超快速液相色谱-三重四级杆/线性离子阱质谱法比较分析竹节参根与根茎中多元指标成分

邹立思; 陈佳丽; 谈梦霞; 刘训红; 陈舒妤; 石婧婧; 王程成; 梅余琪; 魏丽芳

doi:10.14148/j.issn.1672-0482.2023.0265

超快速液相色谱-三重四级杆/线性离子阱质谱法比较分析竹节参根与根茎中多元指标成分

doi: 10.14148/j.issn.1672-0482.2023.0265

南京中医药大学药学院, 江苏南京 210023

基金项目:

江苏高校优势学科建设工程资助项目 YSXK-2014

江苏高校品牌专业建设工程资助项目 PPZY2015A070

详细信息

作者简介:
邹立思, 男, 实验师, E-mail: zlstcm@126.com

通讯作者:
刘训红, 男, 教授, 主要从事中药鉴定与品质评价研究, E-mail: liuxunh1959@163.com

中图分类号: R284
计量
- 文章访问数: 124
- HTML全文浏览量: 12
- PDF下载量: 7
- 被引次数: 0
出版历程
- 收稿日期: 2022-10-11
- 网络出版日期: 2023-03-21
- 发布日期: 2023-03-10

Comparative Analysis of Multiple-Index Constituents in Roots and Rhizomes of Panax japonicus by UFLC-QTRAP-MS/MS

School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China

摘要

摘要: 目的建立超快速液相色谱-三重四级杆/线性离子阱质谱法(UFLC-QTRAP-MS/MS)同时测定竹节参根与根茎中皂苷、氨基酸及核苷类共33种成分含量的方法。方法采用XBridge^®C₁₈(4.6 mm×100 mm, 3.5 μm), 以0.1%甲酸水-0.1%甲酸乙腈为流动相, 梯度洗脱, 流速0.8 mL·min^-1, 柱温30 ℃, 多反应监测离子扫描模式(MRM)测定。根据33种目标成分的含量, 采用聚类、非线性映射及主成分分析进行综合评价。结果 33种成分在一定浓度范围内均呈现良好的线性关系, 相关系数均大于0.999 0;精密度、重复性和稳定性良好; 平均加样回收率为96.90%~101.6%, RSD均≤3.6%。统计分析结果显示从33种成分的含量角度, 竹节参根与根茎中皂苷、氨基酸及核苷类成分较为相似。结论所建立的方法准确、可靠, 可为竹节参药材内在质量的综合评价和全面控制提供新的方法参考, 同时为竹节参根的开发利用提供基础资料。
- 超快速液相色谱-三重四极杆/线性离子阱质谱法 /
- 竹节参 /
- 根 /
- 根茎 /
- 皂苷 /
- 氨基酸 /
- 核苷
Abstract: OBJECTIVE To develop a method for the determination of saponins, amino acids and nucleosides in roots and rhizomes of Panax japonicus by UFLC-QTRAP-MS/MS. METHODS The extract was separated on a XBridge^®C₁₈(4.6 mm×100 mm, 3.5 μm) at 30 ℃ with a gradient elution of 0.1% formic acid solution -0.1% formic acid acetonitrile, and the flow rate was 0.8 mL·min^-1, using multiple-reaction monitoring (MRM) mode. Hierarchical cluster, nonlinear mapping and principal component analysis were adopted for comparison of roots and rhizomes. RESULTS 33 constituents showed good linearity (r>0.999 0) in the range of the tested concentration; the precision, repeatability and stability were good; the average recovery rates were between 96.90% and 101.6%, the relative standard deviations were less than or equal to 3.6%. CONCLUSION The results of data analysis showed that roots were similar to rhizomes according to contents of 33 constituents. The established method was accurate and reliable, which could used as a reference for quality evaluation and comprehensive control of Panax japonicus, and provided basic information for the development and utilization of roots of Panax japonicus.
- UFLC-QTRAP-MS/MS /
- Panax japonicus /
- root /
- rhizome /
- saponin /
- amino acid /
- nucleoside

HTML全文

图 1 33种成分的MRM图

Figure 1. Multi-reaction monitoring (MRM) of 33 constituents

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图 2 竹节参根与根茎的总离子流色谱图

Figure 2. Total ion chromatogram (TIC) of roots and rhizomes of Panax japonicus

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图 3 竹节参根与根茎的总含量(A)及百分含量比较图(B)

