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Abstract
The present work aims at the systematic development of a simple, rapid and highly sensitive densitom-
etry-based thin-layer chromatographic method for the quantification of mangiferin in bioanalytical
samples. Initially, the quality target method profile was defined and critical analytical attributes
(CAAs) earmarked, namely, retardation factor (Rf ), peak height, capacity factor, theoretical plates
and separation number. Face-centered cubic design was selected for optimization of volume loaded
and plate dimensions as the critical method parameters selected from screening studies employing
D-optimal and Plackett–Burman design studies, followed by evaluating their effect on the CAAs. The
mobile phase containing a mixture of ethyl acetate : acetic acid : formic acid : water in a 7 : 1 : 1 : 1 (v/v/v/
v) ratio was finally selected as the optimized solvent for apt chromatographic separation of mangiferin
at 262 nm with Rf 0.68 ± 0.02 and all other parameters within the acceptance limits. Method validation
studies revealed high linearity in the concentration range of 50–800 ng/band for mangiferin. The
developed method showed high accuracy, precision, ruggedness, robustness, specificity, sensitivity,
selectivity and recovery. In a nutshell, the bioanalytical method for analysis of mangiferin in plasma
revealed the presence of well-resolved peaks and high recovery of mangiferin.
Introduction protection against ROS-induced oxidative stress (3, 4). Other potential
Mangiferin, a polyphenolic C-glucosylxanthone, primarily exists as a therapeutic applications of mangiferin include being used as an antidi-
principal phytoconstituent in the leaves and stem bark of Mangifera abetic (5), antiobesitic, antiosteoclastogenic, antiasthmatic (6), antidiar-
indica (family Anacardiaceae). Chemically, 2-C-β-D-gluco-pyranosyl-1, rhoeal (7), immunomodulator, analgesic, antiallergic, antibacterial (5,
3,6,7-tetrahydroxyxanthone (Figure 1) exhibits moderate aqueous sol- 8), antimicrobial (4), antiviral (9, 10) and anticancer (3, 11–13)
ubility, i.e., 1.44 mg/mL and low lipophilicity (log P) of −0.59 (1, 2) agent. Considering the number of its therapeutic benefits, mangiferin
predicted by in silico simulations performed on mangiferin using has proved to have promising utility in clinical treatment and manage-
ADMET Predictor (Version 7.1.0013; Simulations Plus, Inc., USA) ment of multiple disorders.
and GastroPlus Simulation software (Version 8.6; Simulations Plus, Development of a simple, rapid and highly sensitive bioanalytical
Inc.). Presence of the phenolic xanthone moiety owes to the powerful method is highly desirable for quantification and routine chromatographic
antioxidant activity of mangiferin for scavenging free radicals and analysis of mangiferin in pharmaceutical formulations, and bioanalytical
© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com 1
2 Khurana et al.
and toxicological samples. Several literature reports have been document- the CAAs using prioritization and screening studies, method optimiza-
ed for quantification of mangiferin employing techniques like UV–Vis tion using suitable experimental designs, modelization and optimum
spectroscopy (1, 14), spectrofluorimetry (15), thin-layer chromatography search through response surface methodology to embark upon the ana-
(TLC) (16, 17), high-performance liquid chromatography (HPLC) lytical design space and postulation of control strategy for continuous
(18–20), high-performance thin-layer chromatography (HPTLC) (21), liq- improvement in method performance (41). In the past few years, several
uid chromatography–mass spectrometry (LC–MS) (22, 23), tandem mass literature reports have successfully demonstrated the immense utility of
spectrometry (LC–MS-MS) (24, 25) in the bulk drug (26–30) as well as in the AQbD approach for developing efficient and cost-efficient liquid
bioanalytical samples like rat plasma (31, 32), urine (33) and aqueous chromatographic methods for estimating different analytes in pharma-
humor (34, 35). However, none of these methods have been considered ceutical formulations and bioanalytical samples (42–48). Application of
to be highly satisfactory owing to the involvement of complex aqueous AQbD specifically in the current thin-layer densitometry method
and organic solvent mixtures, and monitoring of the complex chromato- development envisioned for identifying method variables plausibly
graphic conditions. associated with high degree of variability and exhibiting maximal
Consequently, in order to obviate the tedious, expensive and pro- influence on method performance.
