药学学报

No.	Compound	t_R/min	Formula	Calculated m/z	Extracted ion m/z
1	Allantoin	1.11	C₄H₆N₄O₃	159.051 3	159.050 5-159.052 1 [M+H]⁺
2	L-Proline	1.13	C₅H₉NO₂	116.070 6	116.070 0-116.071 2 [M+H]⁺
3	Pyroglutamic acid	2.24	C₅H₇NO₃	130.049 9	130.049 2-130.050 6 [M+H]⁺
4	Hordenine	5.95	C₁₀H₁₅NO	166.122 6	166.121 8-166.123 4 [M+H]⁺
5	Adenosine	6.85	C₁₀H₁₃N₅O₄	268.104 0	268.102 7-268.105 3 [M+H]⁺
6	L-Phenylalanine	6.89	C₉H₁₁NO₂	166.086 2	166.085 4-166.087 0 [M+H]⁺
7	Guanosine	8.82	C₁₀H₁₃N₅O₅	284.099 0	284.097 6-284.100 4 [M+H]⁺
8	L-Tryptophan	14.32	C₁₁H₁₂N₂O₂	205.097 1	205.096 1-205.098 1 [M+H]⁺
9	Caffeic acid	20.68	C₉H₈O₄	179.035 0	179.034 1-179.035 9 [M-H]^-
10	Calycosin-7-glucoside	29.77	C₂₂H₂₂O₁₀	447.128 6	447.126 4-447.130 8 [M+H]⁺
11	Verbascoside	31.58	C₂₉H₃₆O₁₅	623.198 1	623.197 0-623.203 2 [M-H]^-
12	Isoacteoside	34.07	C₂₉H₃₆O₁₅	623.198 1	623.197 0-623.203 2 [M-H]^-
13	Ononin	36.65	C₂₂H₂₂O₉	431.133 6	431.131 4-431.135 8 [M+H]⁺
14	Calycosin	40.11	C₁₆H₁₂O₅	285.075 8	285.074 4-285.077 2 [M+H]⁺
15	3-Hydroxy-9, 10-dimethoxyptercarpan	42.74	C₁₇H₁₆O₅	301.107 0	301.105 5-301.108 5 [M+H]⁺
16	Formononetin	46.28	C₁₆H₁₂O₄	269.080 8	269.079 5-269.082 1 [M+H]⁺
17	Atractylenolide Ⅲ	49.12	C₁₅H₂₀O₃	249.148 3	249.147 1-249.149 5 [M+H]⁺
18	Atractylenolide Ⅱ	54.02	C₁₅H₂₀O₂	233.153 4	233.152 2-233.154 6 [M+H]⁺
19	Astragaloside A	62.78	C₄₁H₆₈O₁₄	829.459 0	829.454 9-829.463 1 [M+HCOO]^-

No.	Compound	t_R/min	Formula	Calculated m/z	Extracted ion m/z
1	Allantoin	1.11	C₄H₆N₄O₃	159.051 3	159.050 5-159.052 1 [M+H]⁺
2	L-Proline	1.13	C₅H₉NO₂	116.070 6	116.070 0-116.071 2 [M+H]⁺
3	Pyroglutamic acid	2.24	C₅H₇NO₃	130.049 9	130.049 2-130.050 6 [M+H]⁺
4	Hordenine	5.95	C₁₀H₁₅NO	166.122 6	166.121 8-166.123 4 [M+H]⁺
5	Adenosine	6.85	C₁₀H₁₃N₅O₄	268.104 0	268.102 7-268.105 3 [M+H]⁺
6	L-Phenylalanine	6.89	C₉H₁₁NO₂	166.086 2	166.085 4-166.087 0 [M+H]⁺
7	Guanosine	8.82	C₁₀H₁₃N₅O₅	284.099 0	284.097 6-284.100 4 [M+H]⁺
8	L-Tryptophan	14.32	C₁₁H₁₂N₂O₂	205.097 1	205.096 1-205.098 1 [M+H]⁺
9	Caffeic acid	20.68	C₉H₈O₄	179.035 0	179.034 1-179.035 9 [M-H]^-
10	Calycosin-7-glucoside	29.77	C₂₂H₂₂O₁₀	447.128 6	447.126 4-447.130 8 [M+H]⁺
11	Verbascoside	31.58	C₂₉H₃₆O₁₅	623.198 1	623.197 0-623.203 2 [M-H]^-
12	Isoacteoside	34.07	C₂₉H₃₆O₁₅	623.198 1	623.197 0-623.203 2 [M-H]^-
13	Ononin	36.65	C₂₂H₂₂O₉	431.133 6	431.131 4-431.135 8 [M+H]⁺
14	Calycosin	40.11	C₁₆H₁₂O₅	285.075 8	285.074 4-285.077 2 [M+H]⁺
15	3-Hydroxy-9, 10-dimethoxyptercarpan	42.74	C₁₇H₁₆O₅	301.107 0	301.105 5-301.108 5 [M+H]⁺
16	Formononetin	46.28	C₁₆H₁₂O₄	269.080 8	269.079 5-269.082 1 [M+H]⁺
17	Atractylenolide Ⅲ	49.12	C₁₅H₂₀O₃	249.148 3	249.147 1-249.149 5 [M+H]⁺
18	Atractylenolide Ⅱ	54.02	C₁₅H₂₀O₂	233.153 4	233.152 2-233.154 6 [M+H]⁺
19	Astragaloside A	62.78	C₄₁H₆₈O₁₄	829.459 0	829.454 9-829.463 1 [M+HCOO]^-

Analyte	Regression equation	Linearity range /μg·mL^-1	r	LOQ /μg·mL^-1	LOD /μg·mL^-1
Allantoin	Y = 5 472 828 X + 479 320	4.83-144.96	0.999 7	0.060	0.020
L-Proline	Y = 86 975 923 X + 5 638 716	4.24-127.20	0.999 9	0.085	0.028
Pyroglutamic acid	Y = 64 631 208 X + 2 389 539	1.60-47.88	0.999 8	0.032	0.011
Hordenine	Y = 723 082 195 X + 33 356 735	0.30-8.89	0.999 8	0.005	0.002
Adenosine	Y = 266 200 177 X + 9 443 073	0.32-9.57	0.999 6	0.020	0.007
L-Phenylalanine	Y = 247 574 407 X + 9 293 926	1.41-42.21	0.999 6	0.035	0.012
Guanosine	Y = 40 699 138 X + 2 453 794	0.74-22.18	0.999 8	0.009	0.003
L-Tryptophan	Y = 195 962 721 X + 1 750 113	0.26-7.92	0.999 1	0.005	0.002
Caffeic acid	Y = 169 287 666 X + 8 768 389	0.18-5.46	0.999 5	0.069	0.023
Calycosin-7-glucoside	Y = 57 987 427 X+ 2 827 590	2.68-80.32	1.000 0	0.031	0.010
Verbascoside	Y = 32 726 217 X + 1 202 329	0.41-12.38	0.999 5	0.007	0.002
Isoacteoside	Y = 44 913 759 X - 6 465 736	0.77-23.10	0.999 9	0.011	0.003
Ononin	Y = 86 567 555 X + 60 511 347	0.76-22.86	0.999 6	0.008	0.003
Calycosin	Y = 229 728 032 X + 54 122 589	0.90-27.09	0.999 9	0.023	0.008
3-Hydroxy-9, 10-dimethoxyptercarpan	Y = 284 043 686 X + 11 858 456	0.24-7.16	0.999 8	0.003	0.001
Formononetin	Y = 336 848 761 X + 44 638 709	0.37-11.02	1.000 0	0.013	0.004
Atractylenolide Ⅲ	Y = 65 804 295 X + 4 155 839	0.29-8.64	0.999 7	0.097	0.032
Atractylenolide Ⅱ	Y = 1 117 680 022 X - 1 738 208	0.027 5-0.825	0.999 7	0.010	0.003
Astragaloside A	Y = 2 129 447 X + 143 154	0.885-26.55	0.999 9	0.017	0.006

