Background
Methods
Reagents, chemicals and materials
Sample solutions preparation
Standard solutions preparation
Analytical methods
Method validation
Results and discussion
Optimization of the chromatographic conditions
Identification of chemical compounds
R.T. (min) | Mass | Error (ppm) | Fragment ions (m/z) | Molecular formula | Identification | Herbal origin | Confirmed with reference standard | ||
---|---|---|---|---|---|---|---|---|---|
Observed | Calculated | ||||||||
1 | 2.80 | 166.0871 | 166.0863 | 4.8 | 120.0792 [M+H–COOH]+
| C9H11NO2
|
l-Phenylalanine |
H. diversicolor
| Yes |
2 | 7.50 | 291.0862 | 291.0863 | 5.3 | 139.0396 [M+H–C8H7O3]+, 123.0434 [M+H–C8H7O4]+
| C15H14O6
| Catechin |
U. rhynchophylla
| Yes |
3 | 8.59 | 551.2007 | 551.1970 | 4.3 | 573.2007 [M+Na]+, 227.0753 [M+H–maltose]+
209.0656 [M+H–maltose–OH]+
| C23H34O15
| Genipin 1-beta-gentiobioside |
G. jasminoides
| Yes |
4 | 9.30 | 291.0866 | 291.0863 | 1.0 | 139.0381 [M+H–C8H7O3]+, 123.0422 [M+H–C8H7O4]+
| C15H14O6
| Epi-catechin |
U. rhynchophylla
| Yes |
5 | 9.40 | 389.1452 | 389.1442 | 2.6 | 411.0792 [M+Na]+, 231.0366 [M+H–glucose–2H]+
| C17H24O10
| Geniposide |
G. jasminoides
| Yes |
6 | 11.2 | 312.1554 | 312.1554 | 1.6 | 181.0498 [M–C5H13N3O], 132.1125 [M+H–C9H9N4O]+
| C14H22N3O5
| Leonurine |
L. japonicus
| Yes |
7 | 12.3 | 407.1337 | 407.1351 | 3.4 | 245.0768 [M+H–glucose]+
| C20H22O9
| 2 3 5 4′-tetrahydroxystilbene-2-O-β-d-glucoside |
P. multiflorum
| Yes |
8 | 14.9 | 521.3146 | 521.3109 | 5.2 | 503.2947 [M+H–H2O]+, 485.2896 [M+H–2H2O]+
| C29H44O8
| Cyasteron |
C. officinalis
| Yes |
9 | 15 | 383.1978 | 383.1965 | 3.4 | 383.1978[M+H]+
| C22H26N2O4
| Corynoxeine |
U. rhynchophylla
| Yes |
10 | 15.8 | 385.2131 | 385.2122 | 2.3 | 241.1337 | C22H28N2O4
| Isorhynchophylline |
U. rhynchophylla
| Yes |
11 | 15.9 | 383.1976 | 383.1965 | 2.9 | 383.1976[M+H]+
| C22H26N2O4
| Isocorynoxeine |
U. rhynchophylla
| Yes |
12 | 16.7 | 447.0944 | 447.0922 | 4.9 | 271.0621 [M+H–glucuronide]+
| C21H18O11
| Baicalin |
S. baicalensis
| Yes |
13 | 16.9 | 385.2126 | 385.2122 | 1.0 | 353.1854 [M+H–OCH3]+, 160.0754 | C22H28N2O4
| Rhynchophylline |
U. rhynchophylla
| Yes |
14 | 18.9 | 461.1110 | 461.1078 | 5.1 | 285.0762 [M+H–glucuronide]+, 270.0507 [M+H–glucuronide–CH3]+
| C22H20O11
| OroxylinA-7-O-glucuronide |
S. baicalensis
| Yes |
