INTRODUCTION:

Lung cancer is the leading cause of cancer-related mortality, worldwide, for both men and women¹. Tyrosine kinase receptors are over-expressed or deregulated in various types of solid tumours, including non-small cell lung cancer (NSCLC)². Erlotinib received approval from the US Food and Drug Administration in November 2004 for the treatment of NSCLC after the failure of more than one or two courses of previous chemotherapy^{3, 4}. Erlotinib is a small molecule with the chemical name N-(3-ethynylphenyl)-6,7- bis(2-methoxyethoxy)-4-quinazolinamine that reversibly and selectively inhibits the intracellular autophosphorylation of tyrosine kinase in association with epidermal growth factor receptor (EGFR) (Figure 1).

Erlotinib is available as an oral agent that blocks transduction of propagation signals mediated by the EGFRs in a concentration-dependent manner⁵. Erlotinib has demonstrated inter-patient variability in food and drug pharmacokinetic clinical studies^6,
7; it also has the potential to cause drug-drug interactions when given in conjunction with agents that are classified as CYP3A4 inducers⁸. Based on the above data, the need to develop an analytical method to determine erlotinib concentrations in patients is expedient before attempting to draw any correlation between drug dose and biological effects. Our aim was the development and validation of a simple HPLC method with PDA detection for the determination of erlotinib, which can be implemented easily for routine use in quality control laboratories.

Figure 1: Structure of erlotinib

MATERIALS AND METHODS:

Chemical and reagents:

Acetonitrile of HPLC grade were purchased from Merck. Potassium dihydrogenphosphate, Orthophosphoric acids were purchased from SD fine chem., Mumbai, India. HPLC grade water was prepared in-house. Erlotinib powder was generously donated by Hetero Drugs Ltd.

Chromatography:

Analysis was performed with a Shimadzu chromatograph equipped with an LC-10 AT VP solvent-delivery system, a universal loop injector (Rheodyne 7725 i) of injection capacity of 20 μL, and an SPD-10 AVP UV–visible photodiode-array detector set at 246.0 nm. The equipment was controlled by a PC work station. Compounds were separated on a 250x4.6mm 5µ particle, Inertsil ODS-3V C18 column column under reversed-phase partition conditions.

The mobile phase was 0.03 M Potsssium dihydrogen orthophosphate in water pH 3.2 with Orthophosphoric acid: Acetonitrile (55:45). The flow rate was 0.8 mL min⁻¹and the run time was 20 min. Before analysis both the mobile phase and sample solutions were degassed by the use of a sonicator and filtered through a 0.2 μm filter. The identity of the compounds was established by comparing the retention times of compounds in the sample solution with those in standard solutions. Chromatography was performed in an air-conditioned room at ambient temperature.

Solution preparation:

Stock solutions:

Stock solution of erlotinib (1.5 mg/mL) was prepared by dissolving 150 mg of this compound in 100 mL of methanol. Stock solutions were stored at 4°C.

Standard solutions:

Erlotinib working-standard solutions were prepared in the concentration range of 50 to 500 µg/mL for the construction of calibration curves, evaluation of the precision of the analytical method and estimation of limits of detection and quantification. Calibration curves were performed either with solutions of standards in HPLC grade water.

Data analysis: For determination of erlotinib inject equal volumes of the standard preparation and the assay preparation into the chromatograph, record the chromatograms and measure the responses for the major peaks.

Construction of Calibration Plots:

Solutions of the drug having different concentrations were prepared by dilution of the standard solutions. These solutions (20 μL) were chromatographed and the peak areas were measured. Peak areas were then plotted against the respective concentrations for erlotinib. From the plots it was found that the linear range for erlotinib was between 5 and 50 μg mL⁻¹ Unknown assay samples were quantified by reference to these calibration plots.

Assay of Tablet Formulation:

Six replicates of the required dilutions were prepared from tablet stock solution and sonicated for 10 min. These solutions (20 μL) were injected for quantitative analysis. The amounts of erlotinib per tablet were calculated by extrapolating the peak area from the calibration plot.