Analyte	Regression equation	Linearity range /μg·mL^-1	r	LOQ /μg·mL^-1	LOD /μg·mL^-1
Allantoin	Y = 5 472 828 X + 479 320	4.83-144.96	0.999 7	0.060	0.020
L-Proline	Y = 86 975 923 X + 5 638 716	4.24-127.20	0.999 9	0.085	0.028
Pyroglutamic acid	Y = 64 631 208 X + 2 389 539	1.60-47.88	0.999 8	0.032	0.011
Hordenine	Y = 723 082 195 X + 33 356 735	0.30-8.89	0.999 8	0.005	0.002
Adenosine	Y = 266 200 177 X + 9 443 073	0.32-9.57	0.999 6	0.020	0.007
L-Phenylalanine	Y = 247 574 407 X + 9 293 926	1.41-42.21	0.999 6	0.035	0.012
Guanosine	Y = 40 699 138 X + 2 453 794	0.74-22.18	0.999 8	0.009	0.003
L-Tryptophan	Y = 195 962 721 X + 1 750 113	0.26-7.92	0.999 1	0.005	0.002
Caffeic acid	Y = 169 287 666 X + 8 768 389	0.18-5.46	0.999 5	0.069	0.023
Calycosin-7-glucoside	Y = 57 987 427 X+ 2 827 590	2.68-80.32	1.000 0	0.031	0.010
Verbascoside	Y = 32 726 217 X + 1 202 329	0.41-12.38	0.999 5	0.007	0.002
Isoacteoside	Y = 44 913 759 X - 6 465 736	0.77-23.10	0.999 9	0.011	0.003
Ononin	Y = 86 567 555 X + 60 511 347	0.76-22.86	0.999 6	0.008	0.003
Calycosin	Y = 229 728 032 X + 54 122 589	0.90-27.09	0.999 9	0.023	0.008
3-Hydroxy-9, 10-dimethoxyptercarpan	Y = 284 043 686 X + 11 858 456	0.24-7.16	0.999 8	0.003	0.001
Formononetin	Y = 336 848 761 X + 44 638 709	0.37-11.02	1.000 0	0.013	0.004
Atractylenolide Ⅲ	Y = 65 804 295 X + 4 155 839	0.29-8.64	0.999 7	0.097	0.032
Atractylenolide Ⅱ	Y = 1 117 680 022 X - 1 738 208	0.027 5-0.825	0.999 7	0.010	0.003
Astragaloside A	Y = 2 129 447 X + 143 154	0.885-26.55	0.999 9	0.017	0.006

Analyte	S1	S2	S3	S4	S5	S6	S7	S8	S9	S10	Average
Allantoin	137.04	116.49	94.48	150.01	126.94	133.80	121.15	123.79	136.08	134.31	127.41
L-Proline	78.40	83.66	49.48	72.48	71.10	76.36	73.42	71.93	68.85	65.96	71.16
Pyroglutamic acid	32.42	42.20	24.90	36.66	29.05	35.43	33.28	26.13	33.80	24.97	31.88
Hordenine	5.01	4.52	4.69	4.82	4.98	5.23	5.11	5.82	5.08	6.01	5.13
Adenosine	12.61	11.74	9.03	11.68	11.03	10.91	11.38	10.77	13.37	12.80	11.53
L-Phenylalanine	33.38	27.00	21.71	31.11	28.43	29.72	32.11	25.49	28.00	30.49	28.74
Guanosine	25.23	26.71	17.81	22.38	20.87	22.72	23.00	21.70	25.43	24.45	23.03
L-Tryptophan	6.61	5.71	4.56	5.78	5.47	5.59	6.31	5.78	6.01	6.09	5.79
Caffeic acid	4.45	3.13	2.30	4.08	4.32	3.80	4.20	3.54	4.67	4.31	3.88
Calycosin-7-glucoside	61.19	46.43	38.36	60.40	57.48	60.02	60.29	50.71	48.83	42.93	52.66
Verbascoside	9.73	5.78	5.41	8.49	12.09	9.16	10.24	8.46	8.59	10.97	8.89
Isoacteoside	19.71	14.42	9.67	15.49	22.06	14.18	20.67	19.22	18.67	22.02	17.61
Ononin	44.09	17.01	14.17	21.54	20.50	22.32	20.30	17.17	17.14	16.24	21.05
Calycosin	23.81	16.61	13.09	18.38	20.98	20.04	14.93	13.41	23.52	18.83	18.36
3-Hydroxy-9, 10-dimethoxyptercarpan	7.00	5.52	4.49	5.78	6.91	6.81	4.15	3.93	7.38	5.99	5.80
Formononetin	8.98	6.62	5.21	6.43	7.86	7.89	4.50	3.89	7.55	6.87	6.58
Atractylenolide Ⅲ	10.00	8.42	7.42	8.58	10.40	10.18	7.32	6.78	10.22	11.38	9.07
Atractylenolide Ⅱ	0.59	0.51	0.48	0.49	0.68	0.61	0.30	0.29	0.61	0.62	0.52
Astragaloside A	32.47	27.80	29.08	30.52	30.02	27.92	27.56	27.08	50.18	42.29	32.49
Total	552.72	470.28	356.34	515.10	491.17	502.69	480.22	445.89	513.98	487.53	-

Analyte	S1	S2	S3	S4	S5	S6	S7	S8	S9	S10	Average
Allantoin	137.04	116.49	94.48	150.01	126.94	133.80	121.15	123.79	136.08	134.31	127.41
L-Proline	78.40	83.66	49.48	72.48	71.10	76.36	73.42	71.93	68.85	65.96	71.16
Pyroglutamic acid	32.42	42.20	24.90	36.66	29.05	35.43	33.28	26.13	33.80	24.97	31.88
Hordenine	5.01	4.52	4.69	4.82	4.98	5.23	5.11	5.82	5.08	6.01	5.13
Adenosine	12.61	11.74	9.03	11.68	11.03	10.91	11.38	10.77	13.37	12.80	11.53
L-Phenylalanine	33.38	27.00	21.71	31.11	28.43	29.72	32.11	25.49	28.00	30.49	28.74
Guanosine	25.23	26.71	17.81	22.38	20.87	22.72	23.00	21.70	25.43	24.45	23.03
L-Tryptophan	6.61	5.71	4.56	5.78	5.47	5.59	6.31	5.78	6.01	6.09	5.79
Caffeic acid	4.45	3.13	2.30	4.08	4.32	3.80	4.20	3.54	4.67	4.31	3.88
Calycosin-7-glucoside	61.19	46.43	38.36	60.40	57.48	60.02	60.29	50.71	48.83	42.93	52.66
Verbascoside	9.73	5.78	5.41	8.49	12.09	9.16	10.24	8.46	8.59	10.97	8.89
Isoacteoside	19.71	14.42	9.67	15.49	22.06	14.18	20.67	19.22	18.67	22.02	17.61
Ononin	44.09	17.01	14.17	21.54	20.50	22.32	20.30	17.17	17.14	16.24	21.05
Calycosin	23.81	16.61	13.09	18.38	20.98	20.04	14.93	13.41	23.52	18.83	18.36
3-Hydroxy-9, 10-dimethoxyptercarpan	7.00	5.52	4.49	5.78	6.91	6.81	4.15	3.93	7.38	5.99	5.80
Formononetin	8.98	6.62	5.21	6.43	7.86	7.89	4.50	3.89	7.55	6.87	6.58
Atractylenolide Ⅲ	10.00	8.42	7.42	8.58	10.40	10.18	7.32	6.78	10.22	11.38	9.07
Atractylenolide Ⅱ	0.59	0.51	0.48	0.49	0.68	0.61	0.30	0.29	0.61	0.62	0.52
Astragaloside A	32.47	27.80	29.08	30.52	30.02	27.92	27.56	27.08	50.18	42.29	32.49
Total	552.72	470.28	356.34	515.10	491.17	502.69	480.22	445.89	513.98	487.53	-

