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Spectrophotometric Method Development and Validation for Determination of Carteolol

Hydrochloride Ramchandra N. Chilkawar1*, S. M. Patil1, Vinod S. Panchal1, Basavaraj K. Nanjwade2,

Yallappamaharaj R. Hundekar3

1Department of Quality Assurance, Remidex Pharma Pvt Ltd., 2nd Stage, Peenya Industrial Area, Bengaluru-560058, Karnataka, INDIA

2Department of Pharmaceutics, KLE College of Pharmacy, Nipani-591237, Karnataka, INDIA.

3Department of Pharmaceutics, Omer Al-Mukhtar University, Tobruk, P.O. Box 919, LIBYA. 4Department of Quality Assurance, Elegant Drugs Pvt Ltd., Chalmatti-581204, Karnataka, INDIA.

Abstract The present research work discussed about the development of a UV spectophotometric method for estimation and validation of Carteolol Hydrochloride. It is simple, fast, accurate, cost efficient and reproducible spectrophotometric method, developed for the estimation of Carteolol Hydrochloride as a pure Active Pharmaceutical Ingredients (APIs). Based on measurement of absorption of UV light, the spectra of Carteolol Hydrochloride in simulated tear fluid as a solvent showed maximum absorption wavelength (λ max) at 229 nm. The calibration curve was plotted over the concentration range from 2-20 µg/ml of Carteolol Hydrochloride and Beer’s law was obeyed in this concentration range with correlation coefficient 0.999. Validations were performed as per ICH Q2 guidelines for linearity, accuracy, precision and recovery. Selected method has good reproducibility with % RSD less than one. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.068 and 0.2083 respectively by simple UV spectroscopy method. Proposed validated method can successfully apply for estimation of Carteolol Hydrochloride in the quality control test, routine analytical work, and in pharmaceutical formulations. KEYWORDS: Carteolol Hydrochloride, Spectrophotometric method, ICH Q2 guidelines. Introduction Carteolol is a nonselective beta-adrenergic blocking agent with associated intrinsic sympathomimetic activity and without significant membrane-stabilizing activity. Carteolol hydrochloride reduces normal and elevated intraocular pressure (IOP) whether or not accompanied by glaucoma. The correct mechanism of the ocular hypotensive effect of beta-blockers has not been definitely demonstrated. In general, beta-adrenergic blockers reduce cardiac output in patients in good, poor and very cardiovascular health. In different patients with severe impairment of myocardial function and -blockers may inhibit the sympathetic stimulation necessary to maintain adequate cardiac function. -adrenergic blockers may also increase airway resistance in the bronchi and bronchioles due to unopposed parasympathetic activity1.

Ramchandra N. Chilkawar, Actapharmica. 2014,1(1).,077-083

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Chemically Carteolol Hydrochloride is 5-[(2RS)-3-[(1, 1-Dimethylethyl) amino]-2-hydroxypropoxy]-3, 4-dihydroquinolin-2(1H)-onehydrochloride. It has molecular formula C16H25N2O3Cl and molecular weight is 328.8. Carteolol Hydrochloride is a white, crystalline powder. 4 Carteolol hydrochloride soluble in water, slightly soluble in alcohol, sparingly soluble in methanol and practically insoluble in methylene chloride2. The ocular hypertensive effect caused by β-blocker is probably due to suppression of aqueous humor formation by blockage of the β adrenal receptors in the ciliary body. The β -blockers decrease aqueous humor production by equivalent to one-third. To obtain the desired lowering in Intra ocular pressure (IOP), large quantity of conventional eye drops of Carteolol/Betaxolol hydrochloride is used3. NEED FOR STUDY AND OBJECTIVES In formulation development of any drug molecule, analysis is an important. It becomes essential to develop a very simple, sensitive, accurate, precise, reproducible method for the estimation of drug sample (Carteolol hydrochloride). Our main objective is development and validation of UV spectrometric method as per ICH guideline4, 5.

1. Up to present date there is no valid UV spectrophotometric method available for estimation of Carteolol Hydrochloride.

2. The goal of our present research work was to develop a validated UV spectrometric method for estimation in pure and pharmaceutical formulation.

3. To develop easy, rapid, economical and reproducible UV spectroscopic method for quality control of pharmaceutical formulations containing Carteolol Hydrochloride as the active ingredient.

4. To utilize this method for quality control and analysis of drug in pharmaceuticals containing Carteolol Hydrochloride6.

