RP-HPLC Method Development, Validation Including Stability Indicating and Forced Degradation Studies for the Estimation of Pemetrexed in API and Pharmaceutical Dosage Form

Stability indicating analytical RP-HPLC (Reverse Phase- High performance Liquid Chromatography) method was developed for the analysis of the drug in the presence of its degradation products and subsequent validation of Pemetrexed that was conducted in API and pharmaceutical dosage form. Pemetrexed was subjected to different stress conditions as per International Conference on Harmonization (ICH) guidelines, stress conditions applied were including the effect of acid, base, oxidative, hydrolysis, thermal and photolytic degradation conditions. Chromatographic separation of API (Active Pharmaceutical ingredient) was achieved on Water’s HPLC instrument and ODS C18, 250x4.6 mm, 5µm particle size column with gradient elution of 0.1% ortho phosphoric acid buffer: Acetonitrile 80:20 ratio was taken as the eluent (mobile phase). The buffer pH was maintained at 3, the flow rate was 1.0 ml/min and wavelength were detected at 225 nm. The Retention time (RT) of Pemetrexed was Found at 2.9 minutes, column temperature was kept ambient and the runtime was 6 min. System suitability parameters like theoretical plates, tailing factor, and Asymmetry values were showed at 3391, 1.41, and 1.101 respectively. The percentage drug purity was found to be 99.99%. Validation of the method was proved that linear relationship over the range of 25-150 µg/mL, with linear regression curve (correlation coefficient) r2 value was noticed at 0.999 and LOD (limit of detection), LOQ (limit of quantification) values was established at 0.33µg/mL, 0.99µg/mL respectively. Recovery studies carried out at the concentrations of 50%, 100% and 150% with percentage recovery values of 100.06, 99.8 and 99.87 respectively. The precision of intraday was 0.64% and inter-day was 0.95%. Robustness of flow rate and mobile phase was changed even though the method was Robust. Forced degradation studies were also conducted to check the stability and suitability of this method to resolve the degradation products. Method validation was proved to be accurate, precise, linear, repeatable and robust. Thus, the accepted method can be proceeded for the estimation of Pemetrexed in pharmaceutical dosage form in a daily basis, due to their simplicity, suitability, accuracy, robustness and reproducibility.


Figure 1. Structure of pemetrexed
Extensive literature review reveals that there were reports on Pemetrexed drug determination using analytical methods individually and as combinations.Hardly few techniques were reported methods for Pemetrexed estimation and moreover, stability indicating method for the drug with RP-HPLC technique were not reported yet [3][4][5][6][7][8][9][10][11][12][13][14][15][16].The objective of the current study is to propose simple, economic, quick, sensitive, accurate, precise RP-HPLC method, the method was optimized and validated for the concurrent determination of pemetrexed. .

Reagents and Chemicals required
Pemetrexed and its formulations were gifted samples from Vinkem labs, HPLC graded Water, methanol from Merck.

Instrument specifications
A WATER'S High Performance Liquid Chromatograph system of LC 2695 gradient pump with PDA detector with an EMPOWER SOFTWARE were used, a calibrated DENEVER Electronic balance, LAB INDIA ultra sonicator, Thermo scientific pH meter were used to measure pH, Filter 0.45 and 0.2 µm made by Ultipor N66 nylon, PALL life sciences.

Optimized chromatographic conditions
The analysis was performed by taking ODS C18, 250×4.6mm,5µm column, Mobile phase in the ratio 0.1%OPA Buffer: Acetonitrile (80:20) with a flow rate of 1.0ml/min via column, detected by using UV detector at a wavelength of 225 nm, run time was 6 minutes and temperature was maintained at 300C with an injection volume of 10µL.The retention time of pemetrexed was 2.9 minutes.

Mobile phase preparation
80ml of (0.1%) OPA Buffer and 20 ml of Acetonitrile prior to use it was percolated using 0.45 membrane filter and any gas bubbles present were removed for 10 minutes.

Preparation of Diluent
Diluent used was a mixture of water and Methanol (50:50)

Preparation of standard drug solution
Carefully measured and poured 10mg of pemetrexed working standards into a volumetric flask along with 7ml of diluent, fragmented by sonication for half an hour and diluents were made up to the final volume to 10ml.1ml of the before mentioned stock solution was pipetted into another 10ml volumetric flask followed by the addition of the diluent to reach the final volume up to 10ml.The final concentration of drug is 100µg/ml.

Sample solution preparation
The marketed formulation of pemetrexed Janumet TM tablet (Merck & Co., India) as 20 tablets were taken and powdered finely.500mg of the pemetrexed powder sample was accurately weighed and taken into 100ml volumetric flask.After 30mins of extraction with water, the volume was increased to 100ml with the diluent to reach a concentration of 1000µg/ml, 0.2ml of the before mentioned solution was placed into 10ml volumetric flask and 10 ml of diluent was taken to dilute.The final solution was sieved using a 0.45 syringe filter.

