INTRODUCTION:

Chemically Clonidine Hydrochloride is 2-[(2, 6- Dichlorophenyl) amino]-2-imidazoline hydrochloride¹. Clonidine is an imidazoline derivative that acts as an alpha adrenergic agonist in the central nervous system and has antihypertensive properties. It does this by binding to central alpha2 adrenergic receptors and stimulating them, which leads to a decrease in the amount of norepinephrine (NE) release and a reduction in sympathetic outflow to the heart and kidneys. As a result of the reduction in sympathetic outflow, heart rate, blood pressure and peripheral vascular resistance, all decrease². Chlorthalidone is chemically, (RS)-2-Chloro-5-(3-hydroxy-1- Oxoisoindolin-3- yl) benzene sulphonamide.

In the renal tubular epithelium located in the ascending limb of the loop of Henle's cortical diluting segment, Chlorthalidone inhibits the transport of sodium ions across the epithelium. It does this by increasing the amount of sodium that is delivered to the distal renal tubule, which in turn increases the potassium excretion which is driven by the sodium-potassium exchange mechanism³. Figure 1 and Figure 2 shows the chemical structure of Clonidine Hcl and Chlorthalidone, respectively.

Figure 1: Chemical structure of Clonidine

Figure 2: Chemical structure of Chlorthalidone

MATERIALS AND METHOD:

Apparatus and Instruments:

For the purposes of data collection and analysis, an FT-IR spectrophotometer (IR- Affinity-1) manufactured by Shimadzu Corporation in Japan was used. This instrument was outfitted with a diffuse reflectance sampling interface and was connected to a computer running Shimadzu IR solution software. In addition to that, it has a detector called DLATGS and also utilises a high-energy, long-life ceramic light source. The FT-IR spectrum was recorded with 45 scans and a resolution of 4 cm^-1 in the recording range of 400 to 4000 cm^-1. Model AA-2200 Analytical Weighing Balance is used for weighing purpose.

Chemical and Reagent:

Working standard/drug sample Clonidine Hydrochloride was obtained as gift sample from Neon Laboratories Limited, Andheri (E), Mumbai and Chlorthalidone is obtained as gift sample from Trichem Life Science Ltd. MIDC. Tarapur Dist. Palghar Maharashtra. Analytical grade Pure KBr was used as diluent. All glasswares used throughout the work were calibrated.

EXPERIMENTAL:

Preparation of working standard (1% w/w)

Pure drugs of CLO (10mg) and CHT (10mg) were individually mixed with 990mg KBr (IR Grade) and then triturated well to made uniform mixture to get sample of 1% w/w CLO and CHT respectively, used as working standard.

Selection of analytical wavenumber:

1% w/w of working standards of both drugs were prepared and scanned in the infrared spectrum between 400 and 4000 cm^-1 at resolutions of 4 cm^-1 and 45 scans. Wavenumber (peak intensity) was chosen as the criterion for both drugs. In order to eliminate the possibility of interfering effects of the drugs, the wavenumber was selected in such a way that the functional groups of one drug should not be present in another drug. –C=N (Imine) was the functional group selected for CLO and wavenumber was found to be 1658.78 cm^-1 and –S=O (Sulphonates) as that for CHT and wavenumber was found to be 1346.31cm^-1. Figure 3 and Figure 4 shows the infrared spectum of CLO and CHT, respectively.

Figure 3: FT-IR spectrum of Clonidine Hydrochloride

Figure 4: FT-IR spectrum of Chlorthalidone

Preparation of calibration curve:

In order to get concentrations ranging from 1.5-3.0% w/w for CLO and 15-90% w/w for CHT, pure drug sample was diluted with KBr. Peak intensity of these dilutions was measured at 1658.78 cm^-1for CLO and 1346.31 cm^-1for CHT. Table 1 shows the calibration data of CLO and CHT.

Table 1: Standard calibration data for mixed standard of CLO and CHT

CLO		CHT
Conc. (% mg)	Peak Intensity (1658.78 cm^-1)	Conc. (% mg)	Peak Intensity (1346.31 cm^-1)
1.5	0.311	15	0.517
1.8	0.370	30	0.549
2.1	0.433	45	0.599
2.4	0.491	60	0.641
2.7	0.542	75	0.688
3.0	0.605	90	0.716

Analysis of synthetic mixture:

The synthetic mixture of 1.5mg of CLO and 15mg of CHT was prepared. It has been combined with 983.5 mg of KBr, and then dilutions have been made as 0.15% w/w for CLO and 1.5% w/w for CHT. The dilutions were scanned in IR range of 400-4000 cm^-1with resolution of 4 cm^-1and 45 scans. The process for the analysis was carried out a total of six times with the synthetic combination. Figure 5 shows the FT-IR spectrum of synthetic mixture. Table 2 includes the optical characteristics and other parameters. The findings of the investigation into the synthetic mixture are shown in Table 3.