15 | 19.6 | 447.0951 | 447.0922 | 3.7 | 271.0601[M+H–glucuronide]+
| C21H18O11
| Bacalein-6-O-beta-glucopyranoside |
S. baicalensis
| Yes |
16 | 19.8 | 461.1105 | 461.1078 | 5.9 | 285.0754 [M+H–glucuronide]+
| C22H20O11
| Wogonoside |
S. baicalensis
| Yes |
17 | 25.6 | 285.0773 | 285.0757 | 5.6 | 270.0535 [M+H–CH3]+
| C16H12O5
| Wogonin |
S. baicalensis
| Yes |
18 | 5.6 | 153.0546 | 153.0507 | 4.9 | 175.0380 [M+Na]+
| C8H8O3
| Vanillin |
G. elata
| Not yet |
19 | 10.9 | 683.2537 | 683.2507 | 4.4 | 705.2363 [M+Na]+
| C32H42O16
| Pinoresinol diglucoside |
E. ulmoides
| Not yet |
20 | 12.2 | 549.1646 | 549.1616 | 5.5 | 531.1493 [M+H–H2O]+
| C26H28O13
| Chrysin-6-C-ara-8-C-glu |
S. baicalensis
| Not yet |
21 | 13.1 | 549.1620 | 549.1616 | 0.7 | 531.1482 [M+H–H2O]+
| C26H28O13
| Chrysin-6-C-glu-8-C-ara |
S. baicalensis
| Not yet |
22 | 14.0 | 449.1143 | 449.1178 | 5.4 | 303.0486 [M+H–rhamnose]+
| C21H20O11
| Quercitrin |
L. japonicus
| Not yet |
23 | 18.5 | 447.0946 | 447.0922 | 5.4 | 271.0617 [M+H–glucuronide]+
| C21H19O11
| Norwogonin-7-O-glucuronide |
S. baicalensis
| Not yet |
24 | 18.9 | 477.1065 | 447.1028 | 4.9 | 301.0719 [M+H–glucuronide]+
| C22H20O12
| 5,7,8,-Trihydroxy-6-methoxyflavone-7-glucuronide |
S. baicalensis
| Not yet |
25 | 19.8 | 477.1058 | 477.1028 | 3.7 | 301.0718 [M+H–glucuronide]+
| C22H20O12
| 5,6,7,-Trihydroxy-8-methoxyflavone-7-glucuronide |
S. baicalensis
| Not yet |
Method validation
Analyte | Linearity | LOD (ng/mL) | LOQ (ng/mL) | Repeatability (RSD, %, n = 6) | Spike recovery (RSD, %, n = 3) | Stability (RSD, %, n = 6) | |||||
---|---|---|---|---|---|---|---|---|---|---|---|
Range (ng/mL) | Equation | r2
| Intra-day | Inter-day | High | Middle | Low | ||||
1 | 50.60–404.80 | Y = 148.2X − 704.4 | 0.9996 | 13.67 | 45.58 | 3.45 | 4.81 | 96.37 (2.08) | 96.55 (0.18) | 105.99 (2.65) | 3.16 |
2 | 397.50–3180.00 | Y = 33.8X − 721.8 | 0.9989 | 104.61 | 348.68 | 2.93 | 4.84 | 98.86 (2.16) | 98.67 (0.65) | 94.99 (2.00) | 2.46 |
3 | 329.10–10530.00 | Y = 56.9X + 10454.0 | 0.9997 | 43.38 | 144.60 | 1.20 | 3.37 | 101.88 (2.21) | 101.73 (0.65) | 94.37 (1.72) | 1.81 |
4 | 3187.50–25500.00 | Y = 7.3X + 2059.8 | 0.9997 | 233.80 | 779.34 | 2.53 | 5.54 | 96.96 (2.22) | 104.26 (0.78) | 102.12 (5.48) | 4.59 |
