Journal of Chromatographic Science Advance Access published February 23, 2016

Journal of Chromatographic Science, 2016, 1–13

doi: 10.1093/chromsci/bmw001
Article

Article

Systematic Development and Validation of a

Thin-Layer Densitometric Bioanalytical Method
for Estimation of Mangiferin Employing
Analytical Quality by Design (AQbD) Approach
Rajneet Kaur Khurana1, Satish Rao2, Sarwar Beg1, O.P. Katare1, and
Bhupinder Singh1,3,*
1
University Institute of Pharmaceutical Sciences, UGC Centre of Advanced Studies, Punjab University, Chandigarh
160 014, India, 2Division of Radiation Biology and Toxicology, School of Life Sciences, Manipal University, Manipal,
Karnataka 576 104, India, and 3UGC-Centre of Excellence in Applications of Nanomaterials, Nanoparticles and
Nanocomposites (Biomedical Sciences), Punjab University, Chandigarh 160 014, India
*Author to whom correspondence should be addressed. Email: bsbhoop@yahoo.com; bsbhoop@pu.ac.in
Received 5 July 2015; Revised 7 December 2015

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
Mangiferin, a polyphenolic C-glucosylxanthone, primarily exists as a
principal phytoconstituent in the leaves and stem bark of Mangifera
indica (family Anacardiaceae). Chemically, 2-C-β-D-gluco-pyranosyl-1,
3,6,7-tetrahydroxyxanthone (Figure 1) exhibits moderate aqueous sol-
ubility, i.e., 1.44 mg/mL and low lipophilicity (log P) of −0.59 (1, 2)
predicted by in silico simulations performed on mangiferin using
ADMET Predictor (Version 7.1.0013; Simulations Plus, Inc., USA)
and GastroPlus Simulation software (Version 8.6; Simulations Plus,
Inc.). Presence of the phenolic xanthone moiety owes to the powerful
antioxidant activity of mangiferin for scavenging free radicals and
protection against ROS-induced oxidative stress (3, 4). Other potential
therapeutic applications of mangiferin include being used as an antidi-
abetic (5), antiobesitic, antiosteoclastogenic, antiasthmatic (6), antidiar-
rhoeal (7), immunomodulator, analgesic, antiallergic, antibacterial (5,
8), antimicrobial (4), antiviral (9, 10) and anticancer (3, 11–13)
agent. Considering the number of its therapeutic benefits, mangiferin
has proved to have promising utility in clinical treatment and manage-
ment of multiple disorders.
Development of a simple, rapid and highly sensitive bioanalytical
method is highly desirable for quantification and routine chromatographic
analysis of mangiferin in pharmaceutical formulations, and bioanalytical