注：As.氨基酸; Ns.核苷; Ss.皂苷

Figure 3. The total content (A) and percentage content (B) in roots and rhizomes of Panax japonicus

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图 4 竹节参根与根茎的聚类分析结果

Figure 4. Results of hierarchical cluster analysis of roots and rhizomes of Panax japonicus

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图 5 竹节参根与根茎的非线性映射分析结果

Figure 5. Results of nonlinear mapping analysis of roots and rhizomes of Panax japonicus

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图 6 主成分的方差贡献率累积图

Figure 6. Accumulative variance contribution of principal components

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图 7 7个主成分的特征值

Figure 7. Eigenvalues of seven principal components

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表 1 竹节参样品信息

Table 1. Information of samples of Panax japonicus

样品	产地
R1(根), RM1(根茎)	湖北恩施(栽培)
R2(根), RM2(根茎)	湖北恩施(栽培)
R3(根), RM3(根茎)	湖北恩施(栽培)
R4(根), RM4(根茎)	湖北恩施(栽培)
R5(根), RM5(根茎)	四川巴中(栽培)
R6(根), RM6(根茎)	四川普洱(栽培)
R7(根), RM7(根茎)	四川普洱(栽培)
R8(根), RM8(根茎)	云南文山(栽培)
R9(根), RM9(根茎)	湖北恩施(栽培)
R10(根), RM10(根茎)	湖北恩施(栽培)
R11(根), RM11(根茎)	江西遂川(野生)
R12(根), RM12(根茎)	四川成都(野生)
R13(根), RM13(根茎)	四川乐山(野生)

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表 2 优化的质谱条件参数

Table 2. Optimized MS/MS parameters

化合物	分子式	t_R/min	MRM参数
化合物	分子式	t_R/min	离子对m/z	碰撞电压/V	碰撞能量/eV	离子模式
L-His	C₆H₉N₃O₂	1.15	156.080/110.030	100.00	16.00	ESI⁺
L-Arg	C₆H₁₄N₄O₂	1.17	175.120/70.020	100.00	18.00	ESI⁺
L-Ser	C₃H₇NO₃	1.24	106.050/59.990	100.00	8.00	ESI⁺
L-Ala	C₃H₇NO₂	1.25	90.060/44.020	100.00	10.00	ESI⁺
L-Asp	C₄H₇NO₄	1.25	134.050/87.960	59.00	10.00	ESI⁺
L-Thr	C₄H₉NO₃	1.27	120.070/74.000	100.00	20.00	ESI⁺
L-Pro	C₅H₉NO₂	1.35	116.070/70.020	68.00	10.00	ESI⁺
2'-Deoxycyt	C₉H₁₃N₃O₄	1.45	228.200/112.053	76.00	13.00	ESI⁺
Cyt	C₉H₁₃N₃O₅	1.85	244.090/112.000	61.00	10.00	ESI⁺
L-Met	C₅H₁₁NO₂S	2.02	150.060/104.030	91.00	10.00	ESI⁺
Uri	C₁₀H₁₃N₅O₅	2.51	244.896/113.000	10.00	13.00	ESI⁺
L-Isoleu	C₆H₁₃NO	3.01	132.100/86.050	100.00	10.00	ESI⁺
L-Leu	C₆H₁₃NO₂	3.31	132.100/86.050	100.00	10.00	ESI⁺
Ade	C₁₀H₁₃N₅O₄	3.34	268.100/136.070	86.00	23.00	ESI⁺
Gua	C₁₀H₁₃N₅O₅	3.89	284.300/152.000	62.00	15.00	ESI⁺
Ino	C₁₀H₁₂N₄O₅	3.95	269.000/137.070	46.00	15.00	ESI⁺
2'-Deoxygua	C₁₀H₁₃N₅O₄	4.42	268.100/152.100	61.00	15.00	ESI⁺
L-Phe	C₉H₁₁NO₂	6.45	166.100/120.050	100.00	14.00	ESI⁺
Thy	C₁₀H₁₄N₂O₅	6.59	243.100/127.070	61.00	13.00	ESI⁺
N-R₁	C₄₇H₈₀O₁₈	9.01	932.412/638.300	-225.00	-50.00	ESI^-
G-Rg₁	C₄₂H₇₂O₁₄	9.66	845.680/637.400	-20.00	-35.00	ESI^-
G-Re	C₄₈H₈₂O₁₈	9.68	991.700/799.400	-120.00	-45.00	ESI^-
G-Rb₁	C₅₄H₉₂O₂₃	10.50	1109.600/352.00	111.00	31.00	ESI⁺
G-Rc	C₅₃H₉₀O₂₂	10.70	1077.600/1077.600	-120.00	-60.00	ESI^-
G-Rb₂	C₅₃H₉₀O₂₂	10.80	1077.600/1077.600	-120.00	-60.00	ESI^-
P-F₁₁	C₄₂H₇₂O₁₄	10.90	845.400/653.400	-110.00	-50.00	ESI^-
G-Ro	C₄₈H₇₆O₁₉	11.00	955.585/793.400	-5.00	-60.00	ESI^-
N-R₂(20S)	C₄₁H₇₀O₁₃	11.10	769.427/637.400	-25.00	-38.00	ESI^-
G-Rg₂(20S)	C₄₂H₇₂O₁₃	11.30	829.600/637.400	-20.00	-40.00	ESI^-
P-RT₁	C₄₇H₇₄O₁₈	11.40	949.380/641.300	261.00	55.00	ESI⁺
C-Ⅳ	C₄₇H₇₄O₁₈	11.40	926.000/569.300	-15.00	-60.00	ESI^-
G-Rd	C₄₈H₈₂O₁₈	11.50	991.700/783.400	-120.00	-55.00	ESI^-
C-Ⅳa	C₄₂H₆₆O₁₄	11.90	793.530/631.200	-185.00	-58.00	ESI^-