Stress Degradation studies of Stock solution preparation
A precise weight of 10mg of drug component was accurately taken into 10ml volumetric flask and the volume was makeup to the mark with diluent to give a concentration of 1000µg/ml.The final concentration is 100µg/ml.

Preparation of sample for acid degradation
1ml of pemetrexed stock solution was added to 1ml of 1N HCl and heated for 6 hours at 60ºC.The concentration of the resulting solution was prepared to 100µg/ml.10µL solution was introduced into HPLC instrument and plotted the chromatograms in order to determine the sample stability.

Alkaline degradation sample preparation
1ml of pemetrexed stock solution was added to 1ml of 0.1N NaOH and heated for 6hours at 60ºC.The concentration of the resulting solution was 100µg/ml, solution was introduced into HPLC instrument and plotted the chromatograms in order to determine the sample stability.

Oxidative degradation sample preparation
Separately 1ml of 3% H2O2 was taken and mixed with 1ml of pemetrexed stock solution.The resultant solution was maintained for 6 hours.The concentration of the solution was 100µg/ml.10µL of the solution was introduced into the HPLC system and the chromatograms were plotted to determine the sample stability.

Thermal degradation sample preparation
It was conducted by adding preheated sample on a petri dish in the form of a thin film at 105ºC for solid state stability.At different time intervals, 10mg of the heated samples were appropriately melted and mixed with mobile phase to achieve a concentration of 100µg/ml and was introduced into HPLC instrument.

Photolytic degradation sample preparation
The photochemical stability of the sample was investigated by subjecting the stock solution to UV light for 7 days or placing in a photo stability chamber (200Watt-hours/m2).The concentration of the solution was prepared to 100µg/ml, the solution was introduced into HPLC instrument and plotted the chromatograms in order to determine the sample stability.

Results and Discussions
The main objective of the study was the use of RP-HPLC technique for the analysis of pemetrexed drug and its degradation products.The method for suitable analysis was developed and validated using International Conference of Harmonization (ICH) guidelines.The developed and validated method was then applied for force degradation studies of pemetrexed API and its pharmaceutical dosage form and the results are discussed below:

Validation of the analytical method
Validation was carried out to prove the developed method via parameters viz., specificity, system suitability, linearity, range, LOD (Limit od Detection), LOQ (Limit of Quantification), robustness, and stress degradation studies based on ICH standards.

System suitability
To confirm the system viable criteria such as tailing factor, Asymmetry factor, capacity factor, retention time, USP theoretical plate count, inject about 10µL of the solution to the ODS C18, 250×4.6mm,5µm column and the chromatographic resolution has been observed.Then the mobile phase with a composition of 0.1%OPA Buffer: Acetonitrile (80:20) was allowed to pass along at a circulation time of 1ml/min.Retention time, tailing factor, USP theoretical plate of the particular technique is depicted in table 1. [17,18]

Specificity
Specificity refers to a method's capacity to distinguish between the targeted analyte(s) and other constituents of the sample.The isolation of analytes from other possible constituents such as contaminants, degradation products, or additives demonstrates the HPLC method's specificity, for checking specificity injected standard solution into the instrument.As their peaks were not eluted at the retention time pemetrexed, the suggested approach was appropriate for the identification of drug.Results were shown in table 2. The linearity of the calibration curves was determined in pure solution for drug concentrations ranges from 25µl/ml to 150µl/ml.Each concentration was introduced into the chromatographic system to check the linearity from low to high concentration.The area at each concentration has been measured and calculated to know correlation co-efficient.A graph has been plotted as peak area Vs concentration as shown in Figure 5, correlation coefficient was found to be 0.999.The outcome results were depicted in the following table 4.

. Accuracy
Recovery study was taken to determine accuracy, where three different concentration levels such as 50%, 100%, 150% were executed to know accuracy.At same concentration the standard solutions were introduced to the chromatographic system.The results were portrayed in the table 5.