longed sample preparation, use of intricate mobile phase compositions The present studies were accordingly undertaken to develop an
and optimization of instrumental method variables associated with AQbD-enabled, simple, ultra-fast, robust, sensitive, effective and econom-
the conventional HPLC procedures, HPTLC and TLC techniques ical densitometry-based TLC method for quantification of mangiferin in
have been considered highly useful for ease of quantification and faster the bioanalytical samples.
analysis of drugs, requiring minimal expenditure of developmental ef-
fort, time and resources. Use of a densitometry-based TLC method
during analytical method development offers multiple advantages Materials and methods
over HPLC, like lower mobile phase consumption, higher scanning Standards and reagents
speed, shorter analysis time, reduced sample cleanup, lower operation- Mangiferin was purchased from M/s Sigma-Aldrich Co., Mumbai,
al and maintenance costs, and, above all, ease of detection and optimi- India, and was used as working standard in the concentration of
zation of critical method variables, thus facilitating faster analysis of 1 µg/mL. Analytical grade ethyl acetate, acetic acid, formic acid and
drug by saving a great deal of time and resources (36). Also, it provides methanol were purchased from M/s Merck Ltd., Mumbai, India,
the leverage of avoiding tedious sample preparation, liquid–liquid and were used as received without further purification.
extraction and filtration steps encountered during HPLC analysis. Be-
sides, other stellar merits of TLC techniques encompass the possibility
Preparation of standard solutions
of selecting corrosive solvents with high pH range, minimal chances of
The stock solution was prepared by weighing accurately 10 mg of
sample contamination with the previous ones, high sensitivity from
mangiferin in a 10 mL volumetric flask followed by dilution in meth-
the nanogram to pictogram level and highly reproducibility of the den-
anol to obtain the concentration of 1,000 µg/mL. This stock solution
sitometric method for identifying the concentration of the sample (37).
was further appropriately diluted with methanol in a 10 mL volumet-
Of late, the use of Quality by Design (QbD) paradigms has been per-
ric flask to prepare the final concentration of 1,000 ng/mL.
meating the industrial, academic and federal domains at an alarming
pace with the objective to gain a holistic process and product under-
standing. Implementation of the Analytical Quality by Design (AQbD) Biological matrix
approach, in particular, has been highly popularized for developing ro- Fresh human blood was procured ex gratis from the Rotary Blood
bust methods by specifically understanding the critical method parame- Bank, Chandigarh, India. Harvested plasma samples were further
ters (CMPs) influencing method performance (38, 39). Considered as a employed for the entire method development and validation studies.
science and risk-based approach, AQbD provides rational comprehen- For preparation of calibration standards and quality control samples,
sion of the CMPs affecting the critical analytical attributes (CAAs) of human plasma was extracted by centrifugation of blood treated with
the bioanalytical method by risk assessment and factor screening studies, ethylenediaminetetraacetic acid at 10,000 r.p.m. (5,590 × g) using a
followed by unearthing the plausible interactions among them and op- centrifuge (Remi, Mumbai, India).
timizing them by employing Design of Experiments for enhanced meth-
od performance (40). The AQbD approach of method development Instrumentation and chromatographic conditions
primarily involves defining the quality target method profile (QTMP) Bioanalytical method development for mangiferin was carried out using
and CAAs, identifying those CMPs that are significantly influencing a Camag HPTLC system equipped with Linomat-V semiautomatic
Systematic Development of a Thin-Layer Chromatographic Method for Mangiferin 3
sample applicator. Suitable volumes of standard and sample solutions phase vapors for suitable time before each run. The development of
were applied in triplicate using a Hamilton syringe (100 mL) with slit the plate was carried out by the ascending technique to a migration
dimensions of 6 mm × 0.30 mm, scanning speed of 20 mm/s and data distance of 80 mm. After development, aluminum-backed silica gel
resolution of 100 µm/step, equipped with an optical filter (second TLC 60 plates were dried in a hot air oven at 25°C. Densitometric
order) and filter factor (Savitzky golay 7). Chromatographic separation scanning of mangiferin was performed at 262 nm (deuterium lamp)
was achieved on a precoated silica gel aluminum TLC plate 60 (10 × with a Camag TLC Scanner III in the remission/absorption mode op-
10 cm; Lot No. HX 130,195; Product Catalog No. 1.05554.0007; erated by a user-friendly WinCATS software (version 1.4.2) (Muttenz,