基于UHPLC-Q-exactive orbitrap MS和化学计量学分析的芪龙壮儿口服液质量评价研究

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崔小敏 ¹ , 董明芝 ² , 张红 ¹ , 李建刚 ³ , 任慧 ¹ , 胡静 ¹ , 鲁文静 ¹ , 陈娟 ¹ , 孟雪 ¹ , 陈志永 ¹^,^*

药学学报 | 研究论文 2023,58(6): 1655-1662

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药学学报 |研究论文 2023 , 58 (6) : 1655 -1662

基于UHPLC-Q-exactive orbitrap MS和化学计量学分析的芪龙壮儿口服液质量评价研究

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崔小敏¹, 董明芝², 张红¹, 李建刚³, 任慧¹, 胡静¹, 鲁文静¹, 陈娟¹, 孟雪¹, 陈志永¹^,^*

作者信息

1.陕西省中医药研究院, 陕西西安 710061

2.西安正大制药有限公司, 陕西西安 710043

3.西安培华学院, 陕西西安 710199

通讯作者:

*陈志永, Tel: 86-29-85395696, E-mail: chenzhiyong0612@sina.com

Quality evaluation of Qilong Zhuang'er oral liquid based on UHPLC-Q-exactive orbitrap MS and chemometrics analysis

Xiao-min CUI¹, Ming-zhi DONG², Hong ZHANG¹, Jian-gang LI³, Hui REN¹, Jing HU¹, Wen-jing LU¹, Juan CHEN¹, Xue MENG¹, Zhi-yong CHEN¹^,^*

Affiliations

1. Shaanxi Academy of Traditional Chinese Medicine, Xi'an 710061, China

2. Xi'an C.P. Pharmaceutical Co., Ltd., Xi'an 710043, China

3. Xi'an Peihua University, Xi'an 710199, China

出版时间: 2023-06-12 doi: 10.16438/j.0513-4870.2022-1249

文章导航

摘要

收起

建立同时测定芪龙壮儿口服液中19种化学成分的UHPLC-Q-exactive orbitrap MS定量分析方法, 并结合化学计量学分析比较不同批次样品的质量差异, 为该制剂的质量评价提供依据。采用UHPLC-Q-exactive orbitrap MS方法测定了10批芪龙壮儿口服液中尿囊素、L-脯氨酸、焦谷氨酸、大麦芽碱、腺苷、L-苯丙氨酸、鸟苷、L-色氨酸、咖啡酸、毛蕊异黄酮苷、毛蕊花糖苷、异毛蕊花糖苷、芒柄花苷、毛蕊异黄酮、3-羟基-9, 10-二甲氧基紫檀烷、芒柄花素、白术内酯Ⅲ、白术内酯Ⅱ和黄芪甲苷的含量, 并利用Hiplot开源科研绘图平台及Markerlynx^XS软件对含量数据进行聚类热图分析、主成分分析(principal component analysis, PCA)和偏最小二乘判别分析(partial least squares discriminant analysis, PLS-DA), 综合评价不同批次芪龙壮儿口服液的质量差异。19个化学成分在各自浓度范围内线性关系良好(r ≥ 0.999), 精密度、重复性、稳定性(24 h)试验的RSD均小于1.94%, 平均加样回收率为97.24%~102.75% (RSD均小于2.74%, n = 6)。聚类热图和PCA分析将10批样品分为两大类, PLS-DA法确定了7个差异标志物, 分别为3-羟基-9, 10-二甲氧基紫檀烷、白术内酯Ⅲ、毛蕊异黄酮、白术内酯Ⅱ、芒柄花素、尿囊素和咖啡酸。建立的芪龙壮儿口服液多成分定量方法结合化学计量学分析可以为该制剂质量评价提供参考。

关键词

芪龙壮儿口服液 / 轨道阱高分辨质谱 / 含量测定 / 化学计量学 / 质量评价

Abstract

收起

An UHPLC-Q-exactive orbitrap MS method for the simultaneous determination of 19 chemical components in Qilong Zhuang'er oral liquid was established and the quality differences between different batches of samples was compared by chemometric analysis to provide a basis for the quality evaluation of the preparation. The contents of allantoin, L-proline, pyroglutamic acid, hordenine, adenosine, L-phenylalanine, guanosine, L-tryptophan, caffeic acid, calycosin-7-glucoside, verbascoside, isoacteoside, ononin, calycosin, 3-hydroxy-9, 10-dimethoxyptercarpan, formononetin, atractylenolide Ⅲ, atractylenolide Ⅱ and astragaloside A were analyzed by cluster heat map, principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) using Hiplot platform and Markerlynx^XS software to comprehensively evaluate the quality difference of different batches of Qilong Zhuang'er oral liquid. The 19 chemical compounds showed good linearity in their respective concentration ranges (r ≥ 0.999). The RSD of precision, repeatability and stability (24 h) tests were all less than 1.94%. The average recovery was 97.24%-102.75% (RSD < 2.74%, n = 6). The 10 batches of samples were divided into two categories by cluster heat map and PCA analysis. 3-Hydroxy-9, 10-dimethoxyptercarpan, atractylenolide Ⅲ, calycosin, atractylenolide Ⅱ, formononetin, allantoin and caffeic acid were identified as differential markers by PLS-DA. The established multi component quantitative method of Qilong Zhuang'er oral liquid combined with chemometric analysis can provide reference for the quality evaluation of the preparation.

Key words

Qilong Zhuang'er oral liquid / orbitrap MS / determination / chemometric analysis / quality evaluation

引用本文

崔小敏, 董明芝, 张红, 李建刚, 任慧, 胡静, 鲁文静, 陈娟, 孟雪, 陈志永. 基于UHPLC-Q-exactive orbitrap MS和化学计量学分析的芪龙壮儿口服液质量评价研究. 药学学报, 2023 , 58 (6) : 1655 -1662 . DOI: 10.16438/j.0513-4870.2022-1249

Xiao-min CUI, Ming-zhi DONG, Hong ZHANG, Jian-gang LI, Hui REN, Jing HU, Wen-jing LU, Juan CHEN, Xue MENG, Zhi-yong CHEN. Quality evaluation of Qilong Zhuang'er oral liquid based on UHPLC-Q-exactive orbitrap MS and chemometrics analysis[J]. Acta Pharmaceutica Sinica, 2023 , 58 (6) : 1655 -1662 . DOI: 10.16438/j.0513-4870.2022-1249

正文

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芪龙壮儿口服液是由著名中医儿科专家午雪峤主任医师根据传统中医理论, 结合临床经验, 以黄芪、党参、熟地黄、当归、阿胶、鹿角胶、龟甲胶、龙骨(煅)、白术、山药、鸡内金、牡蛎(煅)、山楂和麦芽(炒)十四味药组方的OTC甲类中成药, 具有健脾补肾、益气养血、填精充髓、消食和胃的功效。药理学研究表明芪龙壮儿口服液能提高脾虚小鼠的免疫功能和机体应激能力, 增加脾虚小鼠、大鼠食欲等作用。

目前芪龙壮儿口服液的质量控制方法是采用薄层扫描法测定其中黄芪甲苷的含量^[1], 中药复方制剂药味复杂, 化学成分种类繁多, 具有多靶点和多作用机制等特征, 单指标的含量控制难以准确评价其综合质量。近年来, 多指标含量测定及化学计量学分析越来越多地应用于中成药复方制剂质控研究中^{[2, 3]}。

