Ilango et al /2014

International Journal of Phytopharmacy Research Article

Vol. 4 (1), pp.11-15, Jan -Feb 2014
ISSN: 2277-2928 (Online)
Journal DOI:10.7439/ijpp
©Scholar Science Journals
www.ssjournals.com

LC-MS Quantification of Mangiferin inhydroalcoholic extract of

Salacia oblonga, Salacia roxburghii and polyherbal formulation
1 1 1 2
Ilango Kaliappan *, Ananth Kumar Kammalla , Mohan Kumar Ramasamy , Agarwal Aruna
3
and Dubey GP
1
Interdisciplinary School of Indian System of Medicine, SRM University, Kattankulathur-603203, Tamil Nadu, India.
2
National Facility for Tribal & Herbal Medicine, Institute of Medical sciences, Banaras Hindu University, Varanasi, India.
3
Faculty of Ayurveda, Institute of Medical sciences, Banaras Hindu University, Varanasi, India.

*Correspondence Info:
Prof. Dr. Kaliappan Ilango,
Dean,
Interdisciplinary School of Indian System of Medicine (ISISM),
SRM University, Kattankulathur-603203, Kancheepuram (Dt), Tamil Nadu, India.
E-Mail: ilangok67@gmail.com

Abstract
Mangiferin is the one of the major active constituents in the tubers of the Salacia species, which is one of the valuable species
in Ayurveda with antidiabetic, antihypertensive, hepatoprotective, anticaries and anticancer potentials. A simple and rapid liquid
chromatography coupled mass spectrometry analysis was developed for the quantification of Mangiferin in two species of Salacia and
also in polyherbal formulation. This method allowed the direct coupling of an electron spray mass selective detector for the LC system
with photo diode array detector, Phenomenex C 18 column, acetonitrile and aqueous formic acid (0.5%) as mobile phase. Under these
conditions, Mangiferin was well separated from the mixture of components present in the extract and detected with mass spectrometry
(mass selective detector). Liquid chromatography coupled with electron spray mass was used in positive ion mode and detected at m/z
423(M+1)+. The proposed method is more accurate and sensitive can be used for the routine quantification of the Mangiferin in the herbal
extracts as well as polyherbal formulations.
Keywords: Salacia oblonga, Salacia roxburghii, Mangiferin, Polyherbal formulation

1. Introduction
Indian System of Medicine (ISM) is one of the most ancient system of traditional medicine and it has been practiced in Indian
peninsula since 5000BC to offer natural ways to treat diseases and to promote healthcare 1. Single active components, herbal extracts and herbal
combinations have played a significant role in the prevention and management of diseases, especially in complicated chronic conditions.
Salacia species are one of the most important species in the traditional system medicine with antidiabetic, antihypertensive,
hepatoprotective, anticaries and anticancer activities. It has been officially listed in the Ayurvedic Pharmacopeia 2. Literature survey revealed that,
Salacia species plays an important role in the management of diabetes by various mechanisms like inhibition of protein kinase, activation of
PPAR γ, inhibition of the α-glucosidase enzyme 3,4. Mangiferin is one of the major active components in the tubers of the Salacia species. It can
also be isolated from the other medicinal plants such as Mangifera indica, Folium pyrrosiae, Swetia chirata and Rhizoma anemmarrhenae5.
Chemically Mangiferin is a (1,3,6,7-tetrahydroxyxanthone-C-2-β-D glycoside having variety of pharmacological effects including anti-
oxidative6, anti-diabetic7, neuroprotective8, gastro protective9, immunomodulatory activities 10. In the present study liquid chromatography
coupled with mass spectroscopy employed for quantification of Mangiferin in the extracts of Salacia oblonga, Salacia roxburghii and its
polyherbal formulated capsules with other plants.