E. Merck, Darmstadt, Germany) using a Camag [CAT No. 022.7400, Switzerland).
Ser No. 130,513, Muttenz, Switzerland (Anchrom Enterprises (I) Pvt.
Ltd., Mumbai, India)]) flat bottom and twin-trough TLC developing
chamber. The position of sample application was fixed at 10 mm Defining the QTMP and CAAs
from the bottom and 10 mm from the side edges with 10 mm as the dis- As per the AQbD approach for the systematic development of a bio-
tance between tracks in the form of bands, with each band of length 6 analytical method of mangiferin, the QTMP was defined encompass-
mm on a precoated silica gel aluminum plate 60 (10 × 10 cm, 100 µm ing the summary of the quality characteristics of the targeted method.
thickness) using a Camag Linomat sample applicator. Table I enlists the QTMP elements setup for developing an efficient
The suitable mobile phase composition was employed consisting thin-layer densitometric method with enhanced chromatographic sep-
of a mixture of organic and aqueous solvents like ethyl acetate : acetic aration of mangiferin. In order to meet the QTMP, various CAAs were
acid : formic acid : water. Mobile phase components were mixed prior earmarked, namely Rf, peak height, capacity factor, theoretical plates
to use and the development chamber was left to saturate with mobile and separation number. Table II enumerates the list of CAAs
Mangiferin Active ingredient/antioxidant For quantitative estimation in diverse biological samples from pharmacokinetic, clinical and
toxicological studies.
Method type Liquid densitometry Liquid densitometric method is primarily desirable for attaining chromatographic
separation of mangiferin owing to its hydrophobic nature. Due to the partitioning
phenomenon, the drug tends to retain easily on the stationary phase composed of silica,
while the latter tends to provide an adsorbent effect for efficient separation owing to its
narrow particle size, small pore volume and high surface area.
Instrument HPTLC system equipped It facilitates measuring the absorbance or fluorescence from the test substances at a very
requirement with a scanner wide wavelength ranging between 200 and 800 nm. It allows detection of 31 wavelengths
following integration of densitometric data for simultaneous estimation.
Sample characteristic Liquid It is mandatory to prepare the samples in liquid form for application on the stationary phase
matrix, which tends to migrate on it for quantitative estimation of the sample
concentration based on the densitometric evaluation.
Biological sample Handling, weighing, sampling, Plasma sample preparation is carried out by spiking the accurate volume of bioactive
preparation admixing with solvents compound each time and mixing with a fixed amount of plasma to obtain the stock
followed by ultracentrifugation and separation of the supernatant.
Retention/retardation Optimum Rf is a function of the partition coefficient and is considered to be constant for a drug substance under a
factor (Rf ) (between given set of chromatographic conditions. Usually, Rf is always less than unity. Larger Rf reveals the
0.5 and 0.7) non-polar nature of the compound, thus shortening the time required for separation. Thus, intermediate
Rf value is desirable for attaining optimal separation and, hence, deemed as highly critical.
Peak height High For most chromatographic analysis, peak height is particularly employed for quantitative estimation of the
analyte. It is more vital as peaks may undergo tailing owing to diverse factors, which cause variation in
the area count leading to weird results. In this context, peak height was considered as highly critical for
estimation of the target compound.
Capacity factor High Capacity factor is best described as a measure of the retentivity of the analyte, which can be used to assess
the column’s efficiency. The longer a component is retained by the column, the greater is the capacity
factor. Hence, it was assumed to be highly critical for the target analytical method.