Validation:

The method was validated for linearity, accuracy, precision, repeatability, selectivity and specificity. Accuracy was assessed by measuring recovery at three different levels, 80, 100, and 120% of the amount expected from analysis of the formulation, in accordance with ICH guidelines^9,
10. Precision assessed by measurement of intra and inter-day precision. In the intra-day study the concentrations of both drugs were calculated three times on the same day at intervals of 1 h. In the inter-day study the concentrations of the drugs were calculated on three different days. Selectivity and specificity of the method were assessed by injecting solutions containing the drug; after chromatography a sharp peak was obtained for erlotinib. LOD and LOQ were measured to evaluate the detection and quantitation limits of the method and to determine whether these were affected by the presence of impurities¹¹. They were calculated by use of the equations LOD = 3.3 σ/S and LOQ = 10 σ/S, where σ is the standard deviation of the response and S is the slope of the calibration plot.

RESULTS AND DISCUSSION:

Chromatographic separation and UV detection of erlotinib:

Erlotinib has a specific and independent chromatographic peak when measured at the 246.0 nm wavelength. The retention time for erlotinib is 4.7 minutes. A representative chromatogram was shown in Figure 2.

Figure 2: Representative chromatogram of erlotinib

Column chemistry, solvent type, solvent strength (volume fraction of organic solvent(s) in the mobile phase and pH of the buffer solution), detection wavelength, and flow rate were varied to determine the chromatographic conditions giving the best separation. The mobile phase conditions were optimized so there was no interference from solvent and excipients. Other criteria, for example time required for analysis, assay sensitivity, solvent noise, and use of the same solvent system for extraction of the drugs from the formulations during drug analysis were also considered.

After trying different columns of different particle size containing C18 and C8, the final choice of the stationary phase giving satisfactory resolution and run time was the reversed-phase 250x4.6mm, 5µ particle, Inertsil ODS-3V C18 column. A series of aqueous RP-HPLC Analysis of erlotinib different mobile phases containing Potassium dihydrogen orthophosphate buffer solutions of different pH in combination and different volume fractions of acetonitrile and orthophosphoric acid as modifier were also tested. The best result was obtained with Potassium dihydrogen orthophosphate buffer of pH 3.2.

From the study it was found that the quality separation in terms of peak symmetry, resolution, reasonable run time, and other factors were obtained by use of with 0.03 M Potassium dihydrogen orthophosphate in water pH 3.2 with Orthophosphoric acid: Acetonitrile (55:45) as mobile phase. The optimum flow rate, determined by testing the effect of flow rate on peak area and resolution, was 0.8 mL min⁻¹. All experiments were performed at ambient temperature.

Linearity:

The linearity was determined for erlotinib. Solutions of the drug at six different concentrations were analyzed and calibration curves were constructed by plotting mean areas against the respective concentrations. The method was evaluated by determination of the correlation coefficient and intercept values. The results are given in Table 1. From the calibration plots it was clear that the linear range for erlotinib was between 5 and 40 μg mL⁻¹ with a correlation coefficient of 0.9995

Table 1: Linearity

Concentration (µg/ml)	Mean Peak area	% RSD
5	4544621	0.2
10	6660386	0.0
20	8774974.5	0.4
30	10998263	0.1
40	13057751	0.2

Accuracy:

The accuracy of the method was confirmed by conducting a recovery study at three different concentrations (80, 100, and 120% of the amount expected from analysis of the formulation) by replicate analysis (n = 6), in accordance with ICH guidelines. Standard drug solutions were added to a preanalysed sample solution and recovery of the drug was calculated. Results from the accuracy study are reported in Table 2.

Table 2: Results from the recovery study

Concentration (µg/ml)	% Recovery	% RSD
20	115.58	0.2
30	104.83	0.1
40	96.41	0.4

From the recovery study it is clear that the method enables very accurate quantitative analysis of erlotinib in the tablet dosage form, because all the statistical results were within the acceptable range, i.e. COV < 2.0% and S.D. < 1.0.

Precision:

Intra-day and inter-day precision were studied. Six replicate sample solutions were prepared from the stock solution. For study of intra-day precision the concentrations of both drugs were calculated three times on the same day at intervals of 1 h. In the inter-day study the drug concentration was calculated on three different days. Accuracy was expressed as relative error and precision was expressed as the COV (%).Intra-day accuracy ranged from −0.480 to 2.880% and precision from 0.234 to 0.890%. Inter-day accuracy ranged from −4.060 to −1.360 and precision from 0.618 to 1.581. Intra and inter-day accuracy were within the acceptable ranges for relative error, ±5% ¹² and in the precision study COV was also within the acceptable range i.e. <2, indicating the method enabled precise quantitative analysis of erlotinib.

