821.398 8; 351.055 9
43	39.98	[M+FA-H]^-	825.468 5	825.470 0	-1.8	C₄₂H₆₈O₁₃	780.47	825.475 5; 779.465 8; 617.403 6
44	40	[M+H]⁺	455.351	455.352	-2.2	C₃₀H₄₆O₃	454.34	455.353 1; 437.348 1; 267.209 5; 191.178 7
45	40.18	[M-H]^-	367.121 5	367.118 7	7.6	C₂₁H₂₀O₆	368.13	367.119 6; 309.039 4; 201.017 2
46	41.25	[M-H]^-	407.284 1	407.280 3	9.3	C₂₄H₄₀O₅	408.29	407.281 6; 343.265 4; 289.220 7
47	41.44	[M-H]^-	391.288 2	391.285 4	7.2	C₂₄H₄₀O₄	392.29	391.286 8
48	41.96	[M+FA-H]^-	825.471 0	825.464 2	8.2	C₄₂H₆₈O₁₃	780.47	825.469 6; 779.462 1; 617.402 9
49	43.46	[M+H]⁺	277.180 1	277.179 8	1.1	C₁₇H₂₄O₃	276.17	137.058 9; 122.035 7
50	43.47	[M+H]⁺	137.059 5	137.059 7	-1.5	C₈H₈O₂	136.05	137.057 8; 122.037 0; 94.040 7
51	43.52	[M+FA-H]^-	829.502 3	829.495 5	8.2	C₄₂H₇₂O₁₃	784.50	829.502 5; 783.493 1; 621.440 4; 459.384 0
52	44.13	[M-H]^-	391.287 1	391.285 4	4.3	C₂₄H₄₀O₄	392.29	391.286 5; 345.278 6
53	44.43	[M+FA-H]^-	723.385 3	723.380 8	6.2	C₃₃H₅₈O₁₄	678.38	723.384 1; 677.380 4; 415.143 9; 397.139 9; 279.232 7
54	44.67	[M-H]^-	471.352 9	471.348 0	10.4	C₃₀H₄₈O₄	472.36	471.350 9
55	45.19	[M-H]^-	469.335 5	469.332 3	6.8	C₃₀H₄₆O₄	470.34	469.335 9; 425.343 1

No.	Retention time/min	Adduction	Actual value of m/z	Theoretical value of m/z	Relative error (×10^-6)	Molecular formula	Molecular weight	MS/MS
1	0.86	[M-H]^-	133.014 9	133.014 2	5.3	C₄H₆O₅	134.02	115.005 2; 71.014 2
2	1.85	[M-H]^-	169.014 8	169.014 2	3.5	C₇H₆O₅	170.02	125.024 9; 81.034 2
3	2.00	[M+FA-H]^-	405.140 8	405.140 2	1.5	C₁₆H₂₄O₉	360.14	359.136 5; 197.085 8; 179.074 1
4	7.32	[M-H]^-	165.056 7	165.055 7	6.1	C₉H₁₀O₃	166.06	165.054 6; 93.036 6
5	9.08	[M-H]^-	353.090 9	353.087 8	8.8	C₁₆H₁₈O₉	354.10	191.056 6
6	11.81	[M+FA-H]^-	525.165 7	525.161 3	8.4	C₂₃H₂₈O₁₁	480.16	525.161 2; 479.157 2; 283.000 0; 121.029 9
7	12.20	[M-H]^-	593.153 7	593.151 2	4.2	C₂₇H₃₀O₁₅	594.16	593.152 8; 503.115 5; 383.075 0; 353.063 7
8	12.79	[M+FA-H]^-	525.165 5	525.161 3	8.0	C₂₃H₂₈O₁₁	480.16	428.159 1; 479.159 8; 449.148 5; 327.110 4; 121.029 9
9	13.87	[M-H]^-	563.142 3	563.140 6	3.0	C₂₆H₂₈O₁₄	564.15	563.139 9; 545.136 9; 443.101 5; 383.073 5
10	13.93	[M+H]⁺	469.168 6	469.170 4	-3.8	C₂₂H₂₈O₁₁	468.16	469.169 6; 397.109 7; 307.116 3; 235.057 4
11	14.40	[M-H]^-	415.104 9	415.103 4	3.6	C₂₁H₂₀O₉	416.11	415.107 0; 253.050 5; 224.055 7
12	15.26	[M-H]^-	417.121 5	417.119 1	5.8	C₂₁H₂₂O₉	418.13	417.117 1; 255.066 7; 135.009 5; 119.050 5
13	15.47	[M-H]^-	549.166 8	549.161 3	10.0	C₂₆H₃₀O₁₃	550.17	549.162 0; 255.066 4; 135.008 4
14	16.27	[M-H]^-	461.075 1	461.072 5	5.6	C₂₁H₁₈O₁₂	462.08	285.041 5; 267.027 7; 175.024 2
15	16.50	[M-H]^-	623.167 4	623.161 7	9.1	C₂₈H₃₂O₁₆	624.17	623.158 0; 314.042 1; 243.031 4
16	16.66	[M+H]⁺	307.116 9	307.117 6	-2.3	C₁₆H₁₈O₆	306.11	307.119 7; 259.060 4; 235.062 7; 221.045 1
17	17.17	[M-H]^-	623.201 9	623.198 1	6.1	C₂₉H₃₆O₁₅	624.21	623.197 8; 461.166 4; 161.024 7
18	18.44	[M-H]^-	623.202 3	623.198 1	6.7	C₂₉H₃₆O₁₅	624.21	623.196 4; 461.162 4; 161.022 5
19	18.63	[M-H]^-	433.114 7	433.114 0	1.6	C₂₁H₂₂O₁₀	434.12	433.113 3; 271.061 5; 151.004 4
20	18.74	[M+FA-H]^-	497.168 5	497.166 4	4.2	C₂₂H₂₈O₁₀	452.17	497.173 2; 451.161 6; 271.097 6
21	20.52	[M+H]⁺	447.127 1	447.128 6	-3.4	C₂₂H₂₂O₁₀	446.12	285.075 5; 269.041 1
22	21.16	[M-H]^-	549.164 3	549.161 3	5.5	C₂₆H₃₀O₁₃	550.17	549.163 9; 255.066 3; 135.010 7
23	21.33	[M+FA-H]^-	475.128 6	475.124 6	8.4	C₂₂H₂₂O₉	430.13	475.133 6; 267.068 2; 252.042 8
24	21.68	[M-H]^-	417.121 0	417.119 1	4.5	C₂₁H₂₂O₉	418.13	417.117 8; 255.067 5; 135.008 8
25	22.02	[M-H]^-	445.081 3	445.077 6	8.3	C₂₁H₁₈O₁₁	446.08	269.046 0; 113.025 4
26	23.54	[M-H]^-	165.057 2	165.055 7	9.1	C₉H₁₀O₃	166.06	136.037 4; 93.033 9
27	24.18	[M-H]^-	445.080 2	445.077 6	5.8	C₂₁H₁₈O₁₁	446.08	269.045 1; 241.052 0; 197.063 1
28	25.58	[M+H]⁺	439.158 2	439.159 9	-3.9	C₂₁H₂₆O₁₀	438.15	277.108 9; 259.097 4; 205.048 1
29	25.76	[M-H]^-	459.097 3	459.093 3	8.7	C₂₂H₂₀O₁₁	460.10	459.092 7; 283.061 8; 268.037 6
30	26.82	[M-H]^-	459.095 6	459.093 3	5.0	C₂₂H₂₀O₁₁	460.10	459.094 8; 283.060 9; 268.038 1
31	27.49	[M-H]^-	271.061 4	271.061 2	0.7	C₁₅H₁₂O₅	272.07	271.063 0; 177.018 0; 151.006 1
32	30.52	[M-H]^-	269.046 6	269.045 5	4.1	C₁₅H₁₀O₅	270.05	269.043 9; 223.043 4
33	30.55	[M+H]⁺	271.060 2	271.060 1	0.4	C₁₅H₁₀O₅	270.05	271.061 4; 169.011 9; 123.006 5
34	30.78	[M+FA-H]^-	845.494 1	845.490 4	4.4	C₄₂H₇₂O₁₄	800.49	845.498 3; 799.490 0; 637.431 1; 475.375 6
35	32.49	[M+H]⁺	269.079 9	269.080 8	-3.3	C₁₆H₁₂O₄	268.07	269.080 2; 254.054 8; 237.055 2; 197.056 6
36	32.99	[M+FA-H]^-	829.499 6	829.495 5	4.9	C₄₂H₇₂O₁₃	784.50	829.502 4; 783.493 5; 637.424 8; 475.375 0
37	35.00	[M-H]^-	955.497 4	955.490 8	6.9	C₄₈H₇₆O₁₉	956.50	955.492 3; 793.436 6
38	36.07	[M-H]^-	283.061 6	283.061 2	1.4	C₁₆H₁₂O₅	284.07	283.060 8; 268.038 6; 163.003 7
39	36.6	[M+H]⁺	255.064 2	255.065 2	-3.9	C₁₅H₁₀O₄	254.06	255.063 7; 153.018 0
40	37.14	[M-H]^-	373.094 7	373.092 9	4.8	C₁₉H₁₈O₈	374.10	373.092 0; 358.066 0; 343.044 2; 328.020 4
41	37.35	[M-H]^-	821.399 1	821.396 5	3.2	C₄₂H₆₂O₁₆	822.40	821.391 7; 351.054 7
42	39.53	[M-H]^-	821.403 2	821.396 5	8.2	C₄₂H₆₂O₁₆	822.40	821.398 8; 351.055 9
43	39.98	[M+FA-H]^-	825.468 5	825.470 0	-1.8	C₄₂H₆₈O₁₃	780.47	825.475 5; 779.465 8; 617.403 6
44	40	[M+H]⁺	455.351	455.352	-2.2	C₃₀H₄₆O₃	454.34	455.353 1; 437.348 1; 267.209 5; 191.178 7
45	40.18	[M-H]^-	367.121 5	367.118 7	7.6	C₂₁H₂₀O₆	368.13	367.119 6; 309.039 4; 201.017 2
46	41.25	[M-H]^-	407.284 1	407.280 3	9.3	C₂₄H₄₀O₅	408.29	407.281 6; 343.265 4; 289.220 7
47	41.44	[M-H]^-	391.288 2	391.285 4	7.2	C₂₄H₄₀O₄	392.29	391.286 8
48	41.96	[M+FA-H]^-	825.471 0	825.464 2	8.2	C₄₂H₆₈O₁₃	780.47	825.469 6; 779.462 1; 617.402 9
49	43.46	[M+H]⁺	277.180 1	277.179 8	1.1	C₁₇H₂₄O₃	276.17	137.058 9; 122.035 7
50	43.47	[M+H]⁺	137.059 5	137.059 7	-1.5	C₈H₈O₂	136.05	137.057 8; 122.037 0; 94.040 7
51	43.52	[M+FA-H]^-	829.502 3	829.495 5	8.2	C₄₂H₇₂O₁₃	784.50	829.502 5; 783.493 1; 621.440 4; 459.384 0
52	44.13	[M-H]^-	391.287 1	391.285 4	4.3	C₂₄H₄₀O₄	392.29	391.286 5; 345.278 6
53	44.43	[M+FA-H]^-	723.385 3	723.380 8	6.2	C₃₃H₅₈O₁₄	678.38	723.384 1; 677.380 4; 415.143 9; 397.139 9; 279.232 7
54	44.67	[M-H]^-	471.352 9	471.348 0	10.4	C₃₀H₄₈O₄	472.36	471.350 9
55	45.19	[M-H]^-	469.335 5	469.332 3	6.8	C₃₀H₄₆O₄	470.34	469.335 9; 425.343 1

