Journal of Pharmaceutics & Drug Delivery ResearchISSN: 2325-9604

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Research Article, J Pharm Drug Deliv Res Vol: 6 Issue: 1

Development and Validation of Stability Indicating HPLC Method for Gefitinib and Its Related Compounds and Characterisation of Degradation Impurities

Siva Kumar R*, Yogeshwara KR, Manish Gangrade, Nitesh Kanyawar, Siva Ganesh and Jeenet Jayachandran
Analytical Development Laboratory, Cipla Limited, Virgonagar, Bangalore (Karnataka), India
Corresponding author : Siva Kumar R
Analytical Development Laboratory, Cipla Limited, Virgonagar, Bangalore (Karnataka), India
+91 9035922638; +91 80 25060385
Received: January 13, 2017 Accepted: February 10, 2017 Published: February 15, 2017
Citation: Siva Kumar R, Yogeshwara KR, Gangrade M, Kanyawar N, Ganesh S, et al. (2017) Development and Validation of Stability Indicating HPLC Method for Gefitinib and its Related Compounds and Characterisation of Degradation Impurities. J Pharm Drug Deliv Res 6:1. doi: 10.4172/2325-9604.1000161


A validated stability indicating RP-HPLC was developed for the estimation of Gefitinib related compounds as well as degradants on Inertsil C8 (250 × 4.6 mm, 5 μ) column using 50 mM aqueous ammonium acetate: acetonitrile as the mobile phase in a gradient mode of elution at a flow rate of 1.0 mL/min at 50°C. The column effluents were monitored by a photo diode array detector set at 300 nm. The method was validated in terms of accuracy, precision and linearity as per the ICH guidelines. The limits of quantification of Gefitinib and impurities were obtained in the range of 0.015-0.05%. The forced degradation of Gefitinib was carried out under acidic, basic, thermal, reduction and oxidation conditions. The degradation products were characterized by MS-MS and 1 H NMR spectroscopy. The method was successfully applied to quantify the related substances and degradation products of Gefitinib in bulk drugs. The recoveries of Gefitinib and impurities were well within the range.

