An improved stability-indicating HPLC method for ...

Available online at www.pharmscidirect.com Int J Pharm Biomed Res 2011, 2(1), 48-55

International Journal of PHARMACEUTICAL AND BIOMEDICAL RESEARCH ISSN No: 0976-0350

Research article

An improved stability-indicating HPLC method for Riluzole hydrochloride in bulk and pharmaceutical dosage forms Sreekanth Nama1*, Bahlul Z.Awen2, Babu rao Chandu2, Mukkanti Kagga3 1

Jawaharlal Nehru Technological University Hyderabad Kukatpally, Hyderabad - 500 085, Andhra Pradesh, India College of Pharmacy, Al-Jabal Al-Gharbi University, Al-Zawia, Libya 3 Centre for Chemical Sciences and Technology, IST, Jawaharlal Nehru Technological University Hyderabad Kukatpally, Hyderabad - 500 085, Andhra Pradesh, India 2

Received: 04 Mar 2011 / Revised: 10 Mar 2011 / Accepted: 21 Mar 2011 / Online publication: 28 Mar 2011

ABSTRACT In the present study, a stability-indicating HPLC method for the determination of Riluzole hydrochloride in bulk and pharmaceutical dosage form has been reported. The separation was achieved on BDS Hypersil C18 column (100 × 4.6 mm i.d, 3µm) in isocratic elution mode with the mobile phase consisting of 5mM ammonium acetate and acetonitrile in the ratio of 50: 50 (v/v) and the column was maintained at 30°C. The detection of eluent from the column was detected using photo diode array detector (PDA) at 220nm and the flow rate was maintained at 1 ml/min. The proposed method has permitted the quantification in the linearity range of 50-400µg/ml. The method was validated in terms of accuracy, precision, linearity, limits of detection, limits of quantitation and robustness. The limit of detection and limit quantification were found to be 0.020µg/ml and 0.093µg/ml, respectively. This method has been successively applied to commercial tablet formulation and no interference from the tablet excipients was found. Riluzole hydrochloride was subjected to different stress conditions such as acid, base and neutral hydrolysis, oxidation, dry heat and photolytic stress conditions and the stressed samples were analyzed by the proposed method. As the proposed method could effectively separate the drug from its degradation products, it can be employed as stability-indicating method for the determination of these drugs in bulk and commercial products. Key words: Riluzole hydrochloride, Stability-indicating assay, Isocratic elution, RP-HPLC method, Forced degradation studies, Method validation.

1. INTRODUCTION Riluzole hydrochloride (RLZ) is a member of the benzothiazole class and chemically it is, 2-amino-6trifluoromethoxybenzothiazole hydrochloride, has the molecular formula C8H5F3N2OS (Fig.1). HCl and molecular weight of 270.66. RLZ is an antiglutamatergic agent, used in the treatment of several diseases including Parkinson’s disease (PD) [1], Amyotrophic lateral sclerosis (ALS) [2], Ischemia [3], and multiple sclerosis (MS) [4]. It is well absorbed (60% oral bioavailability) with peak plasma concentrations after 1–1.5 h. High-fat meals decrease absorption, reducing the area under the concentration–time curve (AUC) by about 20% and peak blood levels by about 45%. The plasma protein binding is 96%. RLZ is extensively *Corresponding Author. Tel: +91 9885070563, Fax: 040 23152331 Email: [email protected]

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metabolized into six major and a number of minor metabolites. Metabolism is mostly hepatic, consisting of cytochrome P450-dependent hydroxylation and glucuronidation. CYP1A2 is the primary isozyme involved in N-hydroxylation. CYP2D6, CYP2C19, CYP3A4, and CYP2E1 are considered unlikely to contribute significantly to RLZ metabolism in humans [4]. RLZ is not official in any of the pharmacopoeias and also not listed in the extra Pharmacopoeia. A detailed survey of the literature for RLZ reveals several methods were established for the determination of RLZ in a variety of matrices such as rat brain [5], mouse plasma [6], in human plasma or serum [7], in human plasma by protein precipitation method and liquid- liquid extraction [8], in human plasma by LC-ESI-MS/MS [9], using HPLC, UPLC [10] and by spectrophotometric method [11]. To the best of our knowledge, there is only one stability-indicating HPLC method for RLZ has been reported in the literature, which