本课题组前期考察了HPLC-UV法测定芪龙壮儿口服液的方法, 发现HPLC-UV色谱图化学信息量很少, 而且黄芪甲苷等没有紫外吸收的化合物还需要建立HPLC-ELSD方法进行检测。UHPLC-Q-exactive orbitrap MS法具有高灵敏度、高分辨率、高选择性等优势, 弥补了液相色谱紫外检测器只能检测有紫外吸收的化合物、灵敏度低等方面的不足。本研究基于芪龙壮儿口服液调节免疫的主要药理功效及成分的可测性, 选择尿囊素、L-脯氨酸、焦谷氨酸、大麦芽碱、腺苷、L-苯丙氨酸、鸟苷、L-色氨酸、咖啡酸、毛蕊异黄酮苷、毛蕊花糖苷、异毛蕊花糖苷、芒柄花苷、毛蕊异黄酮、3-羟基-9, 10-二甲氧基紫檀烷、芒柄花素、白术内酯Ⅲ、白术内酯Ⅱ和黄芪甲苷共19种化学成分作为含量测定的指标, 建立了同时测定19个指标性成分的UHPLC-Q-exactive orbitrap MS定量分析方法, 并结合化学计量学分析, 全面、综合地评价芪龙壮儿口服液的药品质量, 保证临床用药安全和有效。

材料与方法

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仪器液相系统为UltiMate 3000系列高效液相色谱仪(美国戴安公司); Q Exactive Focus静电场轨道阱高分辨质谱仪(美国Thermo Fisher Scientific公司), 配有可加热电喷雾离子源(HESI)及Xcalibur4.0数据处理软件。KQ-400DE型超声波清洗机(昆山市超声仪器有限公司); BT25S型十万分之一电子分析天平和BS210S型万分之一电子分析天平(北京赛多利斯科学仪器有限公司)。

材料尿囊素(批号MUST-22061307)、大麦芽碱(批号MUST-21102104)、腺苷(批号MUST-21070613)、鸟苷(批号MUST-22021408)、毛蕊花糖苷(批号MUST-21122712)、异毛蕊花糖苷(批号MUST-22030810)、白术内酯Ⅱ (批号MUST-21111614)、黄芪甲苷(批号MUST-17022804)购自成都曼思特生物科技有限公司; L-脯氨酸(批号HG7111W3)、焦谷氨酸(批号HR22271W2)、L-苯丙氨酸(批号HR83299W1)、3-羟基-9, 10-二甲氧基紫檀烷(批号HR2182W1)、白术内酯Ⅲ (批号HS20913B1)购自宝鸡辰光生物科技有限公司; 咖啡酸(批号17122804)购自成都普菲德生物技术有限公司; L-色氨酸(批号wkq22011707)、毛蕊异黄酮苷(批号wkq20010904)、芒柄花苷(批号wkq20060507)、毛蕊异黄酮(批号wkq20010906)、芒柄花素(批号wkq20062208)购自四川省维克奇生物科技有限公司, 以上对照品纯度均≥ 98%。甲醇和甲酸为色谱纯(美国Fisher试剂公司), 水为重蒸水, 其他试剂均为分析纯。

芪龙壮儿口服液[规格: 10 mL/瓶; 批号分别为211135 (S1), 211136 (S2), 211237 (S3), 211238 (S4), 211239 (S5), 211240 (S6), 211241 (S7), 2111242 (S8), 211243 (S9), 211244 (S10)], 由西安正大制药有限公司提供。

色谱条件 色谱柱为Thermo Accucore aQ RP18 (2.1 mm × 150 mm, 2.6 µm); 流动相为甲醇(A)-0.1%甲酸水溶液(B)梯度洗脱(0~5 min, 0% A; 5~45 min, 0%~58% A; 45~65 min, 58%~68% A); 柱温为25 ℃, 体积流量为0.3 mL·min^-1, 进样量为2 µL。

质谱条件 Thermo UHPLC-Q-Exactive Focus型液质联用仪。采用加热电喷雾离子源(HESI), 正、负离子同时检测模式, 喷雾电压为+3.5 kV/-2.8 kV, 碰撞电压为阶梯能量20、40、60 eV, 扫描方式为一级全扫描/数据依赖型二级质谱扫描(full MS/dd-MS²), Full MS分辨率为70 000, dd-MS²分辨率为17 500, 扫描范围为m/z 80~1 200, 离子传输毛细管温度为320 ℃, 透镜电压55 kPa, 雾化室温度350 ℃, 鞘气和辅助气流速分别为40和10 arb。

对照品储备溶液的配制 分别称取各对照品适量, 精密称定, 加甲醇制成含尿囊素241.60 μg·mL^-1、L-脯氨酸212.00 μg·mL^-1、焦谷氨酸79.80 μg·mL^-1、大麦芽碱15.96 μg·mL^-1、腺苷70.35 μg·mL^-1、L-苯丙氨酸14.81 μg·mL^-1、鸟苷36.98 μg·mL^-1、L-色氨酸13.20 μg·mL^-1、咖啡酸9.10 μg·mL^-1、毛蕊异黄酮苷133.88 μg·mL^-1、毛蕊花糖苷20.63 μg·mL^-1、异毛蕊花糖苷38.50 μg·mL^-1、芒柄花苷38.15 μg·mL^-1、毛蕊异黄酮45.15 μg·mL^-1、3-羟基-9, 10-二甲氧基紫檀烷11.93 μg·mL^-1、芒柄花素18.38 μg·mL^-1、白术内酯Ⅲ14.40 μg·mL^-1、白术内酯Ⅱ1.38 μg·mL^-1、黄芪甲苷44.25 μg·mL^-1的混合对照品储备溶液。

供试品溶液的制备 精密量取芪龙壮儿口服液2 mL, 置于15 mL离心管中, 加入8 mL乙腈, 于涡旋振荡器中振荡混匀5 min, 于4 ℃静置0.5 h后, 2 500 r·min^-1条件下离心20 min, 取上清液1 mL, 吹干溶剂, 残渣用30%甲醇-水溶解并定容至1 mL, 振摇, 0.22 μm微孔滤膜过滤, 即得。

系统适用性 取混合对照品溶液, 按上述色谱和质谱条件进样, 记录待测成分色谱峰的分离度、理论塔板数和拖尾因子。

专属性 分别按上述色谱和质谱条件进样空白溶液、混合对照品溶液和供试品溶液, 根据各待测成分的理论质荷比(表 1), Mass tolerance设为5 ppm, Mass precision设为4位数, 正模式检测的化合物分两组、负模式检测的化合物为一组, 采用Xcalibur 4.0软件获得各化合物提取离子流色谱图。比较空白溶液、混合对照品溶液和供试品溶液的色谱图。

线性与范围 分别精密量取混合对照品储备溶液0.1、0.2、0.5、1.0、2.0和3.0 mL置5 mL量瓶中并加甲醇稀释至刻度, 摇匀, 用0.22 μm微孔滤膜滤过, 进样。

仪器精密度 取同一混合对照品溶液, 按照色谱和质谱条件进样测定, 连续进样6次, 测定峰面积, 计算19种化学成分峰面积的RSD值。

稳定性 取同一份供试品溶液, 分别于0、2、4、8、12和24 h按照色谱和质谱条件进样测定。

重复性 取同一批次样品, 按“供试品溶液的制备”方法平行制备6份供试品溶液, 按照色谱和质谱条件进样测定, 计算待测成分含量及RSD。

加样回收率 精密吸取已知含量的芪龙壮儿口服液1 mL, 共6份, 分别以1∶1的比例加入各对照品溶液适量, 按“供试品溶液的制备”项下的方法制备供试品溶液, 按照色谱和质谱条件进样测定, 计算加样回收率及RSD。

耐用性 取同一批芪龙壮儿口服液2.0 mL, 按“供试品溶液的制备”项下方法制备供试品溶液, 根据上述色谱和质谱条件, 分别考察不同进样体积(1、2和3 μL), 不同流速(0.25、0.30和0.35 mL·min^-1), 不同柱温(24、25和26 ℃), 不同甲酸浓度(0.05%甲酸、0.1%甲酸和0.2%甲酸), 计算各待测成分的含量并计算不同条件的RSD值。