Figure 5: FT-IR spectrum of synthetic mixture

Table2: Optical characteristics and other parameters

Parameters	CLO	CHT
Wave number (cm^-1)	1658.78 cm^-1	1346.31 cm^-1
Linearity Range	1.5-3.0	15-90
y = mx + c	0.1947x + 0.0207	0.0028x + 0.4729
Slope (m)	0.1947	0.4729
Intercept (c)	0.0207	0.4729
Regression coefficient (R²)	0.9993	0.9949
Limit of Detection (% w/w)	0.076	0.589
Limit of Quantitation (% w/w)	0.230	1.785

Table 3: Assay of pharmaceutical synthetic mixture (CLO 1.5 mg/ CHT 15 mg)

Drug	Label claim (mg)*	Amount Found (mg)*	% Found	SD	% RSD
CLO	1.5	1.49	99.78	1.057	1.0593
CHT	15	14.95	99.72	0.633	0.634

* indicates average of six determinations

Analysis of excipients in synthetic mixture:

In a volumetric flask of 100millilitres, 115.3 milligrammes of powder containing an equivalent weight of 1.5 milligrammes of CLO and 15 milligrammes of CHT was dissolved in 100millilitres of chloroform and this mixture was sonicated for approximately 30 minutes. Immediately following sonication, it was filtered using Whatmann filter paper no. 41. After the residue had been completely dried, one milligramme of it was combined with 99 milligrammes of KBr which is used as a diluent, and then it was scanned in the infrared range of 400-4000 cm^-1. Figure 6 represents the FT-IR spectrum of blank sample containing only KBr. Figure 7 represents the FT-IR spectrum of overlay of synthetic mixture and excipients.

Figure 6: FT-IR spectrum of blank sample containing only KBr

Figure 7: FT-IR spectrum of overlay of synthetic mixture and excipients

ANALYTICAL METHOD VALIDATION:

Following are the parameters by which the developed method was validated in accordance with ICH recommendations.

Linearity:

The linearity study was performed by preparing standard dilutions of 1.5, 1.8, 2.1, 2.4, 2.7 and 3.0% w/w for CLO and 15, 30, 45, 60, 75 and 90% w/w for CHT. For CLO and CHT, respectively, the plotted calibration curve graph was concentration verses peak intensity. The regression coefficient (R²) value was found to be 0.999 for CLO and 0.994 for CHT. Figure 8 and Figure 9 represents the calibration curve of CLO and CHT. Figure 10 shows the absorbance of CLO standard in linearity spectrum and Figure 11 shows the absorbance of CHT standard in linearity spectrum.

Figure 8: Calibration curve of CLO

Figure 9: Calibration curve of CHT

Figure 10: Absorbance of CLO standard in linearity spectrum

Figure 11: Absorbance of CHT standard in linearity spectrum

Precision:

Inter-day and intra-day variance experiments were performed in order to evaluate the method's level of precision. When conducting intraday investigations, the percentage relative standard deviation (% RSD) was computed by analysing the working dilutions of the sample in triplicate within a day. Whereas, working dilutions of the sample were analysed on three different days in a row for the inter-day variation investigations, and the percentage relative standard deviation (% RSD) was computed. The data of repeatability was shown in Table 4. The summary of the findings from the intra-day study may be seen in Table 5 while of the inter-day study may be seen in Table 6.