Sreekanth Nama et al., Int J Pharm Biomed Res 2011, 2(1), 17-21

Fig.1. Chemical structure of Riluzole hydrochloride (RLZ)

was developed by Sivakumari et al. [12]. The stability of a drug substance or drug product is defined as its capacity to remain within established specifications, i.e. to maintain its identity, strength, quality, and purity until the retest or expiry date [13]. Stability testing of an active substance or finished product provides evidence of how the quality of a drug substance or drug product varies with time under a variety of environmental conditions, for example temperature, humidity, and light. Knowledge from stability studies is used in the development of manufacturing processes, selection of proper packaging and storage conditions, and determination of product shelf-life [14, 15]. Although the RP-HPLC method proposed by Sivakumari et al [10] provides the required sensitivity and accuracy for the estimation of RLZ in the presence of their degradants, the excess analysis time (30 min) and complex gradient elution may limit its application to routine analysis. Hence, the aim of this work is to develop a simple, isocratic, stability-indicating HPLC method suitable for the routine quality control analysis of RLZ in a pharmaceutical laboratory.

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waters, used in isocratic elution of mobile phase comprising of 5 mM ammonium acetate and acetonitrile in the ratio of 50:50 (v/v) with flow rate of 1ml/min was performed on C18 column (100x 4.6 mm i.d; 3µm). The run time was set at 5 min and column temperature was maintained at 300C. The volume of injection was 2 μl, prior to injection of analyte, the column was equilibrated for 30-40 min with mobile phase. The eluents were monitored at 220 nm using PDA detector, data were acquired, stored and analysed with Empower-2 software. The mobile phase was premixed, filtered through a 0.45 µm nylon filter and degassed by sonication. 2.3 Preparation of mobile phase The solvents of 5 mM ammonium acetate and acetonitrile were used for the preparation of mobile phase in the ratio of 50:50 (v/v). The contents of the mobile phase were filtered before use through a 0.45μm membrane filter, sonicated and pumped from the solvent reservoir to the column at a flow rate of 1 ml/min. 2.4 Preparation of standard solution

2. EXPERIMENTAL

A stock solution of was prepared by dissolving 100 mg of RLZ in 100 mL volumetric flask containing 70 mL of methanol (HPLC grade), then made up to volume with methanol. Daily working standard solutions of RLZ were prepared by suitable dilution of the stock solution with the mobile phase. Six sets of the drug solution were prepared in the mobile phase containing RLZ at a concentration of 50400µg/mL. Each of these drug solutions (2µl) was injected into the column, the peak area and retention times were recorded in triplicates.

2.1 Materials and methods

2.5 Procedure for pharmaceutical formulation

Pure standard of RLZ (purity 99.98%) was obtained as gift sample from Dr. Reddy’s laboratories Pvt. Ltd, Hyderabad along with certificate of analysis (COA). HPLC grade of Acetonitrile and Methanol (Qualigens), 5mM Ammonium Acetate, Hydrochloric acid, Sodium hydroxide, Hydrogen peroxide (SD fine chemicals limited, Mumbai, India), Electronic analytical balance (DONA), Hot air oven (Sky lab instruments & engineering Pvt. Ltd), Micro pipette (lab net, 2 µL to 1000 µL), 30 W UV-C lamp (Philips Lighting Co., Somerset, NJ) and Rilutek tablets (50 mg, Cipla pharmaceuticals Ltd) purchased from local market were employed. High purity water was prepared using a millipore milli –Q plus water purification system. The chemical structure and purity of the sample obtained were confirmed by IR and DSC studies.

To determine the RLZ content of tablet formulation, ten tablets of RLZ labeled to contain 50 mg were weighed to determine the average weight of the tablets, crushed, and mixed using a mortar and pestle. A sample of the tablet powder equivalent to 100 mg was accurately weighed, mixed with known amount of methanol and the active pharmaceutical ingredient was extracted into the methanol by vortex mixing followed by ultrasonication and then filtered through a 0.45 µm membrane filter. The solution was diluted by adding methanol to obtain a stock solution of 100µg/mL. All determinations were conducted in triplicate.