No.	Retention time/min	Name	The whole prescription	Section A	Section B	Section C
1	0.86	Malic acid	√	/	/	/
2	1.85	Gallic acid	√	√	/	/
3	2.00	Yuheinoside	√	√	/	/
4	7.32	Phloretic acid	√	√	/	/
5	9.08	Chlorogenic acid	√	√	/	/
6	11.81	Albiflorin	√	√	√	√
7	12.20	Vicenin Ⅱ	√	√	√	/
8	12.79	Paeoniflorin	√	√	√	√
9	13.87	Schaftoside	√	√	√	/
10	13.93	Prim-O-glucosylcimifugin	√	√	√	√
11	14.40	Daidzin	√	/	√	/
12	15.26	Liquiritin	√	√	√	√
13	15.47	Liquiritin apioside	√	√	√	√
14	16.27	Scutellarin	√	√	/	/
15	16.50	Isorhamnetin-3-O-neohespeidoside	√	/	√	√
16	16.66	Cimifugin	√	√	√	√
17	17.17	Acteoside	√	/	√	/
18	18.44	Isoacteoside	√	/	√	/
19	18.63	Naringenin 7-O-β-D-glucoside	√	/	/	√
20	18.74	4'-O-β-Glucopyranosyl-5-O-methylvisamminol	√	√	√	√
21	20.52	Calycosin 7-glucoside	√	/	/	√
22	21.16	Isoliquiritin apioside	√	/	√	√
23	21.33	Ononin	√	/	√	√
24	21.68	Isoliquiritin	√	/	√	√
25	22.02	Baicalin	√	√	√	√
26	23.54	Ethyl 4-hydroxybenzoate	√	/	/	√
27	24.18	Apigenin 7-O-β-D-glucuronide	√	/	/	/
28	25.58	Sec-O-glucosylhamaudol	√	/	/	√
29	25.76	Oroxyloside	√	√	√	√
30	26.82	Wogonin 7-O-glucuronide	√	√	√	√
31	27.49	Naringenin	√	/	/	√
32	30.52	4', 6, 7-Trihydroxyisoflavone	√	/	√	/
33	30.55	Apigenin	√	√	√	/
34	30.78	Ginsenoside Rf	√	/	/	√
35	32.49	Formononetin	√	/	/	√
36	32.99	Ginsenoside Rg1	√	/	/	√
37	35.00	Ginsenoside Rg2	√	/	/	√
38	36.07	Oroxylin A	√	/	/	/
39	36.6	Chrysin	√	/	/	/
40	37.14	Chrysosplenetin B	√	/	/	/
41	37.35	Glycyrrhizic acid	√	√	√	√
42	39.53	Glycyrrhizic acid isomer	√	√	/	√
43	39.98	Saikosaponin A	√	/	/	/
44	40	Liquidambaric acid	√	/	/	√
45	40.18	Glycycoumarin	√	/	/	/
46	41.25	Cholan-24-oic acid	√	/	/	√
47	41.44	Ursodeoxycholic acid	√	/	/	/
48	41.96	Saikosaponin D	√	/	/	/
49	43.46	6-Shogaol	√	√	√	√
50	43.47	p-Methoxybezaldehyde	√	/	/	/
51	43.52	Ginsenoside Rg3	√	√	/	/
52	44.13	Ursodeoxycholic acid isomer	√	/	/	/
53	44.43	Gingerglycolipid B	√	√	/	/
54	44.67	Corosolic acid	√	/	/	/
55	45.19	Glycyrrhetinic acid	√	/	/	/

No.	Retention time/min	Name	The whole prescription	Section A	Section B	Section C
1	0.86	Malic acid	√	/	/	/
2	1.85	Gallic acid	√	√	/	/
3	2.00	Yuheinoside	√	√	/	/
4	7.32	Phloretic acid	√	√	/	/
5	9.08	Chlorogenic acid	√	√	/	/
6	11.81	Albiflorin	√	√	√	√
7	12.20	Vicenin Ⅱ	√	√	√	/
8	12.79	Paeoniflorin	√	√	√	√
9	13.87	Schaftoside	√	√	√	/
10	13.93	Prim-O-glucosylcimifugin	√	√	√	√
11	14.40	Daidzin	√	/	√	/
12	15.26	Liquiritin	√	√	√	√
13	15.47	Liquiritin apioside	√	√	√	√
14	16.27	Scutellarin	√	√	/	/
15	16.50	Isorhamnetin-3-O-neohespeidoside	√	/	√	√
16	16.66	Cimifugin	√	√	√	√
17	17.17	Acteoside	√	/	√	/
18	18.44	Isoacteoside	√	/	√	/
19	18.63	Naringenin 7-O-β-D-glucoside	√	/	/	√
20	18.74	4'-O-β-Glucopyranosyl-5-O-methylvisamminol	√	√	√	√
21	20.52	Calycosin 7-glucoside	√	/	/	√
22	21.16	Isoliquiritin apioside	√	/	√	√
23	21.33	Ononin	√	/	√	√
24	21.68	Isoliquiritin	√	/	√	√
25	22.02	Baicalin	√	√	√	√
26	23.54	Ethyl 4-hydroxybenzoate	√	/	/	√
27	24.18	Apigenin 7-O-β-D-glucuronide	√	/	/	/
28	25.58	Sec-O-glucosylhamaudol	√	/	/	√
29	25.76	Oroxyloside	√	√	√	√
30	26.82	Wogonin 7-O-glucuronide	√	√	√	√
31	27.49	Naringenin	√	/	/	√
32	30.52	4', 6, 7-Trihydroxyisoflavone	√	/	√	/
33	30.55	Apigenin	√	√	√	/
34	30.78	Ginsenoside Rf	√	/	/	√
35	32.49	Formononetin	√	/	/	√
36	32.99	Ginsenoside Rg1	√	/	/	√
37	35.00	Ginsenoside Rg2	√	/	/	√
38	36.07	Oroxylin A	√	/	/	/
39	36.6	Chrysin	√	/	/	/
40	37.14	Chrysosplenetin B	√	/	/	/
41	37.35	Glycyrrhizic acid	√	√	√	√
42	39.53	Glycyrrhizic acid isomer	√	√	/	√
43	39.98	Saikosaponin A	√	/	/	/
44	40	Liquidambaric acid	√	/	/	√
45	40.18	Glycycoumarin	√	/	/	/
46	41.25	Cholan-24-oic acid	√	/	/	√
47	41.44	Ursodeoxycholic acid	√	/	/	/
48	41.96	Saikosaponin D	√	/	/	/
49	43.46	6-Shogaol	√	√	√	√
50	43.47	p-Methoxybezaldehyde	√	/	/	/
51	43.52	Ginsenoside Rg3	√	√	/	/
52	44.13	Ursodeoxycholic acid isomer	√	/	/	/
53	44.43	Gingerglycolipid B	√	√	/	/
54	44.67	Corosolic acid	√	/	/	/
55	45.19	Glycyrrhetinic acid	√	/	/	/

No.	Retention time / min	Name	Molecular formula	Measured molecular weight	Main feature fragment
1	9.08	Chlorogenic acid	C₁₆H₁₈O₉	m/z 353.087 8 [M-H]^-	m/z 191.056 6
2	11.81	Albiflorin	C₂₃H₂₈O₁₁	m/z 525.161 3 [M+FA-H]^-	m/z 479.157 2, 283.000 0, 121.029 9
3	12.79	Paeoniflorin	C₂₃H₂₈O₁₁	m/z 449.148 5 [M-H]^-	m/z 327.110 4, 121.029 9
4	13.93	Prim-O-glucosylcimifugin	C₂₂H₂₈O₁₁	m/z 469.170 4 [M+H]⁺	m/z 397.109 7, 307.116 3, 235.057 4
5	15.26	Liquiritin	C₂₁H₂₂O₉	m/z 417.119 1 [M-H]^-	m/z 417.119 1, 255.066 7, 135.009 5
6	15.47	Liquiritin apioside	C₂₆H₃₀O₁₃	m/z 549.161 3 [M-H]^-	m/z 549.162 0, 255.066 4, 135.008 4
7	16.27	Scutellarin	C₂₁H₁₈O₁₂	m/z 461.072 5 [M-H]^-	m/z 285.041 5, 267.027 7, 175.024 2
8	16.66	Cimifugin	C₁₆H₁₈O₆	m/z 307.117 6 [M+H]⁺	m/z 259.060 4, 235.062 7, 221.045 1
9	18.74	4'-O-β-Glucopyranosyl-5-O-methylvisamminol	C₂₂H₂₈O₁₀	m/z 497.166 4 [M+FA-H]^-	m/z 451.161 6, 271.097 6
10	21.16	Isoliquiritin apioside	C₂₆H₃₀O₁₃	m/z 549.161 3 [M-H]^-	m/z 549.162 0, 255.066 4, 135.008 4
11	21.33	Ononin	C₂₂H₂₂O₉	m/z 475.124 6 [M+FA-H]^-	m/z 475.133 6, 267.068 2, 252.042 8
12	21.68	Isoliquiritin	C₂₁H₂₂O₉	m/z 417.119 1 [M-H]^-	m/z 417.117 8, 255.067 5, 135.008 8
13	22.02	Baicalin	C₂₁H₁₈O₁₁	m/z 445.077 6 [M-H]^-	m/z 269.046 0, 113.025 4
14	25.58	Sec-O-glucosylhamaudol	C₂₁H₂₆O₁₀	m/z 439.159 9 [M+H]⁺	m/z 277.108 9, 259.097 4, 205.048 1
15	32.49	Formononetin	C₁₆H₁₂O₄	m/z 269.080 8 [M+H]⁺	m/z 269.080 2, 254.054 8, 237.055 2
16	37.35	Glycyrrhizic acid	C₄₂H₆₂O₁₆	m/z 821.396 5 [M-H]^-	m/z 821.391 7, 351.054 7
17	39.53	Glycyrrhizic acid isomer	C₄₂H₆₂O₁₆	m/z 821.396 5 [M-H]^-	m/z 821.398 8, 351.055 9
18	43.46	6-Shogaol	C₁₇H₂₄O₃	m/z 277.179 8 [M+H]⁺	m/z 137.058 9, 122.035 7
19	45.19	Glycyrrhetinic acid	C₃₀H₄₆O₄	m/z 469.332 3 [M-H]^-	m/z 469.335 9, 425.343 1

