International Journal of Pharmaceutical Sciences

International Journal of Pharmaceutical Sciences INT.J.PH.SCI.,May-August, 2011;3(2):1208-1214 ISSN 0975-4725 www.ijps.info

Original Research Manuscript Date of Submission:07-04-2011 Date of Acceptance:12-05-2011

STABILITY INDICATING HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHIC METHOD FOR ESTIMATION OF PEMETREXED DISODIUM IN BULK DRUG Ankit D. Patel, Dr. D. J. Sen and Dr. C. N. Patel Department of Quality Assurance and Pharmaceutical Chemistry, Shri Sarvajanik Pharmacy College, Gujarat Technological University, Mehsana-384001, Gujarat, India Corresponding author’s E-mail: [email protected]

ABSTRACT To understand the degradation behavior and intrinsic stability of Pemetrexed disodium and to develop and validate HPTLC method for estimation of Pemetrexed disodium, in presence of its degradation products. The best separation was achieved on a pre-coated silica gel aluminum plate 60F254 TLC plate (200×100 mm, layer thickness 2 mm) with ethanol: methanol: ethyl acetate: dil. NH3 (4.2%w/v) (4:3:2:0.6, v/v/v/v) as the mobile phase. The detection wavelength was set kept at 254 nm. The response was a linear function of concentration over the range of 200-1200 ng/spot (R2=0.9984) and the limits of detection and quantitation were 11.01 ng/spot and 33.37 ng/spot, respectively. The method was validated in accordance with the International Conference on Harmonization (ICH) guidelines. The drug was subjected to different stress conditions like acid, base, neutral, oxidation, thermal and UV light. The drug degraded under acidic, alkaline and neutral hydrolytic stress conditions. The degradation products produced as a result of this stress did not interfere with the detection of Pemetrexed disodium and the assay could thus be regarded as stability indicating. The developed and validated HPTLC method is rapid, specific, accurate and precise and can be used for analysis of formulations of Pemetrexed disodium in quality control laboratories. KEY WORDS: High performance thin layer chromatography, powder for infusion, stress testing, validation

INTRODUCTION Pemetrexed disodium has the chemical name N-[4-[2(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo-[2,3-d]pyrimidin-5-yl) ethyl]-benzoyl]-L-glutamic acid disodium salt, heptahydrate. Pemetrexed disodium is a folate analog metabolic inhibitor that exerts its action by disrupting folate-dependent metabolic processes essential for cell replication.[1]

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Structure of Pemetrexed disodium heptahydrate In-vitro studies have shown that Pemetrexed inhibits thymidylate synthase (TS), dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyltransferase (GARFT), which are folatedependent enzymes involved in the de-novo

Int.J.Ph.Sci.,May-August 2011;3(2):

Ankit D. Patel et.al.: STABILITY INDICATING HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHIC METHOD

biosynthesis of thymidine and purine nucleotides.[2,3] Cells that are resistant to antifolates are generally less resistant to Pemetrexed, irrespective of the mechanism of resistance. It is used in treatment of locally advanced or metastatic nonsquamous non-small cell lung cancer (NSCLC), breast cancer, malignant pleural mesothelioma, colorectal cancer and pancreatic cancer.[4,5] Analytical techniques such as gradient HPLC, LC-MS are available for the estimation of Pemetrexed disodium in human plasma and urine.[6,7] So far to our present knowledge no stability indicating HPTLC method was available in the literature. It is felt necessary to perform forced degradation study and to develop and validate stability indicating HPTLC method for the quantitative determination of Pemetrexed disodium.[8,9] The current research work deals with development and validation of stability indicating HPTLC method. MATERIALS AND METHODS Chemicals and reagents Pemetrexed disodium was supplied by Intas pharmaceutical limited, Ahmedabad, as gift sample. Pemetrexed disodium powder for infusion (Pemmet infusion) was purchased from market. Ethanol, methanol, ethyl acetate and ammonia (NH3 aqs.) were purchased from Finar Chemicals Limited, Ahmedabad. Pre-coated silica gel aluminum plate 60F254 TLC plate (200×200 mm, layer thickness 2 mm, Merck, Mumbai) was used in the study as stationary phase. Instrumentation Desaga HPTLC system with AS-30 sample applicator, densitometer CD-60 TLC scanner with ProQuant software, photo chamber with ProviDoc software with Canon power shot G5 digital camera, UV cabinet with dual wavelength 254 nm and 365 nm UV lamp, 100 μl Hamilton applicator syringe and twin trough glass chamber (200×100 mm) was used for analytical purpose. Analytical balance (Acculab ALC-210.4, Huntingdon Valley, PA), ultra sonicator (Fast clean ultrasonic cleaner), hot air oven (TO-90S, Thermolab) and photostability chamber (TH-90S, Thermolab, Mumbai, India) were used in study for different purposes. Preparation of standard solutions Preparation of standard stock solution (1000 μg/ml)

