INTRODUCTION:

Andrographis paniculata known as sambiloto in Indonesia is used as a medicinal plant in many countries, e.g. India, China, Malaysia, and Thailand to treat some diseases, such as HIV, hepatitis, diabetes, cancer, kidney disorders, anti malaria, antifungal, and anti hypertensive^1-5. Andrographis paniculata as a natural antimalarial agent has been developed to be herbal products which are used to overcome the resistance of antimalaria drugs.

We recently developed a phytopharmaceutical product containing major components of ethyl acetate fraction of ethanol extract of Andrographis paniculata (F70 and F96). Our previous study of antimalarial activity showed that tablets containing F70 and F96 inhibited Plasmodium berghei growth with inhibition range by 70-80%⁶. Andrographolide is the major active ingredient in Andrographis paniculata. Therefore, andrographolide is used in this study as a marker compound of the raw materials and tablets⁷^-9.

To ensure the quality, efficacy, and safety of phytopharmaceutical products containing ethyl acetate fractions of the ethanol extract of Andrographis paniculata, a simple and selective analytical method becomes necessary for the determination of andrographolide in both raw materials and products. The method should be developed and validated since raw materials of the herbal products show variation in its composition, quality, and activity as well as influenced by harvest time, part of plants, solvent extraction, and production methods^10-12. Moreover, the composition of the compound in herbal products often changes during process production^13-16. Several methods have been developed for the analysis of andrographolide in Andrographis paniculata, including Flow Injection Spectrophotometric (FIS)¹⁷, Microemulsion Electrokinetic Capillary Chromatography (MEEKC)¹⁸ and Micellar Electrokinetic Capillary Chromatography (MEKC)¹⁹. However, all methods need instruments that are not common in the pharmaceutical laboratory. Thin Layer Chromatography (TLC)²⁰ and High performance Thin Layer Chromatography (HPTLC) was used to analyze extract and product Andrographis paniculata^21-22. However, HPTLC method is tedious and time consuming for routine quantitative purposes. High Performance Liquid Chromatography (HPLC) have been reported for the analysis of andrographolide in Andrographis paniculata extract using different column, mobile phase, injection volume, flow rate and analysis wavelength using UV detector^23-26. The advantages of HPLC method include selectivity, sensitivity, and suitability for the routine analysis of pharmaceutical products.

The present study aims to develop and validate a simple, selective and rapid method of HPLC for the determination of andrographolide in raw materials and tablets containing ethyl acetate fraction of the ethanol extract of Andrographis paniculata. A short column of Poroshell has been used in this study to get advantages including its high sensitivities for small volume samples, low pressure for the lower flow rate of mobile phase and to earn shorter analysis time with high efficiency resolution.

MATERIAL AND METHODS:

Materials:

Andrographolide used as a reference standard in this research was obtained from Sigma Aldrich, other solvents such as methanol HPLC grade, phosphoric acid were purchased from Merck (Darmstadt, Germany). Raw materials (F70 and F96) and tablet formulation (tablet of F70 and F96) were obtained from the project site of Natural Products Medicine Research and Development, Institute of Tropical Disease Airlangga University (Indonesia).

Chromatographic conditions:

HPLC Agilent 1100 series is equipped with an automatic injector, a column oven, and a UV detector. HPLC method was performed using a Poroshell 120 EC-C18 column (3.0 x 50mm i.d., 2.7µm particle size) as a stationary phase. The Column temperature was maintained at 30⁰C, and methanol : water (pH 3.05 with phosphoric acid) (50:50 v/v) was used as a mobile phase with a flow rate of 0.3ml/min. The mobile phase was degassed and filtered through a 0.22µm membrane filter before pumping into the HPLC system. Injection volume was 0.5µl and the detector was set at 228nm.

Preparation of standard solution:

Accurately weighed 10.0mg andrographolide standard was transferred into a 10.0ml volumetric flask, then dissolved and diluted up to mark with methanol. This solution was used as a standard of stock solution (1000 ppm). The standard working solutions were prepared by suitable dilution with methanol from the standard stock solution to cover the concentration range between 50 and 800ppm.

