INTRODUCTION:

Chlorthalidone(CHR) is Chemically is 2-chloro-5[(1RS)-1-hydroxy-3-oxo-2, 3-dihydro- 1H-isoindol-1yl) benzene-1-sulfonamide¹. Chemical structure of chlorthalidone is shown in figure 1. Chlorthalidone is a diuretic drug used to treat hypertension and fluid retention caused by various conditions, including heart diseases. Chlorthalidone is very similar to hydrochlorothiazide and is used as an independent drug or in combination with other antihypertensive agents for lowering arterial blood pressure². Diuretics lower blood pressure by decreasing cardiac output and reducing plasma and extracellular fluid volume³

Chemically clonidine HCl (CLD) is chemically ((2-[2, 6-dichlorophenyl] amino)-2-imidazoline)⁴ preferentially stimulates central alpha (2)-adrenoceptors. Chemical structure of clonidine is shown in figure 2 which leads to inhibition of sympathetic tone, resulting in a lowering of arterial pressure and of heart rate⁵.

Clonidine HCl is a centrally acting alpha agonist hypotensive agent used to treat hypertension (high blood pressure), attention deficit hyperactivity disorder, migraine etc. Clonidine HCl used to treat psychiatric disorders including stress, sleep disorders, other anxiety disorders. Mild sedative nature of Clonidine HCl implies its use as premedication before surgery or procedures⁶.

Literature survey revealed UV-Visible spectrophotometric methods ^{7, 8} RP-HPLC ^{9, 10}, HPTLC¹¹ and UPLC¹² Methods for the estimation of CHR and CLD alone or in combination with other drugs. The validation of methods was carried out as per ICH guidelines ^13,14

Figure 1.Chlorthalidone

Figure 2. Clonidine Hydrochloride

MATERIALS AND METHODS:

Instrument¹⁵:

The Present Work Was Carried out on Shimadzu UV 1800 Double Beam Visible Spectrophotometer Wave length range 190-1100Band Width 2nmm, with a pair of 1 cm matched quartz cells.

Reagents and Materials:

Pure samples of Clonidine HCl and chlorthalidone were provided as gift sample from Neon Lab. Ltd. Mumbai. Tablet Catapres (100mg) purchase from local market.

Clonidine standard stock solution:

Accurately weighed 10 mg of Clonidine HCl was transferred to 10 ml volumetric flask, dissolved in Ethanol by shaking manually. The volume was adjusted with the same solvent up to the mark to give final concentration i.e. 1000 μg/ml.

Preparation of working solution:

Take 1ml from the Clonidine HCl stock solution and transferred to 10ml volumetric flask and volume made up to the mark by Ethanol. Appropriate volume of 1ml of 10µg/ml of Clonidine HCl transferred into 10ml volumetric flask, diluted to mark with Ethanol to give Conc. Of 1µg/ml. The resulting solution was scanned in UV range (200 nm – 400 nm). In spectrum Clonidine HCl showed absorbance maximum at 208 nm. Area was recorded in the range 201nm-211nm.

Fig 3: Clonidine HCl Spectrum by Area Under Curve Method

Preparation of Calibration curve for the Clonidine:

Appropriate volume of aliquots from standard Clonidine HCl stock solutions were transferred to different volumetric flasks of 10 ml capacity. The volume was adjusted to the mark with Ethanol to obtain concentrations of 1- 5μg/ml. Area of each solution against Ethanol as blank were measured in the range 201nm-211nm. The graph of area against concentration was plotted and was shown in Fig no.4.The regression equation and correlation coefficient was determined and were reported in table 1 and 2.

Table 1: Calibration curve for Clonidine HCl

Sr No.	Conc in µg/ml	Area
0	0	0
1	1	1.748
2	2	3.496
3	3	5.244
4	4	6.992
5	5	8.64

Fig 4: Calibration curve of Clonidine HCl

Table no 2:Optical characteristics and Statistical data of the Regression equation in AUC.

Parameter	AUC
λ max (nm)	208nm
Beer’s law limits (μg / ml)	1-5μg/ml
Regression equation (Y*)	Y = 1.733x + 0.019
Slope (b)	1.733
Intercept (a)	0.019
Correlation coefficient (r2)	0.9999

Method Validation:

The linearity for Clonidine HCl was assessed by analysis of standard solution in range of 1-5µg/ml. Correlation co-efficient for calibration curve Clonidine HCl was found to be 0.9999.

Table 3: Result of Tablet Analysis.

Clonidine HCl
Sr. No.	Label Claim mg/tab	% of Label Claim
1	100	99.94
2	100	99.97
3	100	100.03
4	100	100.01
5	100	99.93
	SD %RSD	0.043359 0.043398

Precision:

І. Intra-day:

The intraday precision for Clonidine HCl is shown in table 2. The % RSD for intraday precision was found to be 0.1154 for Clonidine HCl.