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2 Khurana et al.
Figure 1. Chemical structure of mangiferin.
and toxicological samples. Several literature reports have been document-
ed for quantification of mangiferin employing techniques like UV–Vis
spectroscopy (1, 14), spectrofluorimetry (15), thin-layer chromatography
(TLC) (16, 17), high-performance liquid chromatography (HPLC)
(18–20), high-performance thin-layer chromatography (HPTLC) (21), liq-
uid chromatography–mass spectrometry (LC–MS) (22, 23), tandem mass
spectrometry (LC–MS-MS) (24, 25) in the bulk drug (26–30) as well as in
bioanalytical samples like rat plasma (31, 32), urine (33) and aqueous
humor (34, 35). However, none of these methods have been considered
to be highly satisfactory owing to the involvement of complex aqueous
and organic solvent mixtures, and monitoring of the complex chromato-
graphic conditions.
Consequently, in order to obviate the tedious, expensive and pro-
longed sample preparation, use of intricate mobile phase compositions
and optimization of instrumental method variables associated with
the conventional HPLC procedures, HPTLC and TLC techniques
have been considered highly useful for ease of quantification and faster
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
over HPLC, like lower mobile phase consumption, higher scanning
speed, shorter analysis time, reduced sample cleanup, lower operation-
al and maintenance costs, and, above all, ease of detection and optimi-
zation of critical method variables, thus facilitating faster analysis of
drug by saving a great deal of time and resources (36). Also, it provides
the leverage of avoiding tedious sample preparation, liquid–liquid
extraction and filtration steps encountered during HPLC analysis. Be-
sides, other stellar merits of TLC techniques encompass the possibility
of selecting corrosive solvents with high pH range, minimal chances of
sample contamination with the previous ones, high sensitivity from
the nanogram to pictogram level and highly reproducibility of the den-
sitometric method for identifying the concentration of the sample (37).
Of late, the use of Quality by Design (QbD) paradigms has been per-
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)
approach, in particular, has been highly popularized for developing ro-
bust methods by specifically understanding the critical method parame-
ters (CMPs) influencing method performance (38, 39). Considered as a
science and risk-based approach, AQbD provides rational comprehen-
sion of the CMPs affecting the critical analytical attributes (CAAs) of
the bioanalytical method by risk assessment and factor screening studies,
followed by unearthing the plausible interactions among them and op-
timizing them by employing Design of Experiments for enhanced meth-
od performance (40). The AQbD approach of method development
primarily involves defining the quality target method profile (QTMP)
and CAAs, identifying those CMPs that are significantly influencing
the CAAs using prioritization and screening studies, method optimiza-
tion using suitable experimental designs, modelization and optimum
search through response surface methodology to embark upon the ana-
lytical design space and postulation of control strategy for continuous
improvement in method performance (41). In the past few years, several
literature reports have successfully demonstrated the immense utility of
the AQbD approach for developing efficient and cost-efficient liquid
chromatographic methods for estimating different analytes in pharma-
ceutical formulations and bioanalytical samples (42–48). Application of
AQbD specifically in the current thin-layer densitometry method
development envisioned for identifying method variables plausibly
associated with high degree of variability and exhibiting maximal
influence on method performance.
The present studies were accordingly undertaken to develop an
AQbD-enabled, simple, ultra-fast, robust, sensitive, effective and econom-
ical densitometry-based TLC method for quantification of mangiferin in
the bioanalytical samples.
Materials and methods
Standards and reagents
Mangiferin was purchased from M/s Sigma-Aldrich Co., Mumbai,
India, and was used as working standard in the concentration of
1 µg/mL. Analytical grade ethyl acetate, acetic acid, formic acid and
methanol were purchased from M/s Merck Ltd., Mumbai, India,
and were used as received without further purification.
Preparation of standard solutions
The stock solution was prepared by weighing accurately 10 mg of
mangiferin in a 10 mL volumetric flask followed by dilution in meth-
anol to obtain the concentration of 1,000 µg/mL. This stock solution
was further appropriately diluted with methanol in a 10 mL volumet-
ric flask to prepare the final concentration of 1,000 ng/mL.
Biological matrix
Fresh human blood was procured ex gratis from the Rotary Blood
Bank, Chandigarh, India. Harvested plasma samples were further
employed for the entire method development and validation studies.
For preparation of calibration standards and quality control samples,
human plasma was extracted by centrifugation of blood treated with
ethylenediaminetetraacetic acid at 10,000 r.p.m. (5,590 × g) using a
centrifuge (Remi, Mumbai, India).
Instrumentation and chromatographic conditions
Bioanalytical method development for mangiferin was carried out using
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
were applied in triplicate using a Hamilton syringe (100 mL) with slit
dimensions of 6 mm × 0.30 mm, scanning speed of 20 mm/s and data
resolution of 100 µm/step, equipped with an optical filter (second
order) and filter factor (Savitzky golay 7). Chromatographic separation
was achieved on a precoated silica gel aluminum TLC plate 60 (10 ×
10 cm; Lot No. HX 130,195; Product Catalog No. 1.05554.0007;
E. Merck, Darmstadt, Germany) using a Camag [CAT No. 022.7400,
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
from the bottom and 10 mm from the side edges with 10 mm as the dis-
tance between tracks in the form of bands, with each band of length 6
mm on a precoated silica gel aluminum plate 60 (10 × 10 cm, 100 µm
thickness) using a Camag Linomat sample applicator.
The suitable mobile phase composition was employed consisting
of a mixture of organic and aqueous solvents like ethyl acetate : acetic
acid : formic acid : water. Mobile phase components were mixed prior
to use and the development chamber was left to saturate with mobile
phase vapors for suitable time before each run. The development of
the plate was carried out by the ascending technique to a migration
distance of 80 mm. After development, aluminum-backed silica gel
TLC 60 plates were dried in a hot air oven at 25°C. Densitometric
scanning of mangiferin was performed at 262 nm (deuterium lamp)
with a Camag TLC Scanner III in the remission/absorption mode op-
erated by a user-friendly WinCATS software (version 1.4.2) (Muttenz,
Switzerland).
Defining the QTMP and CAAs
As per the AQbD approach for the systematic development of a bio-
analytical method of mangiferin, the QTMP was defined encompass-
ing the summary of the quality characteristics of the targeted method.
Table I enlists the QTMP elements setup for developing an efficient
thin-layer densitometric method with enhanced chromatographic sep-
aration of mangiferin. In order to meet the QTMP, various CAAs were
earmarked, namely Rf, peak height, capacity factor, theoretical plates
and separation number. Table II enumerates the list of CAAs