样品含量测定 分别取混合对照品溶液、10个不同批次的芪龙壮儿口服液按“供试品溶液的制备”方法制备供试品溶液, 按照色谱和质谱条件进样测定。外标法计算19个待测成分的含量。

聚类热图分析 为更直观地体现各批次样品间的质量差异, 对其含量测定结果进行聚类热图分析。将10批芪龙壮儿口服液的19个化学成分的含量数据导入Hiplot开源科研绘图平台(https://hiplot.com.cn/), 聚类方法采用ward法, 距离度量方法设置为euclidean, 标准化选择“row”。

PCA和OPLS-DA分析 PCA属于无监督模式识别方法, 利用方差最大原则, 对原始数据所包含的多个自变量进行线性拟合, 以新的低维变量代替原始高维变量, 即主成分, 进而实现数据的降维。将10批芪龙壮儿口服液样品的19个化学成分含量导入Waters Markerlynx^XS软件进行PCA分析。为明确引起产品质量差异的特征性成分, 进一步采用有监督的模式识别方法对原始数据进行PLS-DA分析, 绘制PLS-DA模型得分图。

结果

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1 系统适用性

混合对照品溶液提取离子流色谱图中各待测成分色谱峰与其相邻色谱峰之间的分离度均大于1.5; 理论塔板数以毛蕊异黄酮苷计不低于5 000; 拖尾因子在0.80~1.20之间, 表明系统适用性良好。

2 方法学考察

2.1 专属性

供试品溶液正、负离子模式下的总离子流基峰图见图 1。空白溶液、混合对照品溶液和供试品溶液的提取离子流色谱图见图 2。供试品溶液中19种待测成分峰形良好, 且保留时间与混合对照品基本一致, 空白样品在待测成分出峰处无干扰, 表明该方法专属性良好。

2.2 线性关系

以各对照品的质量浓度(X, μg·mL^-1)为横坐标, 峰面积值(Y)为纵坐标绘制标准曲线, 并计算得线性方程及相关系数, 表明各对照品在相应浓度范围内线性关系良好, 相关系数均大于0.999; 以信噪比3∶1和10∶1为标准分别测得各化合物的检测限和定量限, 结果见表 2。

2.3 仪器精密度

尿囊素、L-脯氨酸、焦谷氨酸、大麦芽碱、腺苷、L-苯丙氨酸、鸟苷、L-色氨酸、咖啡酸、毛蕊异黄酮苷、毛蕊花糖苷、异毛蕊花糖苷、芒柄花苷、毛蕊异黄酮、3-羟基-9, 10-二甲氧基紫檀烷、芒柄花素、白术内酯Ⅲ、白术内酯Ⅱ和黄芪甲苷的峰面积RSD分别为1.73%、1.88%、1.29%、1.74%、1.68%、1.53%、1.23%、1.67%、1.50%、1.77%、1.66%、1.49%、0.81%、1.55%、1.69%、1.93%、1.73%、0.58%、0.87%, 表明仪器精密度良好。

2.4 稳定性

尿囊素、L-脯氨酸、焦谷氨酸、大麦芽碱、腺苷、L-苯丙氨酸、鸟苷、L-色氨酸、咖啡酸、毛蕊异黄酮苷、毛蕊花糖苷、异毛蕊花糖苷、芒柄花苷、毛蕊异黄酮、3-羟基-9, 10-二甲氧基紫檀烷、芒柄花素、白术内酯Ⅲ、白术内酯Ⅱ和黄芪甲苷的峰面积RSD分别为1.32%、1.71%、1.25%、0.80%、1.62%、1.44%、1.61%、1.44%、0.34%、1.72%、1.84%、1.94%、1.28%、1.74%、1.30%、0.86%、1.29%、1.06%、1.80%, 表明该样品溶液在24 h内稳定性良好。

2.5 重复性

尿囊素、L-脯氨酸、焦谷氨酸、大麦芽碱、腺苷、L-苯丙氨酸、鸟苷、L-色氨酸、咖啡酸、毛蕊异黄酮苷、毛蕊花糖苷、异毛蕊花糖苷、芒柄花苷、毛蕊异黄酮、3-羟基-9, 10-二甲氧基紫檀烷、芒柄花素、白术内酯Ⅲ、白术内酯Ⅱ和黄芪甲苷的平均含量分别为94.71、49.68、24.70、9.42、8.99、21.22、17.58、4.62、2.33、38.28、5.43、9.86、14.35、13.13、4.47、5.24、7.47、0.55和28.71 μg·mL^-1, RSD分别为1.68%、0.94%、2.03%、1.65%、1.77%、1.65%、2.19%、0.93%、1.65%、1.37%、1.90%、2.02%、1.71%、1.43%、1.64%、1.54%、2.23%、1.53%和1.68%。结果表明该方法具有良好重复性。

2.6 加样回收率

尿囊素、L-脯氨酸、焦谷氨酸、大麦芽碱、腺苷、L-苯丙氨酸、鸟苷、L-色氨酸、咖啡酸、毛蕊异黄酮苷、毛蕊花糖苷、异毛蕊花糖苷、芒柄花苷、毛蕊异黄酮、3-羟基-9, 10-二甲氧基紫檀烷、芒柄花素、白术内酯Ⅲ、白术内酯Ⅱ和黄芪甲苷的平均回收率分别为101.40%、98.58%、100.22%、100.32%、101.33%、100.51%、99.62%、97.99%、97.24%、98.17%、100.06%、98.16%、98.90%、100.78%、102.75%、99.25%、98.10%、98.55%和98.87%, RSD分别为1.51%、1.39%、1.67%、2.27%、1.99%、1.99%、2.36%、1.82%、2.53%、2.13%、2.74%、2.27%、1.92%、1.61%、1.37%、2.69%、1.46%、2.33%和1.77%, 表明该方法回收率良好。

2.7 耐用性

结果表明, 各条件下的19种待测成分RSD值均小于2.56%, 说明方法耐用性良好。

3 样品含量测定

10批芪龙壮儿口服液中19个待测成分的含量测定结果见表 3。

4 化学计量学分析

4.1 聚类热图分析

分析结果见图 3。通过横向聚类可将10批样品聚为两大类, 第一类含4个批次(S2, S3, S7, S8), 第二类含6个批次(S1, S4, S5, S6, S9, S10), 第一类样品中的尿囊素、毛蕊异黄酮、黄芪紫檀烷、芒柄花素、白术内酯Ⅲ、白术内酯Ⅱ、黄芪甲苷含量相比第二类样品普遍较低。

4.2 PCA和OPLS-DA分析

PCA分析结果见图 4。10批样品大致可分为两类, 其中S2、S3、S7和S8归为第一类, S1、S4、S5、S6、S9和S10归为第二类, 与聚类热图分析结果一致。PLS-DA模型得分图见图 5A, 结果表明, 累积解释能力参数(RX²、RY²)分别为0.629和0.923, 预测能力参数Q²为0.785, 均大于0.5, 说明表明所建立的模型稳定可靠, 预测能力强, 可用于区分不同批次的芪龙壮儿口服液。根据变量重要性投影值(VIP)筛选10批样品差异化学成分, 以VIP > 1为标准, 其值越大, 表明对质量差异的贡献越大。根据图 5B可知, VIP > 1 (标红色)的有7个成分, 分别为3-羟基-9, 10-二甲氧基紫檀烷(VIP = 1.399 6)、白术内酯Ⅲ (VIP = 1.399 3)、毛蕊异黄酮(VIP = 1.354 4)、白术内酯Ⅱ (VIP = 1.343 6)、芒柄花素(VIP = 1.291 5)、尿囊素(VIP = 1.227 8)、咖啡酸(VIP = 1.121 5), 推断这7个成分为芪龙壮儿口服液产生质量差异的标志性成分。