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Accurately weighed 100 mg quantity of Pemetrexed disodium reference standard was transferred into 100 ml volumetric flask, dissolved and diluted up to the mark with distilled water to give a stock solution having strength 1000 μg/ml. Preparation of working standard solution (40 μg/ml) 40 µg/ml of Pemetrexed disodium solution was prepared by diluting 4 ml of stock solution to 100 ml with methanol. Preparation of sample stock solution Powder for infusion equivalent to 50 mg of Pemetrexed disodium was weighed and transferred into a 50 ml of volumetric flask, dissolved and diluted up to the mark with distilled water. This solution was filtered using whatman filter paper no.42. (1000 μg/ml) Procedure for preparation of sample for forced degradation study Degradation study of Pemetrexed disodium in 0.1N HCl at room temperature (Acidic hydrolysis) Accurately weighed quantity of 50 mg Pemetrexed disodium was transferred into 50 ml volumetric flask, dissolved in 10 ml of methanol and diluted up to the mark with 0.1N HCl (1000 µg/ml). It was kept at room temperature for 6 days in dark place. After 6 days, 1 ml was taken from this solution and diluted up to 10 ml with methanol (100 µg/ml). From this solution, 10 µl was spotted on TLC plate. Degradation study of Pemetrexed disodium in 0.5N NaOH at 70°C (Alkaline hydrolysis) Accurately weighed quantity of 50 mg Pemetrexed disodium was transferred into 50 ml volumetric flask, dissolved and diluted up to the mark with 0.5N NaOH (1000 µg/ml). It was refluxed for 10 hour at 70°C. From this solution, 1 ml was taken and diluted up to 10 ml with methanol (100 µg/ml). From this solution, 10 µl was spotted on TLC plate. Degradation study of Pemetrexed disodium in water at room temperature (Neutral hydrolysis) Accurately weighed quantity of 50 mg Pemetrexed disodium was transferred into 50 ml volumetric flask, dissolved and diluted up to the mark with water (1000 µg/ml). It was kept at room temperature for 6 days in dark place. After 6 days, 1 ml was taken from this solution and diluted up to 10 ml with methanol


Ankit D. Patel, et. al.: STABILITY INDICATING HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHIC METHOD

(100µg/ml). From this solution, 10 µl was spotted on TLC plate. Degradation study of Pemetrexed disodium in 1%w/v H2O2 at room temperature (Oxidation) Accurately weighed quantity of 50 mg Pemetrexed disodium was transferred into 50 ml volumetric flask, dissolved in 10 ml of methanol and diluted up to the mark with 1%w/v H2O2. It was kept at room temperature for 3 days in dark place (1000 µg/ml). After 3 days, 1 ml was taken from this solution and diluted up to 10 ml with methanol (100µg/ml). From this solution, 10 µl was spotted on TLC plate. Degradation study of Pemetrexed disodium under thermal condition Some quantity of Pemetrexed disodium standard powder in 2 mm thickness was exposed in hot air oven at 80°C for 3 days. After this exposure, this powder was mixed and 50 mg powder was accurately weighed, transferred into 50 ml volumetric flask, dissolved and diluted up to the mark with HPLC grade water (1000 µg/ml). From this solution, 1 ml was taken and diluted up to 10 ml with methanol (100µg/ml). From this solution, 10 µl was spotted on TLC plate. Degradation study of Pemetrexed disodium under photolytic condition Some quantity of Pemetrexed disodium standard powder in 2 mm thickness was exposed in photostability chamber under 254 nm for 3 days (515 watt hours/m2). After this exposure, this powder was mixed and 50 mg powder was accurately weighed, transferred into 50 ml volumetric flask, dissolved and diluted up to the mark with HPLC grade water (1000 µg/ml). From this solution, 1 ml was taken and diluted up to 10 ml with methanol (100 µg/ml). From this solution, 10 µl was spotted on TLC plate. HPTLC method development The HPTLC procedure was optimized to establish a stability-indicating assay. A premix of ethanol, methanol, ethyl acetate and dilute ammonia (4.2% w/v) in the ratio of 4:3:2:0.6, v/v/v/v, respectively was optimized for thin layer chromatographic plate development. A run distance was kept about 90 mm. The Rf value of Pemetrexed disodium peak was observed about 0.72. Samples were applied as bands of 4 mm width with the gaps of 10 mm in between. Developed plate was dried in air. Detection was done 1210

