INTRODUCTION:

Terbutaline (bronchodilator), acts through stimulation of beta-adrenergic receptors of intracellular adenyl cyclase, the enzyme which catalyzes the conversion of adenosine triphosphate to cyclic- 3',5'-adenosine monophosphate. Chemically, terbutaline is 5-[2-(tert-butylamino)-1-hydroxyethyl] benzene-1, 3-diol^1-3. Various analytical methods are reported for the analysis of terbutaline sulphate (TER); either individually or in combination with other drug^4-8. Ambroxol hydrochloride (AMB), acts as mucolytic agent is Trans-4-{[(2-Amino-3, 5-dibromophenyl)-methyl] - amino]} - cyclohexanol hydrochloride⁹.

Literature survey revealed that UV, HPLC, HPTLC methods have been reported for AMB either individually or in combination with other drugs^10-17. Antiallergic agent Guaiphenesin (GUA) chemically is (+)-3-(2-methoxyphenoxy)-propane-1, 2-diol¹⁸. Literature survey revealed that UV, HPLC, HPTLC chromatographic methods have been reported for analysis of Guaiphenesin^19-22.

Although the HPLC method is reported for the simultaneous analysis of these drugs, there is a need to develop HPTLC method which offers more flexibility for the selection of stationary phase, mobile phase, developing technique and gives better analytical precision and accuracy. The literature survey showed that the HPTLC method has not been reported for the simultaneous analysis of Terbutaline sulphate, Ambroxol hydrochloride, and Guaifenesin in the combined syrup dosage form. Thus, the current research work undertaken which describes a simple, selective, accurate, precise, and robust normal-phase HPTLC method which is validated according to ICH Q2 (R1) guideline²³. This can be used for concurrent quantification of Terbutaline sulphate, Ambroxol hydrochloride, and Guaifenesin as in bulk drug as well as in their combined syrup dosage form.

MATERIAL AND METHODS:

Chemicals and reagents:

Pharmaceutical grade Ambroxol Hydrochloride and Guaifenesin, were received gifted by Polymed Pharmaceuticals Pvt. Ltd. Jalgaon, Maharashtra, India, and Terbutaline Sulphate was received as a gift sample from Alkem Laboratories Ltd. Mumbai, Maharashtra, India. Marketed formulation Broozeet syrup (Alembic Pharmaceuticals Ltd. Gujarat, India-containing Ambroxol 15mg+Guaifenesin 50mg+Terbutaline 1.5 mg/5mL) was procured from the local market. Analytical grade reagents and chemicals used in the study were procured from Merck Specialities Pvt. Ltd. (Mumbai, India). Precoated silica gel aluminum HPTLC plates 60 F254 (Merck KGaA, Darmstadt, Germany) were used in the study.

Instrumentation:

The HPTLC fingerprint analysis was carried out on GF254 silica gel plates (Merck KGaA, Darmstadt, Germany). The sample was spotted as a band onto a 20.0 × 10.0cm silica gel plate with the help of CAMAG Automatic TLC Sampler 4-241607 and by using a 25μL sampler syringe. Development of the plate was performed in CAMAG 20.0 × 10.0cm Twin and Trough chamber previously saturated with the mobile phase. The Photo documentation was done using a CAMAG TLC Visualizer 2-250204. Densitometric scanning (slit dimensions: 6 × 0.45mm, micro; scanning speed of 20 mm/s; data resolution at 100μm/step) was performed at 200nm using CAMAG TLC Scanner 4–250173, operated in reflectance- absorbance mode. The data were analyzed using Vision CATS Server, version 2.5.18072.1.

Preparation of standard stock solutions:

Standard stock solutions of drugs were prepared individually in methanol by dissolving 10mg in 10mL volumetric flask to get 1000ppm. From this stock solution, further dilutions were done to get 100ppm, 500 ppm, and 50ppm standard solutions of GUA, AMB, and TER, respectively.

Preparation of Sample solution

For analysis of marketed syrup formulation (As per lable claim Each 5mL contains 1.5mg TER, 15mg AMB HCl, and 50mg GUA). The sample solutions were prepared according to the linearity range observed for the drugs. The stock solution of the formulation sample was prepared by accurately pipetting 5mL of the sample formulation in 50ml volumetric flask, 5mL of water was added and the solution was diluted up to mark with methanol. This solution was treated as sample 1. Since the concentration of Terbutaline sulphate was very low-1.5mg/5mL of the formulation, TER was estimated directly using sample 1. The sample stock solution was further diluted by pipetting out 5mL and further diluted to 50mL (Sample 2). Since the concentration of Guaifenesin hydrochloride was high-50mg/5mL formulation, further dilution was necessary for its estimation. Hence, GUA and AMB HCl were estimated by considering sample 2. Appropriate volume was applied to a chromatographic plate. In optimized mobile phases, the plate was developed. This prepared solution was used for the assay and recovery study with further suitable dilutions.

