Available online at www.pharmscidirect.com Int J Pharm Biomed Res 2013, 4(1), 01-04
International Journal of PHARMACEUTICAL AND BIOMEDICAL RESEARCH ISSN No: 0976-0350
Research article
A validated LC method for the estimation of Erlotinib in bulk and tablet dosage form Ravi Kumar Konda1, K.Sankara Babu2*,CH.Nagabhushanam3 1
Department of Pharmaceutical Chemistry, Hindu College of Pharmacy, Amaravathi road, Guntur, Andhra Pradesh-522 002, India. Department of Pharmaceutical Analysis, Vagdevi College of Pharmacy and Research Centre, Brahmadevam(v), Nellore, Andhra Pradesh-524 346, India. 3 Department of Pharmacology, K.V.S.R. Siddhartha College of Pharmaceutical Sciences, Vijayawada, Andhra Pradesh-520 010, India. 2
Received: 06 Dec 2012 / Revised: 16 Dec 2012 / Accepted: 20 Dec 2012 / Online publication: 16 Jan 2013
ABSTRACT A simple, sensitive, precise, specific, selective reverse phase high performance liquid chromatographic method was developed and validated for the estimation of Erlotinib in bulk and tablet dosage form. The separation was achieved on ACE 3 C18 analytical column (150mm x 4.6mm i.d., 3.0µm) using water, THF, acetonitrile and TFA in the ratio 69:7:24:0.15 v/v as mobile phase and at a flow rate of 1.5mL/min. Detection was carried out using a UV detector at 245 nm. The method was validated for accuracy, precision, specificity, linearity and range, stability and robustness. The developed and validated method was successfully applied for the quantitative analysis of TARCEVA® tablets. The total chromatographic analysis time per sample was about 7min with Erlotinib eluting at 6.1min. Validation studies demonstrated that this HPLC method is simple, specific, rapid, reliable and reproducible. The standard curves were linear over the concentration ranges, 10-200µg/mL for Erlotinib. The high recovery confirms the suitability of the proposed method for the determination of Erlotinib in TARCEVA® tablets. Key words: Erlotinib, RP-HPLC, Isocratic elution, Method validation, Tablets.
1. INTRODUCTION Erlotinib (Tarceva, Roche Pharma) (Fig.1) is a drug used for the treatment of cancer [1,2]. It is an epidermal growth factor receptor (EGFR) inhibitor-protein tyrosine kinase inhibitor. Chemically, it is a semisynthetic drug. Its main use is in lung cancer, particularly in combination with other chemotherapy agents. Its chemical name is N-(3ethynylphenyl)-6,7-bis (2-methoxyethoxy) quinazolin-4amine. Literature survey revealed that numerous methods have been reported for the estimation of Erlotinib in pharmaceutical formulations and biological matrices. Rajesh et al [3] and Padmalatha et al [4] developed spectro fluorimetric methods for the estimation of Erlotinib HCl in *Corresponding Author. Tel: +91 9441797744 Fax: Email:
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pure and pharmaceutical formulations, but these methods lacks assay sensitivity and accuracy. Rajesh et al [5] developed a high performace thin layer chromatographic method for the estimation of Erlotinib hydrochloride in pure and pharmaceutical formulations. Although the HPLC methods proposed by fouad chia dmi et al [6] and Lepper ER et al [7] provides required sensitivity and accuracy for the estimation of these drugs in human plasma, the excess analysis time and complex sample preparation procedure may limit its application to routine analysis of tablets. Hence, we developed a simple and specific RP-HPLC method for the determination of erlotinib in tablet dosage forms. Moreover, the analytical methods must be validated prior to use by the pharmaceutical industry. Hence, the proposed HPLC-UV method was validated in accordance with the International conference on Harmonization (ICH) guidelines [8,9].