Keywords: Anti-cancer; Gefitinib; Forced degradation; Related compounds


Anti-cancer; Gefitinib; Forced degradation; Related compounds


Gefitinib (Iressa) is a drug used for the treatment of several types of cancers like breast cancer, lung cancer and pancreatic cancer. Gefitinib inhibits EGFR (epidermal growth factor receptor) tyrosine kinase by binding to the ATP-binding site of the enzyme similar to that of Erlotinib. Epidermal growth factor receptor (EGFR) belongs to a subfamily of Erb-B1, Erb-B2, Erb-B3 and Erb-4 [1-3]. An inappropriate action of the intracellular signaling leads to the uncontrolled cell division and causes carcinoma. Anti-cancer drugs like Gefitinib and Erlotinib inhibits the inappropriate action of intracellular signaling and prevents malignant cells. Synthesis of Gefitinib involves two main steps; in the initial step, reaction of 7-methoxy-4-oxo-3,4- dihydroquinazolin-6-yl acetate with oxalyl chloride at reflux temperature results in 4-chloro-7-methoxyquinazolin-6-yl acetate in situ, which further reacts with 3-chloro-4-fluoro aniline in IPA at reflux temperature to yield 4-(3-chloro-4-fluorophenylamino)-7- methoxyquinazolin-6-yl acetate hydrochloride. In the second step, treatment of the hydrochloride salt with methanolic NaOH followed by the reaction with morpholine propyl chloride yields Gefitinib. The raw materials and intermediates obtained in the above mentioned synthetic scheme (Figure 1) may also be present in the final product as known impurities.
Figure 1: Synthetic Scheme of Gefitinib.
Literature survey revealed that several HPLC methods have been reported for the estimation of Gefitinib, its process related impurities and degradants. Faivre et al. have reported HPLC-UV method for the estimation of Gefitinib in plasma [4]. G C Reddy et al. described separation and estimation of process-related impurities of Gefitinib by Reverse-Phase High-Performance Liquid Chromatography [5]. Madireddy et al. identified the degradant impurities of Gefitinib using RRLC method [6]. Ling-Zhi and co-workers reported rapid determination of Gefitinib and its main metabolite, O-desmethyl Gefitinib in human plasma using liquid chromatography–tandem mass spectrometry [7]. Guetens et al. described sensitive and specific quantification of the anticancer agent ZD1839 (Gefitinib) in plasma by on-column focusing capillary liquid chromatography-tandem mass spectrometry [8].
From the literature, it is evident that all process related impurities and degradants of Gefitinib have not yet been reported. Thus, there is a need for the development of analytical methods, which will be useful to monitor the levels of impurities in bulk of active pharmaceutical ingredient (API) of Gefitinib during process development. Hence, the development of new analytical methods is essential to determine various degradants as well as the related impurities and their characterisation.
The draft EP monograph of Gefitinib describes an HPLC method for the separation of Gefitinib and its related impurities, wherein the co-elution of one of the unknown impurities along with the n-alkylated impurity was seen. The EP chromatographic system used is similar to the one used in the proposed work, in which gradient elution of the mobile phase comprised of ammonium acetate and acetonitrile in order to separate Gefitinib from its related substances.
The proposed work not only separates Gefitinib from its related substances (GFT-N-alkylated, GFT-4 isomer, GFT A and GFT B), but also separates Gefitinib from its degradant peaks (MMPQ, GFT N-oxide) which appears during different forced degradation studies. As a result, the proposed method is considered as a stability indicating method wherein PDA was used to confirm the identity and purity of the parent drug peak (Gefitinib).
The present work also deals with the study of forced degradation of Gefitinib under various conditions including hydrolysis (acid, alkaline and neutral), oxidation, dry heat and photo-decomposition. The method is capable of separating the Gefitinib peak from that of various forced degradation impurities.
This manuscript describes the following (i) minimization of coelution of the unknown impurity as per European Pharmacopeia (ii) degradation behaviour of Gefitinib under hydrolysis (acid, base and neutral), oxidation, reduction and thermal stress conditions, (iii) optimization of LC conditions to separate the drug and its degradation products on a reverse-phase C8 column, (iv) method validation and (iv) characterization of degradation impurities by LC-MS/MS and 1H NMR spectroscopy as well as various path ways of degradation. Overall, the present work describes a simple, precise and economical RP-HPLC method for the simultaneous determination of Gefitinib, its process related impurities and degradants. The developed method can be used for routine analysis of Gefitinib in laboratories as well as the quality control purpose.


Chemicals and reagents
Samples of Gefitinib (>99.5 purity) and its related impurities were obtained from in-house research & development lab, Cipla Limited (Bangalore, India). The HPLC grade acetonitrile (Sigma- Aldrich, Mumbai, India), analytical reagent grade ammonium acetate (Merck Ltd, India), HPLC grade water provided by a Milli-Q water purification system (Millipore Corporation, USA) were used.
High performance liquid chromatography: The HPLC system consisting of G1311A quaternary pump, G1315D diode array detector, G1329A auto sampler, and G1322A degasser (all from Agilent technologies, Germany) were used. A reversed-phase Inertsil C8 column (250 × 4.6 mm i.d.; particle size 5 μm) was used for separation. The chromatographic and integrated data were recorded using Chromeleon data acquiring software (Thermo fisher).
ESI-MS/MS conditions: AB SCIEX-Q TRAP 5500 instrument and 1290 infinity separation module pumps, auto sampler device and photodiode array detection was used for the analysis of degradation products. Electro spray ionization in positive mode of detection was used. Nitrogen was the nebulizer and curtain gas. Collision-induced dissociation was achieved by nitrogen as collision gas. The ion source conditions were set as follows: dry temperature: 450ºC; curtain gas: 35, collision gas: high, ion spray voltage: 4500, ion source gas: 50 (GS1), ion spray gas: 50(GS2), declustering potential: 30, entrance potential: 10, collision energy: 10 for Gefitinib, vaporizer temperature 450ºC and dwell time, 200 ms.
1H NMR spectroscopy: 1H NMR spectra were acquired with Varian - oxford 300 MHz spectrometer using impurities deuterated dimethyl sulfoxide (DMSO-d6) as a solvent. The 1H NMR spectra were recorded under the following conditions: acquisition time: 1.52 s, pulse width: 11.0 degrees, relaxation delay: 1.0 s, number of scans: 256, width: 5390.8 Hz, respectively.
Chromatographic conditions
Chromatographic separation was achieved by a reverse phase Inertsil C8 (250 × 4.6 mm, 5 μm) column using a mobile phase A consisting of 50 mM ammonium acetate adjusted to pH 4.7 ± 0.05 with trifluoroacetic acid and mobile phase B consisting of acetonitrile under gradient mode (TminA:B) T065:35, T1065:35, T3540:60, T4030:70, T40.165:35, T4565:35. The flow rate was set to 1.0mL/min and the injection volume was 20μL for elution of samples prepared in diluent (diluent used was 600 ml of 0.2% v/v trifluoroacetic acid and 400 ml of acetonitrile). Detector wavelength was fixed at 300 nm, and the column temperature was maintained at 50oC throughout the analysis.
Forced degradation
Stress studies were performed for Gefitinib bulk drug according to ICH guidelines Q1A (R2) [9,10] to provide an indication of the stability indicating property and specificity of the proposed method. Forced degradation was attempted to stress conditions of acidic (1N HCl, 65ºC, 2 hr), basic (1N NaOH, 65ºC, 2 hr), neutral (H2O, 65ºC, 2hr) and oxidation (10% H2O2, 2 hr). The Gefitinib substance was spread to about 1.0 mm thickness in a Petri dish and kept at 65ºC for one day for thermal, and exposed to UV light at 254nm for 24 h for photolytic stress. After completion of stress, all the collected samples were kept in refrigerator at 5ºC.
Sample preparation
Sample preparation for RP-HPLC optimization and validation
Solutions (1000 μg/mL) of Gefitinib and its process impurities were prepared by dissolving known amounts of the components in diluent. The solutions were adequately diluted with the mobile phase to study accuracy, precision, linearity, limits of detection and quantification. The specification concentration of Gefitinib was taken as 1000 μg/mL.
Sample preparation for stress studies
The collected samples of acid and base hydrolysis were neutralized with sodium hydroxide and hydrochloric acid respectively. Further dilution (overall 10 times) was carried out with the mobile phase. The remaining stressed samples were diluted to 10 times with the mobile phase. All the samples were filtered through 0.22 μm membrane filter before analysis.