Theoretical plates High Theoretical plate count measures the efficiency of both the method and column. The more the number of
theoretical plates the higher is efficiency of the method. Thus, it is considered as highly critical for the
chromatographic analysis.
Separation number High Separation number provides the basis for evaluating the separation capacity of a chromatographic method.
It describes the number of zones that can be separated with a resolution of 4 s. It must be as high as
possible to depict the separation ability of the method for a mixture of analytes. Hence, it indicates high
method efficiency, and is considered as one of the critical parameters.
4 Khurana et al.
considered during method development along with rational justifica- Table III. Design Matrix as per the D-Optimal Response Surface
tion for each of them. Design Illustrating the Experimental Runs
Table IV. Design Matrix as per the 11-Factor 12-Run Plackett–Burman Design for Screening of Method Variables and Process Parameters at
Their Respective Low and High Levels
Runs Mobile Saturation Volume - Developmental Scanning Slit Distance Migration Application Stationary Plate
phase time loaded phase speed dimension between distance position phase dimension
ratio tracks
1. 1 1 1 −1 −1 −1 1 −1 1 1 −1
2. −1 1 1 1 −1 −1 −1 1 −1 1 1
3. −1 1 −1 1 1 −1 1 1 1 −1 −1
4. 1 1 −1 1 1 1 −1 −1 −1 1 −1
5. 1 1 −1 −1 −1 1 −1 1 1 −1 1
6. −1 −1 −1 −1 −1 −1 −1 −1 −1 −1 −1
7. −1 1 1 −1 1 1 1 −1 −1 −1 1
8. −1 −1 −1 1 −1 1 1 −1 1 1 1
9. 1 −1 −1 −1 1 −1 1 1 −1 1 1
10. 1 −1 1 1 −1 1 1 1 −1 −1 −1
11. 1 −1 1 1 1 −1 −1 −1 1 −1 1
12. −1 −1 1 −1 1 1 −1 1 1 1 −1
Table V. Design Matrix as per the Face-Centered Cubic Design using a numerical optimization desirability function by “trading-off”
(FCCD) Employed for Thin-Layer Densitometry Method various CAAs as per the acceptance criteria, i.e., optimum Rf, and
Optimization maximization of peak height, capacity factor, theoretical plates and
separation number, to obtain efficient method performance. On the
Trial no. Coded factor levels
heels of numerical optimization, graphical optimization was also car-
Factor 1 Factor 2 ried out to embark upon the analytical design space for locating the
optimized solution.
1. 1 −1
2. 1 1
3. 0 0 Method validation
4. 0 0 It is imperative to validate a bioanalytical method meant for analysis
5. 0 0
of drug molecules in the biological samples for ensuring efficient chro-
6. −1 1
matographic separation and recovery. Thus, the developed method
7. 0 −1
was validated as per FDA guidance for validation of a bioanalytical
8. −1 −1
9. 1 0 method (55–58).
10. 0 0
11. 0 1 Preparation of calibration standards and linearity range
12. −1 0 The calibration samples were prepared by spiking mangiferin with a
13. 0 0
fixed amount of plasma. Extraction of mangiferin was established
Coded level −1 0 1 upon the principle of liquid–liquid extraction, i.e., relative solubility
of the solute in the mobile phase, usually a mixture of organic solvents
Translation of coded levels in actual units and water. Linearity of the developed method was determined by an-
Volume loaded (µL) 2 4 6 alyzing serial dilutions of mangiferin between the concentration range
Plate dimension (cm) 10 × 10 15 × 10 20 × 10
of 50 and 1,000 ng/band, and plotting the peak height versus concen-
tration to obtain a linear correlation plot. Further, linearity of the
was carried out employing 3D response surface plots and 2D contour method was confirmed by least square regression analysis on the
plots to discern the factor–response relationship and plausible interac- obtained data by comparing the predicted and observed responses at
tion(s) among them. Search for the optimum solution was carried out 95% confidence intervals.