Material and Methods Material Carteolol Hydrochloride was kindly supplied by GSK Pvt. Ltd. Mumbai, India. Sodium chloride (Thermo Fischer scientific India Pvt. Ltd), Sodium bicarbonate and Calcium chloride (Loba Chemicals Pvt. Ltd Mumbai, India) other chemicals and reagents were used analytical grade. Preparation of Simulated Tear Fluid: Simulated Tear Fluid (STF) is a composition of accurately weighed sodium chloride 0.67gm, sodium bicarbonate 0.20 gm and calcium chloride 0.008 gm dissolved in 100 ml distilled water7. Preparation of standard stock solution I Stock solution Standard drug solution of Carteolol hydrochloride was prepared by dissolving 100 mg of pure Carteolol Hydrochloride in a small amount of Simulated Tear Fluid in 100ml volumetric flask and then the volume was adjusted with simulated tear fluid as a solvent. The resultant solution were gives the concentration of 1mg/ml that’s equivalent to 1000 µg/ml. II Stock solution From I stock solution: 10 ml solution were taken and then diluted up to100 ml with the same solvent in a volumetric flask and then concentration of this stock was 100 µg/ml. Determination of Lambda Max The 10µg / ml solution was prepared by withdrawing 10 ml of solution from II stock solution and further diluted with simulated tear fluid to get required concentration. Prepared solution was scanned at a wavelength of 200-400 nm against the blank. The lambda max was found to be at 229 wavelengths

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where observance was maximum at 229 wavelengths. Hence this is considered as absorption maxima which were used for preparation of calibration curve (Figure 2). Preparation of Calibration Curve I Stock solution Standard drug solution of Carteolol HCl was prepared by dissolving 100 mg of pure Carteolol HCl in small amount of STF in 100 ml volumetric flask and then the volume was adjusted with simulated tear fluid (STF) then the volume was adjusted with simulated tear fluid (STF) the resultant solution gives a concentration of 1mg/ml that’s equivalent to 1000 µg/ml. II stock solution From I stock solution 10 ml solution was taken and then diluted up 100 ml with the same solvent in a volumetric flask and then the concentration of this stock was 100 µg/ml. From this II stock solution 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 ml solutions were pipetted and volume was made to 100 ml using simulated tear fluid (STF) as a solvent to get concentrations of 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 µg/ml respectively. The absorbance of these selected solutions was measured at 229 nm (lambda max of Carteolol HCl). The standard calibration curve was obtained from data of concentration v/s absorbance; standard calibration curve data reported in (Table no. 1, Figure3). Instrument Instruments used were Electronic balance, Model-AJ- Vibra manufactured by Essae-Teraoka Ltd., Japan and UV-Visible double beam spectrophotometer model Shimadzu UV1800 with one cm matching quartz cells. ISI standard glass wares were used in each procedure were soaked overnight in a mixture of chromic acid and sulfuric acid rinsed with double distilled water and it should be dried before use. Found absorption spectra of reference and test solution were carried out in a one cm quartz cell over the range of 200-400 nm. Method validation Linearity and Range The linearity of the response of the drug was obtained at 2 to 20 µg/ml concentrations and calibration curve was obtained by plotting the absorbance v/s concentration data and was treated by linear regression analysis (Table no.3). The equation of the calibration curve for Carteolol HCl obtained was y = 0.096x+0.0071, the calibration curve was found to be linear in the fore mentioned concentrations. Precision Precision of the method was analyzed by repeatability8, determined by analyzing 10 μg/ml of Carteolol HCl for six times the results are reported in (Table 2). The precision of the method was studied as intra-day and inter-day variations. Intra-day precision were determined by analyzing 4, 8, 12 µg/ml of Carteolol hydrochloride for three times within a day. Inter-day precision was determined by analyzing the same concentration of solutions daily for three days9, the results are reported in (Table 3). Limit of Detection and Quantification (LOD and LOQ) Determination of the detection and quantification limits was performed based on the standard deviations of y-intercept and the slope of the least square line parameters as defined in the International Conference on Harmonization (ICH) guidelines10,11. The LOD and LOQ were 0.068 and 0.2083 respectively and data is reported in (Table 4). Recovery Study To analyse the accuracy of developed method, it were applied to analyse commercially available Carteolol hydrochloride ophthalmic solution USP (15ml- BAUSCH+LOMB). The amount of eye solution