Ravi Kumar K Konda et al, a Int J Pharm Biomed B Res 2013, 4(1), 01-04
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Fig. 1: Structure of Erlotinib hydrochloride
2.. MATERIAL LS AND METHODS
Fig.2.Typical cchromatogram off blank
2..1. Instrumentt The liquid chromatograph c hic system consisted of Shiimadzu HPLC model LC-2010 H L CHT T containing LC-VP L series pump, vaariable wave length l program mmable UV/vvisible detectorr SPD100AVP and rhheodyne injecttor (7725i) wiith 10µL fixedd loop. C Chromatograph hic analysis was w performedd using ACE E 3 C18 coolumn with 1550 x 4.6mm innternal diametter and 3µm particle p siize. Sartorius electronic baalance (CPA2225D) was ussed for w weighing purpoose. 2..2. Chemicalss and reagentss Water and Acetonitrile of HPLC grade, g Tetra Hydro Furan (THF) and a Tri Fluoroo Acetic acid (TFA) of GR R grade w purchasedd from E.Meerck, Mumbaii, India. LC grader was w water was obttained by douuble distillation and puriffication thhrough Milli – Q water puriification systeem.
Fig.3.Typical HPLC H chromatoggram of calibratio on standard (100µ µg/mL)
2..3. Chromatoggraphic condiitions The mobilee phase contaaining water: THF: Acetoonitrile: TFA in the ratio T r of 69:77:24:0.15 (v/vv/v/v) were filtered f thhrough Ultipoor N66 Nylonn 3.2 membbrane solvent filter, deegassed and were w pumped from the solvvent reservoirr in the raatio of 69:7:224:0.15 (v/v/vv/v), and waas pumped innto the coolumn. The flow f rate of mobile m phasee was maintaiined at 1.5mL/min andd detection wavelength waas set at 245nm m with a run time of 10min. The voolume of injecction loop wass 10µL prrior to injection of the drug solutionn the colum mn was eqquilibrated forr at least 30m min with the mobile m phase flowing fl thhrough the sysstem. The collumn and the HPLC system m were keept in ambiennt temperature. 2..4. Preparatioon of diluent The mixturee of water andd acetonitrile in the ratio off 50:50 (vv/v) used as diluent d for thee study. The chromatogram c m of the bllank is presennted in Fig.2. 2..5. Preparatioon of standardd stock solutioon Accurately weighed andd transferred about 27.300mg of Erlotinib HCl standard E s into 50mL 5 volumeetric flask,
Fig. 4: Tyypical HPLC chroomatogram of sam mple (100µg/mL L)
Ap pproximately 15mL of dilluent was ad dded, sonicateed for 2m min with occaasional shakinng and finallly made up to t the vo olume with thee same diluennt to get a stan ndard stock soolution off 500µg/mL. Filtered F a porttion of solutio on through 0.45µm GH HP membranne filter andd used for the t preparatioon of calibration standdards. 2.6 6. Preparationn of calibratioon curve Aliquots of 0.5, 1.25, 5.0, 7.5, 10.0m mL standard stock solution (500µgg/mL) were diiluted to 25m mL with the diiluent, to obtain Erlotinnib standard cconcentrationss of 10, 25, 500, 100, 15 50 and 200µ µg/mL, respeectively. Thesse solutions were injjected into chhromatographiic system, ch hromatograms were
Ravi Kumar Konda et al, Int J Pharm Biomed Res 2013, 4(1), 01-04
obtained and peak areas were noted for each drug concentrations (Fig.3). Calibration curve of Erlotinib was constructed by plotting peak areas versus drug concentrations and regression equation was computed. 2.7. Preparation of sample solution Grinded 10 tablets by using mortar and pestle and accurately weighed and transferred about 632.13mg of powdered sample into 200mL volumetric flask. To this added approximately 150mL of diluent, sonicated for 15min with occasional shaking, made up to the volume with the same diluent and mixed well. Filtered a portion of solution through 0.45µm GHP membrane filter, pipetted out 5.0mL of the above filtered solution into 50mL volumetric flask, made up to the volume with the same diluent, injected into HPLC system (Fig.4). The concentration of Erlotinib in tablet sample was found out using the calibration curve. 2.8. Method validation The method was validated for, accuracy, precision, specificity, linearity and range and robustness by following procedures. 2.8.1. Accuracy The accuracy of the method was determined by calculating recovery of Erlotinib by the method of standard addition. Known amount of Erlotinib (50%, 100% & 200%) was added to a pre quantified sample solution and the amount of Erlotinib was estimated. The recovery studies were carried out three times over the specified concentration range and amount of Erlotinib was estimated by measuring the peak area ratios by fitting these values to the straight line equation of calibration curve. From the above determination, percentage recovery and standard deviation of percentage recovery were calculated. 2.8.2. Precision The intra-day precision study of Erlotinib was carried out by estimating the correspondence responses six times on the same day with 100µg/mL concentration and inter-day precision study of Erlotinib was carried out by estimating the correspondence responses six times next day with 100µg/mL concentration. 2.8.3. Specificity Commonly used excipients (Microcrystalline cellulose, Sodium starch glycolate, Sodium lauryl sulphate, Lactose monohydrate, Magnesium stearate) were spiked into a preweighed quantity of drug. The chromatogram was taken by appropriate dilutions and the quantity of drug was determined.