Table I. QTMP for Bioanalytical Thin-Layer Densitometry Method of Mangiferin

QTMP elements Target Justification

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.

Table II. CAAs for Bioanalytical Thin-Layer Densitometric Method of Mangiferin

CAAs Target Justification

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

Experimental run Ethyl acetate Acetic acid Formic acid Water

Preparation of bioanalytical samples
To prepare the standard calibration curve and quality control samples, 1 6 1 1 2
2 6 1 2 1
an aliquot of 200 µL of the blank plasma was added to different dilut-
3 6 2 1 1
ed working solutions (200 µL) in a centrifuge tube. The contents of the
4 7 1 1 1
tube were subjected to vortex mixing for 30 s in order to ensure com- 5 8 0 1 1
plete mixing. 6 6.5 0.5 1 2
7 6 2 1 1
Preliminary screening studies 8 7 0 2 1
9 6.25 1.25 1.25 1.25
Preliminary screening studies were carried out for identifying suitable
10 6 0 3 1
solvent systems for apt chromatographic separation of the mangiferin. 11 7 0 1 2
As reported in the literature, diverse combinations of organic and 12 7 0 2 1
aqueous solvents were explored as the potential mobile phase(s), 13 7.5 0 1 1.5
and analytical trials were performed. After thoroughly scrutinizing 14 6.5 0 1.5 2
various reports in the literature, rational combinations of ethyl acetate : 15 6 1 2 1
methanol (49), toluene : acetone : glacial acetic acid (50), ethyl acetate : 16 6 0.5 1.5 2
glacial acetic acid : formic acid : water, methanol : ethyl acetate : glacial 17 7 1 1 1
acetic acid, ethyl acetate : methanol : toluene, ethyl acetate : methanol : 18 6 0 3 1
19 6.25 0.25 2.25 1.25
chloroform and hexane : methanol : ethyl acetate : glacial acetic acid
20 6 0 2 2
(51) were identified as the suitable mobile phases for estimating mangi-
ferin in plasma. At a fixed concentration of the analyte (1,000 ng/band), Name of the factors Low High
all the mobile phases were visually examined for band migration dis-
tance, peak tailing, Rf, migration distance of the solvent front for ruling Translation of coded levels in actual units
Ethyl acetate 6 8
out the less pragmatic mobile phases and drawing a final inference to
Acetic acid 0 2
select the best solvent system with higher resolution for mangiferin.
Formic acid 1 3
Water 1 2
AQbD-based method development and optimization
studies
As per the AQbD approach of method development, endeavor was interaction term(s) (52). The quantitative factor effects and their
made to carry out primary and secondary screening studies for identi- statistical levels were analyzed employing the Pareto charts, based
fying the CMPs critically influencing the method CAAs, and subse- on the principle of “factor sparsity” (53, 54).
quently optimizing the same employing suitable experimental designs.

