ree simple, precise and cost-effective spectrophotometric methods have been developed for the determination of Mycophenolate in bulk and its pharmaceutical formulations. Mycophenolate shows max at 250.0 nm in zero-derivative spectrum (method A), 258.0 nm in �rst-derivative spectrum (method B) and method C is based on the calculation of area under curve (A�C) for analysis of Mycophenolate in the wavelength range of 240.0-260.0 nm. e drug follows the Beer-Lambert&apos;s law in the concentration range of 1.0-150.0 g/mL for all the methods. e methods were validated by following the analytical performance parameters suggested by the International Conference on Harmonization. All validation parameters were within the acceptable range. e developed methods were successfully applied to estimate the amount of Mycophenolate in bulk and pharmaceutical dosage forms

A Narendra

D Deepika

M Mathrusri Annapurna

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Journal of Chemistry
Volume 2013, Article ID 523193, 4 pages
http://dx.doi.org/10.1155/2013/523193
Research Article
Validated Spectrophotometric Methods for
the Determination of Mycophenolate: An Anti-Neoplastic
Agent in Bulk and Pharmaceutical Dosage Forms
A. Narendra,1 D. Deepika,2 andM.Mathrusri Annapurna3
1 Department of Pharmaceutical Analysis and Quality Assurance, Roland Institute of Pharmaceutical Sciences,
Berhampur 760010, India
2Department of Pharmaceutical Analysis and Quality Assurance, Royal College of Pharmacy and Health Sciences,
Berhampur 760002, India
3Department of Pharmaceutical Analysis and Quality Assurance, GITAM Institute of Pharmacy, GITAM University,
Visakhapatnam 530045, India
Correspondence should be addressed to A. Narendra; naren_angirekula@yahoo.com
Received 28 November 2011; Revised 16 May 2012; Accepted 17 May 2012
Academic Editor: Irene Panderi
Copyright © 2013 A. Narendra et al. is is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ree simple, precise and cost-effective spectrophotometric methods have been developed for the determination ofMycophenolate
in bulk and its pharmaceutical formulations. Mycophenolate shows 𝜆𝜆max at 250.0 nm in zero-derivative spectrum (method A),
258.0 nm in �rst-derivative spectrum (method B) andmethod C is based on the calculation of area under curve (A�C) for analysis
of Mycophenolate in the wavelength range of 240.0–260.0 nm.e drug follows the Beer-Lambert’s law in the concentration range
of 1.0–150.0 𝜇𝜇g/mL for all the methods.emethods were validated by following the analytical performance parameters suggested
by the International Conference on Harmonization. All validation parameters were within the acceptable range. e developed
methods were successfully applied to estimate the amount of Mycophenolate in bulk and pharmaceutical dosage forms.
1. Introduction
Mycophenolate (MPH) chemically, 2-(morpholin-4-yl)ethyl
(4E)-6-(4-hydroxy-6-methoxy7-methyl-3-oxo-1,3-dihydro-
2-benzofuran-5-yl)-4-methyl hex-4enoate (Figure 1). Myco-
phenolate mofetil is the 2-morpholino ethyl ester of
mycophenolic acid (MPA), an immunosuppressive agent,
inosine monophosphate dehydrogenase (IMPDH) inhibitor.
It is a potent, selective, uncompetitive, and reversible
inhibitor of inosine mono phosphate dehydrogenase and
therefore inhibits the de novo pathway of guanosine
nucleotide synthesis without incorporation into DNA. Since
T- and B-lymphocytes are critically dependent for their
proliferation on de novo synthesis of purines, whereas other
cell types can utilize salvage pathways, it has potent cytostatic
effects on lymphocytes. It inhibits proliferative responses of
T- and B-lymphocytes to both mitogenic and allospeci�c
stimulation. Addition of guanosine or deoxyguanosine
reverses the cytostatic effects of mycophenolic acid on
lymphocytes. Mycophenolic acid also suppresses antibody
formation by B-lymphocytes and prevents the glycosylation
of lymphocyte and monocyte glycoproteins that are involved
in intercellular adhesion to endothelial cells and may inhibit
recruitment of leukocytes into sites of in�ammation and
gra rejection.
e different analytical methods that are reported for its
determination include HPLC [1–14], LC/MS [15], HPTLC
