UPLC-qTOF-MS/MS法测定透骨草中有效成分

doi:10.3969/j.issn.2097-2806.2023.11.016

Abstract

Abstract:

An ultra-high performance liquid chromatography （UPLC） coupled with electrospray quadrupole time-of-flight mass spectrometry （ESI-qTOF-MS） method was developed for the rapid separation and identification of the components in Phryma leptostachya L.. The samples were eluted on an ACQUITY UPLC HSS T3 C₁₈ column （2.1 mm×100 mm, 1.8 μm） with a gradient of 0.1% formic acid （solvent A） and acetonitrile （solvent B） as the mobile phases at a flow rate of 0.25 mL/min. The chromatographic analysis of Phryma leptostachya L. by UPLC-qTOF-MS/MS identifies the characterization of 47 compounds, 15 of which are clearly identified by comparison with reference standards. For quantitative analysis, 15 major compounds are simultaneously detected in 12 batches of Phryma leptostachya L. samples using UPLC-DAD at 210 nm, 260 nm and 326 nm. The method is validated in terms of precision, reproducibility, stability and accuracy. In conclusion, this study provides a potential method for the overall quality control of Phryma leptostachya L..

Key words: Phryma leptostachya L., UPLC-qTOF-MS/MS, quality assessment

CLC Number:

TQ658

Chen Pingping, Xu Quansheng, Jiang Rui, Feng Songhao. Determination of the active ingredients in Phryma leptostachya L. by UPLC-qTOF-MS/MS[J].China Surfactant Detergent & Cosmetics, 2023, 53(11): 1351-1360.

Figures/Tables 5

Fig. 1

Tab. 1

Identification of the chemical composition of Phryma leptostachya L. by UPLC-qTOF-MS/MS in negative ion mode"

