INTRODUCTION:

Guaifenesin is an orally administered expectorant drug intheDPP-4inhibitorclass.MolecularformulaC₁₀H₁₄O₄ with IUPAC name (RS) – 3 – (2 – methoxy phenoxy) – 1 - 2 – propanediol^1-7. It acts as an expectorant by increasing the volume and reducing the viscosity of secretions in the trachea and bronchi. It has been said to aid the flow of respiratory tract secretions, allowing ciliary movements to carry the loosened secretions upward towards the pharynx, thus it may increase the efficiency of the cough reflex and facilitate removal of the secretions. Guaifenesin has muscle relaxant and anticonvulsant properties and may be acting as NMDA receptor antagonist.

Few methods have been reported^8-10 for Guaifenesinin spectrophotometry, HPLC^11-15. To date, there have been no published reports about the estimation of Guaifenesin by HPLC in bulk drug with faster retention time. This present study reports for the estimation of Guaifenesin by HPLCinbulk dosage form. The proposed method is validate dasper ICH guidelines.

MATERIALSANDMETHODS¹⁶

Instrumentation:

Isocratic HPLC system with Analytical technologies HPLC-Chrom work station Soft ware containing (cosmosil 250x4.6mm, 5µm) column with UV detection. Analytically pure guaifenesin was kindly provided by MSN Laboratories, Hyderabad.

Chemical and reagents

S. No.	Chemicals/standards and reagents	Grade	Laboratories
1	Guaifenesin tablets	AR	Sanzyme
2	Guaifenesin API	NA	Granules
2	Acetonitrile	HPLC	SDFCL
3	Water	HPLC	SDFCL
4	Dipotassium hydrogen phosphate	LR	Merck
5	KOH	LR	Molychem
6	Orthophosphoric acid	LR	Molychem

Chromatographic conditions:

Glasswares used in each procedure were soaked overnight in a mixture of chromic acid and sulphuric acid rinsed thoroughly with double distilled water and dried in a hot air oven. The mobile phase consisted of Acetonitrile: Phosphate buffer PH 5(70:30). The contents of the mobile phase were filtered before use through a 0.45μm membrane and degassed for 20min. The mobile phase was pumped from the solvent reservoir to the column at a flow rate of 1.0 ml/min and the injection volume was 25μL. The column temperature was maintained at ambient temperature. The eluents were monitoredat260nm using UV lamp.

Preparation of standard stock solutions:

Accurately weigh and transfer 100mg of Guaifenes in into 100ml of volumetric flask and add 100ml of mobile phase and sonicated 10min (or) shaken 5min and made with water. Transfers the above 10ml solution into 100ml volumetric flask dilute to volume with water

RESULTS AND DISCUSSION:

Calibration of standards:

The standard calibration curve was constructed for guaifenesin different volumes of stock solutions of each were accurately transferred in to 10mL volumetric flask sand diluted to mark to yield a concentration range of 10-35μg/ml solutions. The calibration line was obtained by plotting the peak area against the concentration of the drug. The results were shown in table2.

Robustness, the limit of detection and the limit of quantification, precision was calculated by percentage relative standard deviation. The accuracy was expressed in terms of percent recovery of the known amount of the standard drugs added to the known amount of the pharmaceutical dosage forms. Various validation parameters are performed.

1. System Suitability:

It is defined as tests to measure the method that can generate the result of acceptable accuracy and precision. The system suitability was carried out after the method development and validation have been completed. The system suitability was assessed by triplicate analyses of the drugs at a concentration of 20μg/mL of Guaifenesin forth is, parameters like plate number, tailing factor, peak asymmetry of samples was measured, and shown in Table1.

Table1: System Suitability data of Guaifenesin

S. No	Sample name	RT	Area	Theoretical plate count	USP tailing
1.	Injection1	3.518	17115746	737	1.17
2.	Injection 2	3.518	14920550	793	0.97
3.	Injection 3	3.035	18425189	652	0.85
4.	Injection 4	3.677	14449086	2159	0.83

1. Specificity:

The specificity of the method was evaluated by injecting the blank injection, working standard samples into the chromatograph to check the co-elution, if any, at the retention time of 3.677 min. the proposed method was specific for the detection of Guaifenesin. There were no peaks at the retention time of guaifenesin.

