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

Abstract

Objective

Apigenin and gallic acid are natural compounds that are useful as antioxidant, anti-inflammatory and anticancer agents, especially when used together in combination. Therefore, the development and validation of a simultaneous method of analysis for both compounds in pure form and when encapsulated in an advanced delivery system such as liposomes would be useful.

Methods

Analysis was performed using C18 column under isocratic conditions. The mobile phase was acetonitrile: water containing 0.2% orthophosphoric acid at a ratio of 67:33, flow rate 1 ml/min, and detection wavelength 334 nm for apigenin and 271 nm for gallic acid.

Results

The assay method was linear at the concentration range (5–600 µg/mL) with R² of 1 for both drugs. The method was also shown to be precise and robust with RSD less than 2% with LOD (0.12, 0.1 µg/mL) and LOQ (4.14, 3.58 µg/mL) for apigenin and gallic acid respectively. The method was also applicable for the determination of the entrapment efficiency of both drugs when co-loaded in a nanoliposomal formulation.

Conclusion

The described HPLC method was shown to be suitable, sensitive, and reproducible for the simultaneous identification and quantification of apigenin and gallic acid. The analytical results were accurate and precise, with good recovery, low limit of detection, and the chromatographic assay was accomplished in less than 3 min, suggesting the suitability of the method for routine analysis of both drugs in pharmaceutical formulations.

Graphical abstract

Keywords:

• Apigenin
• gallic acid
• HPLC
• liposomes
• isocratic

Introduction

Apigenin is a naturally occurring flavonoid (5,7-Trihydroxyflavone) found abundantly in many fruits and vegetables (Figure 1). It exhibits several biological and pharmacological activities such as anti-inflammatory, antioxidant, antiviral, and anticancer [1–3]. Apigenin was recently used as a dietary supplement for cancer patients, and several researchers have encapsulated it in many dosage forms for its promising anticancer activity in inhibiting tubulin binding, induction of apoptosis and autophagy, and modulation of the cell cycle [2,4–6]. Apigenin has a molecular weight of 270.05 g/mol and log P of 3.1. It is poorly soluble in water and soluble in ethanol and dimethyl sulfoxide (DMSO), therefore it is classified as a class two drug according to the Biopharmaceutical Classification System (BCS) [7]. Therefore, the encapsulation of apigenin in lipidic self-assembled systems is expected to efficiently solubilize it, for enhanced and sustained therapeutic activity.

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

Gallic acid (Figure 1) is another phenolic acid-based nutraceutical found in several sources, especially grapes and tea. It exhibits many pharmacological activities as a powerful antioxidant, anticancer, anti-inflammatory, and antibacterial. It is soluble in most solvents (water, ethanol, and methanol) with a molecular weight of 170.12 g/mol [8,9]. Despite its complete oral absorption, gallic acid would benefit from encapsulation in lipidic self-assembled systems to enhance its therapeutic activity and sustain its release [10].

Several reports in the literature have proven that combining two nutraceuticals in one dosage form or a delivery system mostly maximize their therapeutic efficacy [11,12]. In particular, co-loading of nutraceuticals alone or in combination with chemotherapeutic agents have been proven efficacious in the treatment of many cancer types [13–17]. Therefore, the combination of apigenin and gallic acid in one delivery system is expected to synergize their anticancer activity by different mechanistic pathways, such as induction of apoptosis and autophagy, inhibition of proliferation, inhibition of cancer cell migration and invasion, cell cycle arrest, and increased reactive oxygen species [3,18].

Among the promising delivery systems reported till current date is liposomes, which is a self-assembled lipidic system based on phospholipids, effectively known to load both water soluble and insoluble drugs [19–24], and therefore, it would be an optimum system for co-loading of the water insoluble apigenin, and the water soluble gallic acid. In addition, when either apigenin or gallic acid was loaded in liposomes, an increased cellular uptake and cytotoxic activity was reported compared to their free forms [5,18,25–27].

From an analytical perspective, several methods have been developed for the determination of apigenin and gallic acid alone, with the disadvantage of long run time for apigenin owing to its hydrophobicity [28–31]. Therefore, the current method reported in this paper aims to shorten the run time and elute both apigenin and gallic acid either when separately analyzed or when encapsulated in a nanoliposomal formulation. The analytical method was validated, followed by application in the analysis of the entrapment of the co-loaded drugs in the nanoliposomal formulation.

Materials and methods

Materials

Apigenin was purchased from Skin actives scientific company, United States. Gallic acid, orthophosphoric acid, hydrochloric acid, cholesterol, and sodium hydroxide were purchased from Loba Chemical, Jehangir Villa, India. L-α-Phosphatidylcholine soybean lecithin (EPIKURON 200) was kindly gifted by Cargill, Texturizing solutions, Germany. Dimethyldioctadecylammonium bromide (DODAB), Ethanol (HPLC grade > 99.9%), and acetonitrile (HPLC grade > 99.9%) were purchased from Sigma Aldrich Company, USA. Deionized water was obtained from a direct Q-UV 3 millipore device, Millipore SAS, France.