编号	t_R/min	M.F.	M.W./Da	质量误差/ppm	MS/MS碎片（m/z）	鉴定
1	1.036	C₂₁H₃₀O₁₁	458.178 8	503.176 5 [M+HCOO] ^-/（-1.0）	[503]: 221, 191, 179, 161, 89	油菜花苷A^（Oleuropein A^）
2	3.616	C₈H₈O₃	152.047 3	197.045 1 [M+HCOO] ^-/（2.5）	[197]: 163	6-羟基-7, 7a-二氢苯并呋喃-2（6）H-酮（R1）（6-Hydroxy-7, 7α-dihydrobenzofuran-2（6）H-one）
3	3.810	C₁₄H₂₀O₈	316.115 8	361.113 6 [M+HCOO] ^-/（1.9）	[361]: 343, 315, 297, 269, 253, 217, 201, 175, 161, 135, 131, 101, 71	羟基酪蛋白（R2）（Hydroxycasein）
4	4.269	C₇H₆O₄	154.026 6	153.019 6 [M-H] ^-/（2.0）	[153]: 109, 91, 81, 65	原儿茶酸（R3）（Protocatechuic acid）
5	4.900	C₁₆H₁₈O₉	354.095 1	353.088 8 [M-H] ^-/（2.8）	[353]: 191, 179, 135	新绿原酸（C1）（Neochlorogenic acid）
6	5.901	C₁₆H₁₈O₉	354.095 1	353.088 6 [M-H] ^-/（2.3）	[353]: 191, 179	绿原酸（C2）（Chlorogenic acid）
7	6.095	C₁₆H₁₈O₉	354.095 1	353.0 886 [M-H] ^-/（2.3）	[353]: 191, 179, 173, 135, 93	隐绿原酸（C3）（Cryptochlorogenic acid）
8	6.587	C₉H₈O₄	180.042 3	179.033 8 [M-H] ^-/（-3.4）	[179]: 135	咖啡酸（C4）（Caffeic acid）
9	7.326	C₁₇H₂₀O₉	368.110 7	367.103 4 [M-H] ^-/（-0.1）	[367]: 301, 193, 161, 135, 133	3-FQA
10	7.815	C₁₇H₂₀O₉	368.110 7	367.103 0 [M-H] ^-/（-1.4）	[367]: 301, 191, 161, 135, 133	5-FQA
11	8.046	C₂₇H₃₀O₁₆	610.153 4	609.145 7 [M-H] ^-/（-0.7）	[609]: 301, 271, 255, 151, 149	芦丁（R4）（Rutin）
12	8.212	C₂₁H₂₂O₁₀	434.121 3	433.115 3 [M-H] ^-/（2.8）	[433]: 271, 179, 161, 133, 109	香草酸4-O-β-D-6-苯甲酰基吡喃葡萄糖苷（Vanillic acid 4-O-beta-D-6-benzoylglucopyranoside）
13	8.665	C₂₇H₃₀O₁₅	594.158 5	593.150 8 [M-H] ^-/（-0.5）	[593]: 285, 255, 229	烟酸苷（Nicotinic acid glycosides）
14	8.779	C₂₅H₂₄O₁₂	516.126 8	515.120 0 [M-H] ^-/（1.0）	[515]: 353, 335, 191, 179, 173, 161, 135	异绿原酸B （C5）（Isochlorogenic acid B）
15	9.154	C₂₅H₂₄O₁₂	516.126 8	515.119 7 [M-H] ^-/（0.4）	[515]: 353, 191, 179, 173, 135	异绿原酸A （C6）（Isochlorogenic acid A）
16	11.176	C₁₇H₂₀O₉	368.110 7	367.102 3 [M-H] ^-/（-3.0）	[367]: 161, 135, 133	－
17	11.925	C₁₇H₂₀O₉	368.110 7	367.102 3 [M-H] ^-/（-3.0）	[367]: 193, 173, 155, 137, 134, 93, 67	4-FQA
18	12.848	C₂₆H₂₆O₁₂	530.142 4	529.136 9 [M-H] ^-/（3.4）	[529]: 367, 349, 179, 161, 135	Macroantoin G
19	13.060	C₆₅H₁₀6O₃₁	1 382.671 8	1 427.667 9 [M+HCOO] ^-/（-0.8）	[1 427]: 1 103, 1 057, 937, 791, 749	Kudinoside O
20	13.184	C₅₉H₉₆O₂₆	1 220.619 0	1 265.618 2 [M+HCOO] ^-/（1.7）	[1 265]: 1 219, 911, 749, 471	Latifoloside G （K1）
21	13.304	C₁₅H₁₀O₇	302.042 7	301.035 3 [M-H] ^-/（3.3）	[301]: 151, 149, 121, 107, 93, 83, 65	槲皮素（Quercetin）
22	13.467	C₅₃H₈₄O₂₃	1 088.540 3	1 133.536 5 [M+HCOO] ^-/（-1.8）	[1 133]: 1 087, 925, 701, 555	牡丹苷C （Peonidin C）
23	13.683	C₄₇H₇₆O₁₇	912.508 3	957.508 1 [M+HCOO] ^-/（1.8）	[957]: 911, 749	乳清糖苷D （Lactuloside D）
24	13.745	C₅₃H₈₆O₂₂	1 074.561 1	1 119.561 9 [M+HCOO] ^-/（2.3）	[1 119]: 1 073, 911, 749, 603, 471	Peonidin G （K2）
25	13.830	C₄₂H₆₆O₁₅	810.440 2	855.434 6 [M+HCOO] ^-/（-4.3）	[855]: 809	－