No.	Retention time / min	Name	Molecular formula	Measured molecular weight	Main feature fragment
1	9.08	Chlorogenic acid	C₁₆H₁₈O₉	m/z 353.087 8 [M-H]^-	m/z 191.056 6
2	11.81	Albiflorin	C₂₃H₂₈O₁₁	m/z 525.161 3 [M+FA-H]^-	m/z 479.157 2, 283.000 0, 121.029 9
3	12.79	Paeoniflorin	C₂₃H₂₈O₁₁	m/z 449.148 5 [M-H]^-	m/z 327.110 4, 121.029 9
4	13.93	Prim-O-glucosylcimifugin	C₂₂H₂₈O₁₁	m/z 469.170 4 [M+H]⁺	m/z 397.109 7, 307.116 3, 235.057 4
5	15.26	Liquiritin	C₂₁H₂₂O₉	m/z 417.119 1 [M-H]^-	m/z 417.119 1, 255.066 7, 135.009 5
6	15.47	Liquiritin apioside	C₂₆H₃₀O₁₃	m/z 549.161 3 [M-H]^-	m/z 549.162 0, 255.066 4, 135.008 4
7	16.27	Scutellarin	C₂₁H₁₈O₁₂	m/z 461.072 5 [M-H]^-	m/z 285.041 5, 267.027 7, 175.024 2
8	16.66	Cimifugin	C₁₆H₁₈O₆	m/z 307.117 6 [M+H]⁺	m/z 259.060 4, 235.062 7, 221.045 1
9	18.74	4'-O-β-Glucopyranosyl-5-O-methylvisamminol	C₂₂H₂₈O₁₀	m/z 497.166 4 [M+FA-H]^-	m/z 451.161 6, 271.097 6
10	21.16	Isoliquiritin apioside	C₂₆H₃₀O₁₃	m/z 549.161 3 [M-H]^-	m/z 549.162 0, 255.066 4, 135.008 4
11	21.33	Ononin	C₂₂H₂₂O₉	m/z 475.124 6 [M+FA-H]^-	m/z 475.133 6, 267.068 2, 252.042 8
12	21.68	Isoliquiritin	C₂₁H₂₂O₉	m/z 417.119 1 [M-H]^-	m/z 417.117 8, 255.067 5, 135.008 8
13	22.02	Baicalin	C₂₁H₁₈O₁₁	m/z 445.077 6 [M-H]^-	m/z 269.046 0, 113.025 4
14	25.58	Sec-O-glucosylhamaudol	C₂₁H₂₆O₁₀	m/z 439.159 9 [M+H]⁺	m/z 277.108 9, 259.097 4, 205.048 1
15	32.49	Formononetin	C₁₆H₁₂O₄	m/z 269.080 8 [M+H]⁺	m/z 269.080 2, 254.054 8, 237.055 2
16	37.35	Glycyrrhizic acid	C₄₂H₆₂O₁₆	m/z 821.396 5 [M-H]^-	m/z 821.391 7, 351.054 7
17	39.53	Glycyrrhizic acid isomer	C₄₂H₆₂O₁₆	m/z 821.396 5 [M-H]^-	m/z 821.398 8, 351.055 9
18	43.46	6-Shogaol	C₁₇H₂₄O₃	m/z 277.179 8 [M+H]⁺	m/z 137.058 9, 122.035 7
19	45.19	Glycyrrhetinic acid	C₃₀H₄₆O₄	m/z 469.332 3 [M-H]^-	m/z 469.335 9, 425.343 1

基于MIF-HEMT生物传感技术的同仁牛黄清心丸关键质量属性测量研究

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马朝富 ¹^,² , 王子健 ³ , 马丽娟 ¹^,² , 胡小艳 ¹^,² , 魏宇楠 ¹^,² , 张晓梦 ¹^,² , 郭新雨 ¹^,² , 郇星月 ¹^,² , 赵静 ¹^,² , 许谨帆 ¹^,² , 李楠 ¹^,² , 王志斌 ³ , 杜菁 ⁴ , 吴志生 ¹^,²^,^*

药学学报 | 专题报道: 中药制造质量控制与智能制造 2023,58(10): 2853-2861

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药学学报 |专题报道: 中药制造质量控制与智能制造 2023 , 58 (10) : 2853 -2861

基于MIF-HEMT生物传感技术的同仁牛黄清心丸关键质量属性测量研究

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马朝富¹^,², 王子健³, 马丽娟¹^,², 胡小艳¹^,², 魏宇楠¹^,², 张晓梦¹^,², 郭新雨¹^,², 郇星月¹^,², 赵静¹^,², 许谨帆¹^,², 李楠¹^,², 王志斌³, 杜菁⁴, 吴志生¹^,²^,^*

作者信息

1.北京中医药大学中药学院, 北京 102488

2.中药制药与新药开发教育部工程研究中心, 北京 102488

3.北京同仁堂科技发展股份有限公司, 北京 100079

4.北京同仁堂股份有限公司, 北京 100031

通讯作者:

*吴志生, Tel: 86-10-53912101, E-mail: wzs@bucm.edu.cn

The measurement of critical quality attributes of Tongren niuhuang qingxin pills based on MIF-HEMT biosensor technology

Chao-fu MA¹^,², Zi-jian WANG³, Li-juan MA¹^,², Xiao-yan HU¹^,², Yu-nan WEI¹^,², Xiao-meng ZHANG¹^,², Xin-yu GUO¹^,², Xing-yue HUAN¹^,², Jing ZHAO¹^,², Jin-fan XU¹^,², Nan LI¹^,², Zhi-bin WANG³, Jing DU⁴, Zhi-sheng WU¹^,²^,^*

Affiliations

1. School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 102488, China

2. Engineering Research Center of Chinese Medicine Production and New Drug Development, Beijing 102488, China

3. Beijing Tongrentang Technology Development Co., Ltd., Beijing 100079, China

4. Beijing Tongrentang Co., Ltd., Beijing 100031, China

出版时间: 2023-10-12 doi: 10.16438/j.0513-4870.2022-1352

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摘要

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中药复方临床疗效关联的关键质量属性(critical quality attributes, CQAs) 测量是中药制造亟待攻克的关键难题之一。为此, 以同仁牛黄清心丸为载体, 提出生物传感结合超高效液相色谱/串联质谱(ultra-performance liquid chromatography/tandem mass spectrometry, UPLC-MS/MS) 的集成策略。首先, 以干预缺血性脑卒中的潜在关键靶点巨噬细胞移动抑制因子(macrophage migration inhibitory factor, MIF) 为视角, 采用高电子迁移率晶体管(high electron mobility transistor, HEMT), 创建了MIF-HEMT生物传感, 开展了同仁牛黄清心丸不同柱分离组分干预脑卒中作用的测量研究, 发现其3段组分均具有较强的脑卒中治疗作用, 尤其A段, 作用强度达8.722×10^-10 g·mL^-1。进一步采用MIF-HEMT生物传感集成UPLC-MS/MS, 实现了同仁牛黄清心丸不同组分干预脑卒中的CQAs分离和鉴定研究。测量得到了芍药内酯苷(albiflorin)、芍药苷(paeoniflorin)、升麻素苷(prim-O-glucosylcimifugin)等19个干预MIF发挥脑卒中治疗作用的潜在CQAs。这为同仁牛黄清心丸干预MIF产生脑卒中治疗作用的CQAs研究提供了关键测量技术与方法借鉴, 为优质名方高效利用和质量控制测量提供了重要参考。

关键词

巨噬细胞移动抑制因子 / 高电子迁移率晶体管 / 生物传感 / 同仁牛黄清心丸 / 关键质量属性

Abstract

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There is still a serious challenge of the measurement of critical quality attributes (CQAs) related to clinical efficacy for Chinese materia medica manufacturing. To overcome this challenge, an integrated strategy of biosensor and ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) was proposed using Tongren niuhuang qingxin pills as a trial. Firstly, an original biosensor was created using a semiconductor chip material high electron mobility transistor (HEMT) as the transducer and the macrophage migration inhibitory factor (MIF) as the identification element. By this MIF-HEMT biosensor, the efficacy on stoke of different components from Tongren niuhuang qingxin pills was measured. It was clear that all three components of Tongren niuhuang qingxin pills had strong therapeutic effects on stroke, especially the section A, the K_D of which reached to 8.722×10^-10 g·mL^-1. Furthermore, MIF-HEMT biosensor integrated UPLC-MS/MS was introduced to identify the efficacy CQAs of different components of Tongren niuhuang qingxin pills. As a result, 19 potential CQAs, such as albiforin, paeoniflorin, and prim-O-glucosylcimifugin, were measured as the efficacy CQAs of Tongren niuhuang qingxin pills on stroke treatment by MIF. These results provided vital measurement techniques and methodological guidance for the CQAs study of Tongren niuhuang qingxin pills intervention in MIF-induced stroke treatment. This also provided an essential guideline for the efficient utilization and quality control measurement of high-quality classical recipes.

Key words

macrophage migration inhibitory factor / high electron mobility transistor / biosensor / Tongren niuhuang qingxin pill / critical quality attribute

引用本文

马朝富, 王子健, 马丽娟, 胡小艳, 魏宇楠, 张晓梦, 郭新雨, 郇星月, 赵静, 许谨帆, 李楠, 王志斌, 杜菁, 吴志生. 基于MIF-HEMT生物传感技术的同仁牛黄清心丸关键质量属性测量研究. 药学学报, 2023 , 58 (10) : 2853 -2861 . DOI: 10.16438/j.0513-4870.2022-1352

Chao-fu MA, Zi-jian WANG, Li-juan MA, Xiao-yan HU, Yu-nan WEI, Xiao-meng ZHANG, Xin-yu GUO, Xing-yue HUAN, Jing ZHAO, Jin-fan XU, Nan LI, Zhi-bin WANG, Jing DU, Zhi-sheng WU. The measurement of critical quality attributes of Tongren niuhuang qingxin pills based on MIF-HEMT biosensor technology[J]. Acta Pharmaceutica Sinica, 2023 , 58 (10) : 2853 -2861 . DOI: 10.16438/j.0513-4870.2022-1352

正文

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《人用药物注册技术要求国际协调委员会药物研发指导原则》中明确指出, 药品关键质量属性(critical quality attributes, CQAs) 是指在适当的限度、范围或分布之内, 确保所需产品质量的物理、化学、生物或微生物性质或特征。其中, 药效关联的CQAs是保障中药制造质量“安全、有效、稳定可控”的重要基础^{[1, 2]}。然而, 目前中药行业普遍存在质量评价和过程控制指标与临床疗效缺乏关联的行业痛点难题^[3]。

中药制造测量学是研究中药制造相关的测量策略、原理、方法与技术, 研制各类仪器、装置及相关软件, 以精准获取中药物质组成、分布、结构与性质的时空变化规律, 是中药制造质量“安全、有效、稳定可控”的重要保障^[4]。运用中药制造测量学通过测量获得制造信息是解决中药制造过程控制指标与临床疗效缺乏关联的关键^[5]。前期研究发现^[6], 半导体材料制备的砷化铝镓/砷化镓(AlGaAs/GaAs) 高电子迁移率晶体管(high electron mobility transistor, HEMT) 生物传感器, 因其灵敏度高, 检测速度快, 为生物传感筛选药效关联的CQAs的分离、鉴定提供了重要技术支撑。