Chromatographic conditions

The chromatographic separation was done on a C18 column (Luna® 3 µm C18 100 Å, LC Column 150 × 4.6 mm, Ea, Phenomenex). Apigenin and gallic acid were chromatographed with a mobile phase consisting of acetonitrile and water containing phosphoric acid 0.2% (67:33, v/v %) using high-performance liquid chromatography (Waters 2690 Alliance HPLC system equipped with a Waters 996 photodiode array detector). The flow rate was 1 ml per minute, the injected volume was 2.5 µL and the chromatographic run time was 2.5 min per sample. The method was monitored at 334 nm for apigenin [32] and 271 nm for gallic acid [33]. All analyses were carried out at 25 °C.

Preparation of standard solutions

An amount of 100 mg of each (apigenin and gallic acid) was transferred to a 100 ml volumetric flask to create a mixture of the two drugs. They were dissolved in ethanol followed by sonication for 10 min, yielding a stock solution of 1 mg/mL of both drugs. Further appropriate dilutions were made to yield the required concentrations (5,10,25,50,100,150,200,250,300,600 μg/mL) for the construction of the calibration curves. The measured areas under the curves were plotted against the corresponding concentrations, and the regression equations were computed.

Validation procedure

The International Conference on Harmonization (ICH) guidelines [34] and the United States Pharmacopeia (USP) requirements were adopted for the validation studies. Various parameters including selectivity, specificity, linearity, accuracy, robustness, precision, and ruggedness (intermediate precision) were evaluated.

To study linearity, the aforementioned dilutions were prepared from a stock drug standard solution. Precision was determined by analyzing a sample six times in two consecutive days (intra-day and inter-day), and calculation of the relative standard deviation (RSD). Ruggedness (intermediate precision) was evaluated by two different analysts, who analyzed the same sample at different day intervals for two days. The accuracy of the method which expresses the closeness of the expected values to the found values was evaluated by calculating the percent recovery of the drugs. The limit of quantification (LOQ) and limit of detection (LOD) were estimated from the standard deviation of residuals (SD) and slope (S) using the following equations: LOD = 3.3(SD/slope) and LOQ = 10 (SD/Slope). The robustness of the method was estimated by testing its tendency to be unaffected by a slight change of the mobile phase concentrations (0.2% phosphoric acid to 0.3% phosphoric acid). The selectivity and specificity were evaluated by comparison of the chromatograms of the test solution and that of a solution exposed to stress conditions by 0.1 N of either HCl or NaOH [35], to test the ability of the method to measure the analyte response in the presence of its potential degradation products.

Preparation of nanoliposomes co-loaded with apigenin and gallic acid

Nanoliposomes were prepared using the ethanol injection method [36,37]. A phospholipid concentration of 20 mg/mL, 0.5 mg/mL cholesterol, 2 mg/mL Dimethyldioctadecylammonium bromide (DODAB), ethanol volume of 1.25 ml containing 0.3 mg of apigenin and 12.5 mg of gallic acid injected by hand press with the antisolvent flow direction, and 5 ml of PBS pH 7.4 were used. The formulations were evaporated using a rotary evaporator (model IKA RV 10 basic, Germany) till complete evaporation of ethanol.

Determination of the entrapment efficiency of apigenin and gallic acid in nanoliposomes

The entrapment efficiency of apigenin and gallic acid in the nanoliposomes was calculated after centrifugation using a Nanosep tube (PALL laboratory Company, USA) [38,39]. The formulations were centrifuged for 30 min at a speed of 15,000 g at room temperature (NEYA 16 R, REMI laboratory instruments, India) [40–42]. The supernatant was collected from the lower compartment, diluted with ethanol and the unentrapped drugs were quantified using the HPLC method described previously. The entrapment efficiency percentage of the drugs was calculated in supernatant according to the following equation: $$\mathit{\text{Entrapment}}\hspace{0.33em}\mathit{\text{Efficiency}}\%=\frac{\mathit{\text{total}}\mathit{\text{drug}}-\mathit{\text{unentrapped}}\mathit{\text{drug}}}{\mathit{\text{Total}}\mathit{\text{drug}}}\times 100$$

Results

Chromatograms of apigenin and gallic acid

As shown in Figure 2, both apigenin and gallic acid displayed symmetric peaks using the described assay method, with retention times 2.06 and 1.52 min, respectively. The tailing factors for the apigenin and gallic acid peaks were 1.36 and 1.43 respectively. The relationship between the tested concentrations and peak areas are shown in Figure 3. As also shown in Figure 4, there was no interference from formulation components affecting the peak areas of each drug, as the peaks attributed to the lipidic components of liposomes were well-resolved from the apigenin peak.