Preparation of sample solution:

Preparation of F70 and F96 solution:

Accurately weighed 10.0mg of F70 was transferred into 10.0ml volumetric flask, 5ml of methanol was added, sonicated for 15 min., and finally made up the volume to mark with methanol. This solution was filtered through a 0.22µm membrane filter before injected into HPLC system. The same procedure of the preparation was followed in the case of F96.

Preparation of tablet of F70 and F96:

Total of 20 tablets of F70 were weighed and finely powdered. About 10mg of tablet powder was accurately weighed, transferred into 10.0ml volumetric flask, then 5 ml of methanol was added, sonicated 15 min. and volume was made up to mark with methanol. This solution was filtered through a 0.22µm membrane filter before injected into the HPLC system. Preparation tablet of F96 solution used the same procedure of tablet of F70 solution.

System suitability test:

The system suitability test was performed by five times the injection of the standards solution using a 200ppm standard solution. Percent RSD of retention times and peak areas were calculated.

Stability of standards solution:

The stability of the standard solution was carried out by injecting the standard solution at 0, 1, 2, 3, 5 and 24 h at room temperature. The changes in retention time and peak area of test results were examined by about 2% of each standard response time compared to the standard solution at 0 h.

Selectivity:

The selectivity of the method presented the ability to separate the peak of andrographolide from other components in the sample. The selectivity in this study was demonstrated by injection of blank solvent, matrix solvent, standard solution, and samples solution (F70, F96, tablet of F70 and tablet of 96) to the HPLC system.

Limit of detection and Limit of Quantification:

Limit of detection (LOD) and limit of quantification (LOQ) were determined by injecting the low concentration of standard solutions (10, 20, 30, 50 and 60) ppm to the HPLC system. Linear regression analysis was calculated to get the slope and standard deviation of y-intercepts of linear regression (Sy). LOD and LOQ were calculated using formula

and

Linearity:

The linearity was performed by standard solutions with a concentration of 50, 100, 200, 400, 800 and 1000ppm. A series of andrographolide standard solutions were injected to the HPLC system after filtered with a 0.22µm nylon membrane. Linear regression analysis was evaluated from the concentration of standard solution versus peak area.

Accuracy:

Recovery studies were carried out by spiking three different known amounts of andrographolide standard solution (80%, 100%, and 120%) according to concentration in the target sample to the tablet matrix. The study was done in three replicates of each concentration. The recovery studies were also carried out by adding a known amount of andrographolide solution to the sample at three different levels (80%, 100%, and 120%) of the initial sample concentration. Each level was made three replicates. The percent recovery was calculated using formula

Concentration of Fortified (Cf)

% Recovery= --------------------------------------------------------- x 100%

Concentration of unfortified (Cu)+Concentration added (Ca)

Precision:

The precision calculated as RSD was carried out by spiking the matrix tablet with a 100% known amount of standard andrographolide prepared in six replicates. Repeatability (the intraday precision) was determined within one day, whereas intermediate precision (the interday precision) was determined on different days for two days. The repeatability and intermediate precision were also carried out by analyzing samples in six replication within one day and on different days.

Robustness:

Robustness was studied by changing the small procedural parameters. In this study, robustness was determined by changing wavelength of 228nm to 224nm and 230nm, the flow rate of 0.3ml/min to flow rate 0.35 ml/min, and column temperature of 30⁰C to 28⁰C and 32⁰C.

Figure 1: The Chromatogram of andrographolide used poroshell column (A) (andrographolide tR=2.6 min) and used lichrospher column (B) (andrographolide tR = 8.0 min)

RESULTS AND DISCUSSION:

HPLC Method and optimization:

HPLC was developed using Poroshell 120 EC-C18 column (3.0 x 50mm i.d., 2.7µm particle size) as stationary phase and isocratic mobile phase of methanol : water (pH 3.05 with phosphoric acid) (50:50 v/v). Volume injection, flow rate, and temperature column were optimized to analyze andrographolide in the sample. The Mobile phase of methanol: water (pH 3.05 with phosphoric acid) (50:50 v/v) was chosen because it showed good resolution for andrographolide in the sample and showed good shape peak. An Injection volume of 0.5ml was chosen because it improved column efficiency (N).