5 | 1659.40–53100.00 | Y = 16.9X + 18376.0 | 0.9989 | 174.67 | 582.64 | 4.06 | 4.64 | 102.71 (1.99) | 96.39 (0.31) | 102.31 (1.94) | 2.59 |
6 | 59.48–237.93 | Y = 1203.4X + 9810.8 | 0.9992 | 11.65 | 38.84 | 3.78 | 4.66 | 101.38 (5.10) | 104.00 (0.69) | 104.50 (1.23) | 3.98 |
7 | 126.28–5387.76 | Y = 493.9X + 11718.0 | 0.9999 | 25.25 | 84.18 | 2.58 | 4.17 | 97.14 (2.19) | 95.46 (1.27) | 95.10 (0.37) | 2.58 |
8 | 116.50–1864.00 | Y = 170.7X + 5289.2 | 0.9996 | 17.83 | 59.44 | 1.05 | 4.54 | 96.77 (0.99) | 101.81 (0.77) | 104.52 (0.51) | 1.56 |
9 | 13.02–208.33 | Y = 378.9X + 1346.2 | 0.9994 | 2.77 | 9.24 | 1.55 | 3.66 | 101.45 (4.45) | 97.26 (1.42) | 104.54 (2.29) | 1.29 |
10 | 13.27–106.12 | Y = 12974.0X − 33626.0 | 0.9994 | 2.14 | 7.13 | 1.19 | 4.71 | 101.76 (2.68) | 103.99 (1.79) | 105.29 (1.15) | 1.51 |
11 | 52.95–211.80 | Y = 592.1X + 6742.6 | 0.9993 | 9.57 | 31.05 | 2.42 | 3.97 | 102.07 (1.53) | 95.27 (0.78) | 105.62 (0.58) | 2.37 |
12 | 1273.44–81500.00 | Y = 44.0X + 62221.0 | 0.9996 | 49.97 | 166.57 | 2.50 | 4.59 | 100.78 (2.65) | 98.85 (3.36) | 101.55 (3.70) | 4.67 |
13 | 33.04–198.21 | Y = 2767.1X − 52144.0 | 0.9988 | 7.62 | 25.41 | 3.46 | 4.43 | 95.89 (0.71) | 99.58 (3.47) | 94.63 (0.77) | 2.85 |
14 | 204.10–13062.50 | Y = 132.0X + 17621.0 | 0.9992 | 28.98 | 96.62 | 1.69 | 4.89 | 97.72 (3.65) | 98.26 (0.72) | 99.54 (3.98) | 4.28 |
15 | 259.38–4150.00 | Y = 47.4X − 1173.4 | 0.9989 | 71.38 | 237.95 | 2.64 | 3.89 | 102.64 (2.18) | 99.14 (0.64) | 106.11 (1.39) | 2.29 |
16 | 1767.19–28275.00 | Y = 117.5X + 127121.0 | 0.9989 | 26.61 | 88.71 | 1.89 | 4.29 | 97.29 (2.79) | 98.27 (0.29) | 96.19 (2.40) | 1.87 |
17 | 118.64–2548.47 | Y = 1291.1X − 78900.0 | 0.9998 | 27.23 | 90.76 | 1.07 | 1.69 | 96.64 (1.39) | 101.78 (0.77) | 94.88 (1.05) | 1.73 |
Quantification of compounds in commercial samples
Analyte | TGY-01 | TGY-02 | TGY-03 | TGY-04 | TGY-05 | TGY-06 | TGY-07 | TGY-08 | TGY-09 | TGY-10 | TGY-11 |
---|---|---|---|---|---|---|---|---|---|---|---|
1a
| 0.03b ± 2.57c
| –d
| – | – | 0.04 ± 2.50 | 0.08 ± 2.82 | – | – | – | – | 0.05 ± 3.45 |
2 | 0.39 ± 3.42 | 0.38 ± 2.27 | 0.40 ± 1.53 | 0.27 ± 2.72 | 0.25 ± 0.56 | – | – | 0.18 ± 1.09 | – | – | 0.93 ± 2.93 |
3 | 1.02 ± 1.95 | 0.99 ± 2.50 | 1.42 ± 0.13 | 1.27 ± 3.00 | 1.89 ± 2.48 | 1.65 ± 2.65 | 2.02 ± 3.16 | 2.54 ± 4.39 | 1.84 ± 0.67 | 2.03 ± 1.71 | 0.23 ± 1.20 |
4 | – | – | – | – | – | – | – | – | – | – | 4.59 ± 2.53 |