at 254 nm using mercury lamp in extinctionreemission mode. The slit dimension of detection was kept 0.02 mm×0.4 mm. The various statistical reports were generated according to the standard formulae and parameters were validated as per ICH guideline. Table 1: Chromatographic conditions Pre-coated silica gel aluminum plate Stationary 60F254 TLC plate (200×100 mm, phase layer thickness 2 mm) Ethanol: Methanol: Ethyl acetate: Mobile phase Dil. NH3 (4.2% w/v) (4:3:2:0.6, v/v/v/v) 30 min Chamber saturation 90 mm Migration distance 27 ± 2 min Run time Validation of proposed method Specificity The specificity of the method was ascertained by analyzing standard drug and sample. The band for Pemetrexed disodium in sample was confirmed by comparing the Rf and spectra of the band with those obtained from standard. The peak purity of Pemetrexed disodium was assessed in spectrum mode of densitometer by comparing spectra acquired at three different positions on the band, i.e. peak start (S), peak apex (M) and peak end (E). The specificity of the method was also checked by separation of Pemetrexed disodium in the presence of its degradation products. Linearity and range The linearity was determined by analyzing 6 independent levels of calibration curve in the range of 200-1200 ng/spot. From the working standard solution (40 μg/ml), aliquots of 5, 10, 15, 20, 25 and 30 µl were spotted on the TLC plate. The plate was developed in mobile phase, dried in air, scanned and quantified at 254 nm. The calibration curve of peak area vs. concentration was plotted and correlation co-efficient and regression line equations for Pemetrexed disodium were determined. Precision Intra-day precision was determined by analyzing Pemetrexed disodium (200-1200 ng/spot) at three different time points on the same day and inter-day precision was determined by analyzing Pemetrexed Int.J.Ph.Sci.,May-August 2011;3(2):

Ankit D. Patel, et. al.: STABILITY INDICATING HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHIC METHOD

disodium (200-1200 ng/spot) at three different time points on different days and %RSD was calculated. Accuracy Accuracy was determined by performing recovery studies by spiking different concentrations of pure drug in the pre-analyzed powder for infusion samples within the analytical concentration range of the proposed method at three different set at level of 50%, 100% and 150%. The amount of Pemetrexed disodium was calculated at each level and % recoveries were computed. Limit of Detection (LOD) and Limit of Quantitation (LOQ) The LOD and LOQ were estimated from the set of 5 calibration curves used to determine method linearity. LOD= 3.3*σ/S and LOQ= 10*σ/S Where, σ = the standard deviation of y-intercepts of regression lines S = the slope of the calibration curve Analysis of marketed formulation (powder for infusion) by proposed method From the sample stock solution, 1 ml solution was taken in 10 ml volumetric flask and diluted up to the mark with methanol. From this solution, 10 μl was spotted on the TLC plate. RESULT AND DISCUSSION Method validation Specificity Photograph of developed TLC plate was shown in Figure-1. The drug and degradation chromatograms of forced degraded samples were shown from Figure-2 to Figure-5.

Figure-2: Representative HPTLC chromatogram of Pemetrexed disodium standard (1000 ng/spot)

Figure-3: HPTLC chromatogram of Pemetrexed disodium under acidic condition

Figure-4: HPTLC chromatogram of Pemetrexed disodium under alkaline condition

Figure-1: Photograph of developed TLC 1211


Ankit D. Patel, , et. al.: STABILITY INDICATING HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHIC METHOD

Figure-5: HPTLC chromatogram of Pemetrexed disodium under neutral condition Linearity 3D Overlay chromatogram of Pemetrexed disodium was shown in Figure-6. The linearity of Pemetrexed disodium was found to be in the range of 200-1200 ng/spot with correlation co-efficient 0.9984 as shown in Table-2 and Figure-7.

