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

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.


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).Mycophenolate 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.

Experimental
2.1.Chemicals and Reagents.Mycophenolate working standard 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 solution was prepared in Milli-Q water.

Instrumentation.
A double-beam UV-VIS spectrophotometer (UV-1800, Shimadzu, Japan) connected to computer 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 10 mm 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.

Preparation of (0.02 M) Octane 1-Sulfonic Acid Buffer
Solution.4.35 grams of octane 1-sulfonic acid was dissolved in 800 mL of distilled water and the pH was adjusted to 3.5 with o-phosphoric acid in a 1000 mL volumetric �ask.

Preparation of Standard Stock
Solution.e standard solution of MPH was prepared by dissolving accurately weighed 10 mg of the drug in methanol and diluted to 10 mL with methanol to obtain a �nal concentration of 1000 g/mL.From this solution, 2.5 mL was taken and diluted with 0.02 M octane 1-sulfonic acid buffer in a 25 mL volumetric �ask to prepare a working standard solution (100 g/mL).

Method A
3.1.Zero-Derivative Spectrometry.Series dilutions of standard solutions were prepared in 10 mL volumetric �asks and diluting to volume with 0.02 M Octane 1-sulfonic acid buffer (pH 3.5 ± 0.05) to produce the concentrations ranging from 1.0-150.0g/mL.e above solutions were scanned over the range of 400 nm to 200 nm against blank.e  max 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.

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 () 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  values (maxima, at 258.0 nm) on the y-axis.

Method C
5.1.Area-Under-Curve Method.e AUC (area-undercurve) 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  1 (240 nm) and  2 (260 nm).In method A the  max was observed at 250 nm, and therefore for method C the AUC was selected between the wavelength 240-260 nm (i.e.,  max ± 5 or 10 nm;  1 and  2 = 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.

Method Validation
6.1.Linearity.For all the methods, (1.0-150.0g/mL) calibration 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.

Assay of Mycophenolate Tablets.
Mycophenolate is available in the local market with different brand names in India such as Reno�x (500 mg, UNICHEM Laboratories Ltd.), MYCEPT (500 mg, PANACEA Biotech Ltd.), and CELL-CEPT (500 mg, ROCHE Pharmaceuticals).Twenty tablets of Mycophenolate from three different brands were collected from pharmacy store, weighed and �nely powdered.Powder equivalent to 10 mg of the drug was transferred to a 100 mL volumetric �ask and dissolved in about 40 mL 0.02 M 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.

Precision. e intraday and interday precision of the
proposed methods was performed on the same day with three different concentration levels (  3) and on three different days at three different concentration levels (  3) of MPH (10.0, 20.0, and 50.0 g/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 g/mL) was spiked with pure drug solution (80, 100, and 120%) in different 10 mL volumetric �asks, and the absorbance was measured (18, 20, and 22 g/mL).e % recovery as well as the % RSD was calculated (Table 1).

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 99.87-100.05for method A, 99.82-100.02for method B, and 99.89-100.04 for method C. e proposed methods 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.

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.

F 1 :
Chemical structure of Mycophenolate.
T 1: Optical characteristics and validation parameters of Mycophenolate.