INTRODUCTION:

Mangiferin obtained from Mangifera indica belonging to Anacardiaceae family has numerous therapeutic values. It is a xanthonoid with multiple phenolic groups and chemically known as C-2-β-D-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone or C-glucosyl xanthone¹. It shows high antioxidant activity²and multiple therapeutic actions like cardioprotective, neuroprotective, analgesic, immunomodulatory, antipyretic, antitumor, anti-inflammatory, antidiabetic³and antiviral⁴. As a result, mangiferin can be recognized as a more promising, new medicine at the clinic with the advancement of biotechnology and research practice.

Mangiferin can be quantified using several methods. This study's primary goal is to create a HPLC assay method and validate it as per ICH guidelines⁵. Analytical process validation ensures that the same output is produced consistently and reproducibly using various HPLC analytical methods. Since analytical technique validation provides the researcher with information on precision, linearity, detection, robustness, and quantitation limitations, it is an extremely important stage in the development of new methods. Validating an analytical method's suitability for its intended use is the aim of the "International Conference on Harmonisation" (ICH) guideline. The relevant authorities now require validation data to be provided during the medication development process. Utilising the USP and ICH standards, validation processes are verified.⁶ Through this study, a novel, quick, affordable, and sensitive HPLC approach for identifying mangiferin in pharmaceutical dosage forms was created and validated.

MATERIALS AND METHODS:

Materials:

Mangiferin (95% pure) CAS. No: 4773-96-0 was purchased from Nanjing Nutriherb Biotech CO.LTD, China. Chromatography-grade acetonitrile was purchased from Merck Ltd. in Mumbai, while the 0.22-micron nylon filter membrane was obtained from Pvt. Ltd company i.e Riviera Glass., also situated in Mumbai. Ultrapure water from Merck, Millipore Direct-QR 3 UV-purification system based in the USA was used in the process. Potassium dihydrogen orthophosphate was procured from SRL, orthophosphoric acid was purchased from Merck, and triethylamine was obtained from Molychem, all of which are of GR grade.

Instruments and software:

The analytical method for Mangiferin separation was done by using HPLC system of Shimadzu (Kyoto, Japan) pressure gradient pump at binary high or low, PDA detector of SPD-M20A, autosampler of SIL-20A/C HT and degasser. The software used for the process of chromatographic output data is LC solution 5.57 control system. The stationary phase utilized is the Phenomenex KinetexR 5-micron C18. Standard substances are precisely weighed using the analytical balance Saffron SES204 procured from Saffron Electronics scale, after filtering the potassium dihydrogen orthophosphate buffer through a 0.22-micron filter membrane using a vacuum filtration unit equipped with a membrane holder from Tarsons, an ultrasonic bath sonicator obtained from Oscar Ultrasonics is used for degassing. The electronic pH meter, model number 335 from Systronics, is utilized for adjusting the pH of the potassium dihydrogen orthophosphate buffer. The ultrapure water has undergone treatment via the Direct-Q3 purification system.

Conditions for chromatography:

Mangiferin is effectively separated and detected at a wavelength of 258nm by employing an isocratic elution method using a mixture of 10mM potassium dihydrogen phosphate buffer (pH adjusted to 2.4±0.05 with orthophosphoric acid) and Acetonitrile in a volumetric ratio of 90:10. After conducting a thorough screening of pH variations and various ratios within the mobile phase, this particular composition was identified. The injection volume for Mangiferin is set at 20 microliters, while the total run time is 20 minutes, maintaining a flow rate of 1.3ml/min. The autosampler temperature is controlled at 20°C, and the room temperature is regulated within a range of 24±2°C.

Standard and Sample Dilutions Preparations:

The normal stock of ten milligrams of mangiferin were precisely weighed and dissolved in ten millilitres of 30% ethanol. The mixture was then vortexed for three to five minutes and sonified for ten minutes at room temperature.One milligram per millilitre of stock was diluted to create the second solution stock B, which has a concentration of 100μg/ml. The samples of different concentrations from 100ng to 25000ng/ml are prepared by diluting standard solution of Mangiferin with the mobile phase to check the linearity.

Analytical Method Validation:

For determining the validity of the optimised approach, ICH rules were followed. The subsequent validation criteria were assessed⁷:

Specificity:

It was evaluated to see if the developed approach could precisely resolve Mangiferin without interference at its Rt⁸.

System suitability:

The suitability of the improved procedure for the intended purpose was tested by injecting six duplicates of Mangiferin (10 micrograms per millilitre) into the system.

Linearity:

If an analytical method has the capability to produce test results directly proportional to the concentration (quantity) of the tested component within the sample, particularly within a specific range, it is deemed to be linear. A linearity plot was generated by adding Mangiferin in triplicates at concentrations ranging from 100 to 25,000ng/mL.Plotting the substance amounts over the relevant peak areas was the first step in creating the regression equation. Plotting the substance amounts over the relevant peak areas was the first step in creating the regression equation^9-10.

Precision:

To assess the method's ability to produce consistent results under specific conditions, six replicates were injected into HPLC at a concentration of ten micrograms per millilitre. This was done for both intra- and inter-day precision testing. The areas were used to compute the % relative standard deviation¹¹.

Accuracy:

Three replicates of quality control samples containing Mangiferin were injected into the High-Performance Liquid Chromatography (HPLC) system at concentrations of 8micrograms per millilitre, 10 micrograms per millilitre, and 12 micrograms per millilitre^12-14. This was conducted as part of an accuracy assessment aimed at evaluating the agreement between the obtained values and established reference values for determining the accuracy of the analysis. The calculated recovery percentage was assessed against predefined acceptance criteria to validate the accuracy of the results^15-16.