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表 3 33种成分的回归方程、相关系数、检测限和定量限

Table 3. Linear regression equations, correlation coefficients, limits of detection and limits of quantification of 33 compounds

化合物	标准曲线	r	线性范围/(ng·mL^-1)	LOD/(ng·mL^-1)	LOQ/(ng·mL^-1)
L-His	Y=128X+1.74×10³	0.999 6	4.5~900	0.27	0.81
L-Arg	Y=44.2X+1.08×10⁴	0.999 4	21~3.36×10⁵	2.08	6.25
L-Ser	Y=2.32X+2.54×10³	0.999 1	1.4~5.6×10⁴	0.33	0.99
L-Ala	Y=2.57X+553	0.999 7	32.7~1.044×10⁵	3.76	11.28
L-Asp	Y=206X+1.44×10⁴	0.999 7	8.5~1.36×10⁴	1.07	3.22
L-Thr	Y=5.39X+537	0.999 3	0.67~2.7×10⁴	0.13	0.39
L-Pro	Y=142X+1.14×10⁵	0.999 6	12.8~2.56×10⁵	3.39	10.16
2’-Deoxycyt	Y=393X-946	0.999 7	1.5~300	0.25	0.75
Cyt	Y=209X+274	0.999 7	0.85~6.78×10³	0.25	0.75
L-Met	Y=72X+697	0.999 2	0.75~2.4×10³	0.15	0.45
Uri	Y=1 740X-4.83×10⁴	0.999 7	29.9~5.98×10³	1.50	4.50
L-Isoleu	Y=188X+2.27×10⁴	0.999 4	2.33~1.862×10⁴	0.23	0.69
L-Leu	Y=79.5X+3.33×10⁴	0.999 3	25.6~4.08×10⁴	5.12	15.36
Ade	Y=3 950X+4.11×10⁴	0.999 6	1.56~1.248×10⁴	0.17	0.51
Gua	Y=359X+2.28×10⁵	0.999 3	12.8~1.28×10⁵	2.02	6.06
Ino	Y=4 650X+1.12×10⁵	0.999 7	12.4~4.97×10³	2.48	7.44
2’-Deoxygua	Y=3 950X-2.88×10⁵	0.999 7	1.5~300	0.15	0.45
L-Phe	Y=124X+891	0.999 4	0.65~2.58×10⁴	0.13	0.39
Thy	Y=178X-403	0.999 4	4.12~1.65×10³	0.82	2.46
N-R₁	Y=8.96X+222	0.999 6	12.7~1.014×10⁴	2.54	7.62
G-Rg₁	Y=54.2X+1.15×10⁴	0.999 3	7.23~2.312×10⁵	1.45	4.35
G-Re	Y=3.85X+3.17×10³	0.999 4	33.3~5.32×10⁵	6.65	19.95
G-Rb₁	Y=54.3X+3.95×10³	0.999 9	12.5~1.002×10⁵	2.51	7.53
G-Rc	Y=27.5X-280	0.999 7	12.5~4.99×10³	0.25	0.75
G-Rb₂	Y=6.32X+234	0.999 8	3.7~7.4×10³	1.11	3.33
P-F₁₁	Y=140X+1.85×10⁴	0.999 4	19.2~1.232×10⁵	3.84	11.52
G-Ro	Y=15.4X+2.34×10⁴	0.999 1	61.2~2.44×10⁵	4.90	14.70
N-R₂(20S)	Y=3.47X+1.1×10³	0.999 5	6.78~2.71×10⁴	1.36	4.08
G-Rg₂(20S)	Y=202X+4.26×10³	0.999 1	3.35~1.072×10⁵	0.67	2.01
P-RT₁	Y=7.97X+1.29×10⁴	0.999 1	31.2~1×10⁶	6.24	18.72
C-Ⅳ	Y=16.9X+1.17×10³	1.000 0	110~4.4×10⁵	6.13	18.40
G-Rd	Y=15.6X+1.04×10⁴	0.999 9	31.4~1.26×10⁵	6.28	18.84
C-Ⅳa	Y=64.2X+8.91×10⁴	0.999 3	8.29~3.32×10⁵	0.81	2.43