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equivalent to 10 mg of Carteolol hydrochloride accurately measured and transferred to 10 ml volumetric flask13. Then sufficient quantity of simulated tear fluid added and kept for 10-20 min with frequent shaking and volume made up to the mark with given solvent, solution utilized to get 10µg/ml concentration. Absorbance was measured against the blank solution and recovery was performed at three different levels which are 80%, 120%, and180%. To the preanalysed sample solution a known amount of standard drug solution was added at three different levels and absorbance were recorded. Then drug content of preparation was calculated using the standard calibration curve and amount of drug estimated by this method is given in (Table 5). Results and Discussion: The proposed method showed molar absorptivity 4.054834*104 L/Mol. cm. The calibration curve of Carteolol Hydrochloride plotted at 229 nm (Figure 3) a linear relationship was obtained between 2-20 µg/ml. The accuracy of the method was determined by calculating mean percentage recovery it was found to be 99.78% (Table 5). Further precision was calculated as repeatability, inter and intra-day variations and % Relative Standard Deviation (RSD) was less than one (Table 3). The Limit of Detection (LOD) value was found to be 0.068 and Limit of Quantification (LOQ) value were found to be 0.2083. Conclusion The developed method (UV-spectroscopy) was found to be simple, accurate, sensitive and reproducible can be used for routine quality control analysis of Carteolol Hydrochloride.

Figure 1: structure of Carteolol Hydrochloride

Figure 2: Determination of lambda max of Carteolol Hydrochloride.

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Figure 3: Calibration Curve of Carteolol HCl in Simulated Tear Fluid

Table 1: Calibration data for the method development for Carteolol HCl

Sr. No. Concentration (μg/ml) Absorbance at 229 nm ± standard deviation

1 2 0.226±0.0026

2 4 0.386±0.00360

3 6 0.581±0.00556

4 8 0.768±0.00503

5 10 0.958±0.0036

6 12 1.165±0.0020

7 14 1.326±0.00305

8 16 1.555±0.00360

9 18 1.745±0.0055

10 20 1.931±0.002636

Table 2: Data showing Repeatability of Absorbances

Sr. No.

Conc. (µg/ml)

Wavelength (nm)

Absorbance Mean± S.D. %R.S.D

1

229 0.956

2 229 0.964

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3 10 229 0.96 0.958±0.0431

0.449

4 229 0.962

5 229 0.953

6 229 0.955

S.D- Standard Deviation, R.S.D- Relative Standard Deviation

Table 3: Results for Intra-day and Inter-day precision of Carteolol HCl

Drug

Conc. (µg/ml)

Intra-day Mean Absorbance

± S.D.

%RSD Inter-day Mean Absorbance

± S.D.

%RSD

Carteolol Hydrochloride

04 0.380±0.001732 0.5263 0.391±0.001927

0.5294

08 0.756±0.003082

0.3968 0.774±0.002741

0.2583

12 1.152±0.002645

0.1475 1.169±0.00456

0.2737

Mean %RSD 0.3568 1 0.3538 S.D- Standard Deviation, R.S.D- Relative Standard Deviation

Table 4: Validation parameters for Carteolol Hydrochloride

Sr. No. Parameters Results 1 Absorption maxima (nm) 229nm 2 Linearity range (μg/ml) 2-20µg/ml 3 Standard Regression Equation y = 0.096x +0.007 4 Correlation coefficient (R2) R² = 0.999 5 Specificity A 10 µg /ml solution of candidate drug

in solvent (simulated tear fluid) at UV detection lambda max of 229 nm will show an absorbance value of 0.958 ± 0.003742

6 Accuracy (% Recovery) 99.78% 7 Precision RSD Repeatability (n=6)

Intra-day(n=3)

0.449 0.3569

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Inter-day(n=3)

0.3538

8 Molar Absorptivity[12] 4.054834*104 L/Mol. cm. 9 LOD 0.068µg/ml

10 LOQ 0.2083µg/ml

n=no. Of determinations, LOD=Limit of Detection, LOQ =Limit of Quantification, RSD= Relative Standard Deviation.

Table 5: Determination of Accuracy by Percentage Recovery Method

Drug Eye Drop amount (µg/ml)

Level of addition (%)

Amount added (µg/ml)

Amount recovered (µg/ml)

% Recovery

Average% Recovery

Carteolol Hydrochloride

10 80 8 17.96 99.78 99.78 10 120 12 21.96 99.83

10 180 18 27.92 99.74 Acknowledgement The authors are thankful to Pravin Belavi (GSK Pvt. Ltd. Mumbai, India), providing Carteolol Hydrochloride drug as a gift sample. The authors are also thankful to Principal KLE’S College of Pharmacy, Nipani and Management KLE, Nipani, Karnataka for providing the necessary facilities, guidance and support. References

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