5 | 2.10 ± 3.11 | 2.12 ± 0.48 | 3.18 ± 1.90 | 2.92 ± 4.33 | 5.04 ± 4.73 | 5.98 ± 1.94 | 8.00 ± 0.63 | 5.69 ± 1.71 | 5.74 ± 2.10 | 6.67 ± 0.63 | 13.15 ± 4.06 |
6 | – | – | – | – | – | 0.05 ± 0.65 | 0.04 ± 1.68 | 0.04 ± 1.16 | 0.02 ± 4.77 | 0.02 ± 4.16 | 0.053 ± 3.78 |
7 | 0.03 ± 3.40 | 0.03 ± 0.64 | 0.27 ± 4.12 | 0.24 ± 2.86 | 0.09 ± 1.95 | 1.79 ± 1.83 | 1.79 ± 3.25 | 1.67 ± 1.96 | 1.72 ± 3.28 | 1.44 ± 2.65 | 0.10 ± 2.58 |
8 | – | – | – | – | – | – | – | – | – | – | 0.36 ± 1.05 |
9 | – | – | – | – | – | 0.06 ± 1.34 | 0.02 ± 4.10 | 0.02 ± 4.43 | 0.01 ± 1.51 | 0.01 ± 2.96 | 0.02 ± 1.55 |
10 | – | – | – | – | – | 0.03 ± 4.30 | 0.01 ± 1.51 | 0.01 ± 5.02 | 0.01 ± 2.85 | 0.01 ± 3.09 | 0.01 ± 1.19 |
11 | – | – | – | – | – | 0.14 ± 1.26 | 0.06 ± 1.13 | 0.04 ± 1.90 | 0.03 ± 2.29 | 0.03 ± 0.67 | 0.05 ± 2.42 |
12 | 1.83 ± 2.28 | 1.64 ± 2.29 | 2.08 ± 2.07 | 1.30 ± 4.48 | 5.55 ± 1.81 | 8.33 ± 0.95 | 8.18 ± 3.38 | 9.22 ± 1.83 | 10.94 ± 0.40 | 12.11 ± 3.41 | 31.34 ± 2.50 |
13 | – | – | – | – | – | 0.08 ± 1.84 | 0.03 ± 1.70 | 0.02 ± 1.13 | 0.03 ± 1.50 | 0.02 ± 1.38 | 0.03 ± 3.46 |
14 | 0.24 ± 4.02 | 0.21 ± 1.87 | 0.25 ± 1.41 | 0.22 ± 2.63 | 0.53 ± 0.50 | 0.85 ± 0.62 | 0.90 ± 2.66 | 1.02 ± 1.90 | 1.21 ± 1.29 | 1.38 ± 1.75 | 3.59 ± 1.69 |
15 | – | – | – | – | – | – | – | – | – | – | 0.26 ± 2.64 |
16 | 0.41 ± 3.08 | 0.32 ± 3.72 | 0.52 ± 1.53 | 0.44 ± 3.37 | 0.95 ± 1.29 | 1.33 ± 2.08 | 1.31 ± 4.36 | 1.60 ± 4.13 | 1.74 ± 0.40 | 2.06 ± 0.56 | 5.98 ± 1.89 |
17 | 0.09 ± 2.50 | 0.05 ± 2.12 | 0.06 ± 3.66 | – | – | 0.17 ± 0.20 | 0.08 ± 4.89 | 0.09 ± 3.89 | 0.11 ± 1.69 | 0.12 ± 3.12 | 0.57 ± 1.07 |
Fructose | 38.03 ± 1.45 | 70.92 ± 0.99 | 30.26 ± 3.02 | 41.04 ± 1.03 | 45.46 ± 1.21 | 38.08 ± 1.66 | 80.56 ± 1.86 | 26.79 ± 1.45 | 40.15 ± 3.67 | 38.12 ± 0.71 | 69.60 ± 2.86 |
Glucose | 45.70 ± 1.07 | 46.34 ± 0.39 | 34.31 ± 0.41 | 37.30 ± 0.50 | 46.35 ± 3.21 | 30.64 ± 4.48 | 52.21 ± 1.59 | 32.79 ± 4.38 | 33.07 ± 4.93 | 27.89 ± 2.10 | 67.87 ± 4.12 |
Sucrose | 14.50 ± 0.63 | 14.71 ± 0.04 | 13.52 ± 1.02 | 12.92 ± 4.74 | 16.18 ± 4.79 | 4.72 ± 1.78 | 5.85 ± 1.23 | 5.01 ± 2.13 | 3.83 ± 4.33 | 4.10 ± 4.56 | 4.03 ± 1.02 |
Mac. molecular | 549.36 ± 2.81 | 643.87 ± 4.33 | 559.69 ± 3.69 | 483.25 ± 0.48 | 655.19 ± 0.94 | 618.28 ± 0.02 | 535.61 ± 0.06 | 696.64 ± 4.11 | 611.02 ± 2.92 | 636.59. ± 0.03 | 294.98 ± 1.11 |