Results and Discussion

The synthetic scheme of Gefitinib generally followed in a unit manufacturing active pharmaceutical ingradients is shown in Figure 1. The structures and characterisation data of process related impurities like MMPQ impurity, GFT N-alkylated impurity, GFT N-oxide impurity, GFT 4-isomer , GFT B impurity, GFT A impurity etc., generated at various stages could be seen from Figures 2-12. It is possible that impurities along with the degradation products are generally present in the finished products of Gefitinib. The present study was aimed at developing a chromatographic system capable of performing separation and quantitative determination of not only process related impurities, but also degradation products.
Figure 2: Typical chromatogram of Gefitinib spiked with 0.15% of each impurity.
Figure 3: Typical chromatogram of Gefitinib and impurities in LOQ Level.
Figure 4: Typical chromatogram of Gefitinib European Pharmacopeia method vs In-house sample.
Figure 5: Typical chromatograms of Gefitinib under stress conditions.
Figure 6: NMR and Mass Characterization data of Gefitinib (API).
Figure 7: NMR and Mass Characterization data of GFT A impurity.
Figure 8: NMR and Mass Characterization data of GFT B impurity.
Figure 9: NMR and Mass Characterization data of GFT N-Alkylated impurity.
Figure 10: NMR and Mass Characterization data of GFT N-Oxide impurity.
Figure 11: NMR and Mass Characterization data of GFT 4-Isomer impurity.
Figure 12: NMR and Mass Characterization data of MMPQ impurity.
RP-HPLC method
Gefitinib and its degradation products were determined by an Agilent1200 series HPLC system. The HPLC system consisting of G1311A quaternary pump, G1315D diode array detector, G1329A auto sampler, and G1322A degasser (all from Agilent technologies, Germany) were used. A reverse-phase Inertsil C8 (250 × 4.6 mm, 5 μm) column thermo stated at 50°C was employed for the separation of Gefitinib from its impurities using 50mM aqueous ammonium acetate: acetonitrile as the mobile phase in a gradient mode of elution. The flow rate was maintained at 1 mL/min and the eluents were detected at 300nm by a photo diode array (PDA) detector.
Method development and optimization of the chromatographic conditions
Initially, we have initiated the evaluation of Gefitinib EP Pharmacopeia [11] as per the mentioned chromatographic conditions; however, during evaluation we have encountered some of the specificity problems i.e.,
1) MMPQ impurity almost elute at void,
2) GFT N Alkylated impurity is merging with one of the unknown impurity (Figure 4),
3) GFT B Impurity tailing factor is more than 2.0,
Based on the above complications (observations), we have initiated the optimisation of the chromatographic system. Different compositions of 0.01 M CH3COONH4, methanol and acetonitrile were tried on three different columns, namely, Purosphere star RP18e (250 × 4.6 mm, 5 μm), Merck Inertsil ODS-3V (250 × 4.6 mm, 5 μm) and Zorbax SB Phenyl (250 × 4.6 mm, 5 μm). The peaks were not resolved satisfactorily. In an isocratic elution mode, long retentions and poor resolutions (polar analytes) were observed when lower and higher concentrations of organic modifiers are used. To get good resolutions between polar analytes and decreased retention times for non-polar analytes, isocratic mode was switched to gradient elution mode. In the gradient mode, improved peak parameters were observed on Inertsil C8 column compared to Inertsil ODS-3V, Purosphere star RP18e and Zorbax SB Phenyl (250 × 4.6 mm, 5 μm) columns with mobile phase consisting of 0.01 M CH3COONH4 pH 4.7, and acetonitrile. The impact of system volume on separation using this multistep programme was found to be minimum and did not affect the ruggedness of the method. The HPLC conditions were optimized by studying the effect of organic modifier, column temperature, and the concentration of buffer and buffer pH on peak parameters of analytes.
Effect of organic modifier
Broad peaks were observed and separation was poor using methanol. When acetonitrile was changed as an organic modifier, peaks became sharp and resolutions between GFT N-Alkylated impurity and GFT N-oxide impurity were >2.0. The initial concentration of organic modifier was kept as 35% in the gradient mode of elution.
Effect of temperature
The Inertsil C8 column was maintained at different temperatures ranging from 25 ºC to 50 ºC in a thermo stated oven. GFT N-Alkylated impurity tailing was more with less column temperature, but we achieved a tailing factor < 2.0 with 50 ºC column oven temperature.
Effect of buffer concentration
The effect of concentration of ammonium acetate on separation was studied by varying its concentration from 10 mM to 50 mM on Inertsil C8 column maintained at 50°C. The concentration of ammonium acetate had more effect on the retention of the MMPQ impurity (highly polar), the MMPQ Impurity retention was good with increased buffer concentration. Finally, we achieved a proper retention with 50 mM buffer concentration.