2.2 Instrumentation and chromatographic conditions A HPLC system Alliance (2695), photodiode array detector (2998) with Empower-2 software, manufactured by

2.6 Forced degradation studies of API and tablets of RLZ To determine whether the method was stabilityindicating, RLZ tablets and RLZ active pharmaceutical ingredient (API) powder were stressed under different conditions to promote degradation. Regulatory guidance in ICH Q2A, Q2B, Q3B and FDA 21 CFR section 211 requires the development and validation of stability-indicating


potency assays. The methanol was used as solvent and diluent in all forced degradation studies. All solutions were prepared to use in forced degradation studies were prepared by dissolving RLZ in small volume methanol and later diluted with aqueous hydrochloric acid, aqueous sodium hydroxide and aqueous hydrogen peroxide and refluxed at 600c for about 6h to observe the extent of degradation. After degradation the solutions were diluted and analysed by the proposed method. Solutions for neutral degradation studies were prepared in water and the resulting solution was refluxed for 6h at 60°C .To study the effect of thermal stress, Rilutek tablets and API powder were exposed to dry heat (70°C) in a convection oven for 48 h. The samples were then removed from the oven, ten tablets were crushed and thoroughly mixed, and amounts of powder equivalent to the 100 mg were analysed as per the proposed assay method. To study photo stability of RLZ, the solutions of RLZ were exposed to UV light to determine the effects of irradiation. All samples for photo stability testing were placed in a light cabinet and exposed to a 30 W UV-C lamp (Philips Lighting Co., Somerset, NJ) at 100–280 nm, for 6 h. Approximately 50 mg API was spread on a glass dish in a layer less than 2 mm thick. After removal from the light cabinet, all samples were analysed. With the objective of evaluating stability of RLZ, the drug at a concentration of 100µg/mL was used and subjected to forced degradation for the detection of RLZ. After the degradation these solutions were diluted with methanol to get starting concentration of 10μg/mL. All the stressed samples of RLZ were analyzed and peak purities were checked using photodiode array detector (PAD). RLZ was well resolved from its degradation products, indicating the stability indicating assay more specific. 2.6.1 Acid hydrolysis The solutions for acid hydrolysis were prepared in methanol and 0.1 N hydrochloric acid in the ratio of 50:50 (v/v), and refluxed at 60°C about 6 h. 2.6.2 Alkali hydrolysis The solutions for acid hydrolysis were prepared in methanol and 0.1 N sodium hydroxide in the ratio of 50:50 (v/v), and refluxed at 60°C about 6 h. 2.6.3 Neutral hydrolysis The solutions for acid hydrolysis were prepared in methanol and water in the ratio of 50:50 (v/v), and refluxed at 60°C about 6 h. 2.6.4 Oxidation The solutions for acid hydrolysis were prepared in methanol and 3% H2O2 in the ratio of 50:50 (v/v) and refluxed at 60°C about 6 h.

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2.6.5 Photo degradation Photo degradation was induced by exposing samples prepared in methanol to UV light (100–280 nm) for 6 h. For this, samples were placed into an irradiation chamber equipped with a 30 W UV-C lamp (Philips Lighting Co., Somerset, NJ). 2.6.6 Thermal degradation The bulk drug and sample of RLZ were exposed to dry heat in oven at 70°C about 48h. The tablets and bulk drug were removed from the oven and the tablets were powdered. The solutions of the both samples were prepared in methanol and injected to chromatographic system. 3. RESULTS AND DISCUSSIONS 3.1 HPLC method development A simple, rapid, economic stability-indicating RPHPLC method has been developed for determination of RLZ in the presence of its degradation products. The method was optimized to provide a good separation of the components (acceptable theoretical plates and resolution between peaks) with a sufficient sensitivity and suitable peak symmetry (peak tailing factor < 2) in a short run. For this purpose, the analytical column, solvent selection, mobile phase composition, flow rate, and detector wavelength were studied. The use of hydrophobic stationary phases usually provides adequate retention of organic non polar molecules. The chromatographic separation was achieved using an RP C18 column because it was suitable to resolve the degradation products from RLZ with adequate resolution and gave symmetrical peak shapes. For RLZ, methanol was used as diluent and it is a well known solvent for various pharmaceutical compounds. Our experiments and data reported in the literature showed that both the methanol and acetonitrile could be used an organic modifier in the mobile phase. The use of acetonitrile as a mobile phase organic modifier resulted in better sensitivity compared to methanol. Tests involving the use of mixtures of acetonitrile and different buffer solutions (e.g., potassium phosphate or ammonium acetate) were made to optimize the mobile phase with different pH values, finally 5 mM ammonium acetate and acetonitrile in the ratio of 50:50 (v/v) was selected as mobile phase whose combination given good peak symmetry, sensitivity, and shorter retention time. Our experiments revealed that isocratic elution with simple mobile phase were given good results than gradient with complicated mobile phases. The method has many advantages, e.g., simplicity, isocratic conditions, shorter run time, low injection volume, smaller particle size, and less flow rate, inexpensive mobile phases than the method reported in the literature. Under these conditions, the retention time of RLZ was about 2.452 min, with a good peak