讨论

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在制备样品溶液时需要对口服液中的糖浆和大分子蛋白类化合物进行沉淀去除, 分别考察了乙醇、甲醇和乙腈作为沉淀试剂, 结果乙腈的分层、沉淀效果最佳。将上清液吹干后选择复溶溶剂时, 分别考察了30%甲醇和甲醇对试验结果的影响, 结果表明, 30%甲醇作为复溶溶剂时各待测物峰形最佳。

在常规的HPLC检测条件下, 氨基酸类化合物通常需要经过繁琐的衍生化过程后进行紫外检测^{[4, 5]}; 而黄芪甲苷则需要蒸发光检测器来进行检测^[6]; 尿囊素和大麦芽碱仅有末端吸收^{[7, 8]}, 在常规紫外检测条件下干扰较大, 本研究所建立的UHPLC-Q-exactive orbitrap MS方法可在同一色谱条件下实现对样品中氨基酸、核苷、黄酮、皂苷等多种不同类型化合物的同时检测, 采用分辨率精度高达4位数的提取离子色谱峰记录峰面积, 峰形好、专属性高、抗基质干扰能力强。

在正、负离子同时检测模式下对样品溶液和对照品溶液进行扫描, 观察其信号响应, 结果表明, 咖啡酸、毛蕊花糖苷、毛蕊异花糖苷及黄芪甲苷在负离子模式下有更高的质谱响应, 因而选择在负离子模式下对这4个化合物进行检测, 其余氨基酸类、核苷类及其他化合物均在正离子模式下有更好的响应信号, 因此选择正离子模式进行检测。

测定了10批芪龙壮儿口服液中19个化学成分的含量, 19个测定成分的总量为356.34~552.72 μg·mL^-1, 其中尿囊素的含量最高, 平均为127.41 μg·mL^-1; 其次是L-脯氨酸, 平均含量为71.16 μg·mL^-1; 此外君药黄芪中的指标性成分也相对较高, 黄芪甲苷和毛蕊异黄酮苷的平均含量分别为32.49和52.66 μg·mL^-1。根据10批样品中19个成分的含量数据, 采用PLS-DA方法筛选出了3-羟基-9, 10-二甲氧基紫檀烷、白术内酯Ⅲ、毛蕊异黄酮、白术内酯Ⅱ、芒柄花素、尿囊素、咖啡酸7个差异性标志物, 主要来源于黄芪、白术、山药和山楂。黄芪是芪龙壮儿口服液的君药, 具有增强或抑制免疫的双向调节作用, 3-羟基-9, 10-二甲氧基紫檀烷、毛蕊异黄酮和芒柄花素为黄芪中主要的黄酮类物质, 具有免疫调节和抗氧化等药理作用^[9-12], 与其传统功效发挥密切相关; 白术内酯Ⅲ和白术内酯Ⅱ是白术的主要活性成分, 具有抗炎、抗肿瘤、免疫调节等多种药理作用^{[13, 14]}; 主要来源于山楂的咖啡酸具有抗炎、抗癌和抗病毒等诸多活性^[15]; 山药是临床应用中常见的养胃健脾中药, 尿囊素是山药中的重要活性成分^[16]。为保证成品制剂的质量均一性和疗效一致性, 应在原料选用中重点关注黄芪、白术、山药和山楂药材的质量情况。

作者贡献: 崔小敏负责实验研究、数据分析及文稿撰写; 董明芝和任慧负责实验材料收集、文章选题; 李建刚、鲁文静和胡静负责成分检测; 陈娟和孟雪负责数据的统计分析; 陈志永和张红对整个研究工作进行设计、监督和指导。

利益冲突: 本研究内容无任何利益冲突。

基金

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国家自然科学基金面上项目(81973419)
陕西省重点研发计划一般项目(2022SF-557)
陕西省中医药研究院“苗圃培育计划”项目(2021-17)

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：

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Zhang XX, Du DP. Determination of astragaloside A in Qilong Zhuang'er oral liquid by TLC scanning[J]. Northwest Pharm J (西北药学杂志), 2001, 16: 255.

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Si LT, Ni HF, Li Q, et al. Simultaneous determination of five saponins in Yaobitong capsule by HPLC-CAD[J]. Acta Pharm Sin (药学学报), 2020, 55: 2449-2453. http://www.yxxb.com.cn/aps/cn/article/doi/10.16438/j.0513-4870.2020-1129

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Li Q, Ren H, Guo S, et al. Determination of 12 components in Bufei Huoxue Capsule by UHPLC[J]. Chin Tradit Herb Drugs (中草药), 2022, 53: 3991-3996.

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Huang Y, Chen ML, Huang HZ, et al. Comparison of odor and quality of Galli Gigerii Endothelium Corneum derived from domestic chickens and broilers[J]. China J Chin Mater Med (中国中药杂志), 2022, 47: 5434-5442.

[5]

Yin NN, Guo DX. Simultaneous determination of 4 amino acids in Fufang Ejiao Buxue Granules by HPLC[J]. J Pharm Res (药学研究), 2019, 38: 389-392.

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Yang ZC, Sun CH, E XH, et al. Astragali Radix honey roasting process optimization based on response surface and weighted score[J]. Chin Tradit Herb Drugs (中草药), 2021, 52: 2247-2256.

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Liu YJ, Chu SF, Yuan ZY, et al. Simultaneous determination of allantoin, adenosine and phenylalanine in Rhizoma Dioscoreae, brained and fried Rhizoma Dioscoreae by HPLC[J]. Lishizhen Med Mater Med Res (时珍国医国药), 2019, 30: 568-570.

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Yang B, Yu GH, Li MY, et al. Mechanism of flavonoid components in Astragali Radix in inhibiting tumor growth and immunoregulation in C57BL/6 tumor bearing mice based on "invigorating Qi for consolidation of exterior"[J]. China J Chin Mater Med (中国中药杂志), 2019, 44: 5184-5190.

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Chen CY, Zu YG, Fu YJ, et al. Preparation and antioxidant activity of Radix Astragali residues extracts rich in calycosin and formononetin[J]. Biochem Eng J, 2011, 56: 84-93.

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Dai YT, Zhang XY, Wang YX, et al. Research progress on Astragali Radix and prediction of its quality markers (Q-markers)[J]. China J Chin Mater Med (中国中药杂志), 2022, 47: 1754-1764.

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Jiang WY, Han SL, Tang MZ, et al. Spectrum-effect relationship analysis between HPLC fingerprint and immunomodulatory activity of Zhenqi Fuzheng Granules[J]. Chin J Exp Tradit Med Form (中国实验方剂学杂志), 2022, 28: 123-130.

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Yuan MY, Zhang XX, Xie XD, et al. Observation on effect of atractylodesin Ⅱ on gastric cancer cells based on macrophage polarization[J]. Chin J Exp Tradit Med Form (中国实验方剂学杂志), 2020, 26: 100-108.

