Development and Validation of UV-Visible Spectrophotometric and RP-HPLC Methods for Quantification of Mangiferin in Bulk Drug According to ICH Q2(R1) Guidelines

Mangiferin is a bioactive xanthone C-glycoside with the IUPAC name 1,3,6,7-tetrahydroxy-2-[(2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]xanthen-9-one [1,2]. It was first isolated from Mangifera indica L. (Anacardiaceae) [2,3,7]. 

It is found in at least 16 plant families, such as Anacardiaceae, Iridaceae, and Gentianaceae [2,3,4,6]. It is especially common in mango extracts [1-4,6]. Mangiferin functions as a strong plant metabolite with antioxidant, anti-inflammatory, antidiabetic, and neuroprotective effects [1,2,4,5]. It works by scavenging free radicals, reducing oxidative stress, inhibiting inflammatory substances, and influencing cellular pathways linked to apoptosis and immune responses [1,2,4,5,8]. 

There are only few studies reported in UV-visible spectrophotometric and RP-HPLC methods with existing challenges include time consuming optimization, limited specificity in UV-Vis and high equipment dependency in HPLC. UV-Vis studies provide rapid, non-destructive absorbance sbased analysis but lacks separation for impurities, whereas RP-HPLC provides high resolution for polar compounds but often used C18 columns and acidic mobile phases with complex mobile phases [9-11]. 

This study aims to develop and validate UV-Vis and RP-HPLC methods for mangiferin estimation which include method optimization, validation per ICH guidelines (linearity, precision, accuracy, LOD/LOQ, robustness) with enhanced sensitivity, linearity, reduced run times (~10 min), low %RSD (<2%) for precision and eco-friendly solvent utilization, addressing demerits like high LOD in UV methods and reproducibility/ruggedness by integrating with hybrid UV-HPLC for cost-efficiency.

Chemical structure:

Figure 1: Chemical Structure of Mangiferin

MATERIALS AND METHODS

Chemicals:

Mangiferin was purchased from BLD Pharmatech (India) Pvt. Ltd, Hyderabad. Methanol AR grade was procured from Sigma Aldrich Chemicals Pvt. Ltd, Mumbai. Acetonitrile HPLC grade was purchased from Sigma Aldrich Chemicals Pvt. Ltd, Mumbai. Ammonium acetate was bought from Titan Biotech Ltd., New Delhi. Acetic acid was obtained from Amichem Research Lab LLP, Dehradun, Uttarakhand.

Apparatus and conditions

Spectroscopic conditions

Shimadzu UV-1650PC UV spectrophotometer, which has a silicon photodiode detector and UV probe software was used for this study.

1. Selection of solvent

Methanol was used to prepare the initial stock solution of Mangiferin and distilled water was used for subsequent serial dilutions.

2. Selection of wavelength

Based on absorbance shown in UV scanning for Mangiferin from 200 to 400 nm, 257 nm is found to be the appropriate wavelength for detection and that λmax is used for the study.

Chromatographic conditions

The Shimadzu (CBM-20A) HPLC device which was equipped with Shimadzu-SPD-M20A diode array detector and LC lab solution software was used. The Shim pack solar C8 column with dimensions 250 mm x 4 mm x 5 µm was used.

1. Selection of wavelength

The UV spectra were used to determine the wavelength and 257 nm was chosen because of its good absorbance. Therefore, based on the spectrum it was determined that the detection wavelength under study is 257 nm.

2. Fixed chromatographic conditions

Method development was carried out with mobile phase 10mM ammonium acetate pH 3: acetonitrile (80: 20). At a detection wavelength of 257 nm, the flow rate, column temperature, and sample volume were adjusted to 1 ml/min, 25?C, and 20µl, respectively.

Preparation of stock solutions:

UV-visible Spectroscopy

Stock 1

10 mg of Mangiferin was weighed and transferred into 10ml volumetric flasks and the volume was made up to 10 ml using a 1:1 ratio of methanol to water. The mixture was then sonicated for 15 minutes until thoroughly dissolved. The final concentration of Mangiferin was 1000 µg/ml.

Stock 2

A 1 ml aliquot of Mangiferin from stock solution 1 was transferred to 10 ml volumetric flasks and the volume was made up to 10 ml with water. The solution was sonicated for 15 minutes, and the final concentration of Mangiferin was found to be 100 µg/ml.

Construction of Calibration curve:

To achieve a final concentration range of 1-5 µg/ml, aliquots of 0.1, 0.2, 0.3, 0.4, and 0.5 ml of Mangiferin solutions from stock 2 were transferred into 10 ml volumetric flasks and filled with water up to the mark. The standard solutions of Mangiferin were scanned at 257 nm and calibration curve was created by plotting concentration on the x-axis and absorbance on the y-axis.

RP-HPLC

Preparation of 10mM ammonium acetate buffer (pH 3)

770 mg of ammonium acetate was weighed and dissolved with about 800ml water (HPLC grade) in a beaker. pH was adjusted to the target value by using dilute acetic acid solution. The solution was filtered through a membrane of 0.45µm into a measuring flask and sufficient water was added to produce 1000 ml.

Preparation of mobile phase

A mixture of 10mM ammonium acetate buffer (pH 3) and acetonitrile in an 80:20 ratio was prepared and degassed in an ultra sonicator for 15 minutes.

Preparation of standard stock solution

10 mg of Mangiferin standard was taken in a 10 ml volumetric flask and the volume was made up to 10 ml with mobile phase (10mM ammonium acetate buffer of pH 3 and acetonitrile in 80:20 ratio). Then the mixture was sonicated for 15 min to dissolve it completely. From the stock solution, working standard solutions were prepared by appropriate dilutions with mobile phase. Calibration curve was plotted from the concentration range of 1-5 µg/ml for Mangiferin.