26	13.887	C₅₃H₈₆O₂₁	1 058.566 2	1 103.567 7 [M+HCOO] ^-/（3.1）	[1 103]: 1 057, 937, 791, 749, 731	油菜素苷A （Oleuropein A）
27	13.952	C₄₇H₇₆O₁₉	944.498 1	989.495 9 [M+HCOO] ^-/（ 0.7）	－	－
28	13.979	C₄₉H₇₈O₁₉	970.513 7	1 015.514 3 [M+HCOO] ^-/（3.3）	[1 015]: 926, 764	菊苣皂苷V甲酯（Chicory Saponin V methyl ester）
29	14.322	C₄₇H₇₆O₁₉	944.498 1	989.495 9 [M+HCOO] ^-/（0.7）	－	－
30	14.385	C₄₇H₇₄O₁₈	926.487 5	971.486 8 [M+HCOO] ^-/（2.2）	[971]: 925, 763, 701, 555, 493	Chrysin A （K3）
31	15.167	C₅₉H₉₆O₂₅	1 204.624 1	1 249.621 8 [M+HCOO] ^-/（0.5）	[1 249]: 1 203, 895, 733, 455	－
32	15.292	C₄₇H₇₆O₁₉	944.498 1	989.495 9 [M+HCOO] ^-/（0.7）	[989]: 911.5, 62.0	－
33	15.394	C₅₃H₈₂O₂₂	1 070.529 8	1 115.530 9 [M+HCOO] ^-/（3.6）	[1 115]: 1 069, 907, 745, 555	Kudinoside E （R5）
34	15.456	C₄₇H₇₆O₁₉	944.498 1	989.495 89 [M+HCOO] ^-/（0.7）	[989]: 911.5, 62.0	－
35	15.600	C₄₈H₇₈O₁₈	942.518 8	987.514 84 [M+HCOO] ^-/（-1.2）	[987]: 939, 777	－
36	15.864	C₄₁H₆₄O₁₃	764.434 7	809.432 6 [M+HCOO] ^-/（0.4）	[809]: 763, 701, 555	Ilekudinoside B
37	15.935	C₄₂H₆₄O₁₃	776.434 7	821.431 8 [M+HCOO] ^-/（0.0）	[775]: 629	Ilekudinoside K
38	16.347	C₃₅H₅₄O₉	618.376 8	663.385 0 [M+HCOO] ^-/（1.7）	[663]: 617, 599, 555, 529, 461, 131	Kudinoside I
39	16.460	C₃₅H₅₄O₉	618.376 8	663.376 5[M+HCOO] ^-/（3.9）	[663]: 617, 599, 555, 543, 501, 483, 439	Kudinoside I-异构体（Kudinoside I-Isomers）
40	16.649	C₄₇H₇₄O₁₇	910.492 6	909.489 0 [M-H] ^-/（4.1）	[909]: 909, 747, 601	－
41	16.717	C₄₉H₇₈O₁₉	970.513 7	969.504 4 [M-H] ^-/（-2.1）	[969]: 923, 761	菊苣皂苷V甲酯（Chicory Saponin V methyl ester）
41	16.719	C₄₇H₇₄O₁₇	910.492 6	955.490 2 [M+HCOO] ^-/（0.4）	[955]: 909, 745	Ilekudinoside T （K4）
42	17.220	C₄₈H₇₈O₁₈	942.518 9	941.511 6 [M-H] ^-/（0.1）	[941]: 895, 733	Latifoloside J
43	17.336	C₄₁H₆₆O₁₄	782.445 3	781.407 2 [M-H] ^-/（-0.9）	[781]: 745	皂角苷C （Saponin C）
44	17.348	C₄₂H₆₄O₁₄	792.429 6	791.425 5 [M-H] ^-/（4.0）	[791]: 745	油菜花苷N （Oleuropein N）
45	19.956	C₃₀H₄₈O₆	504.345 1	503.336 5 [M-H] ^-/（-2.6）	503.3	（+）-Arjungenin
46	20.219	C₃₀H₄₆O₅	486.334 5	531.331 5 [M+HCOO] ^-/（-0.4）	[531]: 485, 467, 423, 397, 381, 329	β-丁内酯（β-butyrolactone）
49	21.082	C₃₅H₅₆O₈	604.397 5	649.395 4 [M+HCOO] ^-/（1.1）	[649]: 469, 179, 161, 135, 106	伊乐藻苷A或伊乐藻苷B （Elaiophylin A or Elaiophylin B）
50	21.225	C₃₀H₄₆O₅	486.334 5	485.325 9 [M-H] ^-/（-2.7）	[485]: 423, 383, 353, 87, 57	β-丁内酯（β-butyrolactone）
51	21.442	C₃₀H₄₈O₄	472.355 3	471.347 51 [M-H] ^-/（-0.8）	[471]: 441	坡模酸（Pozzolanic acid）
52	21.545	C₃₀H₄₄O₄	468.324 0	513.319 6 [M+HCOO] ^-/（-2.9）	[513]: 468, 277, 253	α-丁内酯（α-butyrolactone）
53	21.710	C₂₉H₄₂O₄	454.308 3	453.302 8 [M-H] ^-/（3.9）	[453]: 255, 195	Ilekudinnol A
54	22.218	C₃₅H₅₈O₆	574.423 3	619.420 2 [M+HCOO] ^-/（-0.5）	[619]: 453, 163, 119	谷甾醇（Glutathione）
55	22.380	C₃₀H₄₆O₃	454.344 7	453.335 6 [M-H] ^-/（-4.0）	[453]: 407, 405, 255, 79	油脂酸C （Oleic acid C）
56	22.369	C₃₀H₄₈O₅	488.350 2	487.341 0 [M-H] ^-/（-3.7）	[487]: 453, 407	丙烯酸（Acrylic acid）