Method optimization studies

Primary parameter selection
Selection of apposite combinations of the solvents for the mobile
phase is highly desirable for efficient chromatographic analysis and
migration of the analyte in the stationary phase. The solvent system
exhibiting higher affinity for separation of the analyte was used to
identify the optimum ratio of the solvents for the selected mobile
phase. In the present studies, a D-optimal design was employed for
selecting the apt combination of the ratios of ethyl acetate, glacial ace-
tic acid and formic acid as the mobile phase mixture for separating
mangiferin, and evaluating the method CAAs like Rf and peak height.
Table III illustrates the design matrix depicting the experimental runs
obtained as per the D-optimal design along with the actual and coded
values of the levels of CMPs.
Secondary parameter selection
Following primary parameter selection, the secondary parameters in-
fluencing method performance were selected. An 11-factor 12-run
Plackett–Burman design was employed for screening studies for iden-
tifying the “vital few” CMPs influencing the method CAAs viz. Rf,
peak height, capacity factor, theoretical plates and separation number.
Table IV illustrates the design matrix enlisting the studied factors
along with the translation of their coded low and high levels into ac-
tual units. Model development was carried out by fitting the experi-
mental data to the linear polynomial equations by obviating the
Based on the preliminary screening studies, followed by primary and
secondary parameter selection, the CMPs actually affecting method
performance were embarked upon for method optimization. A face-
centered cubic design (FCCD) with degree of rotatability (i.e., α = 1)
was employed for optimization of the CMPs, viz. volume loaded
(X1) and plate dimension (X2), at three different levels, i.e., low
(−1), intermediate (0) and high (+1) levels. Table V outlines the design
matrix as per the FCCD with 13 experimental runs including quintu-
plicate studies at the center point runs (0, 0). The standard concentra-
tions of 200, 400 and 600 ng/band were used for all the experimental
runs, for the selected CAAs as per the design.
Optimization data analysis and validation studies
The optimization data analysis was carried out by multiple linear
regression analysis using Design Expert 9.0 software (M/s Stat-Ease
Inc., Minneapolis, USA) to fit the experimental data into the second-
order quadratic polynomial model with added interaction terms, fol-
lowed by establishing the relationship among the studied CMPs with
the chosen CAAs. Only the coefficients with high statistical signifi-
cance (P < 0.001) as per analysis of variance were considered in fram-
ing the polynomial equations for the CAAs, while evaluating the
model aptness by lack of fit analysis, coefficient of correlation (r)
and predicted error sum of squares (PRESS). Response surface analysis
Systematic Development of a Thin-Layer Chromatographic Method for Mangiferin 5

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

Name of the factors Levels

Low (−1) High (+1)

Mobile phase ratio 6 : 1.5 : 1.5 : 1 8 : 0.5 : 0.5 : 1

Saturation time (min) 2 4
Volume loaded (µL) 2 6
Developmental phase (mL) 10 15
Scanning speed (mm/sec) 10 15
Slit dimension (mm) 5 6
Distance between tracks (mm) 5 10
Migration distance (mm) 70 80
Application position (mm) 5 10
Stationary phase Silica-coated aluminum-based Silica-coated aluminum-based 60F254
60 plates plates
Plate dimension (cm) 10 × 10 20 × 10

