Quantitive analysis of flavonoids in ainaxiang tablets by high-performance liquid chromatography

TAN, Daopeng; WANG, Guopan; WANG, Jianmei; FENG, Yidong; HE, Yuqi

doi:10.1590/fst.130422

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Food Science and Technology

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Original Article • Food Sci. Technol 43 • 2023 • https://doi.org/10.1590/fst.130422 copy

Quantitive analysis of flavonoids in ainaxiang tablets by high-performance liquid chromatography

Authorship SCIMAGO INSTITUTIONS RANKINGS

Abstract:

Blumea balsamifera is a very famous medicinal plant and functional tea in many countries of Asia. Ainaxiang tablets are a preparation consisting of flavonoids from B. balsamifera. Today, a high performance liquid chromatography method was developed for simultaneous quantification of 15 flavonoids in Ainaxiang Tablets. The analysis was performed on a Kromasil C18 column (250 × 4.6 mm, 5 µm) with a binary gradient mobile phase of acetonitrile and 0.2% aqueous acetic acid. The method was validated in terms of linearity, sensitivity, stability, precision and accuracy. It was found that this method had linearity with R² at 0.9992- 0.9999 in the test range of 0.15-276.40 µg/mL. The limit of detection (LOD) and limit of quantification (LOQ) for 15 tested reference compounds were 0.01-0.28µg/mL and 0.02 - 0.55µg/mL, respectively. The relative standard deviations (RSD%) for intra-day and inter-day repeatability were not more than 3.18%. The analyzed samples were stable for at least 18 h. The spike recoveries for 15 analyzed samples were 106.17 - 95.02%. The results suggested that this newly developed HPLC method could be used for quantitative analysis of flavonoids in Ainaxiang Tablets and its raw materials.

Keywords:
Blumea balsamifera ; Ainaxiang tablets; HPLC quantification; flavonoids

No.	Compound name	Calibration curve^a a y is the peak area and x is the concentration of compound (µg/mL).	R²	Linear range(μg/mL)	LOD^b b LOD refers to the limit of detection, S/N = 2.3-3.6:1. (μg/mL)	LOQ^c c LOQ refers to the limit of quantification, S/N = 8.1-10.3:1. (μg/mL)
1	Rutin	y=37.49x+5.05	0.9998	0.24~24.00	0.02	0.04
2	Hyperoside	y=48.77x-2.30	0.9999	0.25~25.26	0.03	0.05
3	Isoquercitrin	y=48.71x+2.49	0.9994	0.26~26.10	0.03	0.05
4	3,3',5,7-Tetrahydroxy-4'-methoxyflavanone	y=7.18x-5.97	0.9998	2.76~276.40	0.28	0.55
5	3',5,5',7-Tetrahydroxyflavanone	y=8.29x-9.56	0.9998	2.94~58.82	0.12	0.24
6	Quercetin	y=68.14x-23.02	0.9995	0.82~16.38	0.07	0.16
7	3,3',4',5-Tetrahydroxy-7-methoxyflavanone	y=5.82x-4.66	0.9994	3.04~60.80	0.24	0.61
8	Chrysosplenol C	y=44.92x-7.82	0.9992	0.38~38.23	0.03	0.08
9	Diosmetin	y=79.71x-4.94	0.9994	0.17~17.17	0.01	0.03
10	Tamarixetin	y=72.72x-13.44	0.9994	0.17~16.75	0.07	0.17
11	3,5,7-Trihydroxy-3',4'-dimethoxyflavone	y=70.14x-4.17	0.9996	0.16~40.20	0.01	0.02
12	3,3',5-Trihydroxy-4',7-dimethoxyflavanone	y=7.99x-1.24	0.9998	0.69~34.50	0.07	0.14
13	Blumeatin	y=8.26x-1.57	0.9997	0.81~16.29	0.06	0.16
14	Rhamnetin	y=57.54x-12.94	0.9994	0.30~29.79	0.01	0.02
15	Ombuin	y=108.25x-7.86	0.9994	0.15~14.55	0.01	0.02