6 Khurana et al.
Results
Repeatability
Studies were performed to investigate the influence of different plates, Preliminary screening studies
accuracy of the applicator, effect of the developing phase and operator The preliminary screening studies were aimed at identifying the suit-
on the chromatographic separation of mangiferin performed employ- able mobile phase composition for efficient chromatographic separa-
ing different quality control samples. These samples at four different tion of mangiferin. In this regard, various combinations of solvent
concentration levels (50, 100, 200 and 400 ng/band) were applied in systems were explored based on extensive literature survey. At a
triplicate in 12 tracks on 3 different TLC plates on the same day. The fixed concentration of the analyte (100 ng/band), the peaks obtained
repeatability of the method was considered to be acceptable if all the at all the explored mobile phases were visually examined for band
bioactive compounds on each plate were identical with respect to migration distance, peak tailing, Rf, migration distance of the solvent
position, intensity and formation of parallel lines on the chromato- front, etc. Table VI enlists various mobile phase combinations
gram. It was seen that the Rf value for each of these bioactive com- employed for obtaining “the best possible” resolution of mangiferin
pounds on three plates did not vary by more than 0.02 (59). and the observations are made therein. Among the eight mobile
Table VI. Comparative Preliminary Screening of the Various Mobile Phase Mixtures for Chromatographic Separation of Mangiferin
Ethyl acetate : methanol 4:6 0.12 Poor resolution of bands with a high solvent front
Ethyl acetate : methanol (with four drops of glacial 4:6 0.15 Solvent front decreased with no significant improvement
acetic acid) in the resolution
Toluene : acetone : glacial acetic acid 6:3:1 0.14 Poor resolution of the bands
Methanol : ethyl acetate : glacial acetic acid 5:4:1 0.46 Better resolution of the bands with presence of tailing
Ethyl acetate : methanol : toluene 6:3:1 0.15 No apt resolution of the bands
Ethyl acetate : methanol : chloroform 6:3:1 0.17 No apt resolution of the bands
Hexane : methanol : ethyl acetate : glacial acetic acid 2:2:5:1 0.21 Low band resolution with tailing
Ethyl acetate : glacial acetic acid : formic acid : water 5:2:2:1 0.56 Efficient band resolution with improved Rf
Systematic Development of a Thin-Layer Chromatographic Method for Mangiferin 7
Figure 2. 3D response surface plot using D-optimal design depicting the influence of the mobile phase ratio on CAAs, i.e., Rf and peak height, where (A) ethyl acetate,
(B) acetic acid, (C) formic acid and (D) water. This figure is available in black and white in print and in color at JCS online.
Table VII. Frequency of Various Studied Factors Falling Above t Value and Depicting Their Influence on the Method CAAs
CAAs CAAs
Rf Peak height Capacity factor Theoretical plates Separation time
Mobile-phase concentration – ✓ – – –
Saturation time – ✓ – – –
Volume loaded – ✓ ✓ – ✓
Developmental phase – ✓ – – –
Scanning speed – ✓ – – –
Slit dimension – – – ✓ ✓
Distance between tracks – – – ✓ ✓
Migration distance – – – – –
Application position – ✓ – – –
Stationary phase – – – – –
Plate dimension ✓ ✓ ✓ ✓ ✓
phase combinations studied, ethyl acetate : glacial acetic acid : formic Among the studied combinations of the mobile phase solvents, higher
acid : water was found to be ideal one for efficient chromatographic influence of the concentrations of ethyl acetate was observed on the Rf,
separation of mangiferin. From the findings, it was very well deduced while both the concentrations of ethyl acetate and acetic acid exhibited
that being polar in nature, both precoated silica plates as well as man- higher influence on the peak height. Based on the above observations,
giferin, require more polar composition of the mobile phase to resolve the search for the optimum mobile phase combination was identified
mangiferin better. Thus, the selected mobile phase composition was employing numerical optimization to meet the desired objectives for
finalized for the entire method development study. the CAAs, i.e., optimum value of Rf and maximizing the peak height.
The optimized chromatographic solution was observed using ethyl
Selection of primary parameters acetate : acetic acid : formic acid : water in the ratio of 7 : 1 : 1 : 1.