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表 4 竹节参根与根茎样品的主成分得分、综合得分(从低到高)

Table 4. Principal component scores, comprehensive evaluation in roots and rhizomes of Panax japonicus (from low to high)

样品	主成分得分							综合得分
样品	PC1	PC2	PC3	PC4	PC5	PC6	PC7	综合得分
R7	-3.882	-1.972	-2.568	1.078	-0.883	-0.659	-1.074	-1.862
RM7	-3.816	-3.094	-4.928	2.656	0.141	-0.615	0.542	-1.270
RM13	-3.747	0.424	0.247	0.018	1.684	-1.276	-1.727	-0.878
R13	-3.704	1.464	0.195	1.666	1.714	-1.561	1.112	-0.617
R4	-3.175	-1.800	3.536	-0.227	-0.828	-0.098	-0.079	-0.390
RM12	-2.849	4.628	0.799	0.918	-0.719	-0.309	-0.790	-0.326
RM4	-2.832	-2.537	3.909	-0.709	1.274	1.215	-1.118	-0.281
R6	-2.801	2.009	-1.471	0.068	-0.608	0.177	0.859	-0.249
RM6	-1.877	5.367	-1.637	-0.160	2.075	2.606	0.991	-0.202
RM3	-1.204	-1.815	2.460	-1.098	-0.019	-0.228	1.051	-0.160
R10	-0.997	-1.434	3.092	-0.963	0.386	-0.818	-0.376	-0.121
RM11	-0.725	2.147	-0.440	0.075	-1.082	-0.432	-0.609	-0.100
R12	-0.535	3.189	0.209	-0.085	-2.694	0.499	0.111	-0.086
RM8	-0.390	-1.127	0.417	-0.303	1.329	0.076	2.892	-0.068
R8	0.190	-1.167	0.596	-1.302	-1.286	0.030	0.913	-0.054
RM10	0.467	-0.661	0.840	-0.791	0.174	-0.164	0.500	-0.041
RM5	0.857	-1.567	-1.814	-2.117	1.466	1.960	-1.808	-0.029
R11	1.231	4.324	0.051	-0.574	-1.473	-0.387	-1.262	-0.023
R3	1.676	-1.039	1.764	-1.103	-1.294	-0.217	0.674	-0.017
R9	2.127	-1.508	-3.654	-3.485	1.143	-0.875	-0.387	-0.012
R5	2.586	-1.014	-1.738	-3.293	-0.655	1.042	-0.384	-0.009
R2	2.739	-2.012	-1.482	0.556	-2.042	0.766	0.978	-0.007
RM2	3.247	-1.268	0.424	3.086	0.016	1.928	0.416	-0.004
R1	4.016	-2.331	-0.969	1.897	-0.373	-1.730	-0.688	-0.002
RM1	5.008	-0.710	1.485	4.579	0.794	1.224	-1.211	0.000
RM9	8.391	3.504	0.677	-0.386	1.759	-2.154	0.472	0.000

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