Development and Validation of Stability Indicating LC-PDA Method for Mycophenolate Mofetil in Presence of Mycophenolic Acid and Its Application for Degradation Kinetics and pH Profile Study

Factorial design tool applied for development of isocratic reversed-phase stability-indicating HPLC method for the analysis of mycophenolate mofetil (MMF) and its degradation products. MMF stress degradation products mycophenolate acid (MPA) and DP3 (USP impurity H) were isolated and used for quantitation. Separation achieved on a Symmetry C18 (250mm × 4.6mm, 5.0 μ) column using a methanol: acetate buffer (75 : 25 v/v), pH 6.0 (adjusted with acetic acid), at 0.5mL flow rate, column maintained at 55C, and data integrated at 251 nm. MMF is subjected to hydrolysis, oxidation, heat degradation, and so forth; under all these conditions degraded products are well separated. The method validation characteristics included accuracy, precision, linearity, range, specificity, and sensitivity. Robustness testing is conducted to evaluate the effect of minor changes to the chromatographic conditions and to establish appropriate system suitability parameters. The proposed method is used to investigate kinetics of acid, alkali hydrolysis and oxidation process. Major degradation productsMPA andDP3 were isolated and quantitated. Characterization of MPA by NMR and LC-MS/MS and other degraded products by LC-MS/MS is attempted successfully. The method is used successfully for the quality assessment of three MMF drug commercial formations and its acid, alkali, and oxidative degradation kinetics study.

Its degradation products in aqueous solution are studied [11].Stability-indicating HPLC and LC-MS/MS methods [12,13] are reported where structural elucidation of degraded product and separation of the stress degraded product is lacking.MMF is included in BP, EP, and USP and assayed by potentiometry [14,15].Isocratic LC method was described in proposed USP monograph [16,17] for MMF tablet and capsule assay using mobile phase acetonitrile : water containing 0.3% triethylamine (30 : 20 v/v), pH 5.3 (adjusted with phosphoric acid) with run time of 10 min.Proposed isocratic RP-HPLC-PDA method has many advantages, which includes mass spectrometry compatible and simple methanol and acetate buffer mixture used as mobile phase and the method is stability indicating and highly sensitive.Quantitation of MMF, MPA, and DP3 (USP reported impurity H) is achieved (Figure 1); proposed method is capable of separating all the stress degraded products with short run time.Accordingly, the aim of the present study was to establish inherent stability of MMF through stress studies under a variety of ICH recommended test conditions [18] and to develop a sensitive stability-indicating assay method with MS compatible and simple mobile phase and structural elucidation of the stress degraded products.

Method Validation.
The HPLC method was validated in terms of precision, accuracy, specificity, sensitivity, robustness, and linearity according to ICH guidelines.Assay method precision (interday and intraday) was determined using nine-independent test solutions and precision of repeatability was performed by six-time injection of the solution.Assay method was evaluated with the recovery of the standards from excipients by spiking standards at three levels (50%, 100%, and 150%) to preanalyzed formulation.
Values of limit of detection (LOD) and limit of quantification (LOQ) were calculated by using  (standard deviation of response) and  (slope of the calibration curve) and by using equations, LOD = (3.3× )/ and LOQ = (10 × )/.
Calculated values of LOD and LOQ were confirmed by repeated injections of samples containing amounts of analyte in the range of LOD and LOQ.To determine the robustness of the method, the final experimental conditions were purposely altered and the results were examined.The flow rate was varied by (±)5%.Column temperature was varied by (±)2 ∘ C and effect of column from different suppliers was studied.Measurement wavelength was varied by (±)1 nm, injection volume was changed (±)2 L, % organic was changed by (±)5%, and buffer strength was changed by (±)5 mM.Change in one parameter was considered for study at a time.Each parameter was studied by triplicate injection of the sample containing HTZ, MMF, MPA, and DP3.Robustness was evaluated by studying the effect of parameter change on assay values and resolution of the analytes injected and its effect on other system suitability parameters (retention time, number of theoretical plates, and peak symmetry).Long term (4 ∘ C for 5, 10, 15, and 30 days) and autosampler stability (6, 12, and 24 hrs in autosampler) of the drug in solution were studied.

Procedure for Forced Degradation Study.
Forced degradation of the drug product was carried out under thermolytic, photolytic, acid/base hydrolytic, and oxidative stress conditions.For photolytic stress, drug product in the solid state was irradiated with UV radiation with peak intensities at 254 and 366 nm.The UV dose from the lamp of peak intensity at 366 nm was measured by use of a quinine monohydrochloride (2% solution in water) chemical actinometer as mentioned in the ICH guidelines [18].Minimum desired exposure (200 Wh/m 2 ) was observed after irradiation for 26 h.Sample solution containing 1000 g/mL MMF was subjected to selected stressed conditions, neutralized, appropriately diluted, and injected onto column.Samples except for photo oxidation were protected from light.For acid, base, and water induced degradation, solutions containing 1000 g/mL of the drug were prepared in 0.  Mobile phase used for the TLC separation of DP3 was chloroform : toluene : methanol : acetic acid (7 : 3 : 0.    obtained (Table 1(c)), it can be concluded that there is no interaction between the variables and flow rate does not play an important role.Methanol % affects   and is considered as main effect.Target values of   for MPA (6.0) and MMF (7.0) were fixed and weight = 1 was assigned to it to obtain optimum conditions.HPLC runs were carried out by varying the % of methanol in mobile phase and final chromatographic conditions selected were Symmetry C18 (250 mm × 4.6 mm, 5.0 ) column maintained at 55 ∘ C using column oven.Mobile phase composed of methanol: acetate buffer (pH 6.0) (75 : 25 v/v).The flow rate was 0.5 mL/minutes, the PDA detector was set at 251 nm, and injection volume was 20 L.