同仁牛黄清心丸具有益气养血、活血化瘀等功效, 尤其适用于急性脑血管病及脑卒中后遗症等。研究发现^{[7, 8]}, 在生理状态下, 巨噬细胞移动抑制因子(macrophage migration inhibitory factor, MIF) 在人脑的表达通常处于不可检测水平, 并且缺血性脑卒中会引起MIF在大脑中多区域的高表达现象。此外, MIF对小鼠心脏缺血再灌注损伤具有重要的保护作用, 其表达水平与缺血性脑损伤严重程度及预后关系密切。不仅如此, MIF还可通过触发丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK) 和磷脂酰肌醇3-激酶(phosphatidylin-ositol-3-kinase, PI3K) 信号通路, 参与脑卒中治疗的多个环节^{[9, 10]}。因此, MIF是同仁牛黄清心丸干预脑卒中CQAs辨识研究的一个关键视角, 具有重要的科学价值和产业价值。

因此, 本研究以同仁牛黄清心丸为研究载体, 创建MIF-HEMT生物传感器, 并集成超高效液相色谱/串联质谱(ultra-performance liquid chromatography/tandem mass spectrometry, UPLC-MS/MS) 技术, 实现同仁牛黄清心丸与MIF作用的CQAs测量, 解决其质量控制指标与疗效关联的问题, 从而建立一套中药复方CQAs辨识的集成方法, 为疗效关联质量控制指标的测量提供关键技术支撑。

材料与方法

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试剂与药品 MIF蛋白(批号10246-H08H, 规格100 μg)、磷酸盐缓冲溶液(phosphate buffered saline, PBS, 批号20220805, 规格500 mL, pH 7.4)、疏基丙酸(3-mercaptopropanoic acid, 3-MPA, 批号BCBX0278, CAS号: 107-96-0, 质量分数≥ 99.0%)、碳酰二亚胺盐酸盐[1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, EDC, 批号5006M12X, CAS号: 25952-53-8, 质量分数≥ 98.0%]、N-羟基丁二酰亚胺(N-hydroxysuccinimide, NHS, 批号WXBD1766V, CAS号: 6066-82-6, 质量分数≥ 97.0%) (北京中农博信科技有限公司); 苦杏仁苷(批号1649, CAS号: 29883-15-6, 质量分数≥ 97.7%)、5-O-甲基维斯阿米醇苷(批号2075, CAS号: 84272-85-5, 质量分数≥ 99.9%) 标准品(上海诗丹德技术有限公司); 甲酸(批号151871, CAS号: 64-18-6, 质量分数≥ 95.0%)、甲醇(批号R141931, CAS号: 67-56-1, 质量分数≥ 99.9%)、乙腈(批号WXBB5870V, CAS号: 75-05-8, 质量分数≥ 99.9%) (赛默飞世尔科技有限公司); 同仁牛黄清心丸(批号19010573, 北京同仁堂股份有限公司)。

仪器 Agilent 1290 UPLC超高效液相色谱仪、Agilent ZORBAX RRHD Eclipse XDB-C18色谱柱(2.1 mm × 100 mm, 1.8 μm) (美国安捷伦科技有限公司); AB Sciex Triple TOF^® 4600高分辨质谱(加拿大SCIEX公司); 固相萃取仪(上海本昂科学仪器有限公司); Milli-Q Synthesis超纯水纯化系统(美国Millipore公司); SepaBean machine T快速液相制备色谱系统(常州三泰科技有限公司); TG16MW台式高速离心机(湖南赫西仪器有限公司); CV200真空浓缩仪(北京吉艾姆科技有限公司)。

同仁牛黄清心丸样品的前处理 取同仁牛黄清心丸30 g, 剪碎, 加入300 mL纯水及数粒沸石, 振摇混合后, 于室温下静置1 h; 回流提取1 h, 离心后上清液作为纯水提取段; 采用50%甲醇溶液对药渣部分进行提取, 取上清液与纯水提取段合并, 作为同仁牛黄清心丸提取液。进一步采用制备液相技术, 对同仁牛黄清心丸提取液部分进行精细化拆分, 划分成不同区段。首先, 以水和乙醇为洗脱溶剂, 进行5%~95%大梯度洗脱考察, 紫外吸收结果表明大多物质在高极性区段出峰。进一步优化分段洗脱方法, 将洗脱液醇浓度设定在0~50%区间。根据紫外吸收结果, 将分离得到的3个柱分离区段作为后续实验的样品, 记为A段、B段和C段。

MIF-AlGaAs/GaAs HEMT生物传感器的创建研究 使用500 nm GaAs缓冲层作为基础。随后, 将15 nm的In_0.3Ga_0.7As沟道层、4 nm的Al_0.3Ga_0.7As间隔层、Si-δ掺杂层、25 nm的Al_0.3Ga_0.7As阻挡层和30 nm的GaAs帽层逐层放置。最后, 在扩展的门控栅极部分镀上一层金膜用于识别元件的修饰。

采用CHI-660E电化学工作站, 测定GaAs/GaAs HEMT器件各通道的源极-漏极间的电流强度(I-V curve of source and drain level, I_DS-V_DS) 曲线, 判断比较各通道性能。以AlGaAs/GaAs HEMT半导体材料为生物芯片载体, 将一个透明的超薄石英玻璃管(高10 mm, 内径4 mm) 黏连在芯片的表面作为样品池; 向样品池中加入3-MPA水溶液, 室温下浸泡反应24 h, 在AlGaAs/GaAs HEMT器件表面生成Au-S键, 形成自组装单层; 以去离子水洗去AlGaAs/GaAs HEMT器件表面的3-MPA, 加入EDC和NHS的混合水溶液, 生成稳定的胺类活化产物用于活化羧基。进一步采用10 mmol·L^-1 PBS清洗AlGaAs/GaAs HEMT器件, 加入MIF蛋白溶液, 置4 ℃反应2 h, 即得MIF-AlGaAs/GaAs HEMT生物传感器。当检测到待测目标物质时, 待测目标物质与蛋白发生相互作用, 使得栅压发挥调控异质结内的二维电子气(two-dimensional electron gas, 2DEG) 度的改变, 导致AlGaAs/GaAs HEMT器件源极和漏极之间的电流发生变化。

在此基础上, 采用X射线光电子能谱(X-ray photoelectron spectroscopy, XPS), 参数值设置为: X射线源单色为Al Kalph 150 W, 光斑尺寸为500 μm, 测量能源为200 eV, 高分辨率扫描能源为30 eV, 镜头模式为LargeAreaXL, 分析仪模式为CAE, 测定AlGaAs/GaAs HEMT器件裸片、3-MPA修饰前后、蛋白修饰前后元素的变化。

基于生物传感技术的同仁牛黄清心丸与MIF相互作用研究 取同仁牛黄清心丸分段所得不同组分, PBS复溶至1 mg·mL^-1, 分别作为不同组分的母液。按10倍梯度稀释母液, 制备11个浓度梯度的同仁牛黄清心丸样品溶液, 备用。进一步以0.1 mol·L^-1 PBS溶液为空白溶液, 按浓度由低到高(0.1 pmol·L^-1~1.0 mmol·L^-1) 依次向MIF-AlGaAs/GaAs HEMT器件上加入同仁牛黄清心丸A段溶液, 采用电化学装置记录I_DS-V_DS。以被测溶液浓度的对数(lg [A_g]) 为横坐标, 以电流变化相对值((I - I₀)/I₀) 为纵坐标, 进行线性拟合, 判断生物传感器的线性范围。根据此线性浓度范围, 以被测溶液的浓度([A_g]) 为横坐标, 以浓度([A_g])/电流变化(I - I₀, ΔI) 为纵坐标, 进行线性拟合, 根据公式(1)~(3) 计算同仁牛黄清心丸不同组分分段样品与MIF相互作用的解离常数K_D。

(1)$ \left[{A}_{\mathrm{b}}\right]+\left[{A}_{\mathrm{g}}\right]\leftrightarrow [{A}_{\mathrm{b}}-{A}_{\mathrm{g}}] $

(2)$ K={K}_{\mathrm{A}}=1/{K}_{\mathrm{D}}=[{A}_{\mathrm{b}}-{A}_{\mathrm{g}}]/\left[{A}_{\mathrm{b}}\right]\cdot \left[{A}_{\mathrm{g}}\right] $

(3)$ \left[{A}_{\mathrm{g}}\right]/\mathrm{\Delta }I=\left[{A}_{\mathrm{g}}\right]/\mathrm{\Delta }{I}_{\mathrm{m}\mathrm{a}\mathrm{x}}+{K}_{\mathrm{D}}/\mathrm{\Delta }{I}_{\mathrm{m}\mathrm{a}\mathrm{x}} $

其中, [A_b]为MIF浓度, [A_g]为被测溶液浓度[C], K及K_A为结合常数, K_D为解离常数, ΔI为电流变化值, ΔI_max为电流最大变化值。

进一步采用PBS作为非特异性洗脱剂, 将结合于MIF-HEMT生物传感器件之上的样品进行洗脱, 洗脱3次; 再采用特异性洗脱剂5-O-甲基维斯阿米醇苷进行洗脱, 洗脱3次, 每次反应5 min, 合并洗脱液; 最后, 采用特异性洗脱剂苦杏仁苷进行洗脱3次, 每次反应5 min, 合并洗脱液。采用固相萃取技术, 洗去2种特异性洗脱液中的磷酸盐, 并对其进行富集, 作为样品溶液, 备用。

基于UPLC-MS/MS技术的同仁牛黄清心丸组分干预MIF的CQAs解析 取同仁牛黄清心丸提取液和3个不同组分分段样品溶液及“MIF-AlGaAs/GaAs HEMT生物传感器的创建研究”项中干预MIF后的洗脱液, 12 000 r·min^-1离心30 min, 取上清液备用。采用UPLC-MS/MS, 以水和乙腈为流动相, 对以上供试品进行分离分析。其中, 液相色谱条件: 色谱柱为Agilent ZORBAX RRHD Eclipse XDB-C18 (2.1 mm × 100 mm, 1.8 μm), 柱温为30 ℃, 进样量为1 μL, 检测波长为280 nm。流动相比例和流速分别为: A相0.1%甲酸水溶液, B相0.1%甲酸乙腈; 梯度洗脱(0~4 min, 5% B; 4~10 min, 15% B; 10~25 min, 30% B; 25~40 min, 50% B; 40~44 min, 95% B)。

质谱条件: 正负离子模式检测条件; 离子源HESI; 毛细管电压3 500 V; 毛细管温度350 ℃; 鞘气45 arb; 辅助气10 arb; 源内温度350 ℃; 质谱扫描范围m/z 80~1 200; 分辨率70 000; 碰撞能量为15、25、35 eV。进一步通过对比同仁牛黄清心丸全方的成分信息, 鉴定A、B、C段样品及2种洗脱液中所含的化学成分, 确定同仁牛黄清心丸通过干预MIF蛋白发挥脑卒中治疗作用的CQAs。

结果与讨论

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1 MIF-HEMT生物传感的构建及性能考察

采用XPS测定AlGaAs/GaAs HEMT上修饰MIF抗体前后元素的变化, 图 1展示了HEMT裸片、修饰3-MPA后及修饰MIF后的XPS谱图, 其中图 1A~D分别为C1s、N1s、O1s和S1s的高分辨XPS谱图。N1s峰的峰高及半峰宽在修饰3-MPA后基本无变化, 而在蛋白固定后显著增加, 从其高分辨谱中分解出两个组分, 峰中心位于399.5和402.1 eV, 分别对应于蛋白的中性氨基(NH₂) 和质子化的氨基(NH₃⁺)。未经处理的HEMT芯片表面C1s高分辨谱可归属为两个明显的峰, 分别在285.0 eV (对应于C-H) 和288.8 eV (对应于C-O)。碳元素的信号来自于基底的有机物污染。自主装单层形成后, 在286.4 eV处出现了一个新的羰基(O-C=O) 峰; 羰基由NHS/EDC激活, 并与蛋白的氨基生成肽键(-CONH-), C1s位于288.8 eV处的高分辨峰被展宽。从图 1D中可看出, 3-MPA修饰后, S元素含量稍有增加, 而蛋白修饰之后, S元素的含量显著增加, 表明蛋白中含有巯基。以上元素测定结果表明蛋白成功修饰于HEMT器件之上。