Selection of detection wavelength:

The developed HPTLC plate was scanned in the range of 190 - 400nm, and the overlain spectra were taken. All compounds showed significant absorbance at 200nm and thus selected it for densitometric analysis (Figure 1).

Figure 1: Overlain UV spectrum of TER, AMB, and GUA

Assay validation:

The developed densitometric method was validated as per the International Council of Harmonization [(ICH) Q2 (Rl)] guideline for the following validation parameters,

1. Linearity and Range:

The linearity was studied in the range of 200 - 700ng band^-1, 1000 - 7000ng band^-1, and 400 - 1200ng band^-1 for TER, AMB, and GUA, respectively. Peak area versus concentration was plotted. The data were subjected to least-square linear regression analysis. The linearity was evaluated by determining the correlation coefficient, slope, intercept and residual analysis was also carried out.

2. Sensitivity:

The limit of detection and limit of quantitation was calculated using formula 3.3σ/S and 10σ/S, respectively, where S is the slope of the linearity plot and σ is the standard deviation of the response.

3. Specificity:

The peak purity of each drug was checked for the standard and sample solutions by comparing the UV spectrum of TER, AMB, and GUA at peak start (S), peak apex (M) and peak end (E) positions of the band. The correlation (r) value between S-M and M-E was determined for the sample and compared with the standard.

4. Precision studies:

The precision of the optimized method was verified by intra and inter-day precision studies of the formulation sample [TER (300ng band^-1), AMB (3000ng band^-1) and GUA (1000ng band^-1]. The intra-day precision was studied by analysis of the formulation six times on the same day in order to trace any deviations. For inter-day precision, the drug samples were evaluated on three successive days. The results obtained are expressed as % RSD.

5. Accuracy studies:

Accuracy was assessed by recovery studies from marketed syrup formulation at three levels-80 %, 100 % and 120% of the test concentration using the standard addition method. The sample concentration 300 ng band-1 of TER, 3000ng band-1 of AMB, and 1000 ng band-1of GUA were spiked with 80%, 100% and 120% of standard solutions.

6. Robustness studies:

In the robustness evaluation, small, deliberate changes in the analytical parameters of the proposed method were done and its effect on the peak areas of the drugs were studied. The single parameter was varied at a time. Factors changed were the amount of solvent system (± 5%), solvent system (Chloroform) composition (±0.1 mL), time from band application to chromatographic development (+ 10 min) and time from chromatography to scanning (+ 10 min). The concentration of 300ng band^-1 of TER, 3000ng band^-1 of AMB and 1000ng band^-1 of GUA in six replicates were used to study the robustness of the method. The standard deviation of peak areas and the % relative standard deviation (% RSD) were determined.

RESULTS AND DISCUSSION:

Optimization of HPTLC method:

Solvent systems with varying polarities were tried. Solvents such as ethanol, toluene, n-hexane, acetone, methanol, water, dichloromethane, and ethyl acetate were tried to achieve the Rf value of the drugs in the range 0.2 - 0.8, and having a minimum resolution Rs≥1.5. Finally, the mobile phase consisting of chloroform: methanol: ethyl acetate: acetic acid: formic acid (7.0: 1.4: 0.8: 1.0: 0.5, v/v) was selected for obtaining well-resolved peaks. The optimum wavelength for densitometric analysis used was 200nm. The retention factors were found to be 0.32±0.02, 0.55± 0.02, and 0.72 ±0.02, for TER, AMB, and GUA, respectively (Figure 2).

Figure 2: Densitogram obtained from the mixed standard solution of TER, AMB, and GUA

HPTLC method validation:

1. Linearity and Range:

The Linearity assessment results are presented below in Table 1. The correlation value of 0.999 was obtained for all three drugs indicating that the peak area responses for the concentrations studied were linear (Figure 3).

Table1: Linearity data for TER, AMB, and GUA (n = 6)

Parameters	TER	AMB	GUA
Linearity range (ng band^-1)	200 - 700	1000 - 7000	400 - 1200
r2	0.999	0.999	0.999
Slope	0.669142857	0.153086	0.393
Intercept	68.88	230.6897	34.2
Confidence limit of slope^a	0.6477-0.6904	0.1440-0.1621	0.3713-0.4146
Confidence limit of intercept^a	58.6121-79.1593	192.8245-268.5548	15.8187-52.5812
S.D ^b	2.8765	11.8303	3.7282

a -confidence limit (95%); b - Standard deviation of residuals from the line.

Figure 3: Calibration curve of TER, AMB, and GUA

The residual analysis was also performed to ascertain the linearity. The slope was found to be significantly different from zero.

2. Sensitivity:

The limit of detection and limit of quantitation were found to be 14.18 ng band^-l and 42.98 ng band^-l for TER, 255.02 ng band^-l and 772.78 ng band^-l for AMB and 31.30 ng band^-l and 94.86 ng band^-l for GUA.