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.
Accuracy
Accuracy of the developed method was estimated as mean percentage
of recovery from four different quality control samples containing
100 ng/band of mangiferin and a fixed amount of plasma (200 µL),
followed by spiking with 50% lower quality control (LQC), 100% me-
dium quality control (MQC) and 150% higher quality control (HQC)
of additional amount of mangiferin. Samples were subjected to chro-
matographic analysis to estimate the peak height, the values of recovery
and % Relative Standard Deviation (RSD) to validate whether the ac-
curacy of data is falling within the acceptance limits.
Precision
Precision was assessed by analyzing three different concentrations of
mangiferin each (LQC: 100, MQC: 150 and HQC: 200 ng/band) at dif-
ferent time intervals on the same day (i.e., intra-day precision or
repeatability), and repetition on the next day (i.e., inter-day or interme-
diate precision). All the samples were subjected to analysis and the peak
height was noted for calculating the standard deviation (SD) and %RSD
to investigate the accuracy of data within the acceptance limit.
Limit of detection and limit of quantification
Limit of detection (LOD) and limit of quantification (LOQ) were deter-
mined from the slope (S) of the linear calibration plot and the SD of the
response to the blank sample (σ), as per the following formulae:
σ σ
LOD ¼ 3:3 × ; LOQ ¼ 10 × ð1Þ
S S
Specificity
The specificity of the method was determined in relation to confirm the
absence of any interference of the components of the biological matrix
with the chromatogram of the drug substance. For this purpose, a
comparison of the chromatographic peaks of the standard sample
tracks (i.e., at LOQ: 100 ng/band) and the tracks representing mangi-
ferin in plasma preparations were performed and compared to check
the prevalence of any interference (59).
Repeatability
Studies were performed to investigate the influence of different plates,
accuracy of the applicator, effect of the developing phase and operator
on the chromatographic separation of mangiferin performed employ-
ing different quality control samples. These samples at four different
concentration levels (50, 100, 200 and 400 ng/band) were applied in
triplicate in 12 tracks on 3 different TLC plates on the same day. The
repeatability of the method was considered to be acceptable if all the
bioactive compounds on each plate were identical with respect to
position, intensity and formation of parallel lines on the chromato-
gram. It was seen that the Rf value for each of these bioactive com-
pounds on three plates did not vary by more than 0.02 (59).
Table VI. Comparative Preliminary Screening of the Various Mobile Phase Mixtures for Chromatographic Separation of Mangiferin
Developmental phase Ratio (v/v)
Ethyl acetate : methanol 4:6
Ethyl acetate : methanol (with four drops of glacial 4:6
acetic acid)
Toluene : acetone : glacial acetic acid 6:3:1
Methanol : ethyl acetate : glacial acetic acid 5:4:1
Ethyl acetate : methanol : toluene 6:3:1
Ethyl acetate : methanol : chloroform 6:3:1
Hexane : methanol : ethyl acetate : glacial acetic acid 2:2:5:1
Ethyl acetate : glacial acetic acid : formic acid : water 5:2:2:1
System suitability
System suitability was assessed by hexaplicate analyses of standard
concentration of mangiferin 1,000 ng/band, followed by estimation
of the SD and %RSD for peak height and retention time.
Robustness
Robustness of the method was evaluated by analyzing the system suit-
ability parameters after alteration in the volume of the mobile phase
(±0.5%v/v), scanning speed (±10%), volume loaded (±0.2%) and sat-
uration time (±10 s). A working standard of 1,000 ng/band was used
during the experimentation for assessing the values of mean percent-
age of recovery, and %RSD.
Ruggedness
The ruggedness of the method was determined by chromatographic
analysis of the three different concentrations of the mangiferin (i.e.,
LQC: 100 ng/band, MQC: 150 ng/band and HQC: 200 ng/band)
by two different analysts. The obtained data were analyzed for calcu-
lating the mean percentage of recovery and %RSD to corroborate the
method ruggedness.
Matrix effect
The matrix effect was investigated by comparing the heights of the
analyte in spiked quality control samples with or without biological ma-
trix, at three different concentrations (i.e., LQC: 100 ng/band, MQC:
150 ng/band and HQC: 200 ng/band) by fixing the concentrations of
the biological matrix. The corresponding peak heights of the analyte
in spiked QC samples in plasma (A) were compared with those of the
aqueous standards in the mobile phase (B) at equivalent concentrations.
The matrix effect was calculated as per the following equation:
A
Matrix effect ¼ × 100 ð2Þ
B
Results
Preliminary screening studies
The preliminary screening studies were aimed at identifying the suit-
able mobile phase composition for efficient chromatographic separa-
tion of mangiferin. In this regard, various combinations of solvent
systems were explored based on extensive literature survey. At a
fixed concentration of the analyte (100 ng/band), the peaks obtained
at all the explored mobile phases were visually examined for band
migration distance, peak tailing, Rf, migration distance of the solvent
front, etc. Table VI enlists various mobile phase combinations
employed for obtaining “the best possible” resolution of mangiferin
and the observations are made therein. Among the eight mobile
Rf Inference drawn
0.12 Poor resolution of bands with a high solvent front
0.15 Solvent front decreased with no significant improvement
in the resolution
0.14 Poor resolution of the bands
0.46 Better resolution of the bands with presence of tailing
0.15 No apt resolution of the bands
0.17 No apt resolution of the bands
0.21 Low band resolution with tailing
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
acid : water was found to be ideal one for efficient chromatographic
separation of mangiferin. From the findings, it was very well deduced
that being polar in nature, both precoated silica plates as well as man-
giferin, require more polar composition of the mobile phase to resolve
mangiferin better. Thus, the selected mobile phase composition was
finalized for the entire method development study.
Selection of primary parameters
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.
A total of 20 experimental runs carried out as per the selected design were
fitted to the polynomial equations for each of the CAAs. A quadratic
model was employed for analysis of the individual responses, followed
by framing the polynomial model by considering the model terms with
highly significant statistical difference (P < 0.001). Evaluation of the suit-
ability of the selected model was confirmed from higher values of coeffi-
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
lower values of PRESS (ranging between 0.091 and 0.17; 1.239E + 007).
The rational understanding of the factor–response relationship
was established by the response surface analysis. Figure 2 illustrates
the 3D response surface plot for the CAAs, Rf and peak height.
Among the studied combinations of the mobile phase solvents, higher
influence of the concentrations of ethyl acetate was observed on the Rf,
while both the concentrations of ethyl acetate and acetic acid exhibited
higher influence on the peak height. Based on the above observations,
the search for the optimum mobile phase combination was identified
employing numerical optimization to meet the desired objectives for
the CAAs, i.e., optimum value of Rf and maximizing the peak height.
The optimized chromatographic solution was observed using ethyl
acetate : acetic acid : formic acid : water in the ratio of 7 : 1 : 1 : 1.
Selection of secondary parameters
Factor screening studies were carried out for selecting secondary param-
eters using the Plackett–Burman design. The first-order polynomial
models for estimating the main effect(s) was performed by analysis of
coefficients as per the following equation, where β1 to βn represent the
coefficients of the model terms, and β0 represents the intercept term.
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
The evaluation of polynomial equations generated for each CAA select-
ed during screening studies indicated the absence of any significant in-
teraction effect(s) among the factors. Table VII enlists the frequency of
influence of various studied factors identified from the Pareto charts on
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