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2023年第58卷第6期

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文章信息

doi: 10.16438/j.0513-4870.2022-1249

接收时间：2022-11-18
首发时间：2025-11-21
出版时间：2023-06-12

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文章信息

作者

出版历史

收稿日期：2022-11-18
修回日期：2023-03-10

基金

国家自然科学基金面上项目(81973419)

陕西省重点研发计划一般项目(2022SF-557)

陕西省中医药研究院“苗圃培育计划”项目(2021-17)

作者信息

1.陕西省中医药研究院, 陕西西安 710061

2.西安正大制药有限公司, 陕西西安 710043

3.西安培华学院, 陕西西安 710199

通讯作者:

*陈志永, Tel: 86-29-85395696, E-mail: chenzhiyong0612@sina.com

参考文献

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https://castjournals.cast.org.cn/joweb/yxxb/CN/10.16438/j.0513-4870.2022-1249

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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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No.	Compound	t_R/min	Formula	Calculated m/z	Extracted ion m/z
1	Allantoin	1.11	C₄H₆N₄O₃	159.051 3	159.050 5-159.052 1 [M+H]⁺
2	L-Proline	1.13	C₅H₉NO₂	116.070 6	116.070 0-116.071 2 [M+H]⁺
3	Pyroglutamic acid	2.24	C₅H₇NO₃	130.049 9	130.049 2-130.050 6 [M+H]⁺
4	Hordenine	5.95	C₁₀H₁₅NO	166.122 6	166.121 8-166.123 4 [M+H]⁺
5	Adenosine	6.85	C₁₀H₁₃N₅O₄	268.104 0	268.102 7-268.105 3 [M+H]⁺
6	L-Phenylalanine	6.89	C₉H₁₁NO₂	166.086 2	166.085 4-166.087 0 [M+H]⁺
7	Guanosine	8.82	C₁₀H₁₃N₅O₅	284.099 0	284.097 6-284.100 4 [M+H]⁺
8	L-Tryptophan	14.32	C₁₁H₁₂N₂O₂	205.097 1	205.096 1-205.098 1 [M+H]⁺
9	Caffeic acid	20.68	C₉H₈O₄	179.035 0	179.034 1-179.035 9 [M-H]^-
10	Calycosin-7-glucoside	29.77	C₂₂H₂₂O₁₀	447.128 6	447.126 4-447.130 8 [M+H]⁺
11	Verbascoside	31.58	C₂₉H₃₆O₁₅	623.198 1	623.197 0-623.203 2 [M-H]^-
12	Isoacteoside	34.07	C₂₉H₃₆O₁₅	623.198 1	623.197 0-623.203 2 [M-H]^-
13	Ononin	36.65	C₂₂H₂₂O₉	431.133 6	431.131 4-431.135 8 [M+H]⁺
14	Calycosin	40.11	C₁₆H₁₂O₅	285.075 8	285.074 4-285.077 2 [M+H]⁺
15	3-Hydroxy-9, 10-dimethoxyptercarpan	42.74	C₁₇H₁₆O₅	301.107 0	301.105 5-301.108 5 [M+H]⁺
16	Formononetin	46.28	C₁₆H₁₂O₄	269.080 8	269.079 5-269.082 1 [M+H]⁺
17	Atractylenolide Ⅲ	49.12	C₁₅H₂₀O₃	249.148 3	249.147 1-249.149 5 [M+H]⁺
18	Atractylenolide Ⅱ	54.02	C₁₅H₂₀O₂	233.153 4	233.152 2-233.154 6 [M+H]⁺
19	Astragaloside A	62.78	C₄₁H₆₈O₁₄	829.459 0	829.454 9-829.463 1 [M+HCOO]^-

No.

Compound

t_R/min

Formula

Calculated m/z

Extracted ion m/z

Allantoin

1.11

C₄H₆N₄O₃

159.051 3

159.050 5-159.052 1 [M+H]⁺

L-Proline

1.13

C₅H₉NO₂

116.070 6

116.070 0-116.071 2 [M+H]⁺

Pyroglutamic acid

2.24

C₅H₇NO₃

130.049 9

130.049 2-130.050 6 [M+H]⁺

Hordenine

5.95

C₁₀H₁₅NO

166.122 6

166.121 8-166.123 4 [M+H]⁺

Adenosine

6.85

C₁₀H₁₃N₅O₄

268.104 0

268.102 7-268.105 3 [M+H]⁺

L-Phenylalanine

6.89

C₉H₁₁NO₂

166.086 2

166.085 4-166.087 0 [M+H]⁺

Guanosine

8.82

C₁₀H₁₃N₅O₅

284.099 0

284.097 6-284.100 4 [M+H]⁺

L-Tryptophan

14.32

C₁₁H₁₂N₂O₂

205.097 1

205.096 1-205.098 1 [M+H]⁺

Caffeic acid

20.68

C₉H₈O₄

179.035 0

179.034 1-179.035 9 [M-H]^-

Calycosin-7-glucoside

29.77

C₂₂H₂₂O₁₀

447.128 6

447.126 4-447.130 8 [M+H]⁺

Verbascoside

31.58

C₂₉H₃₆O₁₅

623.198 1

623.197 0-623.203 2 [M-H]^-

Isoacteoside

34.07

C₂₉H₃₆O₁₅

623.198 1

623.197 0-623.203 2 [M-H]^-

Ononin

36.65

C₂₂H₂₂O₉

431.133 6

431.131 4-431.135 8 [M+H]⁺

Calycosin

40.11

C₁₆H₁₂O₅

285.075 8

285.074 4-285.077 2 [M+H]⁺

3-Hydroxy-9, 10-dimethoxyptercarpan

42.74

C₁₇H₁₆O₅

301.107 0

301.105 5-301.108 5 [M+H]⁺

Formononetin

46.28

C₁₆H₁₂O₄

269.080 8

269.079 5-269.082 1 [M+H]⁺

Atractylenolide Ⅲ

49.12

C₁₅H₂₀O₃

249.148 3

249.147 1-249.149 5 [M+H]⁺

Atractylenolide Ⅱ

54.02

C₁₅H₂₀O₂

233.153 4

233.152 2-233.154 6 [M+H]⁺

Astragaloside A

62.78

C₄₁H₆₈O₁₄

829.459 0

829.454 9-829.463 1 [M+HCOO]^-

Analyte	Regression equation	Linearity range /μg·mL^-1	r	LOQ /μg·mL^-1	LOD /μg·mL^-1
Allantoin	Y = 5 472 828 X + 479 320	4.83-144.96	0.999 7	0.060	0.020
L-Proline	Y = 86 975 923 X + 5 638 716	4.24-127.20	0.999 9	0.085	0.028
Pyroglutamic acid	Y = 64 631 208 X + 2 389 539	1.60-47.88	0.999 8	0.032	0.011
Hordenine	Y = 723 082 195 X + 33 356 735	0.30-8.89	0.999 8	0.005	0.002
Adenosine	Y = 266 200 177 X + 9 443 073	0.32-9.57	0.999 6	0.020	0.007
L-Phenylalanine	Y = 247 574 407 X + 9 293 926	1.41-42.21	0.999 6	0.035	0.012
Guanosine	Y = 40 699 138 X + 2 453 794	0.74-22.18	0.999 8	0.009	0.003
L-Tryptophan	Y = 195 962 721 X + 1 750 113	0.26-7.92	0.999 1	0.005	0.002
Caffeic acid	Y = 169 287 666 X + 8 768 389	0.18-5.46	0.999 5	0.069	0.023
Calycosin-7-glucoside	Y = 57 987 427 X+ 2 827 590	2.68-80.32	1.000 0	0.031	0.010
Verbascoside	Y = 32 726 217 X + 1 202 329	0.41-12.38	0.999 5	0.007	0.002
Isoacteoside	Y = 44 913 759 X - 6 465 736	0.77-23.10	0.999 9	0.011	0.003
Ononin	Y = 86 567 555 X + 60 511 347	0.76-22.86	0.999 6	0.008	0.003
Calycosin	Y = 229 728 032 X + 54 122 589	0.90-27.09	0.999 9	0.023	0.008
3-Hydroxy-9, 10-dimethoxyptercarpan	Y = 284 043 686 X + 11 858 456	0.24-7.16	0.999 8	0.003	0.001
Formononetin	Y = 336 848 761 X + 44 638 709	0.37-11.02	1.000 0	0.013	0.004
Atractylenolide Ⅲ	Y = 65 804 295 X + 4 155 839	0.29-8.64	0.999 7	0.097	0.032
Atractylenolide Ⅱ	Y = 1 117 680 022 X - 1 738 208	0.027 5-0.825	0.999 7	0.010	0.003
Astragaloside A	Y = 2 129 447 X + 143 154	0.885-26.55	0.999 9	0.017	0.006