RESULTS

Method development and optimization of Spectroscopic conditions

To determine concentration of an analyte, with a cuvette containing a sample solution, a UV- Visible spectrophotometer detects the light intensity and compares it to the light intensity prior to the sample passage through the cuvette. A light source, a sample holder, a dispersive element to separate the various wavelengths and an appropriate detector are the essential parts of a UV-Visible spectrophotometer [12]. The analytical technique measures the absorption of monochromatic light by colourless substances in the 200–400 nm near ultraviolet region of the spectrum [13]. The UV scanning at 200-400nm for Mangiferin showed that 257 nm is the suitable wavelength for detection as per maximum absorbance. The spectrum is shown in Figure 2.

Method development and optimization of chromatographic conditions

To achieve good resolution different proportions of solvents like acetonitrile and 10mM ammonium acetate buffer of different pH conditions were tested. However, in 10mM ammonium acetate buffer with pH 3 and acetonitrile in the ratio of 80:20 achieved satisfactory results at a flow rate of 1 ml/ min measured at a detection of 257nm. The chromatogram of optimized standard is shown in Figure 5. The system suitability parameters such as retention time, resolution, tailing factor and theoretical plates for optimized standard mixture chromatogram is tabulated in Table 8.

Method Validation

Linearity

Linearity was studied by analysing five standard solutions in the range of 1 – 5 µg/ml for Mangiferin by UV-Visible Spectrophotometry and RP-HPLC. Calibration curves with concentration versus absorbance or peak area were plotted for each method. Linearity was measured by using calibration data, regression analysis as well as coefficient of correlation. The regression data is displayed in Table 1. The correlation coefficient (r²) of Mangiferin was found to be 0.999 and 0.998 for UV and RP-HPLC methods respectively. The linearity graphs were shown in Figure 4 & 6. This shows better detector response in different concentration for both methods.

Precision

A minimum of three determinations per concentration should be used to measure precision [14]. Precision of the analytical method was demonstrated by, intra-day precision, inter-day precision and repeatability. % RSD was calculated for both methods and results are represented in Table 2, 3 & 4. The coefficient of variation (%RSD) was less than 2% which shows the methods were precise.

Limit of detection (LOD) and Limit of quantitation (LOQ)

The limit of detection and quantitation of the drugs for both methods were calculated with standard deviation and slope. The LOD and LOQ of each method was calculated and represented in Table 5.

LOD and LOQ of were calculated by using the following formula,

LOD = 3.3*σ / S

LOQ = 10*σ / S 

where, σ = standard deviation and S = slope

Robustness

Robustness of the UV spectrophotometry analytical method was determined by the analysis of appropriate concentrations of Mangiferin at different wavelengths (257±2nm). Values of % RSD were calculated and are shown in Table 6.

Ruggedness

Ruggedness is the ability to replicate results from an analyst and instrument under normal and expected operational conditions [15]. Appropriate concentrations of Mangiferin were analysed by different analyst. % RSD was calculated using regression coefficients obtained on different analysts and values are shown in Table 7.

System suitability

The determination of the system suitability of RP-HPLC method was accomplished by using the concentration of 3 μg/ml. The system suitability was assessed based on the retention time, peak area, theoretical plates, and asymmetry factor [16]. The optimized method is acceptable as system suitability parameters are valid as they passed the criteria of acceptability, as shown in Table 8.

DISCUSSION

UV-Visible spectrophotometry

For Mangiferin, the parameters of the UV-visible spectrophotometric method were determined. For Mangiferin, the correlation coefficient (r²) was 0.999. This demonstrates improved detector response across a range of concentrations. This proposed method was validated as per ICH Q₂R₁ guidelines. LOD and LOQ indicates the sensitivity of the method. The coefficient of variation (% RSD) was less than 2% which shows the method was precise. As a result, a simple, sensitive, and precise analytical method for estimating Mangiferin by UV-visible spectrophotometry has been developed and validated in accordance with guidelines.

RP-HPLC

The working conditions for the RP-HPLC method were established for Mangiferin in bulk form. The correlation coefficient (r²) for Mangiferin was 0.998. This demonstrates an improved detector response at various drug concentrations. This proposed method was validated as per ICH Q₂R₁ guidelines. LOD and LOQ indicates the sensitivity of the method. The coefficient of variation (% RSD) was less than 2% which shows the method was precise. According to the cited research article, the retention time of Mangiferin was in the range of 6.1 to 18.9 mins [16-25]. But as per the newly developed analytical method, retention time was found to be 2.9 min for Mangiferin by using flow rate of 1 ml/ min and the elution was completed within 5 min. Hence, Simple, sensitive and accurate analytical method has been developed and validated as per guidelines for the estimation of Mangiferin in bulk form by RP-HPLC.

Table 1: Regression data

Table 2: Intra-day precision data

Table 3: Inter-day precision data

Table 4: Repeatability data

Table 5: LOD and LOQ data

Table 6: Robustness

Table 7: Ruggedness

Table 8: System suitability parameters of RP-HPLC

Table 9: UV spectrophotometry analytical method validation parameters

Figure 2: Spectrum of Mangiferin (3 μg/ ml) at 257 nm

Figure 3: Overlain spectra of standard solution of Mangiferin

Figure 4: Linearity graph of Mangiferin at 257nm for UV-visible spectroscopy

Figure 5: Representative chromatogram of Mangiferin at 257nm for RP-HPL

Figure 6: Linearity graph of Mangiferin at 257nm for RP-HPLC