After identifying the ideal mobile phase, the next step was to optimize the
suitable ratio of the solvents in the mobile phase employing the D-optimal
design for attaining superior chromatographic separation of mangiferin. Selection of secondary parameters
A total of 20 experimental runs carried out as per the selected design were Factor screening studies were carried out for selecting secondary param-
fitted to the polynomial equations for each of the CAAs. A quadratic eters using the Plackett–Burman design. The first-order polynomial
model was employed for analysis of the individual responses, followed models for estimating the main effect(s) was performed by analysis of
by framing the polynomial model by considering the model terms with coefficients as per the following equation, where β1 to βn represent the
highly significant statistical difference (P < 0.001). Evaluation of the suit- coefficients of the model terms, and β0 represents the intercept term.
ability of the selected model was confirmed from higher values of coeffi-
Y ¼ β 0 þ β 1 X1 þ β 2 X2 þ β 3 X3 þ β 4 X4 þ β 5 X5 þ β 6 X6 þ β 7 X7 ð3Þ
cient of correlation (ranging between 0.8006 and 0.9362), insignificant
values for the lack of fit (ranging between 0.3863 and 0.8978) and The evaluation of polynomial equations generated for each CAA select-
lower values of PRESS (ranging between 0.091 and 0.17; 1.239E + 007). ed during screening studies indicated the absence of any significant in-
The rational understanding of the factor–response relationship teraction effect(s) among the factors. Table VII enlists the frequency of
was established by the response surface analysis. Figure 2 illustrates influence of various studied factors identified from the Pareto charts on
the 3D response surface plot for the CAAs, Rf and peak height. the method CAAs. The factors, i.e., volume loaded and plate dimension,
8 Khurana et al.
showed statistically significant influence on all the studied CAAs, as the design revealed miniscule/less influence, and thus were fixed as cons-
these were found to be above the t-value limit and Bonferroni limit tant for further method development studies. Based on the critical ob-
with maximum frequency. On the contrary, other factors studied in servations on the highly influential factors obtained from the Pareto
Table VIII. Values of the Coefficients of the Polynomial Equations and r 2 for Various CAAs
Figure 3. 3D response surface plot depicting the interactions among chosen CMPs, i.e., volume loaded and plate dimension on CAAs, i.e., (A) Rf, (B) peak height, (C)
capacity factor, (D) theoretical plates and (E) separation number. This figure is available in black and white in print and in color at JCS online.
Systematic Development of a Thin-Layer Chromatographic Method for Mangiferin 9
Figure 4. Overlay contour plot depicting the optimal analytical design space region. This figure is available in black and white in print and in color at JCS online.
charts, the volume loaded and plate dimension were finally selected as
the CMPs for optimizing the method performance.
The response surface plot depicted in Figure 3C shows significant Figure 3D and E portraying the 3D response surface plot for theoretical
influence of the plate dimension on the capacity factor when compared plate and separation number reveals almost an analogous trend for influ-
with the volume loaded. The 3D plot reveals a sharp declining trend in ence of both volume loaded and plate dimension on the respective CAAs.
the peak height with increase in the levels of plate dimension; in The plate dimension shows higher influence on both the CAAs from low to
contrast increase in the levels of volume loaded indicates miniscule intermediate levels, followed by a dip. However, the volume loaded exhib-
change in the capacity factor. its a negative influence on both the CAAs.
Method validation
Calibration curves
The linear regression data for the calibration curves (n = 3) depict a
good linear relationship over the concentration range of 50–800 ng/
band with respect to height, as shown in Figure 6. No significant dif-
ference was observed in the slopes of standard curves. Table IX depicts
various parameters of the linear calibration plot of mangiferin in
human plasma in the developed densitometric method with the
value of coefficient of correlation being 0.996 ± 0.0012 (P < 0.001).