Analyte

Regression equation

Linearity range /μg·mL^-1

LOQ /μg·mL^-1

LOD /μg·mL^-1

Allantoin

Y = 5 472 828 X + 479 320

4.83-144.96

0.999 7

0.060

0.020

L-Proline

Y = 86 975 923 X + 5 638 716

4.24-127.20

0.999 9

0.085

0.028

Pyroglutamic acid

Y = 64 631 208 X + 2 389 539

1.60-47.88

0.999 8

0.032

0.011

Hordenine

Y = 723 082 195 X + 33 356 735

0.30-8.89

0.999 8

0.005

0.002

Adenosine

Y = 266 200 177 X + 9 443 073

0.32-9.57

0.999 6

0.020

0.007

L-Phenylalanine

Y = 247 574 407 X + 9 293 926

1.41-42.21

0.999 6

0.035

0.012

Guanosine

Y = 40 699 138 X + 2 453 794

0.74-22.18

0.999 8

0.009

0.003

L-Tryptophan

Y = 195 962 721 X + 1 750 113

0.26-7.92

0.999 1

0.005

0.002

Caffeic acid

Y = 169 287 666 X + 8 768 389

0.18-5.46

0.999 5

0.069

0.023

Calycosin-7-glucoside

Y = 57 987 427 X+ 2 827 590

2.68-80.32

1.000 0

0.031

0.010

Verbascoside

Y = 32 726 217 X + 1 202 329

0.41-12.38

0.999 5

0.007

0.002

Isoacteoside

Y = 44 913 759 X - 6 465 736

0.77-23.10

0.999 9

0.011

0.003

Ononin

Y = 86 567 555 X + 60 511 347

0.76-22.86

0.999 6

0.008

0.003

Calycosin

Y = 229 728 032 X + 54 122 589

0.90-27.09

0.999 9

0.023

0.008

3-Hydroxy-9, 10-dimethoxyptercarpan

Y = 284 043 686 X + 11 858 456

0.24-7.16

0.999 8

0.003

0.001

Formononetin

Y = 336 848 761 X + 44 638 709

0.37-11.02

1.000 0

0.013

0.004

Atractylenolide Ⅲ

Y = 65 804 295 X + 4 155 839

0.29-8.64

0.999 7

0.097

0.032

Atractylenolide Ⅱ

Y = 1 117 680 022 X - 1 738 208

0.027 5-0.825

0.999 7

0.010

0.003

Astragaloside A

Y = 2 129 447 X + 143 154

0.885-26.55

0.999 9

0.017

0.006

Analyte	S1	S2	S3	S4	S5	S6	S7	S8	S9	S10	Average
Allantoin	137.04	116.49	94.48	150.01	126.94	133.80	121.15	123.79	136.08	134.31	127.41
L-Proline	78.40	83.66	49.48	72.48	71.10	76.36	73.42	71.93	68.85	65.96	71.16
Pyroglutamic acid	32.42	42.20	24.90	36.66	29.05	35.43	33.28	26.13	33.80	24.97	31.88
Hordenine	5.01	4.52	4.69	4.82	4.98	5.23	5.11	5.82	5.08	6.01	5.13
Adenosine	12.61	11.74	9.03	11.68	11.03	10.91	11.38	10.77	13.37	12.80	11.53
L-Phenylalanine	33.38	27.00	21.71	31.11	28.43	29.72	32.11	25.49	28.00	30.49	28.74
Guanosine	25.23	26.71	17.81	22.38	20.87	22.72	23.00	21.70	25.43	24.45	23.03
L-Tryptophan	6.61	5.71	4.56	5.78	5.47	5.59	6.31	5.78	6.01	6.09	5.79
Caffeic acid	4.45	3.13	2.30	4.08	4.32	3.80	4.20	3.54	4.67	4.31	3.88
Calycosin-7-glucoside	61.19	46.43	38.36	60.40	57.48	60.02	60.29	50.71	48.83	42.93	52.66
Verbascoside	9.73	5.78	5.41	8.49	12.09	9.16	10.24	8.46	8.59	10.97	8.89
Isoacteoside	19.71	14.42	9.67	15.49	22.06	14.18	20.67	19.22	18.67	22.02	17.61
Ononin	44.09	17.01	14.17	21.54	20.50	22.32	20.30	17.17	17.14	16.24	21.05
Calycosin	23.81	16.61	13.09	18.38	20.98	20.04	14.93	13.41	23.52	18.83	18.36
3-Hydroxy-9, 10-dimethoxyptercarpan	7.00	5.52	4.49	5.78	6.91	6.81	4.15	3.93	7.38	5.99	5.80
Formononetin	8.98	6.62	5.21	6.43	7.86	7.89	4.50	3.89	7.55	6.87	6.58
Atractylenolide Ⅲ	10.00	8.42	7.42	8.58	10.40	10.18	7.32	6.78	10.22	11.38	9.07
Atractylenolide Ⅱ	0.59	0.51	0.48	0.49	0.68	0.61	0.30	0.29	0.61	0.62	0.52
Astragaloside A	32.47	27.80	29.08	30.52	30.02	27.92	27.56	27.08	50.18	42.29	32.49
Total	552.72	470.28	356.34	515.10	491.17	502.69	480.22	445.89	513.98	487.53	-

Analyte

S10

Average

Allantoin

137.04

116.49

94.48

150.01

126.94

133.80

121.15

123.79

136.08

134.31

127.41

L-Proline

78.40

83.66

49.48

72.48

71.10

76.36

73.42

71.93

68.85

65.96

71.16

Pyroglutamic acid

32.42

42.20

24.90

36.66

29.05

35.43

33.28

26.13

33.80

24.97

31.88

Hordenine

5.01

4.52

4.69

4.82

4.98

5.23

5.11

5.82

5.08

6.01

5.13

Adenosine

12.61

11.74

9.03

11.68

11.03

10.91

11.38

10.77

13.37

12.80

11.53

L-Phenylalanine

33.38

27.00

21.71

31.11

28.43

29.72

32.11

25.49

28.00

30.49

28.74

Guanosine

25.23

26.71

17.81

22.38

20.87

22.72

23.00

21.70

25.43

24.45

23.03

L-Tryptophan

6.61

5.71

4.56

5.78

5.47

5.59

6.31

5.78

6.01

6.09

5.79

Caffeic acid

4.45

3.13

2.30

4.08

4.32

3.80

4.20

3.54

4.67

4.31

3.88

Calycosin-7-glucoside

61.19

46.43

38.36

60.40

57.48

60.02

60.29

50.71

48.83

42.93

52.66

Verbascoside

9.73

5.78

5.41

8.49

12.09

9.16

10.24

8.46

8.59

10.97

8.89

Isoacteoside

19.71

14.42

9.67

15.49

22.06

14.18

20.67

19.22

18.67

22.02

17.61

Ononin

44.09

17.01

14.17

21.54

20.50

22.32

20.30

17.17

17.14

16.24

21.05

Calycosin

23.81

16.61

13.09

18.38

20.98

20.04

14.93

13.41

23.52

18.83

18.36

3-Hydroxy-9, 10-dimethoxyptercarpan

7.00

5.52

4.49

5.78

6.91

6.81

4.15

3.93

7.38

5.99

5.80

Formononetin

8.98

6.62

5.21

6.43

7.86

7.89

4.50

3.89

7.55

6.87

6.58

Atractylenolide Ⅲ

10.00

8.42

7.42

8.58

10.40

10.18

7.32

6.78

10.22

11.38

9.07

Atractylenolide Ⅱ

0.59

0.51

0.48

0.49

0.68

0.61

0.30

0.29

0.61

0.62

0.52

Astragaloside A

32.47

27.80

29.08

30.52

30.02

27.92

27.56

27.08

50.18

42.29

32.49

Total

552.72

470.28

356.34

515.10

491.17

502.69

480.22

445.89

513.98

487.53