Robustness
Method robustness is assessed by examining system suitability parameters and check the method capacity by minute changes in circulation late, mobile phase composition, and temperature variation were made to access the impact on the method.The flow rate ranged from 0.9ml/min to 1.1ml/min.The following table 8 shows the results.From the results, it is observed that flow rate variation affected the method.Therefore, it indicates the method to be robust through flow rate change.At low flow rate condition, the method is robust.Also observed that the mobile phase composition variation affected the method well.Eventually it is that even on mobile phase variations the method is robust.The values were displayed in table 8.In order to estimate the RP-HPLC sensitivity, LOD as well as LOQ has to be considered which were calculated from the calibration curves by the formulas below based on ICH standards and values were shown in table 9. LOD = 3.3 σ/S, LOQ = 10 σ/S Where, σ = standard deviation of the Y-intercept of the regression line S = slope of the calibration curve Table 9. LOD and LOQ Name LOD LOQ Pemetrexed 0.33μg/ml 0.99μg/ml

Stress Degradation Studies
The therapeutically active substance Pemetrexed was subjected to accelerated disintegration under acidic, alkaline, neutral, heat treatment, oxidation, as well as photolytic stress conditions.Under stress investigation, the portions of the stress sample were diluted using mobile phase to obtain a concentration of 100µg/ml and then injected under optimal conditions with an appropriate blank.

Acid degradation studies
Acid degradation studies are conducted for a period of 6 hours, at 60ºC temperature in 1N HCL.The given drug showed proneness to acidic degradation at ambient temperature.Degradation was done in 6h and the degradation was found to be 7.95% in 1N HCl respectively.The values were shown in table 10.The drug showed liability to Neutral degradation.Degradation was done in 7 days and the % of drug degraded was 0.6%.The values were shown in table 15. 2.9 min -----

Conclusion
Stability suggesting HPLC technique developed and validated is determined to be linear, precise, accurate, specific, and robust.As a result, the approach may be employed to quantify Pemetrexed in tablet formulations on a regular basis.Pemetrexed was subjected to a stability investigation, and an absolute HPLC technique for quantifying and identifying its degradants in bulk drug was devised and confirmed.Under various stress conditions, degradants were generated, according to the findings of stress degradation of bulk drugs conducted as per ICH criteria.Summary of the validation parameters were mentioned in table 16.

Figure 5 .
Figure 5. Linearity and range graph of Pemetrexed

Figure 6 .Table 11 .
Figure 6.Acidic chromatogram 1N HCl 6hrs 4.1.10.2.Alkaline Degradation Alkaline degradation of Pemetrexed was conducted in 0.1 NaOH at 6 hrs.The given drug was found to be sensitive to basic degradation at room temperature.6.88% of the drug product was degraded in 0.1N NaOH in 6 hr.The values were shown in table 11.Table 11.Alkaline degradation report of pemetrexed in 0.1 NaOH S. No. Time (Hrs.) Peak area Mean± S.D. (n=3) % Degradation

Figure 7 .
Figure 7. Basic chromatogram 0.1N NaOH 6hrs 4.1.10.3.Oxidative stress study Oxidative stress study of Pemetrexed was performed in 3% H2O2 at 6hrs is tabulated in Table 19.The given drug has shown susceptibility to oxidative stress at ambient temperature.Degradation was done

Figure 8 .Table 13 .Figure 9 .Figure 10 .
Figure 8. Oxidative chromatogram 3%H2O2 6hr 4.1.10.4.Thermal degradation Thermal degradation of Pemetrexed was performed at a temperature of 105°C for 6 hrs.The given drug in solid form was prone to degradation at temperature at 150°C.Degradation was shown 4.3% in 6h at 105°C respectively.The values were shown in table 13.

Table 1 .
System suitability parameters

Table 2 . Specificity values Injection Rt of Analyte Degradation peak Rt Remarks
In order to determine pemetrexed in its tablet dosage form the verified method has proved efficient.The obtained results were shown in the table 3.

Table 3 .
Assay of pemetrexed

Table 4 .
Linearity and Range of Pemetrexed

Table 5 .
Accuracy report of tablet dosage Evaluation of precision was done with 6 replicate injections prepared and injected in to the instrument.For 6 injections, used the same concentration of sample, the injection of the standard solution was done in HPLC.The %RSD for the area of 6 replicate injections.The results are displayed in table 6.

Table 6 .
Precision Reproducibility) 4.1.7.Intermediate precision / RuggednessAt Different days, different analysts but same lab, the precision was performed in order to review the approach intermediate precision.The standard solution was introduced 6 times and the area for all the 6 introduced was measured in HPLC.The %RSD for the area of 6 replicates introduced was discovered and the results were shown in table7.

Table 19 .
The given drug has shown susceptibility to oxidative stress at ambient temperature.Degradation was done in 6h and the degradation was found to be 5.97% in 3%H2O2 respectively.The values were shown in table12.

Table 15 . Neutral Condition report in drug solution S. No. Time (days) Peak area Mean± S.D. (n=3) % Degradation
Figure 11.Neutral chromatogram 7 days 4.1.10.7.Summary of validation parametersThe Acceptance criteria and the validated results of pemetrexed drug for all the conducted validation parameters within the acceptance criteria and the values were shown in table16.

Table 16 .
Summary of validation parameters