[16], spectrophotometry [17], and gas chromatography [18].
e aim of the present work is to develop and validate for
the estimation of Mycophenolate in bulk and pharmaceutical
formulations by derivative spectrophotometry [19] and to
validate as per the ICH guidelines [20]. Very few methods
are available for the determination of Mycophenolate using
spectrophotometry, which is very sensitive and o cost-
effective. e present proposed method was developed using
0.02M Octane 1-sulfonic acid buffer solution and validated.
2 Journal of Chemistry
O
O
O
O
O
O
N
OH
F 1: Chemical structure of Mycophenolate.
2. Experimental
2.1. Chemicals and Reagents. Mycophenolate working stan-
dard was kindly provided by Alembic Ltd., (Vadodara, India)
and was used as received. A commercial formulation was
purchased from the local market. Octane 1-sulphonic acid
sodium salt of analytical grade solutionwas prepared inMilli-
Q water.
2.2. Instrumentation. A double-beam UV-VIS spectropho-
tometer (UV-1800, Shimadzu, Japan) connected to com-
puter loaded with spectra manager soware UV Probe was
employed with spectral bandwidth of 1 nm and wavelength
accuracy of ±0.3 nm with a pair of 10mm matched quartz
cells. All weights were taken on electronic balance (Denver,
Germany). For scanning, the wavelength range selected was
from 400 nm to 200 nm with medium scanning speed.
2.3. Preparation of (0.02M) Octane 1-Sulfonic Acid Buffer
Solution. 4.35 grams of octane 1-sulfonic acid was dissolved
in 800mL of distilled water and the pH was adjusted to 3.5
with o-phosphoric acid in a 1000mL volumetric �ask.
2.4. Preparation of Standard Stock Solution. e standard
solution of MPH was prepared by dissolving accurately
weighed 10mg of the drug in methanol and diluted to 10mL
withmethanol to obtain a �nal concentration of 1000𝜇𝜇g/mL.
From this solution, 2.5mL was taken and diluted with 0.02M
octane 1-sulfonic acid buffer in a 25mL volumetric �ask to
prepare a working standard solution (100 𝜇𝜇g/mL).
3. Method A
3.1. Zero-Derivative Spectrometry. Series dilutions of stan-
dard solutions were prepared in 10mL volumetric �asks and
diluting to volume with 0.02M Octane 1-sulfonic acid buffer
(pH 3.5 ± 0.05) to produce the concentrations ranging from
1.0–150.0 𝜇𝜇g/mL. e above solutions were scanned over
the range of 400 nm to 200 nm against blank. e 𝜆𝜆max was
found to be at 250.0 and 305.0 nm (Figure 2). But the present
study was carried out at 250.0 nm where the Beer-Lambert’s
law was following properly and statistical data was shown
in Table 1. A calibration curve was constructed by plotting
the concentration on the x-axis and the corresponding
absorbance (at 250 nm) on y-axis.
A
b
so
rb
an
ce
0.3
0.059
−0.102
220 272 350
Wavelength (nm)
F 2: UV absorption spectrum of Mycophenolate (D0)
(10 𝜇𝜇g/mL).
A
b
so
rb
an
ce
0.019
−0.016
−0.039
220 272 350
Wavelength (nm)
F 3: First-derivative absorption spectrum of Mycophenolate
(D1) (20 𝜇𝜇g/mL).
4. Method B
4.1. First-Derivative Spectrometry. e zero-order spectrum
was derivatised to get �rst-order derivative spectra (Figure
3) by the inbuilt spectra manager soware “UV Probe.”
e derivative spectrum shows maxima at 258 nm, and the
derivative absorbance (𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝜆𝜆) was noted at 258 nm for all
the analytical calculations [21].
A graph was drawn by taking the concentration of the
drug solution on the x-axis and the corresponding 𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝜆𝜆
values (maxima, at 258.0 nm) on the y-axis.
5. Method C
5.1. Area-Under-Curve Method. e AUC (area-under-
curve) method is applicable where there is no sharp peak or
when broad spectra are obtained. It involves the calculation
of integrated value of absorbance with respect to the
wavelength between two selected wavelengths 𝜆𝜆1 (240 nm)
and 𝜆𝜆2 (260 nm). In method A the 𝜆𝜆max was observed at
250 nm, and therefore for method C the AUC was selected
between the wavelength 240–260 nm (i.e., 𝜆𝜆max ± 5 or 10 nm;
Journal of Chemistry 3
T 1: Optical characteristics and validation parameters of Mycophenolate.
Parameters Method A Method B Method C
Beer-Lambert’s range (𝜇𝜇g/mL) 1–150 1–150 1–150