Decreasing flow rate to 0.3ml/min and maintaining the column temperature of 30⁰C increased the resolution of andrographolide peak with other components in the sample, giving change about 0.5 min in retention time. The retention time andrographolide was 2.6 min. The proposed method demonstrated a shorter analysis time compared to the previous study.^{4,13,14,15,16} Optimization test was accomplished with column Lichrospher (Merck) RP-18 (250 x 4.0mm, 5µm). The test result showed retention time 8.0min. Figure 1 showed The Chromatogram of andrographolide used poroshell column (A) and used lichrospher column (B)

System suitability test:

The result of the system suitability test passed the requirement of AOAC where the value percent of RSD ≤ 1%.²⁷ Percent RSD for retention time and peak area were 0.099% and 0.725%, respectively.

Stability standards solution:

The retention time and peak area assay results were within a 2% change in standard response at 1, 2, 3, 5 and 24 h in which it related to the standard solution at 0 h. This shows the stability of the test solution during the test.²⁷ The results of the stability standards solution listed in table 1.

Table 1: The results of stability standards solution

Temperatur and solution prepared (hours)	tR (min)	% change	Area	% change
Room temperature Freshly prepared 0 h	2.5	-	777	-
Room temperature at 1 h	2.5	0.16	772	0.48
Room temperature at 2 h	2.5	0.32	774	0.69
Room temperature at 3 h	2.5	0.43	780	0.45
Room temperature at 5 h	2.5	0.39	778	0.85
Room temperature at 24 h	2.5	0.16	773	0.52

Selectivity:

The HPLC method was successful to separate the analyte andrographolide from another components in sample F70, F96, tablet of F70 and tablet of F96. Table 2 showed the result of the selectivity test. Using short HPLC column, the analysis time was reduced, generating the retention time of analyte only 2.6 min, with good resolution (Rs>1.5), high separation efficiency (N> 2000) and good shape peak (T < 2). This result indicated that the method meets AOAC requirement for selectivity.²⁷

Table 2: The result of the selectivity test

Solution sample	tR	Rs > 1.5	N> 2000	α > 1	T < 2
Andrographolide standard	2.6	5.16	2395	1.67	0.76
Methanol blank	-	-	-	-	-
Tablet of matrix blank	-	-	-	-	-
F70	2.6	2.99	2467	1.32	0.77
F96	2.6	2.91	2340	1.32	0.78
Tablet of F70	2.6	5.17	2353	1.69	0.77
Tablet of F96	2.6	5.09	2492	1.67	1.02

The selectivity was evaluated by using Photodiode array (PDA) to show the peak similarity and peak purity. Match factor (MF) and peak purity were used in this study and both values are required not less than 990. The results in this study demonstrated that the values of both MF and peak purity of all peaks of andrographolide in all samples are more than 990. Methanol blank and tablet matrix appeared in different retention time of andrographolide and other components in the sample. Chromatogram of sample F70, F96, tablet of F70, tablet of F96 showed in figures 2, 3, 4, 5, respectively.

Figure 2: Chromatogram of sample F70 (andrographolide tR = 2.6 min)

Figure 3: Chromatogram of sample F96 (andrographolide tR=2.6 min)

Figure 4: Chromatogram of sample tablet F70 (andrographolide tR=2.6 min)

Figure 5: Chromatogram of sample tablet F96 (andrographolide tR=2.6 min)

3.5 Limit of Detection and Limit of Quantitation:

Limit of detection and limit of quantitation were found to be 4.89 and 16.19 ppm, respectively.