2 基于生物传感技术的同仁牛黄清心丸与MIF相互作用强度研究

图 2A~C展示的是同仁牛黄清心丸A、B、C段不同浓度样品与MIF-AlGaAs/GaAs HEMT生物传感器相互作用的I_DS-V_DS信号变化情况。从图中可看出, 加入PBS溶液后, 以MIF蛋白修饰裸片为参照物, 期间的电流变化不大, 表明溶剂对测试结果的影响较小。以PBS溶液为空白对照, 可见10^-10 mg·mL^-1即可产生明显的响应。随着同仁牛黄清心丸不同组分样品溶液浓度的增加, I_DS呈现规律性递减, 且始终保持良好的半导体特性。以同仁牛黄清心丸3段组分样品溶液浓度的对数(lg [A_g]) 为横坐标, 以电流变化相对值((I - I₀)/I₀) 为纵坐标, 进行线性拟合, 结果如图 2D~F。以A段样品为例, 当A段样品浓度范围为10^-9~10^-6 mg·mL^-1时, 线性关系良好, 方程为y = -0.011 1x - 0.142 7, R² = 0.967 0。在此基础上, 以各组分样品溶液浓度([A_g]) 为横坐标, 以浓度与电流变化相对值的比值([A_g]/(I - I₀)) 为纵坐标, 进行线性拟合, 结果如图 2G~I所示。根据公式(1)~(3) 计算其解离常数, 得同仁牛黄清心丸A段样品溶液与MIF相互作用的解离常数K_D = 8.722×10^-10 g·mL^-1。同理, 同仁牛黄清心丸B段样品溶液与MIF相互作用的解离常数K_D = 1.375×10^-7 g·mL^-1, C段样品与MIF相互作用的解离常数K_D = 2.693×10^-8 g·mL^-1。以上结果可看出, A、B和C段样品均能与MIF产生较强的结合, 其中A段与MIF的相互作用最强, 其对脑卒中可能具有更强的治疗作用。

3 基于UPLC-MS/MS的同仁牛黄清心丸干预MIF的CQAs解析

3.1 同仁牛黄清心丸全方质谱分析

在正、负离子扫描模式下, 采用UPLC-MS/MS对同仁牛黄清心丸总提取物进行质谱分析。通过对同仁牛黄清心丸全方提取液UPLC-MS/MS基峰负离子流图和正离子流图、280 nm处紫外光谱进行分析, 相关图谱见图 3, 通过质谱数据解析共鉴定出同仁牛黄清心丸全方55个化合物, 鉴定结果见表 1。

3.2 同仁牛黄清心丸分段质谱分析

在正、负离子扫描模式下, 采用UPLC-MS/MS对同仁牛黄清心丸A、B、C分段样品进行质谱分析, 通过对3段样品UPLC-MS/MS基峰正、负离子流图进行分析, 与全方质谱分析结果表 1进行对比, A、B、C段样品共鉴定出42个化学成分, 其中A、B、C段样品分别包含22、21、20个化合物, 鉴定结果见表 2。由结果可知, A、B、C段样品中存在12个相同的化合物, 其中包括6-姜烯酚(6-shogaol)、芍药内酯苷(albiflorin)、芍药苷(paeoniflorin)、升麻素苷(prim-O-glucosylcimifugin)、甘草苷(liquiritin)、芹糖甘草苷(liquiritin apioside)、升麻素(cimifugin)、5-O-甲基维斯阿米醇苷(4'-O-β-glucopyranosyl-5-O-methylvisamminol)、黄芩苷(baicalin)、千层纸素A-7-O-β-D-葡萄糖醛酸苷(oroxyloside)、汉黄芩苷(wogonin 7-O-glucuronide) 和甘草酸(glycyrrhizic acid), 其他成分则分布于一段或两段样品中。

结合表 2中的潜在CQAs信息可看出, A、B两段辨识的成分主要来源于川芎、当归及白芍。三者在同仁牛黄清心丸干预脑卒中疾病过程中共同发挥疏通血滞、活血化瘀之功效。C段中主要包括皂苷类、胆汁酸类及黄酮类成分, 其中主要包括以甘草素、人参皂苷Rg1、人参皂苷Rg3等为代表性潜在益气补血功效潜在CQAs。C段辨识的成分主要来源于人参和甘草, 人参可大补元气、复脉固脱、生津养血; 甘草可补脾益气、止咳祛痰、调和药性。两者在同仁牛黄清心丸干预脑卒中疾病过程中共同发挥补虚扶弱、益气补血之功效。综合分析推测A和B段是发挥活血化瘀功效的重要组分, C段为发挥益气补血功效的主要组分。

3.3 同仁牛黄清心丸洗脱液质谱分析

在正、负离子扫描模式下, 采用UPLC-MS/MS对5-O-甲基维斯阿米醇苷洗脱液、苦杏仁苷洗脱液进行质谱分析, 通过对两种洗脱液UPLC-MS/MS基峰正负离子流图进行分析。与全方质谱分析结果(表 1) 对比, 共鉴定出19个化学成分, 鉴定结果见表 3。与A、B、C分段样品质谱分析结果(表 2) 进行对比, 其中升麻素苷(prim-O-glucosylcimifugin)、升麻素(cimifugin)、5-O-甲基维斯阿米醇苷(4'-O-β-glucopyranosyl-5-O-methylvisamminol)、黄芩苷(baicalin)、6-姜烯酚(6-shogaol)、甘草酸(gly-cyrrhizic acid) 6个化学成分在3段样品的洗脱液中均被检测, 芒柄花素(formononetin)、甘草酸异构体(gly-cyrrhizic acid isomer) 来自于A、C段样品洗脱液, 绿原酸(chlorogenic acid)、芍药内酯苷(albiflorin)、芍药苷(paeoniflorin)、野黄芩苷(scutellarin)、甘草次酸(gly-cyrrhetinic acid) 则来源于A段样品洗脱液, 甘草苷(liquiritin)、芹糖甘草苷(liquiritin apioside)、异甘草苷(isoliquiritin)、芒柄花苷(ononin)、芹糖异甘草苷(isoliquiritin apioside)、亥茅酚苷(sec-O-glucosylhamaudol) 存在于C段样品洗脱液。

此19个潜在CQAs主要包括4大类成分: 单萜类、黄酮类、三萜皂苷类和酚酸类。其中以芍药内酯苷、芍药苷为代表的单萜类成分及以升麻素苷、5-O-甲基维斯阿米醇苷为代表的黄酮类成分主要来源于活血化瘀的中药芍药和防风等, 而以甘草苷、芒柄花苷、芒柄花素等为代表的三萜皂苷类和黄酮类成分主要来源于益气养血类中药甘草和人参等。这与同仁牛黄清心丸3段样品解析所得潜在CQAs的规律一致, 即A段和B段样品筛选所得的CQAs主要发挥活血化瘀的功效, C段样品筛选所得的CQAs主要发挥益气补血的功效。其中, A段和C段样品筛所得的CQAs包含了B段样品, 且前部分的研究也表明A段样品与MIF相互作用最强, C段次之, B段最弱, 这表明A段和C段可能是同仁牛黄清心丸通过干预MIF发挥脑卒中治疗作用的关键组分, 筛选所得19种潜在CQAs是同仁牛黄清心丸干预MIF发挥脑卒中治疗作用的疗效关联的潜在CQAs。这也进一步证明了通过MIF-HEMT生物传感筛选所得的潜在CQAs确实与临床疗效关联, 这为以疗效为指导的CQAs辨识提供了关键的测量技术支撑。

结论

收起

本研究以自主构建的MIF-AlGaAs/GaAs HEMT生物传感器及超高效液相色谱-高分辨质谱联用为技术支持, 开展了同仁牛黄清心丸干预脑卒中的临床疗效关联CQAs的测量研究。发现了同仁牛黄清心丸通过干预MIF发挥脑卒中治疗作用的潜在CQAs共有19个, 分别是芍药内酯苷、芍药苷、升麻素苷、升麻素、甘草苷、异甘草苷、芹糖苷草苷、芹糖异甘草苷、野黄芩苷、黄芩苷、5-O-甲基维斯阿米醇苷、芒柄花苷、芒柄花素、亥茅酚苷、甘草酸、异甘草酸、甘草次酸、绿原酸、6-姜烯酚。研究形成了一套基于生物传感集成液质联用的同仁牛黄清心丸干预MIF发挥脑卒中治疗作用的潜在CQAs测量技术, 为优质名方高效利用和质量控制测量提供重要参考。

作者贡献: 马朝富、王子健、马丽娟负责研究思路设计、分析数据和文章撰写; 胡小艳、魏宇楠、张晓梦负责实验实施; 郭新雨、郇星月、赵静、许谨帆负责论文修改; 王志斌、杜菁负责样品提供; 李楠、吴志生负责思路指导和对文章的知识性内容进行审阅并提供研究经费。

利益冲突: 所有作者均声明没有利益冲突。

基金

收起

国家自然科学基金优秀青年科学基金项目(82022073)
国家重点研发计划项目(2022YFC3501902)
国家自然科学基金项目(82274110)
中央高校基本科研业务费北京中医药大学揭榜挂帅项目(2022-JYB-JBZR-018)
中央高校基本科研业务费北京中医药大学揭榜挂帅项目(2022-JYB-JBZR-019)

参考文献

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文献

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参考文献引证文献

排序方式：

[1]

Lei

, Ma

, Wei

, et al . Critical quality attribute assessment of big brand traditional Chinese medicine: modular identification of Tongren Niuhuang Qingxin Pills based on efficacy with chemical properties[J]. China J Chin Mater Med (中国中药杂志), 2021, 46: 1606-1615.

[2]

Zhang

, Lin

, Zeng

, et al . Critical quality attribute assessment of big brand traditional Chinese medicine: visualization of blending process for rare medicines in Tongren Niuhuang Qingxin Pills based on spatial distribution uniformity[J]. China J Chin Mater Med (中国中药杂志), 2021, 46: 1585-1591.

[3]

, Xu

, Wang

, et al . Development strategy of pharmaceutical industry of Chinese materia medica toward process system engineering[J]. J Tradit Chin Med (中华中医药杂志), 2016, 31: 3417-3419.

[4]

, Qiao

. Manufacturing Measurement of Chinese Materia Medica (中药制造测量学)[M]. Beijing: Science Press, 2021: 1-10.