𝜆𝜆max/wavelength range (nm) 250 258 240–260
Molar absorptivity (L/mol⋅cm) 9.71 × 103 7.37 × 102 4.68 × 104
Sandell’s sensitivity (𝜇𝜇g cm2/0.001 absorbance unit) 0.05 — —
Slope 0.022 0.001 0.107
Standard deviation of slope 0.0252 0.0002 0.0036
Intercept 0.001 0.000 0.025
Standard deviation of intercept 0.0003 0.000 0.0025
Correlation coefficient 0.999 0.999 0.999
Precision (RSD, %)
Intraday (𝑛𝑛 𝑛 3) 0.61–1.25 0.78–0.90 0.92–1.11
Interday (𝑛𝑛 𝑛 3) 0.96–1.09 0.66–1.12 0.87–1.04
Accuracy (% recovery) (% RSD) 99.87–101.80 (0.23) 98.56–100.54 (0.41) 99.75–100.90 (0.38)
T 2: Assay results of Mycophenolate in pharmaceutical dosage form.
Formulation (tablets) Labelled amount (mg) % Recovery
Method A Method B Method C
Reno�x 500 100.05 99.82 100.04
Mycept 500 99.87 100.02 99.89
Cellcept 500 99.96 99.97 99.93
𝜆𝜆1 and 𝜆𝜆2 = 240−260 nm). A graph can be plotted by taking
the concentration of the drug solution on the x-axis and the
corresponding AUC value on the y-axis.
6. Method Validation
6.1. Linearity. For all the methods, (1.0–150.0 𝜇𝜇g/mL) cal-
ibration curves were prepared on three different days. e
results obtained were used to calculate the equation of the
line by using linear regression by the least-squares regression
method.
6.2. Assay of Mycophenolate Tablets. Mycophenolate is avail-
able in the local market with different brand names in India
such as Reno�x (500mg, UNICHEM Laboratories Ltd.),
MYCEPT (500mg, PANACEA Biotech Ltd.), and CELL-
CEPT (500mg, ROCHE Pharmaceuticals).
Twenty tablets of Mycophenolate from three different
brands were collected from pharmacy store, weighed and
�nely powdered. Powder equivalent to 10mg of the drug
was transferred to a 100mL volumetric �ask and dissolved
in about 40mL 0.02M octane-sulphonic acid buffer,
ultra sonicated for 30 minutes, �ltered through �hatman
�lter paper (number 41), and suitably diluted as per the
requirement. e results obtained with three brands were
summarised in Table 2.
6.3. Precision. e intraday and interday precision of the
proposedmethodswas performed on the same daywith three
different concentration levels (𝑛𝑛 𝑛 3) and on three different
days at three different concentration levels (𝑛𝑛 𝑛 3) of MPH
(10.0, 20.0, and 50.0 𝜇𝜇g/mL), and the results (% RSD) were
reported (Table 1).
6.4. Accuracy. is parameter was evaluated by the percent
recovery studies at concentration levels of 80, 100, and 120%,
which consisted of adding known amounts of MPH pure
drug solution to a preanalysed formulation solution. An
extracted formulation solution containing Mycophenolate
(10.0 𝜇𝜇g/mL) was spiked with pure drug solution (80, 100,
and 120%) in different 10mL volumetric �asks, and the
absorbance was measured (18, 20, and 22 𝜇𝜇g/mL). e %
recovery as well as the% RSD was calculated (Table 1).
7. Results and Discussion
Beer-Lambert’s law was obeyed in the concentration range
of 1–150 μg/mL for all the three methods A, B, and C. e
regression equations were given in Table 1.
e% recovery from the formulation (Table 2) was found
to be 99.87–100.05 formethodA, 99.82–100.02 formethodB,
and 99.89–100.04 for method C.e proposedmethods were
validated as per the ICH guidelines. e % RSD in precision
and accuracy was found to be less than 2.0 indicating that the
proposed methods are precise and accurate. e % recovery
was found to be within the acceptable range in all the three
methods. erefore, the present methods can be employed
for the determination of Mycophenolate in pharmaceutical
formulations successfully.
4 Journal of Chemistry
8. Conclusion
e three validated methods can be successfully applied for
the determination of Mycophenolate in tablet dosage forms.
e three methods are very simple and cost-effective for the
routine analysis of pharmaceutical formulations.
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Validated Spectrophotometric Methods for the Determination of Mycophenolate: An Anti-Neoplastic Agent in Bulk and Pharmaceutical Dosage Forms

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Validated Spectrophotometric Methods for the Determination of Mycophenolate: An Anti-Neoplastic Agent in Bulk and Pharmaceutical Dosage Forms

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