Coefficient code Polynomial coefficient values for response variables

Rf Peak height Capacity factor Theoretical plates Separation number

β0 +0.80 +5732.17 +0.41 +43677.94 +103.02

β1 −0.045 +11693.45 −0.025 −1882.10 −3.52
β2 +3.333E−003 −1678.52 −0.033 +539.50 −0.95
β3 −0.015 −3656.53 −0.069 +812.70 +9.88
__
β4 −0.11 +8150.01 +1159.59 +1.42
β5 −0.013 −1930.49 __
−8235.63 −15.78
R2 0.8960 0.9571 0.9777 0.9422 0.9747

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.

Method optimization studies and response

surface analysis
Based on the highly influential factors selected from the factor screening
studies, the method optimization studies were conducted employing
FCCD. After conducting the experiments as per the suggested design,
the optimization data analysis was carried out by fitting the data to
the second-order quadratic polynomial model for detecting the main ef-
fects as well as the interaction effects. Table VIII illustrates the coeffi-
cients of the polynomial model equations, indicating highly significant
model terms (P < 0.001) and higher values of r 2 (ranging between
0.8960 and 0.9777), thus ratifying the apt selection of model terms
and high goodness of fit of the data to the selected model. The coeffi-
cient analysis was performed by analyzing the quadratic polynomial
model equations generated as per the following equation for each CAA.

