Development and Validation of RP-HPLC Method for Estimation of Mycophenolate Mofetil in Bulk and Pharmaceutical Dosage Form

 

Rajendra Prasad. K.* and Kathirvel S.

Department of Pharmaceutical Analysis, Hindu College of Pharmacy, Amaravathi road, Guntur. A.P.

 

 

ABSTRACT:

A simple, rapid, sensitive and precise high performance liquid chromatography (HPLC) method has been developed for the estimation of Mycophenolate mofetil in bulk and pharmaceutical dosage form. In this method RP-C18 column (250mmx4.6mm I.D and5μm particle size) with mobile phase consisting of acetonitrile and 0.02M phosphate buffer in the ratio of 50:50 v/v in isocratic mode was used. The detection wavelength is 230 nm and the flow rate is 1 ml/min. In the range of 10-60 μg/ml, the linearity of Mycophenolate mofetil shows a correlation coefficient of 0.999. The proposed method was validated by determining sensitivity, accuracy, precision and system suitability parameters. The mobile phase selected for the method is simple, fast, accurate and precise and hence can be applied for routine quality control analysis of Mycophenolate mofetil in bulk and its pharmaceutical dosage form. The method was fully validated according to ICH guidelines.

 

KEYWORDS: Mycophenolate mofetil, High performance liquid chromatography, Validation

 

 

INTRODUCTION:

Mycophenolate mofetil (MMF) is chemically 2-(morpholin-4-yl) ethyl (4E)-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro isobenzofuran-5-yl)-4-methylhex-4-enoate1,2. Mycophenolate mofetil is an immunosuppressant and prodrug of mycophenolic acid, extensively used to prevent rejection in organ transplantation. It acts as a non-competitive, selective and reversible inhibitor of inosine monophosphate dehydrogenase (IMPDH) 3,4 in purine biosynthesis, to be specific guanine synthesis, which is necessary for the growth of T cells and B cells.

 

Analysis plays an important role in the formulation development of any drug molecule. A suitable and validated method has to be available for the analysis of drugs in bulk, in drug delivery systems, in dissolution studies (in vitro), and in biological samples (in vivo). Few HPLC5-8 and LC-MS9-12 methods for its determination have been reported. A simultaneous determination of mycophenolic acid and valproic acid in human plasma by HPLC13 reported.  Thus the present study was undertaken to develop and validate a simple, sensitive, accurate, precise, and reproducible HPLC method for estimation of mycophenolate mofetilin bulk and pharmaceutical dosage form as per ICH guidelines.

 

EXPERIMENTAL:

Instrumentation:

The separation was carried out on isocratic HPLC system (Shimadzu) with Shimadzu Binary HPLC pump, Shimadzu LC- 10ATP UV-Visible Detector, Spinchrom SPD-10AVP software and RP-C18 Xterra column (250mmx4.6mm I.D and particle size 5μm).

 

Drugs and chemicals:

Acetonitrile of HPLC grade was purchased from E.Merck (India) Ltd., Mumbai. Sodium dihydrogen phosphate was obtained from Qualigens Fine Chemicals Ltd., Mumbai. Mycophenolate mofetil was a gift sample by Intas Pharmaceuticals Ltd., Ahmedabad. The commercially available mycophenolate mofetil tablets were procured from the local market.

 

Chromatographic conditions:

The mobile phase consisting of acetonitrile (HPLC grade) and 0.02M Sodium dihydrogen ortho phosphate buffer pH-3 adjusted with orthophosphoric acid, were filtered through 0.45μ membrane filter before use, degassed and were pumped from the solvent reservoir in the ratio of 50:50 v/v was pumped into the column at a flow rate of 1 ml/min. The detection was monitored at 230 nm and the run time was 10 min. The volume of injection loop was 20 μl prior to injection of the drug solution the column was equilibrated for at least 30 min. with the mobile phase flowing through the system. The column and the HPLC system were kept in ambient temperature.

 

Standard Procedure:

Stock solution of Mycophenolate mofetil was prepared by dissolving 100 mg of Mycophenolate mofetil in 100 ml standard volumetric flask containing 50 ml of mobile phase and the solution was sonicated for 20 min. and then made upto the mark with mobile phase to get a concentration of 1 mg/ml. Subsequent dilutions of this solution were made with mobile phase to get concentration of 10-60 μg/ml. The standard solutions prepared as above were injected into the 20 μl loop and the chromatogram was recorded shown in Figure 3. The retention time of Mycophenolate mofetil was found to be 4.100 min. The calibration curve was constructed by plotting concentration vs peak area. It was shown in Figure 2. The amount of Mycophenolate mofetil present in sample was calculated through the standard calibration curve. The linearity experiment was carried out in triplicate to ascertain accuracy and precision of the method.

 

Assay:

Twenty tablets were weighed and powdered. A quantity equivalent to 100 mg of Mycophenolate mofetil was weighed accurately and transferred to 100 ml volumetric flask, dissolved in mobile phase and made upto 100 ml with mobile phase. From this solution, further dilutions were made in mobile phase to get 20 μg/ml. This solution was injected and the chromatogram was recorded. The amount of Mycophenolate mofetil was determined from the regression equation.