[5]

, Zhang

, Lin

, et al . Data-driven engineering framework with AI algorithm of Ginkgo Folium tablets manufacturing[J]. Acta Pharm Sin B, 2023, 13: 2188-2201.

https://doi.org/10.1016/j.apsb.2022.08.011

[6]

, Zheng

, Wang

, et al . Development of MIF/IL-1 beta biosensors for discovery of critical quality attributes and potential allergic rhinitis targets from clinical real-world data by intelligent algorithm coupled with in vitro and vivo mechanism validation[J]. Biosens Bioelectron, 2021, 194: 113608.

https://doi.org/10.1016/j.bios.2021.113608

[7]

, Xiao

, He

, et al . Comparative study on the effects of total ginkgo flavonolglycosides and total ginkgolides on neurofunctional recovery in mice with subacute stroke[J]. Acta Pharm Sin (药学学报), 2021, 56: 231-243.

[8]

Pei

, Shen

, Yan

, et al . The two-way regulation molecular network mechanism of Taohechengqi decoction in prevention and treatment of stroke based on vascular endothelial growth factor signaling pathway/tumor necrosis factor signaling pathway[J]. Acta Pharm Sin (药学学报), 2020, 55: 898-906.

[9]

Osipyan

, Chen

, Dekker

. Epigenetic regulation in macro-phage migration inhibitory factor (MIF)-mediated signaling in cancer and inflammation[J]. Drug Discov Today, 2021, 26: 1728-1734.

https://doi.org/10.1016/j.drudis.2021.03.012

[10]

, Ma

, Wang

, et al . Original end-to-end smart diagnosis framework of systematic critical quality attributes benchmarking FDA standard of phytomedicine by biosensor and multi-information fusion coupled with AI algorithm[J]. Green Chem, 2023, 25: 384-398.

https://doi.org/10.1039/D2GC03835H

2023年第58卷第10期

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文章信息

doi: 10.16438/j.0513-4870.2022-1352

接收时间：2022-12-09
首发时间：2025-11-21
出版时间：2023-10-12

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文章信息

作者

出版历史

收稿日期：2022-12-09
修回日期：2023-01-10

基金

国家自然科学基金优秀青年科学基金项目(82022073)

国家重点研发计划项目(2022YFC3501902)

国家自然科学基金项目(82274110)

中央高校基本科研业务费北京中医药大学揭榜挂帅项目(2022-JYB-JBZR-018)

中央高校基本科研业务费北京中医药大学揭榜挂帅项目(2022-JYB-JBZR-019)

作者信息

1.北京中医药大学中药学院, 北京 102488

2.中药制药与新药开发教育部工程研究中心, 北京 102488

3.北京同仁堂科技发展股份有限公司, 北京 100079

4.北京同仁堂股份有限公司, 北京 100031

通讯作者:

*吴志生, Tel: 86-10-53912101, E-mail: wzs@bucm.edu.cn

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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TxT

No.	Retention time/min	Adduction	Actual value of m/z	Theoretical value of m/z	Relative error (×10^-6)	Molecular formula	Molecular weight	MS/MS
1	0.86	[M-H]^-	133.014 9	133.014 2	5.3	C₄H₆O₅	134.02	115.005 2; 71.014 2
2	1.85	[M-H]^-	169.014 8	169.014 2	3.5	C₇H₆O₅	170.02	125.024 9; 81.034 2
3	2.00	[M+FA-H]^-	405.140 8	405.140 2	1.5	C₁₆H₂₄O₉	360.14	359.136 5; 197.085 8; 179.074 1
4	7.32	[M-H]^-	165.056 7	165.055 7	6.1	C₉H₁₀O₃	166.06	165.054 6; 93.036 6
5	9.08	[M-H]^-	353.090 9	353.087 8	8.8	C₁₆H₁₈O₉	354.10	191.056 6
6	11.81	[M+FA-H]^-	525.165 7	525.161 3	8.4	C₂₃H₂₈O₁₁	480.16	525.161 2; 479.157 2; 283.000 0; 121.029 9
7	12.20	[M-H]^-	593.153 7	593.151 2	4.2	C₂₇H₃₀O₁₅	594.16	593.152 8; 503.115 5; 383.075 0; 353.063 7
8	12.79	[M+FA-H]^-	525.165 5	525.161 3	8.0	C₂₃H₂₈O₁₁	480.16	428.159 1; 479.159 8; 449.148 5; 327.110 4; 121.029 9
9	13.87	[M-H]^-	563.142 3	563.140 6	3.0	C₂₆H₂₈O₁₄	564.15	563.139 9; 545.136 9; 443.101 5; 383.073 5
10	13.93	[M+H]⁺	469.168 6	469.170 4	-3.8	C₂₂H₂₈O₁₁	468.16	469.169 6; 397.109 7; 307.116 3; 235.057 4
11	14.40	[M-H]^-	415.104 9	415.103 4	3.6	C₂₁H₂₀O₉	416.11	415.107 0; 253.050 5; 224.055 7
12	15.26	[M-H]^-	417.121 5	417.119 1	5.8	C₂₁H₂₂O₉	418.13	417.117 1; 255.066 7; 135.009 5; 119.050 5
13	15.47	[M-H]^-	549.166 8	549.161 3	10.0	C₂₆H₃₀O₁₃	550.17	549.162 0; 255.066 4; 135.008 4
14	16.27	[M-H]^-	461.075 1	461.072 5	5.6	C₂₁H₁₈O₁₂	462.08	285.041 5; 267.027 7; 175.024 2
15	16.50	[M-H]^-	623.167 4	623.161 7	9.1	C₂₈H₃₂O₁₆	624.17	623.158 0; 314.042 1; 243.031 4
16	16.66	[M+H]⁺	307.116 9	307.117 6	-2.3	C₁₆H₁₈O₆	306.11	307.119 7; 259.060 4; 235.062 7; 221.045 1
17	17.17	[M-H]^-	623.201 9	623.198 1	6.1	C₂₉H₃₆O₁₅	624.21	623.197 8; 461.166 4; 161.024 7
18	18.44	[M-H]^-	623.202 3	623.198 1	6.7	C₂₉H₃₆O₁₅	624.21	623.196 4; 461.162 4; 161.022 5
19	18.63	[M-H]^-	433.114 7	433.114 0	1.6	C₂₁H₂₂O₁₀	434.12	433.113 3; 271.061 5; 151.004 4
20	18.74	[M+FA-H]^-	497.168 5	497.166 4	4.2	C₂₂H₂₈O₁₀	452.17	497.173 2; 451.161 6; 271.097 6
21	20.52	[M+H]⁺	447.127 1	447.128 6	-3.4	C₂₂H₂₂O₁₀	446.12	285.075 5; 269.041 1
22	21.16	[M-H]^-	549.164 3	549.161 3	5.5	C₂₆H₃₀O₁₃	550.17	549.163 9; 255.066 3; 135.010 7
23	21.33	[M+FA-H]^-	475.128 6	475.124 6	8.4	C₂₂H₂₂O₉	430.13	475.133 6; 267.068 2; 252.042 8
24	21.68	[M-H]^-	417.121 0	417.119 1	4.5	C₂₁H₂₂O₉	418.13	417.117 8; 255.067 5; 135.008 8
25	22.02	[M-H]^-	445.081 3	445.077 6	8.3	C₂₁H₁₈O₁₁	446.08	269.046 0; 113.025 4
26	23.54	[M-H]^-	165.057 2	165.055 7	9.1	C₉H₁₀O₃	166.06	136.037 4; 93.033 9
27	24.18	[M-H]^-	445.080 2	445.077 6	5.8	C₂₁H₁₈O₁₁	446.08	269.045 1; 241.052 0; 197.063 1
28	25.58	[M+H]⁺	439.158 2	439.159 9	-3.9	C₂₁H₂₆O₁₀	438.15	277.108 9; 259.097 4; 205.048 1
29	25.76	[M-H]^-	459.097 3	459.093 3	8.7	C₂₂H₂₀O₁₁	460.10	459.092 7; 283.061 8; 268.037 6
30	26.82	[M-H]^-	459.095 6	459.093 3	5.0	C₂₂H₂₀O₁₁	460.10	459.094 8; 283.060 9; 268.038 1
31	27.49	[M-H]^-	271.061 4	271.061 2	0.7	C₁₅H₁₂O₅	272.07	271.063 0; 177.018 0; 151.006 1
32	30.52	[M-H]^-	269.046 6	269.045 5	4.1	C₁₅H₁₀O₅	270.05	269.043 9; 223.043 4
33	30.55	[M+H]⁺	271.060 2	271.060 1	0.4	C₁₅H₁₀O₅	270.05	271.061 4; 169.011 9; 123.006 5
34	30.78	[M+FA-H]^-	845.494 1	845.490 4	4.4	C₄₂H₇₂O₁₄	800.49	845.498 3; 799.490 0; 637.431 1; 475.375 6
35	32.49	[M+H]⁺	269.079 9	269.080 8	-3.3	C₁₆H₁₂O₄	268.07	269.080 2; 254.054 8; 237.055 2; 197.056 6
36	32.99	[M+FA-H]^-	829.499 6	829.495 5	4.9	C₄₂H₇₂O₁₃	784.50	829.502 4; 783.493 5; 637.424 8; 475.375 0
37	35.00	[M-H]^-	955.497 4	955.490 8	6.9	C₄₈H₇₆O₁₉	956.50	955.492 3; 793.436 6
38	36.07	[M-H]^-	283.061 6	283.061 2	1.4	C₁₆H₁₂O₅	284.07	283.060 8; 268.038 6; 163.003 7
39	36.6	[M+H]⁺	255.064 2	255.065 2	-3.9	C₁₅H₁₀O₄	254.06	255.063 7; 153.018 0
40	37.14	[M-H]^-	373.094 7	373.092 9	4.8	C₁₉H₁₈O₈	374.10	373.092 0; 358.066 0; 343.044 2; 328.020 4
41	37.35	[M-H]^-	821.399 1	821.396 5	3.2	C₄₂H₆₂O₁₆	822.40	821.391 7; 351.054 7
42	39.53	[M-H]^-	821.403 2	821.396 5	8.2	C₄₂H₆₂O₁₆	822.40	821.398 8; 351.055 9
43	39.98	[M+FA-H]^-	825.468 5	825.470 0	-1.8	C₄₂H₆₈O₁₃	780.47	825.475 5; 779.465 8; 617.403 6
44	40	[M+H]⁺	455.351	455.352	-2.2	C₃₀H₄₆O₃	454.34	455.353 1; 437.348 1; 267.209 5; 191.178 7
45	40.18	[M-H]^-	367.121 5	367.118 7	7.6	C₂₁H₂₀O₆	368.13	367.119 6; 309.039 4; 201.017 2
46	41.25	[M-H]^-	407.284 1	407.280 3	9.3	C₂₄H₄₀O₅	408.29	407.281 6; 343.265 4; 289.220 7
47	41.44	[M-H]^-	391.288 2	391.285 4	7.2	C₂₄H₄₀O₄	392.29	391.286 8
48	41.96	[M+FA-H]^-	825.471 0	825.464 2	8.2	C₄₂H₆₈O₁₃	780.47	825.469 6; 779.462 1; 617.402 9
49	43.46	[M+H]⁺	277.180 1	277.179 8	1.1	C₁₇H₂₄O₃	276.17	137.058 9; 122.035 7
50	43.47	[M+H]⁺	137.059 5	137.059 7	-1.5	C₈H₈O₂	136.05	137.057 8; 122.037 0; 94.040 7
51	43.52	[M+FA-H]^-	829.502 3	829.495 5	8.2	C₄₂H₇₂O₁₃	784.50	829.502 5; 783.493 1; 621.440 4; 459.384 0
52	44.13	[M-H]^-	391.287 1	391.285 4	4.3	C₂₄H₄₀O₄	392.29	391.286 5; 345.278 6
53	44.43	[M+FA-H]^-	723.385 3	723.380 8	6.2	C₃₃H₅₈O₁₄	678.38	723.384 1; 677.380 4; 415.143 9; 397.139 9; 279.232 7
54	44.67	[M-H]^-	471.352 9	471.348 0	10.4	C₃₀H₄₈O₄	472.36	471.350 9
55	45.19	[M-H]^-	469.335 5	469.332 3	6.8	C₃₀H₄₆O₄	470.34	469.335 9; 425.343 1

No.