Method Validation.
The method was validated according to ICH guidelines.The following validation characteristics were addressed: linearity, range, accuracy, precision, specificity, sensitivity (LOQ and LOD), and robustness.Among drotaverine, HTZ, diclofenac sodium, etoricoxib, and atenolol, hydrochlorothiazide (HTZ) was observed as the most appropriate internal standard (IS) with values of peak symmetry, theoretical plates, and resolution well within limit.Assay value determined for three formulations was in the range of 100 ± 1.33% with %RSD <1.2.Specificity of the method was determined by analyzing samples containing a mixture of the drug product excipients, samples containing MMF, MPA, and DP3, and stress degraded samples.All chromatograms were examined to determine if MMF, MPA, and DP3 coeluted with each other or with any excipient peak.Linear least square regression and LOD and LOQ data summarized in Table 2 indicate linearity and sensitivity of the method.Accuracy and precision were established for MMF, MPA, and DP3.Results for the accuracy of analytes tested in drug products by     respectively.During various stress conditions, extent of degradation, peak purity, recovery of MMF, and mass balance (always >98.58) were calculated.Peak purity of stressed samples of MMF was checked by using PDA detector.The purity angle within the purity threshold limit obtained in all stress degraded samples demonstrated analyte peak homogeneity.Results of stress degradation, peak purity, and mass balance study are presented in Table 3. MPA, MMF degradation product, was isolated and identified by NMR and LC-MS/MS study and by comparing   and UV spectrum of standard and degraded products.DP3 formed under all the stress conditions was isolated; its structure was determined by LC/MS study and found to be USP impurity H and is eluted at   7.9.It has MS fragmentation pattern like MPA except intensities of the MS peaks.It is less polar than MPA and was retained on C18 column for more time; its proposed structure by MS-MS study is shown in Figure 4. Mass fragmentation pattern and proposed structures of MPA and DP1 are shown in Figures 5(a

Conclusion
A simple and efficient stability-indicating reverse-phase HPLC method was developed and was found to be accurate,

Figure 1 :
Figure 1: Structure of MMF and its major degraded products.
) and5(b).Mass fragmentation patterns of proposed impurity DP2, impurity G, and mycophenolate Factorial design was used as tool for optimization of the chromatographic conditions to develop a stability-indicating method.Various pilot trials were carried out to investigate different stationary phases, for example, C18 column (Symmetry and Kromasil), different mobile phases containing organic modifiers (methanol, acetonitrile, and THF), different buffers of different pH (3-6.5), and temperature (30 to 65 ∘ C).Our objectives were to achieve a peak tailing factor <1.2, retention times (  ) from 4 to 9 min, and MMF and MPA resolution >2.From this experiment pH 6.0 was selected as most appropriate because at lower pH there is increase in baseline disturbances and peaks were not symmetric.Column was maintained at 55 ∘ C and methanol as organic modifier was selected to be the most appropriate.Effect of mobile phase flow rate and % of methanol was studied by full factorial design (Table 1(a)) and 2 2 full factorial design experiments were carried out to study the effect on 3.1.MethodDevelopment.R of MMF and MPA (Table 1(b)).By using full factorial design data linear equations obtained for MMF and MPA were  = 7.113 − 0.4343 1 + 1.0478 2 − 0.0528 1  2 and = 6.1515 − 0.5265 1 + 0.9135 2 − 0.008 1  2 , respectively.From the data

Table 1 :
Results of method optimization.The 1 H NMR and13C NMR data confirmed that the isolated compound is mycophenolic acid.
and DP3 was always within 100 ± 1.5%.Values of other system suitability parameters were always within the limits indicating robustness of the method.The %RSD of assay values of MMF and MPA during solution and autosampler stability experiments were within 1.6 and no significant changes were observed.3.3.NMR Study.The PMR spectrum of MPA in deuterated chloroform showed protons signals  H at 1.

Table 2 :
Results of method sensitivity, linearity, precision, system suitability, and recovery of analytes.

Table 3 :
[11]lt of stress degradation study, peak purity, and mass balance,  = 3.Values of peak angle which are less than peak threshold indicate homogenous peak.mofetil are shown in Figures6(a)-6(c), respectively.The formation of MPA due to thermal degradation at various pH reported in literature[11]was confirmed.Peroxide catalyzed formation of MPA and N-oxide of MMF (USP impurity G) was supported by the experimental findings during proposed study.3.5.Degradation Kinetics.Forced degradation of MMF byHCl, NaOH, and H 2 O 2 resulted in decrease in peak area by time and it was found to be temperature dependent.Apparent first order degradation constant (Figure7) and half-life at each temperature and Arrhenius equations were calculated.Apparent first order degradation constant (2 + log   /  ± standard deviation) for HCl, NaOH, and H 2 O 2 degradation at 60 ∘ C was found to be 1.799 ± 0.08455, 1.884 ± 0.0645, and 1.944 ± 0.0292, respectively.Half-life ( 1/2 , day −1 ) for HCl, NaOH, and H 2 O 2 degradation at 60 ∘ C was found to be 0.7256, 0.7117, and 0.7084, respectively.Calculated Arrhenius equations at each of the selected conditions were Log  = 1.223 − (1.192 × 10 −3 )/ (for 0.1 N HCl), Log  = 1.300 − (1.630143×10 −3 )/ (for 0.05 N NaOH), and Log  = 1.363− (1.906 × 10 −3 )/ (for 10% H 2 O 2 ). *