3. Specificity:

The peak purity for TER, AMB and GUA was estimated by comparing UV spectrum acquired at the start, apex, and end of the peak. The r (S, M) = 0.998, 0.999, 0.998 and r (M, E) = 0.998, 0.998, 0.999 for TER, AMB and GUA respectively indicated non-interference of any compound/impurity.

4. Precision:

According to ICH guideline, RSD < 2 %, in precision studies reflected good precision (Table 2).

Table 2: Intra –day and Inter –day Precision results of HPTLC method (n=3)

Drug	Conc. ^a	% Concentration Intra day	(%) RSD Intra day	% Concentration Inter day	(%) RSD Inter day
TER	300	100.18	0.39	99.38	0.60
AMB	3000	99.59	0.24	100.62	0.57
GUA	500	100.79	0.63	99.29	0.92

a Concentration in ng band^-1; RSD is the relative standard deviation

5. Accuracy:

Around 99.38-100.18 %, 99.59 - 100.62 %, and 99.29 - 100.79 % recoveries of TER, AMB, and GUA, respectively was obtained, which indicate that the proposed simultaneous HPTLC method is reliable for the estimation of TER, AMB, and GUA in the marketed formulation used in the study (Table 3).

Table 3: Results of recovery studies (n=6)

Drug	Amount taken (ng band^-1)	Amount Added (ng band^-1)	Amount found (ng band^-1)	± S.D	% Recovery ± % RSD
TER	300	240	540	2.80	100.12±0.51
	300	300	600	5.74	100.13±0.95
	300	360	660	4.35	99.72±0.66
AMB	3000	2400	5400	12.50	99.98±0.23
	3000	3000	6000	10.99	100.01±0.18
	3000	3600	6600	13.57	100.0±0.20
GUA	500	400	900	7.14	100.38±0.79
	500	500	1000	10.50	99.83± 1.05
	500	600	1100	5.84	100.28±0.52

RSD - relative standard deviation

6. Robustness studies

It was observed that areas of peaks of interest remained unaltered by small changes of the operational parameters (% RSD < 2) which indicated the robustness of the method (Table 4).

Analysis of marketed formulation:

The proposed HPTLC method was used for the analysis of marketed syrup formulation (Broozeet) in six replicate determinations. The percent assay of the formulation was found to be 98.47 %, 98.47 %, and 99.23 % for TER, AMB, and GUA respectively in marketed formulation. Results obtained indicate the reliability of the proposed densitometric method.

CONCLUSION:

A rapid cost-effective, accurate, selective, and robust HPTLC method for simultaneous quantification of Terbutaline sulphate, Ambroxol hydrochloride, and Guaifenesin was developed and validated. The method has good specificity and high recovery and is useful for the simultaneous analysis of Terbutaline sulphate, Ambroxol hydrochloride, and Guaifenesin in bulk and the syrup formulation used in the current research.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGEMENT:

The authors are thankful to Principal Dr. (Mrs.) Kiran Bhise, the Management of M.C.E. Society’s Allana College of Pharmacy, Pune, India, for providing necessary research facilities. Authors wish to thank Dr. Vaishali A. Shirsat - Principal Investigator of ‘National Facility for Research and Training (NFRT) in Integrated Analytical Strategies for Discovery, Development and Testing of Drugs, Pharmaceuticals and Nutraceuticals”, DPRP scheme sanctioned and funded by Department of Science and Technology (Ministry of Science & Technology), Government of India, Project Sanction Order No; VI-D&P/552/2016-17/TDT(G), dated 07.03.2017, and The Indian Pharmaceutical Association-Maharashtra State Branch to Bombay College of Pharmacy, Mumbai- Maharashtra, India, for providing technical support and facilities to carry out research work. Authors also express their gratitude to Polymed Pharmaceuticals Pvt. Ltd. Jalgaon, and Alkem Laboratories Ltd. Mumbai, Maharashtra, India, for providing gift sample of pure drugs.

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Received on 02.12.2020 Modified on 09.08.2021

Research J. Pharm. and Tech. 2022; 15(7):2997-3001.

DOI: 10.52711/0974-360X.2022.00500

Parameter	SD of peak area			% RSD
	TER	AMB	GUA	TER	AMB	GUA
Mobile phase (Chloroform) composition (± 0.1 mL)	2.167948	4.082483	2.160247	0.80743	0.582934	0.928473
Amount of mobile phase (± 5 %)	2.65832	2.94392	2.44949	0.99687	0.42016	1.051283
Time from band application to chromatography (+ 10 min)	1.94079	4.708149	1.94079	0.721931	0.673716	0.826454
Time from chromatography to scanning (+ 10 min)	1.36626	3.868678	2.097618	0.506648	0.5528	0.896418