Y ¼ β 0 þ β 1 X1 þ β 2 X2 þ β 3 X12 þ β 4 X22 þ β 5 X1 X2 þ β 6 X12 X2

þ β 7 X1 X22
Response surface analysis was carried out employing 3D response sur-
face plots and 2D contour plots for each of the CAAs, i.e., Rf, peak
height, capacity factor, theoretical plates and separation time, as
shown in Figure 3A–H.
Figure 3A depicting the 3D response surface plot for Rf reveals
higher influence of volume loaded than the plate dimension. At higher
levels of volume loaded, an initial increase in Rf was observed up to the
intermediate levels, followed by declining values for Rf. The higher
values of Rf are obtained at high levels of plate dimension and
intermediate levels of volume loaded, respectively.
Figure 5. Representative densitogram of mangiferin obtained employing
ð4Þ
chromatographic conditions from design space. This figure is available in
black and white in print and in color at JCS online.
Like Rf, the 3D response surface plots for peak height again show sig-
nificant influence of the volume loaded when compared with the plate
dimension (Figure 3B). An escalating trend for peak height is observed
at all the levels of volume loaded, while plate dimension shows lack of
any significant influence on the peak height. The highest peak height
has been observed at higher levels of volume loaded, thus indicating bet-
ter densitometric separation.
10 Khurana et al.

The response surface plot depicted in Figure 3C shows significant
influence of the plate dimension on the capacity factor when compared
with the volume loaded. The 3D plot reveals a sharp declining trend in
the peak height with increase in the levels of plate dimension; in
contrast increase in the levels of volume loaded indicates miniscule
change in the capacity factor.
Figure 3D and E portraying the 3D response surface plot for theoretical
plate and separation number reveals almost an analogous trend for influ-
ence of both volume loaded and plate dimension on the respective CAAs.
The plate dimension shows higher influence on both the CAAs from low to
intermediate levels, followed by a dip. However, the volume loaded exhib-
its a negative influence on both the CAAs.

Search for the optimum solution

The optimum solution was determined by numerical optimization
while “trading-off” various CAAs for attaining optimum values of
Rf, peak height, separation number, theoretical plates and capacity
factor, all indicative of the efficient densitometric separation and
enhanced resolution of the mangiferin. The numerical optimization
suggested CMPs with volume loaded (i.e., 5.9 µL) and plate dimension
(i.e., 10 × 13.503 cm) as the optimized solution, exhibiting a desirabil-
ity value of 1 and values of the CAAs, i.e., Rf of 0.65, peak height of
26876.2, capacity factor of 0.4127, theoretical plates of 41811.3 and
separation number of 96.9. The obtained optimal solution was demar-
cated in the analytical design space employing the graphical optimiza-
tion method (Figure 4). A typical densitogram of mangiferin obtained
at optimal mobile phase composition with optimal method parame-
ters is illustrated in Figure 5.

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

Table X. Accuracy of the Bioanalytical Thin-Layer Densitometric Method of Mangiferin

Standard concentration Levels (%) Concentration (ng/band) Amount recovered (ng/band) ± SD Recovery (%) RSD (%)

Mangiferin (100 ng/band) LQC: 50 150 146.13 ± 2.52 96.75 1.72

MQC: 100 200 194.69 ± 3.51 97.34 1.80
HQC: 150 250 248.69 ± 3.51 99.47 1.41
Systematic Development of a Thin-Layer Chromatographic Method for Mangiferin 11

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.

Table XI. Intra- and Inter-Day Precision Data of Bioanalytical Robustness

Thin-Layer Densitometric Method of Mangiferin Intra-Day Precision Among varied operable conditions like alteration in the composition
(Within Day) of mobile phase, saturation time, volume loaded and volume of the
Standard Amount recovered Recovery RSD developmental phase, no appreciable difference was perceived in the
concentration (ng/band) ± SD (%) (%) observed parameters like peak height, Rf, capacity factor, theoretical
(ng/band) plates and separation number. The magnitudes of RSD and SEM were
also found to be well within the specification limits.
Intra-day precision (within day)
LQC: 100 95.06 ± 1.10 95.06 1.15
MQC: 150 146.92 ± 1.53 97.94 1.04 Ruggedness
HQC: 200 195.10 ± 1.00 97.55 1.02 As per the ruggedness studies carried out employing two different
Inter-day precision (between day) analysts on the HPTLC instrument, no significant change in the values
LQC: 100 92.06 ± 1.10 92.06 1.19 of the mean percentage of recovery was observed in three QC samples
MQC: 150 144.92 ± 1.53 96.13 1.59 (i.e., LQC, MQC and HQC), thus ratifying through high degree of
HQC: 200 193.10 ± 1.00 96.55 1.03 ruggedness of the developed densitometry method.