Retention time/min

Adduction

Actual value of m/z

Theoretical value of m/z

Relative
error (×10^-6)

Molecular formula

Molecular weight

MS/MS

0.86

[M-H]^-

133.014 9

133.014 2

5.3

C₄H₆O₅

134.02

115.005 2; 71.014 2

1.85

[M-H]^-

169.014 8

169.014 2

3.5

C₇H₆O₅

170.02

125.024 9; 81.034 2

2.00

[M+FA-H]^-

405.140 8

405.140 2

1.5

C₁₆H₂₄O₉

360.14

359.136 5; 197.085 8; 179.074 1

7.32

[M-H]^-

165.056 7

165.055 7

6.1

C₉H₁₀O₃

166.06

165.054 6; 93.036 6

9.08

[M-H]^-

353.090 9

353.087 8

8.8

C₁₆H₁₈O₉

354.10

191.056 6

11.81

[M+FA-H]^-

525.165 7

525.161 3

8.4

C₂₃H₂₈O₁₁

480.16

525.161 2; 479.157 2; 283.000 0; 121.029 9

12.20

[M-H]^-

593.153 7

593.151 2

4.2

C₂₇H₃₀O₁₅

594.16

593.152 8; 503.115 5; 383.075 0; 353.063 7

12.79

[M+FA-H]^-

525.165 5

525.161 3

8.0

C₂₃H₂₈O₁₁

480.16

428.159 1; 479.159 8; 449.148 5; 327.110 4; 121.029 9

13.87

[M-H]^-

563.142 3

563.140 6

3.0

C₂₆H₂₈O₁₄

564.15

563.139 9; 545.136 9; 443.101 5; 383.073 5

13.93

[M+H]⁺

469.168 6

469.170 4

-3.8

C₂₂H₂₈O₁₁

468.16

469.169 6; 397.109 7; 307.116 3; 235.057 4

14.40

[M-H]^-

415.104 9

415.103 4

3.6

C₂₁H₂₀O₉

416.11

415.107 0; 253.050 5; 224.055 7

15.26

[M-H]^-

417.121 5

417.119 1

5.8

C₂₁H₂₂O₉

418.13

417.117 1; 255.066 7; 135.009 5; 119.050 5

15.47

[M-H]^-

549.166 8

549.161 3

10.0

C₂₆H₃₀O₁₃

550.17

549.162 0; 255.066 4; 135.008 4

16.27

[M-H]^-

461.075 1

461.072 5

5.6

C₂₁H₁₈O₁₂

462.08

285.041 5; 267.027 7; 175.024 2

16.50

[M-H]^-

623.167 4

623.161 7

9.1

C₂₈H₃₂O₁₆

624.17

623.158 0; 314.042 1; 243.031 4

16.66

[M+H]⁺

307.116 9

307.117 6

-2.3

C₁₆H₁₈O₆

306.11

307.119 7; 259.060 4; 235.062 7; 221.045 1

17.17

[M-H]^-

623.201 9

623.198 1

6.1

C₂₉H₃₆O₁₅

624.21

623.197 8; 461.166 4; 161.024 7

18.44

[M-H]^-

623.202 3

623.198 1

6.7

C₂₉H₃₆O₁₅

624.21

623.196 4; 461.162 4; 161.022 5

18.63

[M-H]^-

433.114 7

433.114 0

1.6

C₂₁H₂₂O₁₀

434.12

433.113 3; 271.061 5; 151.004 4

18.74

[M+FA-H]^-

497.168 5

497.166 4

4.2

C₂₂H₂₈O₁₀

452.17

497.173 2; 451.161 6; 271.097 6

20.52

[M+H]⁺

447.127 1

447.128 6

-3.4

C₂₂H₂₂O₁₀

446.12

285.075 5; 269.041 1

21.16

[M-H]^-

549.164 3

549.161 3

5.5

C₂₆H₃₀O₁₃

550.17

549.163 9; 255.066 3; 135.010 7

21.33

[M+FA-H]^-

475.128 6

475.124 6

8.4

C₂₂H₂₂O₉

430.13

475.133 6; 267.068 2; 252.042 8

21.68

[M-H]^-

417.121 0

417.119 1

4.5

C₂₁H₂₂O₉

418.13

417.117 8; 255.067 5; 135.008 8

22.02

[M-H]^-

445.081 3

445.077 6

8.3

C₂₁H₁₈O₁₁

446.08

269.046 0; 113.025 4

23.54

[M-H]^-

165.057 2

165.055 7

9.1

C₉H₁₀O₃

166.06

136.037 4; 93.033 9

24.18

[M-H]^-

445.080 2

445.077 6

5.8

C₂₁H₁₈O₁₁

446.08

269.045 1; 241.052 0; 197.063 1

25.58

[M+H]⁺

439.158 2

439.159 9

-3.9

C₂₁H₂₆O₁₀

438.15

277.108 9; 259.097 4; 205.048 1

25.76

[M-H]^-

459.097 3

459.093 3

8.7

C₂₂H₂₀O₁₁

460.10

459.092 7; 283.061 8; 268.037 6

26.82

[M-H]^-

459.095 6

459.093 3

5.0

C₂₂H₂₀O₁₁

460.10

459.094 8; 283.060 9; 268.038 1

27.49

[M-H]^-

271.061 4

271.061 2

0.7

C₁₅H₁₂O₅

272.07

271.063 0; 177.018 0; 151.006 1

30.52

[M-H]^-

269.046 6

269.045 5

4.1

C₁₅H₁₀O₅

270.05

269.043 9; 223.043 4

30.55

[M+H]⁺

271.060 2

271.060 1

0.4

C₁₅H₁₀O₅

270.05

271.061 4; 169.011 9; 123.006 5

30.78

[M+FA-H]^-

845.494 1

845.490 4

4.4

C₄₂H₇₂O₁₄

800.49

845.498 3; 799.490 0; 637.431 1; 475.375 6

32.49

[M+H]⁺

269.079 9

269.080 8

-3.3

C₁₆H₁₂O₄

268.07

269.080 2; 254.054 8; 237.055 2; 197.056 6

32.99

[M+FA-H]^-

829.499 6

829.495 5

4.9

C₄₂H₇₂O₁₃

784.50

829.502 4; 783.493 5; 637.424 8; 475.375 0

35.00

[M-H]^-

955.497 4

955.490 8

6.9

C₄₈H₇₆O₁₉

956.50

955.492 3; 793.436 6

36.07

[M-H]^-

283.061 6

283.061 2

1.4

C₁₆H₁₂O₅

284.07

283.060 8; 268.038 6; 163.003 7

36.6

[M+H]⁺

255.064 2

255.065 2

-3.9

C₁₅H₁₀O₄

254.06

255.063 7; 153.018 0

37.14

[M-H]^-

373.094 7

373.092 9

4.8

C₁₉H₁₈O₈

374.10

373.092 0; 358.066 0; 343.044 2; 328.020 4

37.35

[M-H]^-

821.399 1

821.396 5

3.2

C₄₂H₆₂O₁₆

822.40

821.391 7; 351.054 7

39.53

[M-H]^-

821.403 2

821.396 5

8.2

C₄₂H₆₂O₁₆

822.40

821.398 8; 351.055 9

39.98

[M+FA-H]^-

825.468 5

825.470 0

-1.8

C₄₂H₆₈O₁₃

780.47

825.475 5; 779.465 8; 617.403 6

[M+H]⁺

455.351

455.352

-2.2

C₃₀H₄₆O₃

454.34

455.353 1; 437.348 1; 267.209 5; 191.178 7

40.18

[M-H]^-

367.121 5

367.118 7

7.6

C₂₁H₂₀O₆

368.13

367.119 6; 309.039 4; 201.017 2

41.25

[M-H]^-

407.284 1

407.280 3

9.3

C₂₄H₄₀O₅

408.29

407.281 6; 343.265 4; 289.220 7

41.44

[M-H]^-

391.288 2

391.285 4

7.2

C₂₄H₄₀O₄

392.29

391.286 8

41.96

[M+FA-H]^-

825.471 0

825.464 2

8.2

C₄₂H₆₈O₁₃

780.47

825.469 6; 779.462 1; 617.402 9

43.46

[M+H]⁺

277.180 1

277.179 8

1.1

C₁₇H₂₄O₃

276.17

137.058 9; 122.035 7

43.47

[M+H]⁺

137.059 5

137.059 7

-1.5

C₈H₈O₂

136.05

137.057 8; 122.037 0; 94.040 7

43.52

[M+FA-H]^-

829.502 3

829.495 5

8.2

C₄₂H₇₂O₁₃

784.50

829.502 5; 783.493 1; 621.440 4; 459.384 0

44.13

[M-H]^-

391.287 1

391.285 4

4.3

C₂₄H₄₀O₄

392.29

391.286 5; 345.278 6

44.43

[M+FA-H]^-

723.385 3

723.380 8

6.2

C₃₃H₅₈O₁₄

678.38

723.384 1; 677.380 4; 415.143 9; 397.139 9; 279.232 7

44.67

[M-H]^-

471.352 9

471.348 0

10.4

C₃₀H₄₈O₄

472.36

471.350 9

45.19

[M-H]^-

469.335 5

469.332 3

6.8

C₃₀H₄₆O₄

470.34

469.335 9; 425.343 1

No.	Retention time/min	Name	The whole prescription	Section A	Section B	Section C
1	0.86	Malic acid	√	/	/	/
2	1.85	Gallic acid	√	√	/	/
3	2.00	Yuheinoside	√	√	/	/
4	7.32	Phloretic acid	√	√	/	/
5	9.08	Chlorogenic acid	√	√	/	/
6	11.81	Albiflorin	√	√	√	√
7	12.20	Vicenin Ⅱ	√	√	√	/
8	12.79	Paeoniflorin	√	√	√	√
9	13.87	Schaftoside	√	√	√	/
10	13.93	Prim-O-glucosylcimifugin	√	√	√	√
11	14.40	Daidzin	√	/	√	/
12	15.26	Liquiritin	√	√	√	√
13	15.47	Liquiritin apioside	√	√	√	√
14	16.27	Scutellarin	√	√	/	/
15	16.50	Isorhamnetin-3-O-neohespeidoside	√	/	√	√
16	16.66	Cimifugin	√	√	√	√
17	17.17	Acteoside	√	/	√	/
18	18.44	Isoacteoside	√	/	√	/
19	18.63	Naringenin 7-O-β-D-glucoside	√	/	/	√
20	18.74	4'-O-β-Glucopyranosyl-5-O-methylvisamminol	√	√	√	√
21	20.52	Calycosin 7-glucoside	√	/	/	√
22	21.16	Isoliquiritin apioside	√	/	√	√
23	21.33	Ononin	√	/	√	√
24	21.68	Isoliquiritin	√	/	√	√
25	22.02	Baicalin	√	√	√	√
26	23.54	Ethyl 4-hydroxybenzoate	√	/	/	√
