Developing a rapid analytical method for simultaneous determination of apigenin and gallic acid: validation and application in a nanoliposomal formulation

Figures & data

Figure 1. Structure of A) apigenin and B) gallic acid.

Figure 2. HPLC blank chromatograms A and C at 334 and 271 nm respectively, as well as chromatograms of B) apigenin detected at 334 nm, and D) gallic acid detected at 271 nm.

Figure 3. Calibration curves of A) apigenin B) gallic acid in ethanol.

Figure 4. Chromatograms of encapsulated A) apigenin B) gallic acid in nanoliposomal formulations.

Figure 5. Chromatograms of A) apigenin B) apigenin stressed with HCl C) apigenin stressed with NaOH.

Figure 6. Chromatograms of A) gallic acid B) gallic acid stressed with HCl C) gallic acid stressed with NaOH.

Table 1. Accuracy data for apigenin (n = 3).

Concentration µg/ml	Recovery concentration (µg/mL)			Recovery %			Mean%
	#1	#2	#3	#1	#2	#3
5	5.02	5.01	4.91	100.58	100.23	98.36	99.72 ± 0.97
10	9.72	10.01	10.01	97.22	100.1	100.11	99.14 ± 1.36
25	23.68	23.82	24.82	94.75	95.28	99.31	96.45 ± 2.03
50	49.65	49.45	49.67	99.31	98.91	99.34	99.19 ± 0.19
100	99.31	101.22	100.4	99.31	101.22	100.4	100.31 ± 0.78
150	148.6	149.25	149.08	99.06	99.5	99.38	99.31 ± 0.18
200	199.4	201.69	200.72	99.7	100.84	100.36	100.3 ± 0.46
250	253.43	254.42	249.27	101.37	101.76	99.7	100.95 ± 0.89
300	299.7	300.92	300.53	99.9	100.3	100.17	100.12 ± 0.16
600	597.82	601.39	599.96	99.63	100.23	99.99	99.95 ± 0.24
Mean %		99.54 ± 1.16

Table 2. Accuracy data for gallic acid (n = 3).

Concentration (µg/ml)	Recovery concentration (µg/mL)			Recovery %			Mean%
	#1	#2	#3	#1	#2	#3
5	5.08	5.05	5.07	101.64	101.01	101.54	101.39 ± 0.27
10	10.08	9.89	10.13	100.86	98.97	101.35	100.39 ± 1.02
25	25.76	25.66	24.03	103.04	102.65	96.13	100.61 ± 3.16
50	49.88	48.91	49.39	99.76	97.82	98.79	98.79 ± 0.79
100	100.23	101.23	100.73	100.23	101.23	100.73	100.73 ± 0.4
150	153.59	152.32	150.37	102.39	101.54	100.24	101.39 ± 0.88
200	202.79	202.9	199.75	101.39	101.45	99.87	100.9 ± 0.73
250	250.73	251.44	250.96	100.29	100.57	100.38	100.41 ± 0.11
300	298.95	302.74	298.91	99.65	100.91	99.63	100.06 ± 0.59
600	598.73	603.78	601.25	99.78	100.63	100.2	100.2 ± 0.34
Mean %		100.48 ± 0.70

Table 3. Precision data for apigenin.

Inter-day and intraday Precision
First Day				Second Day
Concentration (µg/mL)	Area under the curve AUC(AU)	Found	Recovery %	Concentration (µg/mL)	Area under the curve AUC(AU)	Found	Recovery %
200.00	2088406.00	202.70	101.350	200.00	2112825	205.07	102.53
200.00	2104571.00	204.27	102.13	200.00	2114411	205.22	102.61
200.00	2076646.00	201.55	100.77	200.00	2142437	207.94	103.97
200.00	2084553.00	202.32	101.16	200.00	2108341	204.63	102.31
200.00	2095611.00	203.40	101.70	200.00	2043060	198.29	99.14
200.00	2077967.00	201.68	100.84	200.00	2048960	198.871	99.43
Average	2087959.00			Average	2095005.67
SD	9782.10			SD	36378.14
RSD%	0.47			RSD%	1.74

Table 4. Precision data for gallic acid.

Inter-day and intraday Precision
First Day				Second Day
Concentration (µg/mL)	Area under the curve AUC(AU)	Found	Recovery %	Concentration (µg/mL)	Area under the curve AUC(AU)	Found	Recovery %
200.00	1576124	198.33	99.16	200.00	1588188.00	199.89	99.94
200.00	1621869	204.23	102.11	200.00	1598506.00	201.22	100.61
200.00	1598390	201.21	100.60	200.00	1592464.00	200.44	100.22
200.00	1619010	203.86	101.93	200.00	1599815.00	201.39	100.69
200.00	1610146	202.72	101.36	200.00	1593149.50	200.53	100.26
200.00	1617991	203.73	101.86	200.00	1597177.00	201.05	100.5265
Average	1607255.00			Average	1594883.25
SD	15932.65			SD	4008.17
RSD%	0.99			RSD%	0.25

Table 5. Ruggedness data for apigenin.

Ruggedness
Analyst one				Analyst Two
Concentration (µg/mL)	Area under the curve AUC(AU)	Found	Recovery %	Concentration (µg/mL)	Area under the curve AUC(AU)	Found	Recovery %
200.00	2113618.00	205.14	102.57	200.00	2015293	195.60	97.80
200.00	2125389.00	206.29	103.14	200.00	2041401	198.13	99.06
200.00	2038519.50	197.85	98.92	200.00	2095381	203.37	101.68
200.00	2084849.50	202.35	101.17	200.00	2058233	199.77	99.88
200.00	2121833.00	205.94	102.97	200.00	2113196	205.10	102.55
200.00	2080599.50	201.94	100.97	200.00	2057388	199.68	99.84
Average	101.63			Average	100.14
SD	1.47			SD	1.58
RSD%	1.44			RSD%	1.58
Mean Total			100.88
SD Total			1.52
RSD% Total			1.51

Table 6. Ruggedness data for gallic acid.

Ruggedness
Concentration (µg/mL)	Area under the curve AUC(AU)	Found	Recovery %	Concentration (µg/mL)	Area under the curve AUC(AU)	Found	Recovery %
Analyst one				Analyst Two
200.00	1641019.00	206.70	103.35	200.00	1582159.00	199.11	99.55
200.00	1597371.00	201.07	100.53	200.00	1587505.00	199.80	99.90
200.00	1593347.00	200.56	100.28	200.00	1593347.00	200.56	100.28
200.00	1596139.50	200.92	100.46	200.00	1596139.50	200.92	100.46
200.00	1600956.00	201.54	100.77	200.00	1619010.00	203.86	101.93
200.00	1600189.00	201.44	100.72	200.00	1588524.00	199.93	99.96
Average	101.02			Average	100.35
SD	1.06			SD	0.76
RSD%	1.05			RSD%	0.76
Mean Total			100.69
SD Total			0.91
RSD% Total			0.90

Table 7. Robustness data for apigenin and gallic acid.

	Apigenin			Gallic acid
Conditions	Peak area (AU)	Found	Recovery%	Peak area (AU)	Found	Recovery%
0.2% phosphoric acid	2064374	200.46	100.23	1603964	201.94	100.97
0.3% phosphoric acid	2089963.65	202.94	101.47	1610846	202.83	101.41

Data Availability Statement

Data will be available upon request.