shape (peak symmetry), and the chromatographic analysis time was 5 min. 3.2 Method validation The method was validated as per the ICH guidelines for validation of analytical procedures for different validation parameters. The method was validated for its specificity, linearity, accuracy, precision, selectivity, robustness, ruggedness, LOD and LOQ. A system suitability test was also carried out to evaluate the reproducibility of the analytical system using five replicate injections of a reference solution. 3.2.1 System suitability To know reproducibility of the method system suitability test was employed to establish the parameters such as tailing factor, theoretical plates, resolution, asymmetry factor, and asymmetry (10%), limit of detection and limit of quantification. The values were shown in Table 1. 3.2.2 Accuracy To ensure the reliability and accuracy of the method, the recovery studies were carried out by adding a known quantity of drug with pre analyzed sample and contents were reanalyzed by the proposed method. Accuracy was evaluated at six different concentrations equivalent to 25, 50, 75, 100, 150 and 200% of the active ingredient, by adding a known amount of RLZ standard to a sample of known concentration and calculating the recovery of RLZ with RSD (%) and % recovery for each concentration. The mean % recoveries were in between 99.59 to 100.97 and were shown in Table 2. There was a high recovery 100.97 of RLZ indicating that the proposed method for the determination of RLZ in the tablet dosage forms was highly accurate.

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Table 1 System suitability parameters Parameter Retention time (min) Theoretical plates Tailing factor Linearity range (µg/mL) Limit of detection (µg/mL) Limit of quantification (µg/mL)

Value 2.452 1068 0.65 50-400 0.020 0.093

Table 2 Accuracy of the method S.No. 1 2 3 Mean (n=3) %RSD

Percentage levels 25 50 98.53 99.94 99.96 99.85 100.3 99.96 99.59 99.91 0.942 0.058

75 98.93 99.99 100.4 99.77 0.759

100 100.2 101.35 99.97 100.5 0.735

150 100.5 101.2 100.3 100.66 0.468

200 101.22 101.43 100.26 100.97 0.617

Table 3 Intraday and inter day precision of the method RLZ

% Recovery Day 1 100.2 99.6 99.7 99.5 100.4 99.3 99.78±0.426 100.02±0.494

1 2 3 4 5 6 Intraday (n=6) Inter dayb (n=18) a

Day 2 101.2 99.9 100.5 100.2 100.3 99.8 100.3±0.503

Day 3a 99.7 99.4 99.6 100.3 100.5 100.4 99.98±0.470

Different analyst, b Mean ± %RSD

Table 4 Linearity of RLZ S.No. 1 2 3 4 5 6

Level % 25 50 75 100 150 200

Concentration % 0.050 0.100 0.150 0.200 0.300 0.400

Mean peak area* 610262 1181974 1806435 2367795 3624544 4836012

3.2.3 Precision

*Mean of three values (r2= 0.999; Slope= 12112921.76; Intercept= -18080.69)

The precision of the method was determined by repeatability and intermediate precision studies. Repeatability was evaluated by performing six determinations (n=6) at the same concentration, during the same day, under the same experimental conditions. Intermediate precision was evaluated by comparing the assays on 3 different days using different analysts. The result revealed the precision with %RSD for intraday and inter day was 0.503, 0.494, respectively. The results were shown in Table 3.

peak areas against the respective concentrations. From the stock reference solution of RLZ, six concentrations were prepared in the mobile phase in the range of 50–400 µg/mL. It was found to be linear with a correlation coefficient (r2) of 0.999, the corresponding linear regression equation being y= 1E+0.7x-18081. The data of linearity was shown in Table 4.

3.2.4 Linearity The linearity was evaluated by linear regression analysis by the least-squares regression method, which was used to calculate the r-value, y-intercept, and slope of the regression line. Three analytical curves were constructed by plotting

3.2.5 Specificity The results from the stress testing studies indicated the method was highly specific for RLZ. The degradation products were completely distinguishable from the parent compound. Based on peak purity data of RLZ, every degradation sample showed that the peaks were homogeneous and there were no co-eluting peaks indicating that the method was stability-indicating and specific.