2. Materials and methods

Polyherbal formulation was obtained from M/s Varanasi Bio Research Pvt. Ltd, Varanasi, India. Mangiferin (99.0% purity) was
obtained from M/s Sigma Aldrich. Acetonitrile of HPLC grade was purchased from the Merck co. (India). Milli-Q water from Millipore
(Massachusetts) was used throughout the study and Formic acid (Rankem, India).
2.1 LC-MS Instrumentation and Analytical Conditions: LC analytical procedure was performed using a system consisting of a LC10ADVP
pump and a single quadruple mass spectrometer with electron spray ionization (ESI) source (LCMS-2010) (Shimadzu, Japan). Data acquisition
and processing software Lab solutions version 7.0 was from Shimadzu.

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Chromatographic separations were carried out using Phenomenex C 18 analytical (5µm, 250 x 4.6mm, i.d.) (Phenomenex, USA). The
analysis was achieved by gradient elution using (A) Water (containing 0.5%formic acid) and (B) Acetonitrile as the mobile phase. The flow rate
was 0.5ml/min. The diode array detector was set at 280nm and column was maintained at ambient temperature.
The ESI was performed using nitrogen gas to assist nebulization (the flow rate was set at 1.5L/min). Selective ion monitoring (SIM)
with positive mode, capillary voltage at 1.6 kV and temperatures of Curved Desolvation Line (CDL) and heat block at 250ºC and 300ºC were
used. Target ion was measured at m/z 423(M+1)+ for Mangiferin.
2.2 Preparation of Mangiferin calibration standards: The standard stock solution was prepared by dissolving 1.5mg of Mangiferinin 10ml of
acetonitrile and water (1:1) to furnish a concentration of 150µg/ml. Then this solution was sonicated for 5min. Calibration standards at six levels
of concentrations were prepared by serial dilution of the stock solution with HPLC grade acetonitrile and water (1:1) from 2µg/ml to 128µg/ml.
Injection of 20µL in triplicate were made from each concentration and chromatographed under the specified conditions described above.
2.3 Sample preparation: Salacia oblonga and Salaciaroxburghii extracts were prepared by dissolving 200mg of the extract powder in 10ml of
Acetonitrile and Water (1:1). The polyherbal formulation capsule powder equivalent to 100mg of the Salacia oblonga was weighed accurately in
10ml of acetonitrile and water (1:1). All the solutions were sonicated cooled and then made up to the volume and filtered through 0.22µ
membrane filter for further analysis.
2.4 Optimization of the LC and MS conditions: On the basis of the structure, solubility and acid-base properties of Mangiferin, the method was
developed with a C18 column and the mobile phase containing acetonitrile and aqueous formic acid. The first mobile phase was 10:90 binary
mixtures of acetonitrile and buffer. When the same binary mixture in different proportions (20:80, 15:85and 10:90) was tested, it was found that,
as the proportions of acetonitrile in the mobile phase was reduced the retention time of the compound gradually increased. As a result, the mobile
phase with gradient program was developed with Acetonitrile and water (0.5% Formic acid) was finally selected to achieve good resolution with
short retention time.
The detection of Mangiferin was set at wavelength 280nm taking above things to in consideration. With these chromatographic
conditions baseline resolution was achieved with reasonable retention time. Typical chromatograms were obtained from a standard, extracts and
herbal formulation.
To select an appropriate ionization mode in LC-MS analysis, the mass spectra were measured in ESI and APCI positive and negative
mode using Mangiferin standard solution. In both ionization modes, the base peak intensities of the positive ion were higher than that of negative
ion, and efficiencies of the ionization in the ESI were higher than APCI. Thus, selective ion monitoring (SIM) mode at 423 (M + 1) + was used for
quantitative analysis of Mangiferin. SIM mode mass spectrum of Mangiferin was shown in Fig. 1

Fig. 1: SIM mode mass spectrum of Mangiferin

Mass spectrometer conditions were optimized by direct infusion of the standard and SIM acquisition mode was used for analysis, in
order to detect only specific mass ions during analysis.
2.5 Application of the method: The validated LC-MS method can be used for routine quantification of Mangiferin in Salacia oblonga,
Salaciarox burghii and its polyherbal formulation. Results obtained are listed in Table 5. The 3D graph representation of the Salacia species and
polyherbal formulation was shown in Fig.6, Fig.7 and Fig.8.