Effect of pH
The pH has much effect on retention of compounds, namely, GFT B impurity, GFT 4-Isomer impurity and Gefitinib. At pH 6.8, the GFT B Impurity was eluted before Gefitinib peak (Principle peak). However, by decreasing the pH from 6.8 to 4.7, the elution order changed i.e., GFT B impurity eluted after Gefitinib (Principle peak), and at pH 2.0 GFT 4-Isomer impurity was co-eluted with Gefitinib. At pH 4.7, symmetrical peaks with good resolutions were obtained. The optimum separation was achieved on Inertsil C8 (250 × 4.6 mm; 5 μm) column using 50mM ammonium acetate and acetonitrile as a mobile phase in a gradient mode of elution at a flow rate of 1.0 mL/min at 50ºC. The column eluents were monitored by a photo diode array detector set at 300 nm with a run time of 45 min. The optimized method was validated with respect to the parameters outlined by ICH guide lines [10].
Method Validation
Specificity: Specificity is the ability of method to measure the analyte response in presence of all potential impurities. For specificity determination, the process related impurities were spiked at 0.15% in to Gefitinib, which showed that the resolution between each peak is more than 3.0. The specificity was also checked by stressing Gefitinib under oxidative conditions such as 10% H2O2, reduction under 10% NaHSO3, hydrolytic conditions such as neutral, 1 N HCl and 1 N NaOH for two hours at 65 ºC. In the presence of acidic, alkaline and oxidative conditions, degradation was observed and the degradants were well separated from Gefitinib. These degradants were confirmed by LCMS, synthesised from our in house research and development laboratory and further characterised by LCMS and 1H NMR spectroscopy. The method was found to be applicable to quantitative determination of Gefitinib related impurities in bulk drugs.
System suitability: The system suitability was checked by making six replicate injections of Gefitinib (1000 μg/mL) spiked with 0.15% (w/w) of all the impurities (Figure 2). The system was deemed to be suitable for use, as the tailing factors and resolutions for Gefitinib and its impurities were less than 1.7 and more than 3.0, respectively. System suitability results are given in Table 1.
Table 1: System suitability data.
Accuracy: The recoveries of all impurities were determined by spiking each impurity at four different levels ranging from LOQ level to 0.23% of Gefitinib at the specified level (1000μg/mL). The recovery range and R.S.D. for all impurities were found to be well within the limits respectively. The results of such studies are given in Table 2.
Table 2: Recovery data.
Precision: The precision of the system was tested by six (n = 6) injections of Gefitinib spiked with 0.15% (w/w) of each impurity and the R.S.D. of retention time and peak areas were determined. The R.S.D. values were found to be <3.0%, which indicated a good repeatability.
The ruggedness of the method is defined as the degree of reproducibility obtained by analysis of the same sample under a variety of conditions at different labs, with different analysts, instruments and lots of reagents. The same samples of three concentrations were analysed in triplicate on 2 days by another instrument (LC-2010 Module HPLC, Shimadzu system containing two pumps and PDA detector) by a different analyst with different lots of reagents and columns. The data obtained were within 2% R.S.D. The results of such studies are presented in Table 3.
Table 3: System Precision Data.
Linearity and range: A linearity test solution for related substance method was prepared by diluting the impurity stock solution to the required concentrations. The solutions were prepared at six concentration levels, i.e., from LOQ level to 150% of the specified level (1000 μg/mL of Gefitinib) at five different levels. The data were subjected to statistical analysis using a linear-regression. The proposed linearity range was useful to quantify impurities at the level of 0.02% and above present in Gefitinib. Thus, the proposed method meets the requirement of ICH guidelines. Similarly we have established the relative response factors also, the results of which are given in Table 4.
Table 4: Linearity, LOD and LOQ data.
Limits of detection and quantification: Limits of detection (LOD) and quantification (LOQ) represent the concentration of the analyte that would yield signal-to-noise ratios of 3 for LOD and 10 for LOQ, respectively. LOD and LOQ were determined by measuring the magnitude of analytical background by injecting a blank, and calculating the signal-to-noise ratio for each compound by injecting a series of dilute solutions of respective impurities. LOD and LOQ values are given in Table 4, and the LOQ chromatogram can be seen in Figure 3.