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Table 5 Robustness of the method Chromatographic condition Mobile phase composition

Mobile phase flow rate (mL/min)

Variation 45: 55 50:50 55:45 0.8 1 1.2

Retention time (Rt) (n=6) Mean±S.D 2.05±0.01 2.44±0.01 3.09±0.01 2.29±0.01 2.48±0.01 2.04±0.01

Table 6 Forced degradation condition Degradation mechanism

Degradation condition

Un degraded Acid hydrolysis Alkali hydrolysis Neutral hydrolysis H2O2 oxidation UV degradation Thermal (bulk) Thermal (tablet)

-0.1 N HCl 0.1 N NaOH Water 3% H2O2 UV light 48 h in oven at 70°C 48 h in oven at 70°C

%RSD 0.34 0.29 0.21 0.35 0.43 0.32

Peak area (n=6) Mean±S.D 2493207.04±37821.88 2586819.47±25499.23 2529984.12±25067.04 2258673.49±24395.55 2377787.70±25899.45 2116934.70±17243.74

Assay (mg/tab) (0 & after 6h) 50.03 49.75, 49.04 49.07, 46.54 49.52, 49.48 41.00, 40.89 49.61, 49.45 49.71, 49.57 49.44, 49.59

%RSD 1.52 0.99 0.99 0.53 1.09 0.81

% Degradation --1.90%, 6.97% -18.26%, 18.04% ----

Table 7 Forced degradation recovery of RLZ S.No. 1

Stress conditions (0 & 6h) Acid hydrolysis: 0.1 N HCl; refluxed at 60°C

2

Alkali hydrolysis: 0.1 N NaOH; refluxed at 60°C

3

Neutral hydrolysis; refluxed at 60°C

4

Oxidation: 3% H2O2; refluxed at 60°C

98.91 98.99 81.74, 81.96

5

Photo degradation: UV light

99.18, 98.85

6

Thermal (bulk drug, at 70°C about 48 h)

99.37, 99.10

7

Thermal (tablet at 70°C about 48 h)

99.14, 98.84

3.2.6 Robustness To determine the robustness of the developed method, experimental conditions were deliberately changed. To study the effect of eluent flow rate (changed from 1.0 to 1.2 mL/min), mobile phase ratio (changed from 50:50 to 55:45 (v/v) and to 45:45(v/v)). In all the above varied conditions, the proposed method indicating that the test method was robust for all variable conditions. Hence the method was sufficiently robust for normally expected variations in chromatographic conditions. The results were shown in Table5. 3.2.7 Selectivity The results of stress testing studies indicated a high degree of selectivity of this method for RLZ. The degradation

% Drug present 98.04 97.45 98.10, 93.03

% Recovery (0& 6 h) 98.04 97.45 98.10 93.03 98.91 98.99 81.74 81.96 99.18 98.85 99.37 99.10 99.14 98.84

Fig No. 2A 2B 3A 3B 4A 4B 5A 5B 6A 6B 7A 7B 8A 8B

of RLZ was found to be similar for both the tablets and API powder. 3.2.8 LOD and LOQ Limits of Detection (LOD) and Quantification (LOQ), the limits of detection and quantitation were calculated by the method based on the standard deviation (σ) and the slope (S) of the calibration plot, using the formulae LOD = 3.3σ/S and LOQ =10σ/S. 3.3 Forced degradation studies RLZ was exposed to different stress conditions and the degradation products were well separated with greater resolution. The conditions of degradation were shown in Table 6. The drug was showed degradation only in alkali and


(A)

(B)

Fig.2. A typical chromatogram of acid hydrolysis degradation of RLZ at (A) 0 h and (B) after 6 h

(A)

(B)

Fig.3. A typical chromatogram of alkali hydrolysis degradation of RLZ at (A) 0 h and (B) after 6 h

(A)

(B)

Fig.4. A typical chromatogram of neutral hydrolysis degradation of RLZ at (A) 0 h and (B) after 6 h

(A)

(B)

Fig.5. A typical chromatogram of H2O2 degradation of RLZ at (A) 0 h and (B) after 6 h

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(A)

(B)

Fig.6. A typical chromatogram of photo degradation of RLZ at (A) 0 h and (B) after 6 h

(A)

(B)

Fig.7. A typical chromatogram of thermal degradation of RLZ bulk at (A) 0 h and (B) after 6 h

(A)

(B)

Fig.8. A typical chromatogram of thermal degradation of RLZ tablet at (A) 0 h and (B) after 6 h

(A)

(B)