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2.8.4. Linearity and range The linearity of the method was determined at six concentration levels ranging from 10-200µg/mL for Erlotinib. The Range shall be established by preparing three samples at three different levels (50%, 100% and 200%) containing the placebo of Erlotinib tablet 150mg. 2.8.5. Robustness Robustness of the method was studied by changing the flow-rate by ±0.2% and wavelength variation ±2.0%, and also by observing the stability of the drugs for 24h at ambient temperature in the mobile phase. 2.8.6. Stability In order to demonstrate the stability of both standard and sample solutions during analysis, both the solutions were analyzed over a period of 8 hours at room temperature. 3. RESULTS AND DISCUSSION The UV spectra of Erlotinib showed that the drug absorbs appreciably at 245nm was selected as the detection wave length in liquid chromatography. Optimization of mobile phase was performed based on asymmetric factor and peak area obtained. Different mobile phases were tried but satisfactory separation, well resolved and good symmetrical peaks were obtained with the mobile phase containing water: THF: Acetonitrile: TFA in the ratio of 69:7:24:0.15 (v/v/v/v) was used. The retention time of Erlotinib was found to be 10min, which indicates a good base line.
Table 1 Regression analysis of the calibration curve Parameters Calibration range (µg/mL) Slope Intercept Correlation coefficient (r2)
Values 10-200 30428.0 -7626 0.9997
The number of theoretical plates was found to be 9912, which indicates efficient performance of the column. The asymmetric factor was found to be 1.40, which indicates asymmetric nature of the peak. The calibration curve for Erlotinib was obtained by plotting the peak area ratio versus the concentration of Erlotinib over the range of 10200µg/mL, and it was found to be linear with r2=0.9997. The regression equation of Erlotinib concentration over its peak area ratio was found to be y = 30428-7626 x, where x is the concentration of Erlotinib (µg/mL) and Y is the respective peak area. The data of regression analysis of the calibration curve was shown in Table 1. The RSD values for accuracy and precision studies obtained were less than 2% which
Ravi Kumar Konda et al, Int J Pharm Biomed Res 2013, 4(1), 01-04
revealed that developed method was accurate and precise. The system suitability and validation parameters were given in Table 2. The percentage of recovery of Erlotinib was found to be 100.2% indicates that the proposed method is highly accurate. Proposed liquid chromatographic method was applied for the determination of Erlotinib in tablet formulation. The result for Erlotinib was comparable with a corresponding labeled amount in Table 3. The absence of additional peaks indicates no interference of the excipients used in the tablets.
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UV detector. The method was validated for accuracy, precision, specificity, linearity and range and robustness. The method has a relatively short run time (6.15min) that allows quantifying a large number of samples in routine and quality control analysis of tablets. Further the method results high percentage of recovery which shows that the method is free from interference of the excipients used in the formulation. Therefore, the proposed method can be used for routine analysis of estimation of Erlotinib in its tablet dosage form. ACKNOWLEDGEMENTS
Table 2 System suitability and validation parameters Parameters Theoretical plates (N) Retention time (min) Asymmetric factor Accuracy (%) RSD (%)
The authors are thankful to the Management of Hindu College of Pharmacy for providing laboratory facilities. Results 9912 6.155 1.4 100.6 0.40
Table 3 Assay of Erlotinib tablets Formulation Erlotinib
Labeled claim (mg) 150
Amount found (mg) 149.70
% Drug 99.80
4. CONCLUSIONS A simple isocratic RP-HPLC method has been developed for the estimation of Erlotinib in tablet dosage form using a
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