27	24.18	Apigenin 7-O-β-D-glucuronide	√	/	/	/
28	25.58	Sec-O-glucosylhamaudol	√	/	/	√
29	25.76	Oroxyloside	√	√	√	√
30	26.82	Wogonin 7-O-glucuronide	√	√	√	√
31	27.49	Naringenin	√	/	/	√
32	30.52	4', 6, 7-Trihydroxyisoflavone	√	/	√	/
33	30.55	Apigenin	√	√	√	/
34	30.78	Ginsenoside Rf	√	/	/	√
35	32.49	Formononetin	√	/	/	√
36	32.99	Ginsenoside Rg1	√	/	/	√
37	35.00	Ginsenoside Rg2	√	/	/	√
38	36.07	Oroxylin A	√	/	/	/
39	36.6	Chrysin	√	/	/	/
40	37.14	Chrysosplenetin B	√	/	/	/
41	37.35	Glycyrrhizic acid	√	√	√	√
42	39.53	Glycyrrhizic acid isomer	√	√	/	√
43	39.98	Saikosaponin A	√	/	/	/
44	40	Liquidambaric acid	√	/	/	√
45	40.18	Glycycoumarin	√	/	/	/
46	41.25	Cholan-24-oic acid	√	/	/	√
47	41.44	Ursodeoxycholic acid	√	/	/	/
48	41.96	Saikosaponin D	√	/	/	/
49	43.46	6-Shogaol	√	√	√	√
50	43.47	p-Methoxybezaldehyde	√	/	/	/
51	43.52	Ginsenoside Rg3	√	√	/	/
52	44.13	Ursodeoxycholic acid isomer	√	/	/	/
53	44.43	Gingerglycolipid B	√	√	/	/
54	44.67	Corosolic acid	√	/	/	/
55	45.19	Glycyrrhetinic acid	√	/	/	/

No.

Retention time/min

Name

The whole prescription

Section A

Section B

Section C

0.86

Malic acid

√

1.85

Gallic acid

√

2.00

Yuheinoside

√

7.32

Phloretic acid

√

9.08

Chlorogenic acid

√

11.81

Albiflorin

√

12.20

Vicenin Ⅱ

√

12.79

Paeoniflorin

√

13.87

Schaftoside

√

13.93

Prim-O-glucosylcimifugin

√

14.40

Daidzin

√

15.26

Liquiritin

√

15.47

Liquiritin apioside

√

16.27

Scutellarin

√

16.50

Isorhamnetin-3-O-neohespeidoside

√

16.66

Cimifugin

√

17.17

Acteoside

√

18.44

Isoacteoside

√

18.63

Naringenin 7-O-β-D-glucoside

√

18.74

4'-O-β-Glucopyranosyl-5-O-methylvisamminol

√

20.52

Calycosin 7-glucoside

√

21.16

Isoliquiritin apioside

√

21.33

Ononin

√

21.68

Isoliquiritin

√

22.02

Baicalin

√

23.54

Ethyl 4-hydroxybenzoate

√

24.18

Apigenin 7-O-β-D-glucuronide

√

25.58

Sec-O-glucosylhamaudol

√

25.76

Oroxyloside

√

26.82

Wogonin 7-O-glucuronide

√

27.49

Naringenin

√

30.52

4', 6, 7-Trihydroxyisoflavone

√

30.55

Apigenin

√

30.78

Ginsenoside Rf

√

32.49

Formononetin

√

32.99

Ginsenoside Rg1

√

35.00

Ginsenoside Rg2

√

36.07

Oroxylin A

√

36.6

Chrysin

√

37.14

Chrysosplenetin B

√

37.35

Glycyrrhizic acid

√

39.53

Glycyrrhizic acid isomer

√

39.98

Saikosaponin A

√

Liquidambaric acid

√

40.18

Glycycoumarin

√

41.25

Cholan-24-oic acid

√

41.44

Ursodeoxycholic acid

√

41.96

Saikosaponin D

√

43.46

6-Shogaol

√

43.47

p-Methoxybezaldehyde

√

43.52

Ginsenoside Rg3

√

44.13

Ursodeoxycholic acid isomer

√

44.43

Gingerglycolipid B

√

44.67

Corosolic acid

√

45.19

Glycyrrhetinic acid

√

No.	Retention time / min	Name	Molecular formula	Measured molecular weight	Main feature fragment
1	9.08	Chlorogenic acid	C₁₆H₁₈O₉	m/z 353.087 8 [M-H]^-	m/z 191.056 6
2	11.81	Albiflorin	C₂₃H₂₈O₁₁	m/z 525.161 3 [M+FA-H]^-	m/z 479.157 2, 283.000 0, 121.029 9
3	12.79	Paeoniflorin	C₂₃H₂₈O₁₁	m/z 449.148 5 [M-H]^-	m/z 327.110 4, 121.029 9
4	13.93	Prim-O-glucosylcimifugin	C₂₂H₂₈O₁₁	m/z 469.170 4 [M+H]⁺	m/z 397.109 7, 307.116 3, 235.057 4
5	15.26	Liquiritin	C₂₁H₂₂O₉	m/z 417.119 1 [M-H]^-	m/z 417.119 1, 255.066 7, 135.009 5
6	15.47	Liquiritin apioside	C₂₆H₃₀O₁₃	m/z 549.161 3 [M-H]^-	m/z 549.162 0, 255.066 4, 135.008 4
7	16.27	Scutellarin	C₂₁H₁₈O₁₂	m/z 461.072 5 [M-H]^-	m/z 285.041 5, 267.027 7, 175.024 2
8	16.66	Cimifugin	C₁₆H₁₈O₆	m/z 307.117 6 [M+H]⁺	m/z 259.060 4, 235.062 7, 221.045 1
9	18.74	4'-O-β-Glucopyranosyl-5-O-methylvisamminol	C₂₂H₂₈O₁₀	m/z 497.166 4 [M+FA-H]^-	m/z 451.161 6, 271.097 6
10	21.16	Isoliquiritin apioside	C₂₆H₃₀O₁₃	m/z 549.161 3 [M-H]^-	m/z 549.162 0, 255.066 4, 135.008 4
11	21.33	Ononin	C₂₂H₂₂O₉	m/z 475.124 6 [M+FA-H]^-	m/z 475.133 6, 267.068 2, 252.042 8
12	21.68	Isoliquiritin	C₂₁H₂₂O₉	m/z 417.119 1 [M-H]^-	m/z 417.117 8, 255.067 5, 135.008 8
13	22.02	Baicalin	C₂₁H₁₈O₁₁	m/z 445.077 6 [M-H]^-	m/z 269.046 0, 113.025 4
14	25.58	Sec-O-glucosylhamaudol	C₂₁H₂₆O₁₀	m/z 439.159 9 [M+H]⁺	m/z 277.108 9, 259.097 4, 205.048 1
15	32.49	Formononetin	C₁₆H₁₂O₄	m/z 269.080 8 [M+H]⁺	m/z 269.080 2, 254.054 8, 237.055 2
16	37.35	Glycyrrhizic acid	C₄₂H₆₂O₁₆	m/z 821.396 5 [M-H]^-	m/z 821.391 7, 351.054 7
17	39.53	Glycyrrhizic acid isomer	C₄₂H₆₂O₁₆	m/z 821.396 5 [M-H]^-	m/z 821.398 8, 351.055 9
18	43.46	6-Shogaol	C₁₇H₂₄O₃	m/z 277.179 8 [M+H]⁺	m/z 137.058 9, 122.035 7
19	45.19	Glycyrrhetinic acid	C₃₀H₄₆O₄	m/z 469.332 3 [M-H]^-	m/z 469.335 9, 425.343 1

No.

Retention time / min

Name

Molecular formula

Measured molecular weight

Main feature fragment

9.08

Chlorogenic acid

C₁₆H₁₈O₉

m/z 353.087 8 [M-H]^-

m/z 191.056 6

11.81

Albiflorin

C₂₃H₂₈O₁₁

m/z 525.161 3 [M+FA-H]^-

m/z 479.157 2, 283.000 0, 121.029 9

12.79

Paeoniflorin

C₂₃H₂₈O₁₁

m/z 449.148 5 [M-H]^-

m/z 327.110 4, 121.029 9

13.93

Prim-O-glucosylcimifugin

C₂₂H₂₈O₁₁

m/z 469.170 4 [M+H]⁺

m/z 397.109 7, 307.116 3, 235.057 4

15.26

Liquiritin

C₂₁H₂₂O₉

m/z 417.119 1 [M-H]^-

m/z 417.119 1, 255.066 7, 135.009 5

15.47

Liquiritin apioside

C₂₆H₃₀O₁₃

m/z 549.161 3 [M-H]^-

m/z 549.162 0, 255.066 4, 135.008 4

16.27

Scutellarin

C₂₁H₁₈O₁₂

m/z 461.072 5 [M-H]^-

m/z 285.041 5, 267.027 7, 175.024 2

16.66

Cimifugin

C₁₆H₁₈O₆

m/z 307.117 6 [M+H]⁺

m/z 259.060 4, 235.062 7, 221.045 1

18.74

4'-O-β-Glucopyranosyl-5-O-methylvisamminol

C₂₂H₂₈O₁₀

m/z 497.166 4 [M+FA-H]^-

m/z 451.161 6, 271.097 6

21.16

Isoliquiritin apioside

C₂₆H₃₀O₁₃

m/z 549.161 3 [M-H]^-

m/z 549.162 0, 255.066 4, 135.008 4

21.33

Ononin

C₂₂H₂₂O₉

m/z 475.124 6 [M+FA-H]^-

m/z 475.133 6, 267.068 2, 252.042 8

21.68

Isoliquiritin

C₂₁H₂₂O₉

m/z 417.119 1 [M-H]^-

m/z 417.117 8, 255.067 5, 135.008 8

22.02

Baicalin

C₂₁H₁₈O₁₁

m/z 445.077 6 [M-H]^-

m/z 269.046 0, 113.025 4

25.58

Sec-O-glucosylhamaudol

C₂₁H₂₆O₁₀

m/z 439.159 9 [M+H]⁺

m/z 277.108 9, 259.097 4, 205.048 1

32.49

Formononetin

C₁₆H₁₂O₄

m/z 269.080 8 [M+H]⁺

m/z 269.080 2, 254.054 8, 237.055 2

37.35

Glycyrrhizic acid

C₄₂H₆₂O₁₆

m/z 821.396 5 [M-H]^-

m/z 821.391 7, 351.054 7

39.53

Glycyrrhizic acid isomer

C₄₂H₆₂O₁₆

m/z 821.396 5 [M-H]^-

m/z 821.398 8, 351.055 9

43.46

6-Shogaol

C₁₇H₂₄O₃

m/z 277.179 8 [M+H]⁺

m/z 137.058 9, 122.035 7

45.19

Glycyrrhetinic acid

C₃₀H₄₆O₄

m/z 469.332 3 [M-H]^-

m/z 469.335 9, 425.343 1