Tab. 1

Tab. 2

Tab. 3

Tab. 4

References 18

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成分	线性方程	相关系数r2	线性范围/（μg/mL）	LOQ/（μg/mL）	t_R/min	RSD/%
R1	y=23 614 x-6 410.8	1.000 0	3.10~ 62.00	0.14	3.535	1.60
R2	y=1 6140 x-38 158	0.999 9	19.00~ 380.00	0.18	4.098	1.40
R3	y=8 772.1 x-691.0	0.999 6	0.51~ 10.20	0.37	4.309	0.95
C1	y=11 769 x-9 493	1.000 0	5.09~ 101.80	0.22	4.780	0.72
C2	y=10 931 x-258.4	0.999 9	37.75~ 755.00	0.42	6.002	0.28
C3	y=10 922 x-13 148	0.999 9	5.05~ 101.00	0.34	6.252	0.22
C4	y=18 876 x-25 782	0.999 8	5.30~ 106.00	0.16	6.742	0.22
R4	y=6 416.7 x-1 738.3	0.999 8	1.58~ 31.60	0.73	8.359	0.08
C5	y=19 677 x-28 546	0.999 9	5.33~ 106.60	0.16	9.214	0.05
C6	y=10 343 x-26 275	0.999 5	68.25~1 365.00	0.28	9.570	0.05
K1	y=580.9 x-1 834.2	0.999 9	52.25~1 045.00	2.53	13.767	0.06
K2	y=200.3 x+877	0.999 7	45.75~ 915.00	5.91	14.317	0.07
K3	y=1 357.3 x-6 721.2	1.000 0	53.25~1 065.00	1.21	15.004	0.07
R5	y=6 912.6 x-3 842.5	0.999 9	3.00~ 60.00	0.46	16.240	0.08
K4	y=1 186.8 x-82.35	0.999 9	1.95~ 39.00	1.01	17.836	0.07