The responses with a percent bias of ±5% were taken into consider-
ation for validating the linearity range, with none of the points
observed as outliers (60). This revealed that all the responses are
present within the desirable statistical limits of 95% confidence inter-
vals, confirming high data reliability and degree of closeness of the pre-
Figure 6. Thin-layer densitometry and the corresponding chromatogram profiles
obtained by the HPTLC instrument, with linearity graph (50–800 ng/band) of
dicted data with those of the observed ones.
mangiferin in triplicate depicting (A) calibration bands on aluminum-based
silica plate and (B) corresponding 3D densitogram tracks. This figure is Accuracy
available in black and white in print and in color at JCS online. Accuracy data for the standard concentrations of mangiferin, i.e.,
LQC (50 ng/band), MQC (100 ng/band) and HQC (150 ng/band)
Table IX. Linear Regression Data for Calibration Curve of Mangiferin indicated good percentage of recovery, ranging between 96.75 and
in Human Plasma (n = 3) 99.47% with %RSD value <2%, respectively. This unequivocally
vouches for a high degree of accuracy of the developed method.
Parameters Values
Table X explicitly enlists the accuracy data for various quality control
Linearity range (ng/band) 50–800 samples of mangiferin, while Figure 7 shows the thin-layer densitometry
Regressed equation Y = 3.046x + 1,066 curves and their corresponding profiles obtained through TLC.
Correlation coefficient 0.996 ± 0.0012
Slope ± SD 3.046 ± 0.557 Precision
LOD 12.06 ng/mL
Intra-day and inter-day precision studies indicated higher values of
LOQ 36.55 ng/mL
percentage of recovery of mangiferin, ranging between 92.1 and
Standard concentration Levels (%) Concentration (ng/band) Amount recovered (ng/band) ± SD Recovery (%) RSD (%)
Figure 7. Thin-layer densitometry and the corresponding chromatogram profiles obtained by HPTLC instrument, for accuracy of mangiferin at LOQ, MOQ and HOQ
at 100, 150 and 200 ng/band, respectively, in triplicate depicting (A) calibration bands on aluminum-based silica plate and (B) corresponding three-dimensional
densitogram tracks. This figure is available in black and white in print and in color at JCS online.
Matrix effect
The matrix effect, calculated using three different QC samples, i.e., 50,
97.9%, respectively. Further, the %RSD values for mangiferin as per 100 and 150 ng/band showed the mean percentage of recovery values
repeatability and intermediate precision were found to be well within of 87.00, 93.03 and 96.75%, respectively. Higher values of the per-
the stipulated limit of <2%. These results confirmed the high degree centage of recovery, i.e., >80%, corroborated efficient densitometric
of precision of the developed method. Table XI illustrates the intra- separation of the mangiferin from the biological matrix. This also
and inter-day precision data for various quality control samples of vouched for the aptness and robustness of the sample preparation
mangiferin. and extraction technique employed throughout the bioanalytical
method development and validation studies.
obtained method. The superiority of the current method can also be 6. Vieira, A.B., Coelho, L.P., Insuela, D.B., Carvalho, V.F., dos Santos, M.H.,
demonstrated on the basis of its high sensitivity, which is evident Silva, P.M., et al. Mangiferin prevents guinea pig tracheal contraction via
from much lower values of the LOQ, i.e., 12.1 ng/band, over the ex- activation of the nitric oxide-cyclic GMP pathway; PLoS One, (2013); 8:
e71759.
isting liquid chromatographic methods showing LOQ of 480 ng/mL
7. Dou, W., Zhang, J., Ren, G., Ding, L., Sun, A., Deng, C., et al. Mangiferin
and 3 µg/mL, respectively (61, 62). In a nutshell, the studies vouch
attenuates the symptoms of dextran sulfate sodium-induced colitis in
for the high degree of utility of the developed method for estimation
mice via NF-kappaB and MAPK signaling inactivation; Intaernational
of mangiferin in bioanalytical samples. Immunopharmacology, (2014); 23: 170–178.
8. Telang, M., Dhulap, S., Mandhare, A., Hirwani, R.; Therapeutic and cos-
metic applications of mangiferin: A patent review; Expert Opinion on
Conclusion Therapeutic Patents, (2013); 23: 1561–1580.