3. Results and discussion

3.1 Method validation
3.1.1 Specificity: Typical chromatograms of standard Mangiferin, Salacia species extract and polyherbal formulation containing Salacia species
are shown in the Fig.2, Fig.3, Fig.4 and Fig.5. The retention time for Mangiferin was found to be at 6.7min with total run time of 30min. A good
separation of the Mangiferin peak from other constituent was obtained under developed chromatographic conditions. Other constituents from
different plants do not interfere with Mangiferin.
3.1.2 Calibration curve: Linearity was determined by constructing calibration plot. Five different concentrations of the standard solutions were
prepared, chromatographed and linearity was obtained by calibration plot. Peak area (A) and concentration (C) for each compound were
subjected to regression analysis to calculate the calibration equations and correlation coefficients. The regression equations obtained for
Mangiferin was 2.19e-005 X-0.436.The linearity range was 1.25 to 20µg/ml. The result shows that there was a first-rate correlation between peak
area and concentration for compound. The correlation coefficient, slope, intercept and % relative standard deviation (%RSD) are suitable as
general acceptable criterion to the linearity performance of an analytical procedure.
3.1.3 Quantification limits (LOD & LOQ): The quantification limit is the lowest concentrations of the compound, that can be accurately and
precisely quantified, which is ten times than noise level. The LOQ of the compound was determined experimentally by performing six injections
of each concentration and it was found to be 9.16ng for Mangiferin.

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3.1.4 Accuracy: Accuracy was determined by developed method and spiking extracts with known amount of the Mangiferin standards and
compared the measured value with true values. Triplicates injections were made with all spiked samples. Table1 summarizes the accuracy results,
expressed as recovery percentage. The method has shown 98.12 ± 1.70 % recovery of sample from extract.

Table 1. Recovery of Mangiferin from the extract of Salacia oblonga (n=3)

Sl. No

1
2
3
Each value is mean of three observations
3.1.5 Precision: The precision of the method as repeatability intra-day assay precision [%CV] was assessed by performing six independent
analysis of sample and qualified reference standards together at 100% of the test concentration. Inter-day precision was determined by repeating
the analysis of the same concentration by repeating the studies by three different analysts on three different days over a period of 1 week also
expressed in terms of %CV in Table 2.

Table 2.Summary of intra-day and inter-day method precision

Amount (µg/ml)

1.25
5.0
20.0
-

System precision is a measure of the method variability that can be expected if a given analyst performs the analysis at three different
concentrations. It was determined by performing three replicate analysis of each standard solution at three different concentrations. The RSD
values for Mangiferin were shown in Table 3.

Table 3.System precision for Mangiferin at different concentrations (n=5)

The method precision was determined by replicate sample solutions and RSD values obtained were 1.1%, 0.97% and 0.47% for 2.5µg/ml,
10µg/ml and 20µg/ml of Mangiferin respectively. The summary of the validated method and results are depicted in Table 4.

Table 4.Summary of method validation parameters

Parameter
Linearity range (µg/ml)
Correlation coefficient
Limit of detection(µg)
Limit of quantification(µg)
Recovery (mean + SD)
Precision (CV)
Intra-day (n=6)
Inter-day (n=3)

Table 5. Percentage of Mangiferin content in the Salacia species and its Polyherbal formulation
S. No
1
2
3

Fig. 2: Chromatogram of Mangiferin standard

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Fig. 3: Chromatogram of Mangiferin in Salaciarox burghii

Fig. 4: Chromatogram of Mangiferin in Salacia oblonga

Fig. 5: Chromatogram of Mangiferin in polyherbal formulation containing Salacia species

Fig. 6: 3D Graph representation of the Salacia oblonga

Fig. 7: 3D Graph representation of the Salaciarox burghii

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Fig. 8: 3D Graph representation of Polyherbal formulation

4. Conclusion
The results described above showed that the developed LC- MS method was highly suitable for rapid determination of standard
Mangiferin, individual extracts and or with a combination of other extracts in the form of formulations. This work also showed that LCMS was a
powerful technique for the quantification of Mangiferin in the complex extracts obtained from the medicinal plants and also in the form of
polyherbal formulations.
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