Table 4: Repeatability data of mixture of standard drugs

Drug	% Found*	SD	% RSD
CLO	100.18	0.794	0.7925
CHT	99.72	0.518	0.519

*Indicates average of six determinations

Table 5: Data for intra-day precision

Drug	Mean*	SD	% RSD
CLO	99.99	1.377	1.377
CHT	99.53	0.915	0.919

*Indicates average of six determinations

Table 6: Data for inter-day precision

Drug	Mean*	SD	% RSD
CLO	99.78	0.747	0.748
CHT	100.19	0.751	0.749

*Indicates average of six determinations

Accuracy:

In accordance with ICH criteria, the recovery studies were conducted at three distinct levels (80%, 100%, and 120%) by standard addition in order to validate the suggested methodologies and determine their degree of precision. The label claims that the mixture contains were 1.5 mg of CLO and 15 mg of CHT. The sample was prepared by using 1.5% weight by volume for CLO and 15% weight by volume for CHT. The percentage of drug recovered and the total amount of drug recovered were calculated. Table 7 is a report of the findings of the recovery studies.

Table7: Results of recovery study data

Level of recovery	% mean recovery*		SD		% RSD
Level of recovery	CLO	CHT	CLO	CHT	CLO	CHT
80 %	98.856	100.07	1.589	0.485	1.607	0.484
100 %	99.53	99.99	0.187	0.324	0.187	0.324
120 %	100.67	100.39	0.221	0.286	0.219	0.284

*Indicates average of three determinations

LOD and LOQ:

ICH guidelines offer multiple ways to calculate detection and quantitation limits. The application of standard deviation of the response, the signal-to-noise ratio, the slope of the calibration curve and the visual inspection are some of the other methods. In the current study, the LOD and LOQ were determined utilising the slope of the calibration curve and the standard deviation of the response, and they were computed with the aid of the following formulae.

LOD = 3.3 𝜎/S.............................. …………………...(1)

LOQ = 10 σ/S............................................................(2)

Where σ refers to the standard deviation of the peak intensity of the drugs, which is used as a measure of noise, and S refers to the slope of the calibration curve that corresponds to the peak intensity.

Force degradation studies:

The ICH guidelines made it possible for new drug substances and products to undergo stability testing. However, these tests had to be accompanied by stress testing in order for them to be accepted. This was done to better understand the active substances' inherent stability characteristics. For six hours, the pure drugs CLO and CHT were subjected to dry heat at 80˚C, in order to carry out the thermal degradation process. After subjecting the samples to heat, KBr was used to dilute them in order to obtain CLO (0.15% w/w) and CHT (1.5% w/w). In this experiment, pure drugs were subjected to UV radiations, and at regular intervals of six hours, the samples were taken. In order to obtain CLO (0.15% w/w) and CHT (1.5% w/w), the samples were diluted with KBr after being subjected to UV light. The pure drugs CLO and CHT are put through the sunlight degradation process by being exposed to sunlight in open space for a period of twelve hours. After exposing the sample to sunlight, it was diluted with KBr to obtain CLO at 0.15% weight by volume and CHT at 1.5% weight by volume. For CLO and CHT, peak intensity was measured at 1658.78 cm^-1 and 1346.31 cm^-1, respectively. Figure 12 displays thermal degradation of CLO, Figure 13 displays thermal degradation of CHT, Figure 14 displays photolytic degradation of CLO, Figure 15 displays photolytic degradation of CHT, Figure 16 displays sunlight degradation of CLO and Figure 17 displays sunlight degradation of CHT. Table 8 displays the findings of the conducted forced degradation study.

Figure12: Thermal degradation of CLO

Figure 13: Thermal degradation of CHT

Figure 14: Photolytic degradation of CLO

Figure 15: Photolytic degradation of CHT

Figure 16: Sunlight degradation of CLO

Figure 17: Sunlight degradation of CHT

Table 8: Data of forced degradation study

Degradation condition	%Assay		% Degradation
Degradation condition	CLO	CHT	CLO	CHT
Thermal	60.41	93.30	39.59	6.7
Photolytic	56.46	84.75	43.54	15.25
Sunlight	59.14	74.48	40.86	25.52

RESULTS AND DISCUSSION:

FT-IR spectrum of pure drug sample of CLO and CHT exhibited absorbance intensity of 1658.78 cm^-1 and 1346.31 cm^-1 respectively. As a result of the fact that the low intensity absorbance bands that resulted from CLO and CHT were not significantly hampered by dilution in dry potassium bromide, we made use of it as a diluent in this work. In dry potassium bromide, for diluted sample of CLO, the most prominent absorbance band corresponding to the C=N (Imine) group was centred in the intensity of 1658.78 cm^-1. Whereas, for diluted sample of CHT, the absorbance band corresponding to the S=O (Sulphonates) group was centred in the intensity of 1346.31 cm^-1. Both of these bands were the most prominent. The calibration curves for CLO and CHT were prepared by using the intensities of 1658.78 cm^-1 and 1346.31 cm^-1, respectively.