Sensitivity:

The LOD was 16.2 and µg mL⁻¹ and the LOQ were 49.0 µg mL⁻¹ for erlotinib.

Selectivity and Specificity:

The selectivity of the method for the drug was established by study of the resolution of the drug peak. Under the proposed chromatographic conditions erlotinib shows a good resolved peak at 4.70 min. Specificity was assessed by comparing the chromatograms obtained from tablet and placebo solutions and from the drug standards. Because the retention times of both drugs from standard solutions and from tablet solution were identical, and no co-eluting peaks from the diluents were observed, the method was specific for quantitative estimation of both drugs in the commercial formulation.

Assay of Tablet Formulation:

Six replicates of the required dilution were prepared from tablet stock solution and sonicated for 10 min. These solutions (20 μL) were injected for quantitative analysis. The amount of erlotinib per tablet was calculated by extrapolating the peak area from the calibration plot. The results are reported in Table 3.

CONCLUSION:

A new, reversed-phase HPLC method has been developed for the quantitative analysis of erlotinib in a tablet formulation. It was shown that the method is accurate, repeatable, linear, precise, specific, and selective, and therefore reliable. The run time is relatively short, i.e. 20 min, which enables rapid quantitation of many samples in routine and quality-control analysis of the tablet formulation. The same solvent was used throughout the experimental work and no interference from any excipients matrix was observed. The method could therefore find practical application as a quality-control tool for simultaneous estimation of both drugs in their combined dosage forms in quality-control laboratories.

ACKNOWLEDGMENT:

The authors are grateful to Hetero Drugs, Hyderabad, India, and for providing gift sample of erlotinib, and to the management of Annamacharya College of Pharmacy, Rajampet, A.P. India, for providing necessary facilities and chemicals.

REFERENCES:

1. Clinical practice guidelines for the treatment of unresectable nonsmall- cell lung cancer. Adopted on May 16, 1997 by the American Society of Clinical Oncology. J Clin Oncol.1997;15: 2996-3018.

2. Baselga J, Hammond LA. HER-targeted tyrosine-kinase inhibitors. Oncology 2002; 63 Suppl 1:6-16.

3. Zielinski SL. Tarceva wins approval from FDA. J Natl Cancer Inst 2004; 96: 1811.

4. Shepherd FA, Rodrigues Pereira J, Ciuleanu T et al. for the National Cancer Institute of Canada Clinical Trials Group. Erlotinib in previously treated non-small-cell lung cancer. N Engl J Med 2005; 353:123- 32.

5. Hidalgo M, Siu LL, Nemunaitis J et al. Phase I and pharmacologic study of OSI-774, an epidermal growth factor receptor tyrosine kinase inhibitor, in patients with advanced solid malignancies. J Clin Oncol 2001;19: 3267-79.

6. Hidalgo M, Bloedow D. Pharmacokinetics and pharmacodynamics: maximizing the clinical potential of erlotinib (Tarceva). Semin Oncol 2003;303 Suppl 7:25-33.

7. Birner A. Pharmacology of oral chemotherapy agents. Clin J Oncol Nurs 2003;7:11-9.

8. Abbas R, Fettner S, Riek M et al. A drug-drug interaction study to evaluate the effect of rifampicin on the pharmacokinetics of the EGF receptor tyrosine kinase inhibitor, erlotinib, in healthy subjects. Proc Am Soc Clin Oncol 2003;22:137.

9. International Conference on Harmonization (ICH), Q2A: Text on Validation of Analytical Procedures: Definitions and Terminology, Vol. 60, US FDA Federal Register, 1995

10. International Conference on Harmonization (ICH), Q2B: Validation of Analytical Procedures: Methodology, Vol. 62, US FDA Federal Register, 1997

11. M. Ribani, C.H. Collins, and C.B.G. Bottoli, J. Chromatogr. A, 1156, 201 (2007)

12. R. Sistla, V.S.S.K. Tata, Y.V. Kashyap, D. Candrasekar, and P.V. Diwan, J. Pharm. Biomed. Anal., 39, 517 (2005)

Received on 28.08.2011 Modified on 06.09.2011

Research J. Pharm. and Tech. 4(11): Nov. 2011; Page 1787-1790

Drug	Label claim (mg/ tablet, n=6)	Amount found (mg)	Drug concentration (%)	S.D	Cov (%)	S.E
Tarceva	150	150.003	100.03	0.494	0.493	0.221