9. Zhu, X.M., Song, J.X., Huang, Z.Z., Wu, Y.M., Yu, M.J.; [Antiviral activ-
A simple, rapid, sensitive and economical bioanalytical thin-layer den-
ity of mangiferin against herpes simplex virus type 2 in vitro]; Zhongguo
sitometric method has been successfully developed employing the Yao Li Xue Bao, (1993); 14: 452–454.
AQbD approach for quantification of mangiferin in bioanalytical sam- 10. Rajendran, P., Jayakumar, T., Nishigaki, I., Ekambaram, G., Nishigaki, Y.,
ples, rat plasma per se. The systematic AQbD tools like risk assessment Vetriselvi, J., et al. Immunomodulatory effect of mangiferin in experimental
and factor screening exercise helped in identifying the “prominent few” animals with benzo(a)pyrene-induced lung carcinogenesis; International
method variables associated with high degree of variability from “plau- Journal of Biomedical Sciences, (2013); 9: 68–74.
sible many” and facilitated optimization of them for attaining superior 11. du Plessis-Stoman, D., du Preez, J., van de Venter, M.; Combination treat-
method performance. Overall, the volume loaded and plate dimension ment with oxaliplatin and mangiferin causes increased apoptosis and down-
showed the maximum influence on all the vital response variables inves- regulation of NFkappaB in cancer cell lines; African Journal of Traditional
and Complementary Alternative Medicine, (2011); 8: 177–184.
tigated like Rf, peak height, capacity factor, theoretical plates and sep-
12. Pal, P.B., Sinha, K., Sil, P.C.; Mangiferin, a natural xanthone, protects mu-
aration number. Method validation studies corroborated the excellent
rine liver in Pb(ii) induced hepatic damage and cell death via MAP Kinase,
linearity, accuracy, precision, high sensitivity, system suitability, robust- NF-kB and mitochondria dependent pathways; PLoS One, (2013); 8:
ness and ruggedness of the developed thin-layer densitometric method. e56894.
Further, stability studies of the drug in plasma revealed lack of any in- 13. Agarwala, S., Mudholkar, K., Bhuwania, R., Satish Rao, B.S.; Mangiferin, a
teraction of the mangiferin with the biological matrix leading to the for- dietary xanthone protects against mercury-induced toxicity in HepG2 cells;
mation of any unwanted peak(s). In a nutshell, the studies indisputably Environmental Toxicology, (2012); 27: 117–127.
vouch for the applicability and utility of the science and risk-based 14. MeiDong, X., BuZhe, Z., Yu, Z., XuQian, Z.; Determination of mangiferin
AQbD approach for developing a bioanalytical method of mangiferin content in extract of mango leaves by UV-spectrophotometry; Guangxi
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to other lipophilic drug substance(s) too.
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Acknowledgments 16. Jutiviboonsuk, A., Sardsaengjun, C.; Mangiferin in leaves of three thai
B.S. gratefully acknowledges the generosity of M/s Stat-Ease Inc., Minneapolis, mango (Mangifera indica L.) varieties; IJPS, (2010); 6: 122–129.
USA, for providing one perpetual license and 10-user annual licence of Design 17. Sethiya, N.K., Trivedi, A., Patel, M.B., Mishra, S.H.; Comparative pharma-
Expert® software, version 9.0, while conferring upon him the “Stat-Ease QbD cognostical investigation on four ethanobotanicals traditionally used as
Performance Award 2014” and “Premier Academic Status” for his unparalleled Shankhpushpi in India; Journal of Adv Pharm Technol Res, (2010); 1:
contribution in the area of QbD-based pharmaceutical research work. Also the 388–395.
coauthors, R.K.K. and S.B. acknowledge the University Grant Commission 18. Geodakyan, S.V., Voskoboinikova, I.V., Kolesnik, J.A., Tjukavkina, N.A.,
(UGC), New Delhi, India for financial grants to them to carry out the present re- Litvinenko, V.I., Glyzin, V.I.; High-performance liquid chromatographic
search work as a Research Fellow under RFMS scheme, F.No. 5-(94)/2007/(BSR). method for the determination of mangiferin, likviritin and dihydroquercetin
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