Characterization of Degradation Products

The samples of Gefitinib stressed under different conditions were subjected to HPLC separation, and the degradation products i.e., MMPQ impurity, GFT N-Oxide impurity and the process impurities such as GFT N-alkylated impurity, GFT 4-isomer impurity and GFT A impurity were characterized by ESI-MS/MS and 1H NMR. The characterization data of all impurities can be seen in Figures 6-12.
Forced degradation studies
Gefitinib was subjected to forced degradation such as hydrolysis (acidic, 1 N HCl, 65°C, 2 h; basic, 1 N NaOH, 65°C, 2 h; neutral, H2O, 65°C, 2h), oxidation (6% H2O2, 2 h,) Reduction (10% NaHSO3, 2 h,) and thermolysis (65°C, 24 h). Significant degradation was observed in acidic, basic and neutral hydrolysis, reduction and oxidative conditions, however, Gefitinib was found to be stable under thermolysis (Figure 5).


A simple and rapid gradient RP-HPLC method was developed and validated for quantifying the process related impurities and degradation impurities in Gefitinib API. The chromatographic conditions were finally optimized on INERTSIL C8 column by studying the effects of temperature, concentration and pH of ammonium acetate buffer etc. on the column. The developed method was found to be selective, sensitive, precise, linear, accurate, and reproducible and stability indicative for determining the Gefitinib potential impurities. Since the method was mass compatible, it is suitable for the identification of major impurities in Gefitinib by LC-ESI-MS/MS. Thus, the method could be of used for process development as well as quality control release of Gefitinib API in bulk drugs, and more importantly, this method can replace the Gefitinib EP pharmacopeia method.


The authors thankful to Dr Y. K. Hameed, Chairman, Cipla Ltd and Mr P. L. Srinivas, Head, Research and Development, Cipla Ltd, Virgonagar, Bangalore, Dr. Srinivasa, Research and Development, Cipla Ltd, Virgonagar, Bangalore for their constant encouragement and permission to communicate the manuscript for publication.


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