成分	含量/（mg/g）															峰值/%
成分	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	峰值/%
R1	1.74	0.31	0.52	0.87	0.46	0.25	0.82	0.46	2.46	2.67	0.66	1.25	0.82	0.24	1.21	98.2
R2	34.3	5.46	2.74	8.54	2.78	1.82	4.04	2.54	1.01	12.88	5.75	10.97	7.01	1.92	7.02	97.8
R3	0.18	0.16	0.41	0.17	0.51	0.33	0.28	0.21	0.47	0.65	0.35	0.22	0.27	0.29	0.24	98.6
C1	4.14	6.63	4.2	4.95	4.57	3.58	2.47	3.9	2.01	1.89	4.11	5.08	2.77	3.46	7.53	100.0
C2	17.4	25.4	13	18.3	12.3	10.4	11.3	15.7	5.58	5.42	19.2	18.6	11.3	19.0	20.1	100.0
C3	0.81	5.56	5.41	3.6	4.76	3.08	3.73	3.92	1.79	2.03	4.15	4.71	3.65	4.31	5.36	100.0
C4	2.37	0.58	1.59	0.89	1.51	1.12	0.98	0.45	0.49	0.6	3.27	0.49	1.45	1.53	0.91	100.0
R4	0.61	0.88	0.42	0.61	0.32	0.35	0.46	0.25	0.24	0.26	1.17	1.72	0.76	0.33	1.18	98.0
C5	1.54	3.22	4.81	4.14	3.88	2.86	3.87	4.14	4.3	2.92	3.69	4.51	4.62	5.54	4.56	100.0
C6	123.8	37.0	34.9	58.9	36.7	28.5	30.9	28.2	71.2	76.4	43.6	70.1	38.5	50.9	58.0	100.0
K1	31.4	11.65	6.38	15.43	9.84	7.45	13.9	11.0	18.7	16.3	28.0	39.2	22.4	18.2	18.1	100.0
K2	30.3	9.81	7.03	11.8	6.65	4.12	13.32	4.42	12.92	15.9	23.6	33.2	25.7	11.0	12.3	100.0
K3	31.4	28.7	22.6	22.2	22.5	21.3	19.1	23.9	22.5	28.2	34.9	36.1	21.3	40.9	35.4	100.0
R5	0.3	0.34	0.38	0.44	0.47	0.38	0.45	0.46	3.27	0.28	0.67	0.9	0.65	0.49	0.61	97.4
K4	0.28	0.23	0.19	0.13	0.44	0.21	0.12	0.3	0.33	0.26	0.96	0.77	0.49	0.46	0.47	99.9
总和-P.A	150.3	63.1	64.3	91.0	59.4	49.82	53.5	56.47	85.8	89.9	78.4	103.7	62.5	85.0	96.7
总和-S	94.3	51.26	37.44	50.36	40.55	34.19	47.49	40.61	58.52	61.57	88.41	110.67	70.81	72.08	67.47
总和-F.O	36.13	6.05	2.81	9.58	2.82	1.32	4.8	2.68	2.99	15.3	7.3	13.51	8.21	1.43	8.78

Determination of the active ingredients in Phryma leptostachya L. by UPLC-qTOF-MS/MS

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成分	精确度		重复性	稳定性	准确度
成分	日内（RSD/%, n=6）	日间（RSD/%, n=3）	RSD/%, n=6	RSD/%, n=6	回收率/%	RSD/%, n=6
R1	2.1	2.7	3.5	2.6	97.7	4.6
R2	1.8	2.5	4.3	4.8	99.2	4.0
R3	3.5	4.3	5.2	3.2	101.5	2.8
C1	2.3	3.4	3.6	0.8	104.1	1.8
C2	1.7	1.9	3.4	1.7	95.7	3.5
C3	1.8	3.7	4.8	1.1	99.7	4.0
C4	2.2	3.2	5.1	3.7	94.7	4.8
R4	3.1	4.2	4.6	2.2	107.5	6.4
C5	1.8	2.3	5.2	2.3	96.5	2.4
C6	1.9	4.3	5.7	1.2	93.3	1.2
K1	2.2	4.5	5.7	1.3	96.6	3.4
K2	4.3	3.2	5.6	2.5	105.1	4.9
K3	1.9	2.6	4.3	3.0	100.8	2.0
R5	4.1	4.6	3.8	3.6	96.4	2.3
K4	4.1	3.9	6.1	3.9	107.5	5.9