3.6 Linearity:

The correlation of concentration of standard solution of andrographolide (ppm) versus peak area showed a linear correlation. The regression equation Y = 3.6538X -12.742, correlation of coefficient =0.9998 and Vxo= 2%. The value of r obtained meets the requirements set by AOAC which the required value of r > 0.99. ²⁷

3.7 Accuracy:

Percent recovery showed the accuracy of the method. The percent recovery of spiking standard solution to the tablet matrix was between 93.76 and 101.72% as described in table 3. These results showed that the method is accurate for analysis of andrographolide without any interference from excipients in the formulation of tablets and other components from raw material samples. The percent recovery of the proposed method meets the AOAC requirements, i.e 92-105%. ²⁷

Table 3 The Result of accuracy

Recovery andrographolide in sample	% recovery at level of addition to target concentration ±SD
Recovery andrographolide in sample	80%	100%	120%
Recovery sample	97.95 ± 0.25	98.02 ± 0.85	101.72 ± 0.42
Raw material F70	99.83 ± 2.81	99.92 ± 4.09	100.82 ± 1.70
Raw material F96	99.24 ± 1.23	100.07 ± 2.68	98.06 ± 1.09
Tablet of F70	99.37 ± 2.39	100.40 ± 1.33	99.27 ± 0.68
Tablet of F96	93.60 ± 0.83	96.94 ± 0.03	96.51 ± 1.74

3.8 Precision:

The Intraday precision was evaluated by six determination concentration andrographolide standard in the matrix tablet and concentration andrographolide in the sample. The interday precision was evaluated by analyzing the result of the same procedure in different days. As shown in Table 4, the intraday and the interday precision resulted %RSD below 2.39 and 2.01, respectively, meet acceptance criteria for precision recommended by AOAC.²⁷

Table 4: The Result intraday and interday precision

Precision andrographolide in sample	Measured concentration (%(w/w) ± RSD (%) ( n = 6 )
Precision andrographolide in sample	Day 1	Day 2
Matrik tablet	9.76 ± 1.60	10.58 ± 1.37
Raw material F70	18.31 ± 2.19	17.79 ± 0.60
Raw material F96	28.64 ± 2.39	28.28 ± 2.01
Tablet of F70	6.54 ± 1.60	6.98 ± 1.53
Tablet of F96	7.47 ± 1.92	7.91 ± 1.73

3.9 Robustness:

The result of robustness study showed that the method can remain unaffected by small deliberate variation method parameters by changing wavelength, flow rate, and temperature column. The result robustness listed in table 5.

Table 5: Robustness data

Variation method parameter		Mean ± SD
Variation method parameter		tR (min) (n=5)	Area (n=5)
Wavelength	224 nm	2.62 ± 0.003	655 ± 4.83
	228 nm	2.62 ± 0.003	660 ± 4.79
	230 nm	2.62 ± 0.003	649 ± 4.81
Flow rate	0.30 ml/min	2.62 ± 0.003	660 ± 4.79
Flow rate	0.35 ml/min	2.25 ± 0.003	580 ± 4.96
Column temperature	28⁰ C	2.67 ± 0.003	684 ± 1.80
	30⁰ C	2.62 ± 0.003	660 ± 4.79
	32⁰ C	2.58 ± 0.004	674 ± 5.03

CONCLUSIONS:

The proposed HPLC method developed in this study is simple, fast, selective, precise, accurate and successfully applied in routine analysis for determination of andrographolide in raw material and tablet formulation containing the fraction of ethyl acetate from Andrographis paniculata ethanolic extract (70% and 96%).

ACKOWLEDGEMENTS:

This Research was funded by MANDAT Research, Airlangga University and also very grateful to the Laboratory of Assessment Service Unit of Faculty of Pharmacy Airlangga University for utilizing HPLC instrument and Central for Natural Product Medicines Research and Development, Institute of Tropical Disease, Airlangga University for the samples.

CONFLICT OF INTEREST:

The authors claim that there is no conflict of interest.

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Received on 28.10.2019 Modified on 10.12.2019

Research J. Pharm. and Tech 2020; 13(9):4291-4296.

DOI: 10.5958/0974-360X.2020.00758.1