97.9%, respectively. Further, the %RSD values for mangiferin as per
repeatability and intermediate precision were found to be well within
the stipulated limit of <2%. These results confirmed the high degree
of precision of the developed method. Table XI illustrates the intra-
and inter-day precision data for various quality control samples of
mangiferin.
Matrix effect
The matrix effect, calculated using three different QC samples, i.e., 50,
100 and 150 ng/band showed the mean percentage of recovery values
of 87.00, 93.03 and 96.75%, respectively. Higher values of the per-
centage of recovery, i.e., >80%, corroborated efficient densitometric
separation of the mangiferin from the biological matrix. This also
vouched for the aptness and robustness of the sample preparation
and extraction technique employed throughout the bioanalytical
method development and validation studies.

LOD and LOQ

The method showed the LOD and the LOQ to be 12.1 and 36.6 ng/
band, respectively, thus indicating quite high sensitivity of the devel-
oped method for quantification of mangiferin.
System suitability
The system suitability results confirmed lack of significant difference in the
peak height, Rf, capacity factor, theoretical plates and separation number
of mangiferin following triplicate injections. The values of RSD and stan-
dard error of the mean (SEM) were found to be <1%, thus corroborating
the high degree of reliability of the HPTLC instrument.
Discussion
The present studies describe the application of QbD principles for es-
tablishment of a bioanalytical thin-layer densitometric method of
mangiferin in human plasma. Initially, the QTMP and CAAs of the
TLC method were defined and CMVs identified by extensive risk as-
sessment followed by factor screening studies. Further, the method
was optimized by response surface optimization methodology for
identifying the optimal solution followed by critical understanding
of the factor–response relationship. Validation studies demonstrated
a high degree of linearity, sensitivity, selectivity and specificity of the
12
obtained method. The superiority of the current method can also be
demonstrated on the basis of its high sensitivity, which is evident
from much lower values of the LOQ, i.e., 12.1 ng/band, over the ex-
isting liquid chromatographic methods showing LOQ of 480 ng/mL
and 3 µg/mL, respectively (61, 62). In a nutshell, the studies vouch
for the high degree of utility of the developed method for estimation
of mangiferin in bioanalytical samples.
Conclusion
A simple, rapid, sensitive and economical bioanalytical thin-layer den-
sitometric method has been successfully developed employing the
AQbD approach for quantification of mangiferin in bioanalytical sam-
ples, rat plasma per se. The systematic AQbD tools like risk assessment
and factor screening exercise helped in identifying the “prominent few”
method variables associated with high degree of variability from “plau-
sible many” and facilitated optimization of them for attaining superior
method performance. Overall, the volume loaded and plate dimension
showed the maximum influence on all the vital response variables inves-
tigated like Rf, peak height, capacity factor, theoretical plates and sep-
aration number. Method validation studies corroborated the excellent
linearity, accuracy, precision, high sensitivity, system suitability, robust-
ness and ruggedness of the developed thin-layer densitometric method.
Further, stability studies of the drug in plasma revealed lack of any in-
teraction of the mangiferin with the biological matrix leading to the for-
mation of any unwanted peak(s). In a nutshell, the studies indisputably
vouch for the applicability and utility of the science and risk-based
AQbD approach for developing a bioanalytical method of mangiferin
employing thin-layer densitometry, a technique that can be extrapolated
to other lipophilic drug substance(s) too.
Acknowledgments
B.S. gratefully acknowledges the generosity of M/s Stat-Ease Inc., Minneapolis,
USA, for providing one perpetual license and 10-user annual licence of Design
Expert® software, version 9.0, while conferring upon him the “Stat-Ease QbD
Performance Award 2014” and “Premier Academic Status” for his unparalleled
contribution in the area of QbD-based pharmaceutical research work. Also the
coauthors, R.K.K. and S.B. acknowledge the University Grant Commission
(UGC), New Delhi, India for financial grants to them to carry out the present re-
search work as a Research Fellow under RFMS scheme, F.No. 5-(94)/2007/(BSR).
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