No.	Original (mg)	Spiked (mg)	Found (mg)	Recovery (%)^a a Recovery% = [(found amount - original amount)/spiked amount] × 100%;	RSD (%)^b b RSD% = (SD/mean) × 100%.
1	0.150	0.096	0.247	101.05 ± 0.70	0.69
	0.111	0.120	0.232	100.28 ± 1.83	1.82
	0.097	0.144	0.243	101.51 ± 2.10	2.07
2	0.155	0.101	0.257	100.71 ± 0.62	0.61
	0.115	0.126	0.242	100.61 ± 2.46	2.44
	0.100	0.152	0.254	101.66 ± 2.03	2.00
3	0.164	0.104	0.267	98.76 ± 0.37	0.37
	0.121	0.130	0.253	100.65 ± 2.28	2.27
	0.105	0.157	0.265	101.79 ± 1.93	1.90
4	1.686	1.106	2.797	100.47 ± 0.74	0.74
	1.251	1.382	2.663	102.22 ± 2.41	2.36
	1.086	1.658	2.786	102.50 ± 2.47	2.41
5	0.393	0.235	0.628	100.22 ± 1.48	1.48
	0.291	0.294	0.589	101.30 ± 3.51	3.46
	0.252	0.353	0.614	102.23 ± 2.00	1.95
6	0.093	0.066	0.158	99.72 ± 2.00	2.00
	0.069	0.082	0.150	98.56 ± 4.72	4.79
	0.060	0.098	0.156	97.54 ± 4.19	4.30
7	0.435	0.243	0.697	108.01 ± 4.76	4.42
	0.322	0.304	0.641	104.90 ± 4.92	4.69
	0.280	0.365	0.662	104.78 ± 2.41	2.30
8	0.057	0.031	0.088	99.93 ± 1.72	1.72
	0.042	0.038	0.082	102.87 ± 2.11	2.05
	0.038	0.046	0.084	102.26 ± 0.89	0.87
9	0.021	0.014	0.034	98.53 ± 1.49	1.51
	0.015	0.017	0.032	99.78 ± 2.15	2.15
	0.013	0.021	0.033	95.52 ± 4.30	4.50
10	0.099	0.067	0.166	100.35 ± 0.32	0.32
	0.073	0.084	0.159	101.95 ± 2.49	2.44
	0.064	0.101	0.167	103.11 ± 2.16	2.09
11	0.052	0.032	0.084	99.04 ± 1.20	1.21
	0.039	0.040	0.079	100.52 ± 3.57	3.55
	0.034	0.048	0.082	100.87 ± 2.18	2.16
12	0.211	0.138	0.352	101.72 ± 1.68	1.65
	0.157	0.172	0.332	101.69 ± 3.50	3.45
	0.136	0.207	0.347	101.96 ± 2.23	2.19
13	0.095	0.065	0.160	99.87 ± 4.86	4.87
	0.071	0.081	0.157	106.17 ± 0.37	0.35
	0.061	0.098	0.154	95.02 ± 1.43	1.50
14	0.102	0.060	0.159	96.21 ± 1.18	1.23
	0.076	0.074	0.148	96.91 ± 2.93	3.02
	0.066	0.089	0.154	98.43 ± 2.10	2.13
15	0.012	0.012	0.024	99.65 ± 1.35	1.35
	0.009	0.015	0.024	102.23 ± 1.95	1.91
	0.008	0.017	0.026	105.20 ± 1.97	1.88

No.	Intra-day^a a Intra-day precision on one day for tested six times; (n=6)		Inter-day^b b Inter-day precision on three consecutive days; (n=6)
No.	Content (μg/mL)	RSD^c c RSD% = (SD/mean) × 100%. (%)	Content (μg/mL)	RSD (%)
1	11.656 ± 0.284	2.44	11.853 ± 0.258	2.18
2	12.364 ± 0.108	0.87	12.392 ± 0.119	0.96
3	13.051 ± 0.098	0.75	13.055 ± 0.111	0.85
4	133.087 ± 0.904	0.68	133.910 ± 1.203	0.90
5	31.328 ± 0.227	0.73	31.549 ± 0.302	0.96
6	8.731 ± 0.040	0.48	8.309 ± 0.083	1.00
7	34.983 ± 1.113	3.18	34.701 ± 0.868	2.50
8	4.502 ± 0.089	1.97	4.619 ± 0.111	2.41
9	1.610 ± 0.016	0.98	1.627 ± 0.017	1.03
10	7.734 ± 0.038	0.49	7.835 ± 0.078	0.99
11	4.203 ± 0.028	0.67	4.197 ± 0.030	0.72
12	17.291 ± 0.339	1.96	17.032 ± 0.141	0.83
13	7.437 ± 0.113	1.52	7.519 ± 0.117	1.56
14	8.037 ± 0.044	0.54	8.067 ± 0.082	1.02
15	0.962 ± 0.017	1.75	0.979 ± 0.019	2.00

No.	20190505	20191102	20191010	20191203
1	347.19 ± 2.36	329.77 ± 1.02	304.95 ± 5.27	366.30 ± 4.09
2	357.41 ± 2.80	362.76 ± 0.88	245.89 ± 3.63	427.98 ± 0.11
3	377.88 ± 2.70	381.35 ± 0.77	271.62 ± 3.58	388.98 ± 4.39
4	3869.18 ± 22.79	4054.43 ± 12.53	4196.32 ± 8.33	4055.99 ± 35.79
5	904.91 ± 12.08	779.94 ± 7.38	994.19 ± 4.09	950.75 ± 22.22
6	198.52 ± 5.57	195.27 ± 0.96	252.66 ± 0.51	253.83 ± 2.90
7	999.60 ± 3.99	455.00 ± 2.41	1037.79 ± 27.18	917.02 ± 4.50
8	130.65 ± 0.69	86.63 ± 0.49	133.13 ± 0.45	141.82 ± 0.50
9	46.00 ± 1.56	47.69 ± 0.17	64.70 ± 0.10	41.22 ± 0.40
10	225.52 ± 3.06	219.37 ± 0.88	221.80 ± 0.41	254.20 ± 1.75
11	120.64 ± 0.64	120.36 ± 0.27	123.52 ± 0.24	107.331 ± 1.35
12	488.41 ± 1.04	445.30 ± 1.07	560.90 ± 24.75	506.26 ± 6.24
13	220.91 ± 2.16	190.53 ± 0.48	233.76 ± 2.54	224.99 ± 2.10
14	235.26 ± 2.10	224.24 ± 0.88	242.34 ± 6.76	241.71 ± 3.60
15	28.50 ± 0.18	16.70 ± 0.35	29.47 ± 0.28	29.44 ± 0.44

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