Linearity was established by making the dilutions in the range of 1.5- 3.0% w/w for CLO and 15- 90% w/w for CHT in dry potassium bromide. The calibration curve can be represented by the equations y= 0.1947x+0.0207 with correlation coefficient 0.999 for CLO, and y= 0.0028x+0.4729 having correlation coefficient 0.994 for CHT.

The precision (%RSD) was expressed by coefficient of variation by the mean of standard deviation. Both intraday and interday precision findings were within the acceptable limits of variable i.e. <2%.

The accuracy of the assay method was evaluated by determining the percentage of pure drug excipients that could be recovered using the method of addition of standard at three different levels: 80%, 100%, and 120%. The mean percentage recovery was found to be 98.85%, 99.53%, and 100.67% for CLO and 100.07%, 99.99%, and 100.39% for CHT at 80%, 100%, and 120% respectively. The method was determined to be accurate as the values fall within the limit.

LOD was 0.076% w/w for CLO and 0.589% for CHT w/w whereas LOQ was found to be 0.230% w/w for CLO and 1.785% w/w for CHT.

Thermal, photolytic, sunlight degradation was found to be 39.59%, 43.54%, 40.86% for CLO and 6.7%, 15.25%, 25.52% for CHT respectively.

CONCLUSION:

The FT-IR spectroscopic method was developed, validated, and found to be suitable for simultaneous estimation of Clonidine Hydrochloride and Chlorthalidone in synthetic mixture form. This was accomplished in accordance with the guidelines established by ICH. The method that was developed is simple while also being sensitive, precise and accurate. The devised method was both cost effective and eco-friendly; it has the potential to be successfully used as a green method in the pharmaceutical sector for the purposes of quality control and routine examination of final products. The findings of the investigation of the combined mixture were discovered to be extremely reliable and reproducible using the proposed method. For both CLO and CHT, studies of forced degradation were conducted, from which the extent of degradation can be proved in chosen experimental conditions.

ACKNOWLEDGEMENT:

Authors are grateful to Neon Laboratories Limited, Andheri (E), Mumbai and Trichem Life Science Ltd. MIDC Tarapur Dist. Palghar, Maharashtra, India for providing the gift sample of pure drug of Clonidine Hydrochloride and Chlorthalidone respectively. For providing the research facilities, the authors would also like to express their gratitude to the Director of School of Pharmacy, Swami Ramanand Teerth Marathwada University, Nanded.

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23. Wadher SJ, Kalyankar TM, Kshirsagar JR, Anitha K. Simultaneous Determination of Famotidine and Dicyclomine HCl in Combined Tablet Dosage Form by UV Spectrophotometer. Research Journal of Pharmacy and Technology. 2017; 10(2): 408-13. doi:10.5958/0974-360X.2017.00082.8

24. Kalyankar TM, Wadher SJ, Bodhankar MR, Anitha K. Development and Validation of Analytical method for Estimation of Cetirizine Hydrochloride and Phenylephrine Hydrochloride in Pharmaceutical Formulation. Research Journal of Pharmacy and Technology. 2019; 12(1): 1-7.

25. Satpute KL, Kalyankar TM. Investigation of Antiacne Activity of some Medicinal Plants Against Pathogenic Bacteria. Research Journal of Pharmacy and Technology. 2018; 11(7): 1-6.

26. Bodhankar MR, Wadher SJ, Kalyankar TM, Anitha K. Stability Indicating Simultaneous Estimation of Cetirizine Hydrochloride and Phenylephrine Hydrochloride in Combined tablet formulation by using HPTLC- Densitometric method. Research Journal of Pharmacy and Technology. 2021; 14(5): 2467-71. doi:10.52711/0974-360X.2021.00434

27. Kalyankar TM, Wadher SJ, Bodhankar MR, Mustasin FS. Stability Indicating Simultaneous Estimation of Phenylephrine HCl and Bromhexine HCl in Combined tablet dosage formby UV-Spectrophotometer. Research Journal of Pharmacy and Technology. 2021; 14(6): 3128-32. doi:10.52711/0974-360X.2021.00545

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Received on 15.03.2023 Modified on 31.07.2023

Research J. Pharm. and Tech 2024; 17(1):284-290.

DOI: 10.52711/0974-360X.2024.00044