Table 4: Intra-day precision for estimation of Clonidine HCl

Sr. No.	Conc. In µg/ml	Area at 208 nm	% Estimation
Morning	1	8.641	99.88
Afternoon	1	8.652	100.01
Evening	1	8.678	99.78
		Mean	99.89
		S.D	0.1153
		% RSD	0.1154

ІІ. Inter-day precision:

The interday precision for Clonidine HCl is shown in table 3. The % RSD for interday precision was found to be 0.1259 for Clonidine HCl.

Table.No.5: Results of Precision Studies (Inter-day)

Sr. No.	Conc. In µg/ml	Area at 208 nm	% Estimation
Day 1	1	8.645	99.77
Day 2	1	8.657	100.02
Day 3	1	8.671	99.87
		Mean	99.88
		.S.D	0.1258
		% RSD	0.1259

Accuracy:

Accuracy of the method was confirmed by recovery study from marketed formulation at three level of standard addition. The results are shown in table 4. Percentage recovery for Clonidine HCl was 0.1907.

Table 6: Recovery for Clonidine HCl and Chlorthalidone

Sr. No.	Amount added μg/ml	%Recovery
1	80	99.87
2	100	99.49
3	120	99.67
	Mean	99.67
	S.D	0.1900
	% RSD	0.1907

Ruggedness:

The Robustness for Clonidine HCl is shown in table 5. The % RSD for Robustness was found to be 0.05655 for Clonidine HCl and 0.042422 for Chlorthalidone.

Table 7 : Result of Ruggedness

Clonidine HCl

Sr. No.

Conc. In µg/ml

Area at 208nm

% Estimation

Analyst 1

Analyst 2

8.642

8.651

0.1559

RESULT AND DISCUSSION:

A new drug Clonidine HCl was selected for Analytical method development as no any method was reported for Clonidine HCl.

For both the methods the percent recovery was determined at 80%,100%,120%levels the percent recovery found in the range of 99.42 to 99.92. for both the methods. The precision was calculated as repeatability and % RSD is less than 2for interday and intraday precision for both the drugs in this method the ruggedness, robustness. Was determined and the %RSD is less than 2 for both drugs.

The proposed methods were found to be simple, accurate and rapid for the routine determination of Aspirin and Omeprazole. To study the validity and reproducibility of proposed methods, recovery studies were carried out. The methods were validated in terms of linearity, accuracy, precision, ruggedness, robustness. Both methods can be successfully used for simultaneous estimation of Aspirin and Omeprazole.

CONCLUSION:

The proposed Area Under Curve method was validated by analysing the laboratory prepared sample. The mean recovery was 99.67% for Clonidine HCl. The Area Under Curve Method is quick and cost effective as compared to Chromatographic techniques.

REFERENCE:

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2. Tripathi KD. Essential of Medical Pharmacology, 6thedition. Jaypee Brothers Medical Publishers (P)Ltd. Edn., Jaypee, 2010,pp.546-47, 565-66.

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8. Parikh P., Sahoo U., Zanvarseth AK. Derivative Spectrophotometric Method for Simultaneous Estimation of Chlorthalidone and Olmesartan Medoxomil in their Tablet Dosage Form, An International Journal of Pharmaceutical Sciences, 2013:4(4), 111-123.

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10. Shah KV., Kapupara PP., GondaliyaKhushboom. Development and Validation of RP-HPLC Method for Simultaneous Estimation of Clonidine HCl and Hydrochlorothiazide in Pharmaceutical Formulation, International Bulletin of Drug Research, 2014; 4(6), 106-115.

11. Teli MS, Sawant SS, Shukla S, Simultaneous Analysis of Clonidine HCl and Hydrochlorthiazide in Bulk and In Tablet By HPTLC With Uv Absorption, World Journal of Pharmacy and Pharmaceutical Sciences, 2016; 5(3), 975-982.

12. Nataraj GD., Natesan SK., Amirtharaj V., Balakrishna A., Analytical Method Development and Validation of Amlodipine-An antihypertensive and Chlorthalidone-A thiazide diuretic By RP-UPLC Method, International Journal of Chemical and Pharmaceutical Sciences. 2016; 7(1), 12-17.

13. ICH, Q2A Text On Validationof Analytical Procedures International Conference On Harmonization, Geneva, Oct, 1994,1-5.

14. ICH Q2B, Validation of Analytical Procedures, Methodology, International conference Harmonization, Geneva, Nov, 1996, 1-8.

15. Instruction Manual User’s System Guide Uv-1800 Shimadzu Spectrophotometer (P/N 206-67001) Shimadzu, Corporation, Analytical Instrument division, Kyoto, Japan.

Received on 16.09.2017 Modified on 05.11.2017

Asian J. Research Chem. 2018; 11(1):40-42.

DOI:10.5958/0974-4150.2018.00009.3