 

Figure 2 :  Linearity plot for Mycophenolate mofetil

 

Table 1: Validation parameters for Mycophenolate mofetil      

System suitability parameters

Results

Linearity (µm/ml)

10-60

Standard regression equation

y = 20.1x + 13.45

Correlation coefficient (R2)

0.999

Retention time (min)

4.100

Theoretical plates (N)

2948

Asymmetric factor

1.000

LOD (µm/ml)

                1.50             

LOQ (µm/ml)

4.56

Accuracy

% Recovery = 99.76-100.50

 

Method Validation 14

Validation is a process of establishing documented evidence, which provides a high degree assurance that a specific activity will consistently produce a desired result, or a product meeting its predetermined specifications and quality characteristics. The method was validated for different parameters like specificity, linearity, precision, accuracy, limit of detection, limit of quantitation, robustness and system suitability parameters has been validated for the determination of Mycophenolate mofetil. The results were shown in Table 2.

 

 

Table 2: Determination of accuracy by the percentage recovery (n=3)

Drug name

Level of addition (%)

Amount of drug added (µg/ml)

(n=3)

Amount recovered

 (µg/ml)  (n=3)

%Recovery*

Mycophenolate mofetil

80

16

16.081

100.50

100

20

19.952

99.76

120

24

24.052

100.21

* Average of three determinations

 

 

Figure 3. Typical chromatogram of Mycophenolate mofetil.

 

 

Specificity:

The specificity was established by preparing a Mycophenolate mofetil standard at 0.5% level of test concentration and injected 5 times into HPLC system as per the test procedure.

 

Linearity:

The standard curve was obtained in the concentration range of 10-60 μg/ml. The linearity was evaluated by linear regression analysis using the least square method. It was found that correlation coefficient and regression analysis are within the limits.

 

Precision:

The precision of the assay was determined in terms of intra-day and inter-day precision. The intra-day and inter-day variation in the peak area of drug solution was calculated in terms of coefficient of variation (C.V.) obtained by multiplying the ratio of standard deviation to mean with 100.

 

Limit of Detection and Limit of quantification:

The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample, which can be detected, but not necessarily quantitated as an exact value. The LOQ is the concentration that can be quantitated reliably with a specified level of accuracy and precision. The LOD and LOQ were calculated using the formula involving the standard deviation of response and the slope of the calibration curve.

 

LOD = Cd × Syx / b and LOQ = Cq × Syx / b.

 

Where Cd and Cq are the coefficients for LOD and LOQ. Syx is the residual Variance of the Regression, and b is the Slope. Calculation   was performed by using values of Cd and Cq of 3.3 and 10.

 

Robustness:

The robustness was checked by changing the temperature to 300 and 350C and the method suits best.

 

RESULTS AND DISCUSSIONS:

The proposed method obeyed beer’s law in the concentration range of 10-60 µg/mL. The regression equation of Mycophenolate mofetil concentration over its peak area was found to be Y= 20.1x + 13.45 (R2=0.999) where Y is the peak area and X is the concentration of Mycophenolate mofetil (μg/ml). The proposed HPLC method was also validated for intra-day and inter-day variation. The coefficient of variation in the peak area of the drug for 5 replicate injections was found to be less than 1%. The asymmetry factor was found to be 1.000, which indicates asymmetric nature of peak. The number of theoretical plates was found to be 2948, which indicates efficient performance of the column. The limit of detection and limit of quantitation was found to be 1.50 μg/mL and 4.56 μg/mL, indicates the sensitivity of the method. To optimize the chromatographic conditions, various combinations of acetonitrile and phosphate buffer were tested. The use of acetonitrile and Sodium dihydrogen ortho phosphate  buffer in the ratio of 50:50 v/v resulted in peak with good shape. The high percentage of recovery of Mycophenolate mofetil ranging from 99.76 to 100.50%  indicates that the proposed method is highly accurate. No interfering peaks were found in the chromatogram indicating that excipients used in tablet formulation did not interfere with the estimation of the drug by proposed HPLC method.

 

CONCLUSION:

Described in the manuscript is HPLC method for the determination of Mycophenolate mofetil in bulk and its pharmaceutical dosage form. The statistical analysis of the results showed that all the proposed procedures had good precision and accuracy. The results of analysis revealed that the proposed method was suitable for the analysis with virtually no interference of the usual additives present in pharmaceutical formulations. This method can be adopted for routine quality control of Mycophenolate mofetil in bulk and pharmaceutical preparations. 

 

ACKNOWLEDGEMENTS:

The authors are thankful to the Intas Pharmaceuticals Ltd., Ahmadabad for providing the gift sample of Mycophenolate mofetil. The authors are also thankful to the Management of Hindu College of Pharmacy for providing necessary facilities to carry out this project.

 

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Received on 28.11.2012

Modified on 20.12.2012

Accepted on 30.12.2012     

© A&V Publication all right reserved

Research Journal of Pharmaceutical Dosage Forms and Technology. 5(1): January- February, 2013, 42-45