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.

Validation of the method of assay

Selectivity

The selectivity of the method was confirmed by comparing the chromatograms of the working standard solution before and after the effect of HCl and NaOH addition. As shown in Figure 5, the retention time, peak resolution, and peak area of apigenin were not affected by treatment with either HCl or NaOH, and likewise for gallic acid in HCl, while showing a significant reduction in the gallic acid area with NaOH only, which was separated without any interference with any peak [43,44], as shown in Figure 6. This indicates that the proposed HPLC chromatographic method for the determination of apigenin and gallic acid is selective and specific for their simultaneous determination.

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.

Linearity

As shown in Figure 3, the linearity curves for both drugs show a strong linear correlation between the concentration and peak areas in the range of (5-600 µg/mL), with coefficients of determination 1 for both drugs. This suggests the suitability of the method for the quantification of both drugs.

Accuracy

The accuracy results of the standard of apigenin and gallic acid were obtained from 10 samples, each conducted in triplicate. As shown in Tables 1 and 2, the mean recovery percentages were 99.54 ± 1.16 and 100.48 ± 0.70 for apigenin and gallic acid respectively. The high average recovery for both apigenin and gallic acid suggests that the described HPLC method was accurate and precise for the determination of both drugs.

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

Precision and Ruggedness (intermediate precision)

Results of interday and intraday precision were evaluated by preparing a certain concentration (200 µg/mL) six times for two consecutive days. Results of interday and intraday precision showed a percentage of relative standard deviation (RSD) less than 2% for both drugs as shown in Tables 3 and 4, hence indicating the precision of the method [45].

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

The ruggedness of the analytical method was evaluated by analyzing the same concentration tested in the precision experiment but by two different analysts on a different day. Six samples were prepared from the same concentration and were injected as previously described. Results are shown in Tables 5 and 6.

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

Determination of the limit of quantification and limit of detection

The limit of detection (LOD) and limit of quantification (LOQ) were calculated to be 0.12 and 4.14 µg/mL respectively for apigenin, and were 0.1 and 3.58 µg/mL respectively for gallic acid, as obtained from the linearity data of both compounds.

Robustness

The effect of variation of mobile phase composition from 0.2% to 0.3% phosphoric acid on apigenin and gallic acid at concentration 200 μg/mL was tested. As shown in Table 7, the peak areas of apigenin and gallic acid were not changed after exposure to changes in mobile phase composition.

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

Application of the analytical method in the quantification of entrapment efficiency of apigenin and gallic acid nanoliposomes

The entrapment efficiency of apigenin and gallic acid in the prepared nanoliposomes was 84.74 ± 2.33% and 60.06 ± 3.94% respectively. As shown in Figure 4, the developed chromatogram showed no interference from liposomal components affecting the peak area of each drug.

Discussion

This work suggests an HPLC method for simultaneous determination of apigenin and gallic acid, with no interference from the liposomal formulation components. The selectivity of the method was confirmed, and the relative standard deviation was less than 2%, indicating the ruggedness of the method [45,46]. The similarity in the peak areas of apigenin and gallic acid after exposure to changes in mobile phase composition suggests the robustness of the method. The tailing factors for the apigenin and gallic acid were within the acceptable range delineated by the ICH guidelines (between 1 and 2) for symmetrical peaks.

Currently, there is an interest in the development of HPLC methods for analysis of drugs within nanoformulations [47]. As obvious from the results, apigenin was entrapped at a higher percent in liposomes owing to its lipophilicity, delineated by its log P value of 3.02 compared with log P value of 0.7 for gallic acid [30]. As a delivery system, liposomes can be considered as one of the most commonly used carriers, owing to their ability to encapsulate both hydrophilic and lipophilic drugs. Owing to the lipophilicity of apigenin, its detection methods suffered from the disadvantage of long run time for apigenin. Therefore, the current method is advantageous in reducing the run time and elute both apigenin and gallic acid either when separately analyzed or when encapsulated in a nanoliposomal formulation, which encourages their co-loading in nanoliposomal formulation while offering means of their simultaneous determination.

Conclusion

As evident from the aforementioned results, the described HPLC method was proven to be convenient, sensitive, and reproducible for the simultaneous identification and quantification of apigenin and gallic acid alone and encapsulated in nanoliposomes. The analytical results were accurate and precise, with good recovery, low limit of detection, and the chromatographic assay was accomplished in less than 3 min.

Acknowledgements

The authors thank Cargill Company for providing free sample of the phospholipids.

Data Availability Statement

Data will be available upon request.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The author(s) reported there is no funding associated with the work featured in this article.