Robustness:

Any changes in the method responses were assessed using variations in pH of the Potassium dihydrogen orthophosphate buffer, flow rate, ACN and Potassium dihydrogen orthophosphate buffer percentage.We next assessed the following variables., pH (2.4±0.1) of the phosphate buffer, flow rate (1.3±0.1ml/min), ACN: phosphate buffer ratio (10:90±1%) The reactions to the technique parameters were evaluated using the percent RSD^17-21.

Limit of Detection (LOD) and Limit of Quantification (LOQ):

Adhering to the directives established by the International Council for Harmonisation (ICH), LOD and LOQfor Mangiferin were established by utilizing the formula: LOD = 3.3(σ)/S. Similarly, LOQ = 10(σ) / S. Here, σ denotes the standard deviation of the response, while S represents the slope of the regression line^22-23.

RESULTS AND DISCUSSION:

Analytical Method Validation:

For determining the validity of the optimised approach, ICH rules were followed. The subsequent validation criteria were assessed.

System suitability:

To assess the suitability of the enhanced method for its intended purpose, the appropriateness of the system was evaluated by injecting six replicate samples of Mangiferin at a concentration of ten micrograms per millilitre. The data obtained was represented in table 1.

Table 1: Data of system suitability

1	Average	728029.5
2	SD	1043.687
3	%RSD	0.14
4	% Recovery	97.6

Specificity:

The chromatograms presented in Figure 1 confirm that the developed method can distinctly separate Mangiferin without any interference from the diluent. This assessment was conducted by injecting three replicates of each parameter into the HPLC system.The Mangiferin was eluted at 14.7 min with Area 728360, tailing factor 1.3 (<2%) and Theoretical plate value 13291 (>2000).

Figure 1: Retention time of (A) Blank and (B) Mangiferin

Linearity and Range:

The ability of an analytical process to generate test outcomes that correspond accurately to the quantity of the test substance in the sample is termed as linearity. To assess linearity, known concentrations of Mangiferin were spiked at levels ranging from 100ng to 25000ng/mL in triplicate. The relationship between peak area and drug concentrations was plotted to form a regression equation, demonstrating the correlation between these variables.

Table 2: Data of linearity results:

Method	Linearity
Equation	y = 75.15x - 5527.9
Correlation coefficient (R²⁾	0.9999

Figure 2: Calibration curve of Mangiferin

Limit of Detection (LOD) and Limit of Quantification (LOQ):

The LOD for Mangiferin was established at 7.03ng/mL, while the LOQ was determined to be 21.32ng/mL, respectively.

Accuracy:

As part of the accuracy assessment, triplicate injections of three different Mangiferin quality control samples (at concentrations of 8 micrograms per millilitre, 10 micrograms per millilitre, and 12 micrograms per millilitre) were carried out using HPLC. This aimed to gauge the agreement between the obtained values and established reference values. The calculated recovery percentages fell within the acceptance criteria, ranging from 98.16% to 99.86%.The accuracy test results are as follows:

Table 3: Data of Accuracy

Addition of standards	% Recovery	% RSD
80%	99.86%	0.95%
100%	99.63%	0.16%
120%	98.16%	0.14%

*Average of three estimations at each level

Precision:

Six replicates were injected into HPLC at a concentration of 10 micrograms per millilitre to perform inter- and intra-day precision testing to ascertain the method's capacity to deliver consistent findings under a given circumstance. The accuracy was evaluated throughout intraday and interday hours. The relative standard deviation was used to report the results (RSD). The precision test results are as follows:

Table 4: Data of Precision:

Day 1 (intraday)	Day 2 (interday)
0.132%	0.364%

RSD: Relative standard deviation, *Average of six determinations

Robustness

It was utilised to identify any changes in method responses when method parameters, such as flow rate, ACN: phosphate buffer ratio, pH of the buffer were changed, and each trial had impacted the results significantly. The % RSD for the parameters changed remained in the acceptable criteria as mentioned in the table 5

Table 5: Data of robustness

Parameters	System suitable parameter	Acceptance Criteria (%RSD)	Observed
Flow rate	1.2mL/min	<2%	0.19%
Flow rate	1.4mL/min		0.72%
Mobile Phase (B %)	89		0.35%
Mobile Phase (B %)	91		0.39%
pH	2.3		0.14%
pH	2.5		0.08%

CONCLUSION:

In this research, a HPLC analytical method was formulated and validated for mangiferin, demonstrating simplicity, accuracy, precision, and linear stability. This method holds promise for routine quality control analysis. The parameters of the analytical method and mobile phase solvents were optimized to ensure excellent resolution of Mangiferin. Validation conducted in line with ICH criteria confirms the reliability of this method to consistently produce precise and accurate results across diverse chromatographic conditions.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGEMENT:

The authors would like to acknowledge the All-India Council for Technical Education (AICTE-QIP) for granting the fellowship to Ms. Shireesha J, enabling her to enhance her knowledge and skills. Additionally, we extend our gratitude to Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education, for their invaluable support in terms of necessary infrastructure and funds, which facilitated the successful execution of this study.

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Received on 04.01.2024 Modified on 23.03.2024

Research J. Pharm. and Tech 2024; 17(11):5230-5234.

DOI: 10.52711/0974-360X.2024.00800