Fig.9. A typical chromatogram of (A) 100 μg/mL Standard RLZ (B) RLZ tablets

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peroxide environment and almost there was no degradation in acid hydrolysis, dry heat, UV and in neutral hydrolysis. The results of forced degradation studies were shown in Table 7. The assay of RLZ was unaffected in presence of degradation products of different conditions indicating the stability indicating capability of the method. The chromatograms of RLZ in different stress conditions along with its degradation products were shown in Fig.2 to Fig.8 3.4 Stability of solutions To demonstrate the stability of standard solutions and tablet sample solutions during analysis, they were analyzed over a period of 60 h at room temperature. The results showed that for both solutions the retention time and peak area of RLZ remained almost unchanged (RSD less than 0.03 and 0.65%, respectively) and no significant degradation was observed during this period, suggesting that both solutions were stable for at least 60 h, which was sufficient for the whole analytical process. 3.5 Assay of the method The assay of commercial tablets was established with present chromatographic condition developed and it was found to be more accurate and reliable. The average drug content was found to be 100.06% of the labeled claim. No interference peaks were found in chromatogram, indicating that the estimation of drug free from interference of excipients. The results were shown in the Table 8. The chromatograms of RLZ standard (100μg/mL) and tablets were shown in Fig.9A and Fig.9B, respectively. Table 8 Assay of the method Brand name RILUTEK

Labeled claim (mg) 50

Assay value Mean ±S.D (n=3) 50.38±0.53

Percent against label claim 100.06

4. CONCLUSIONS An isocratic RP-HPLC stability indicating method developed for determination of RLZ and its degradation

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products in both bulk drug and pharmaceutical dosage form was simple, economic, rapid, precise, accurate and specific. The developed, validated method could separate RLZ from its degradation products with good resolution. The method is stability indicating and can be used for routine analysis and for assessing the stability of RLZ. 5. ACKNOWLEDGEMENTS The authors are thankful to The Management, Ragavendra Institute of Pharmaceutical Education and Research (RIPER) for providing laboratory facilities and authors greatly acknowledge Dr.Reddy’s laboratories, Pvt. Ltd, Hyderabad for providing the gift sample of RLZ. REFERENCES [1] Araki, T., Kumagai, T., Matsubara, M., Ido, T., Imai, Y., Itoyama, Y., Metab Brain Dis 2000, 15, 193-201. [2] Gurney, M.E., Cutting, F.B., Zhai,P., Doble,A., Taylor, C.P., Andrus, P.K., Hall, E.D., Ann Neurol 1996, 39, 147-157. [3] Malgouris, C., Daniel, M., Doble, A., Neuro Sci Lett 1994, 177, 95-99. [4] Gilgun-Sherki, Y., Panet, H., Melamed, E., Offen, D., Brain Res 2003, 989, 196-204. [5] Maltese, A., Maugeri, F., Drago, F., Bucolo, C., J Chromatogr B 2005, 817, 331-334. [6] Colovic, M., Eleonora, Z., Silvio, C., J Chromatogr B 2004, 803, 305309. [7] Van Kan, H.J.M., Spieksma, M., Groeneveld, G.J., Torano, J.S., Van den Berg, L.H., Guchelaar, H.J., Biomed Chromatogr 2004, 18, 723726. [8] Yifan, S.H., Min, M., Yue, Z., Scott, R., Van Horne, K.C., Tandem Labs 2010, 1-12. [9] Babu Rao, C.H., Sreekanth, N., Kanchanamala , K., Balasekhara Reddy, C.H., Parveen Shaik, R., Mukkanti, K., Anal Bioanal Chem 2010, 398, 1367–1374. [10] Siva Kumari, K., Satyanarayana, B., Nageswari, A., Shiva, R., Chromatographia 2009, 69, 513-517. [11] Ahmed, A., Krishnamurthy, G., Reddy, M., Ramesha, S., Bhojya Naik, H.S., Charitha, J.G., J Pharm Res 2010, 3, 1028-1033. [12] Telekone, R.S., Shah, A.N., Khan, M.J., Deshpande, S.V., Mahaparale, S.P., Int J Pharm Res and Dev 2010, 2, 1-6. [13] FDA, Guidance for Industry: Impurities in Drug Product, Draft Guidance, Center for Drug Evaluation and Research (CDER), 1998. [14] International Conference on Harmonization, Guidance for Industry, Q1A (R2). Stability Testing of New Drug Substances and Products, IFPMA, Geneva 2003. [15] Grimm, W., Cartensen, J.T., Rhodes, C.T., Drug Stability, Principles and Practices, Marcel Dekker, New York 2000. [16] International Conference on Harmonization, Validation of Analytical Procedures: Text and Methodology Q2 (R1). International Conference on Harmonization, Geneva 2005.