Sr. No. 1 2 3 4 5 6

Figure-6: 3D Overlay HPTLC chromatogram of Pemetrexed disodium standard

Figure-7: Calibration curve of Pemetrexed disodium by HPTLC Table 2: Calibration data of Pemetrexed disodium by HPTLC Concentration (ng/spot) 200 400 600 800 1000 1200

Peak Area ± S.D. (n=5) 212.22 ± 2.139 441.36 ± 3.655 723.40 ± 3.856 941.10 ± 6.026 1172.68 ± 6.167 1463.81 ± 12.434

%RSD 1.01 0.83 0.53 0.64 0.53 0.85

Rf 0.72 0.72 0.72 0.72 0.71 0.71

Precision In case of intra-day precision, %RSD was found to be in the range of 0.60-1.03. In case of inter-day precision, %RSD was found to be in the range of 0.78-1.31. Accuracy % Recoveries for Pemetrexed disodium was found to be 98.01-101.90. Recovery data was shown in Table-3. Table 3: Recovery data of Pemetrexed disodium from pharmaceutical formulation Amount of sample taken 400

Amount of std. added

Total peak area

0

453.36

400

200

705.78

400

400

944.10

400

600

1181.6 8

Total amount found ± S.D. 399.32±4 .753 603.13±4 .402 795.56±2 .378 987.39±4 .784

Amount recovered

% Recovery

0

0

203.81

101.90

396.23

99.06

588.06

98.01

Limit of Detection and Limit of Quantitation LOD and LOQ were found to be 11.01 ng/spot and 33.37 ng/spot, respectively. 1212



Analysis of pharmaceutical formulation by proposed method The percentage of Pemetrexed disodium in marketed formulation (Pemmet-powder for infusion) was calculated from the calibration curve of Pemetrexed disodium. It was found to be 98.52% as shown in Table-4. Table 4: Estimation of Pemetrexed disodium in pharmaceutical formulation by HPTLC Powder for Label Assay (% of label infusion claim claim*) ± %RSD Pemmet infusion 100 mg 98.52% ± 0.74

6 7 8

Accuracy (%Recovery) LOD LOQ

98.01-101.90 11.01 ng/spot 33.37 ng/spot

CONCLUSION A specific, accurate and precise HPTLC method has been developed for the determination of Pemetrexed disodium in active pharmaceutical ingredient and pharmaceutical formulation. Stress testing showed that all degradation products in all the stressed samples were well separated from Pemetrexed Disodium, confirming its stability-indicating capability. The method can be used for analysis of formulations of Pemetrexed disodium in quality control laboratories.

*average of five estimations Table 5: Summary of forced degradation study by HPTLC Sr. No.

Stress types

Stress Conditions

Rf value Of drug/ degradation products

Peak area

% Degradation

1 2

Standard Acidic Hydrolysis

0.1N HCl at room temperature for 6 days

0.72 0.72,0.85, 0.92

1172.68 366.58

68.74%

3

Alkaline Hydrolysis

0.5N NaOH at 70°C for 10 hr reflux

0.72, 0.83

644.59

45.03%

4

Neutral Hydrolysis

Water at room temperature for 6 days

0.72, 0.88

909.77

22.42%

5

Oxidation

1% w/v H2O2 at room temperature for 3 days

0.72

1163.98

Negligible

6

Thermal (Dry heat) Photolytic

At 80°C for 3 days UV 254 for 3 days

0.72

1169.16

Negligible

0.72

1171.02

Negligible

7

Table 6: Summary of validation parameters of HPTLC Sr. No. 1 2 3 4 5

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Parameters Specificity Linearity range Regression line equation Correlation coefficient (R2) Precision Intra-day precision (%RSD) Inter-day precision

Results Specific 200-1200 ng/spot Y=1.2385X-41.197 0.9984 0.60-1.03 0.78-1.31

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8. International Conference on Harmonization (ICH) (2005) Harmonized Tripartite Guideline on, Topic Q2(R1), Validation of Analytical Procedures: Text and Methodology. 9. International Conference on Harmonization (ICH) (2005) Harmonized Tripartite Guideline on, Topic Q1A(R2), Stability testing of new drug substance and new drug product.

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