Fig1: Typical chromatogram of Guaifenesin

2. Linearity:

The method can elicit test result that is directly proportional to analytic concentration within a given range. It is generally reported guaifenesin regression line. The appropriate volume of an aliquot from Guaifenesin standard stock solution was transferred to a volumetric flask of 10ml capacity. The volumes were adjusted to the mark with mobile phase to give a solution containing 10-35μg/ml Guaifenesin.

The slope, Y-intercept and correlation coefficient were calculated. The regression line relating standard concentrations of drug using regression analysis was calculated. The calibration curves were linear in the studied range and equations of the regression analysis were obtained. The mean and correlation % RSD (N=6) were calculated. The represented data was shown in below figure 2 and Table 2

Table2: Linearity data of guaifenesin

S. No	Concentration (μg/ml)	RT (min.)	Peak area
1.	10	4.102	942008
2.	15	3.730	14131300
3.	20	3.795	1828907
4.	25	3.872	2270006
5.	30	3.027	2660769
6	35	3.048	3163178
Correlation coefficient (r²)		0.998

Fig.2. Calibration curve of Guaifenesin

3. Precision:

It is a measure of degree of repeatability of an analytical method under normal operation and it is normally expressed as % of relative standard deviation (% RSD). The standard solution was injected for five times and measured the area for all five injections in HPLC. The % RSD for the area of five replicate injections was found to be within the specified limits.

Table:4. Precision studies

S. No	RT	Peak area	% Assay
Injection1	2.988	4393802	99.03
Injection2	2.973	4070799	99.09
Injection3	3.027	4147528	98.97
Injection4	3.071	4331478	98.96
Injection5	3.048	4372308	98.99
Injection6	2.988	4234578	99.97
Mean	-	-	99.89
Std. Dev.	-	-	0.32
% RSD	-	-	0.32

Intermediate precision/ruggedness:

To evaluate the intermediate precision (also known as Ruggedness) of the method. Precision was performed on different days by using the same column of the same dimensions.

The standard solution was injected five times and measured the area for all five injections in HPLC. The % RSD for the area of five replicate injections was found to be within the specified limits. (Table no 2). Acceptance Criteria: The % RSD for all the five standard injections results should not be more than2%

4. Accuracy:

The accuracy of the analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value reference value and value found. Accuracy was assessed by determination of the recovery of the method by addition of the standard drug to the pre-quantified placebo preparation at 3 different concentration levels 50, 100 and 150 %, taking into consideration percentage purity of added bulk drug samples. Each concentration was analyzed 3 times and average recoveries were measured. Results of assay and recovery were presented in Table5.

5. Robustness:

As part of the Robustness, deliberate change in the Flow rate was made to evaluate the impact on the method

The flow rate was varied at 0.8 ml/min to 1.2 ml/min:

The Standard solution of Guaifenes in was prepared and analyzed using the varied flow rates along with method developed flow rate. One valuation of the above results, it was concluded that the variation in flow rate does not affected the method significantly. Hence it was indicated that the method was robust even by change in the flow rate.

Table 5: Accuracy of the guaifenesin:

S. No	Accuracy level	Sample name	Sample Weight (g)	μg/ml added	μg/ml found	% Recovery	% Mean
1	50%	1	0.3	20	19.5	100	100
		2	0.3	20	20	100
		3	0.3	20	21	101
2	100%	1	0.6	40	40	100	100
		2	0.6	40	40	100
		3	0.6	40	40	100
3	150%	1	0.9	60	60	100	100
		2	0.9	60	58	99
		3	0.9	60	60	100

Values are expressed in % recovery of the three replicate samples

LOD and LOQ

LOD and LOQ were calculated from the formula 3.3 x(σ/S) and 10 x (σ/S), respectively where σ is the standard deviation of intercept and S is the mean of slope. The LOD and LOQ can also be determined by S/N. The value for LOD should be 3-5 whilst for LOQ10-15. Results of LOQ and LOD studies are shown in Table

CONCLUSION:

The study is focused to develop and validate HPLC methods for estimation of Guaifenesin in the tablet dosage form. For routine analytical purposes, it is desirable to establish methods capable of analyzing the huge number of samples in a short time with good robustness, accuracy and precision without any prior separation steps. HPLC method generates a large amount of quality data, which serves as a highly powerful and convenient analytical tool. The method shows good reproducibility and good recovery. From the specificity studies, it was found that the developed methods were specific for Guaifenesin.

ACKNOWLEDGMENT:

The authors are very thankful to the management and principal Chilkur Balaji College of Pharmacy, Hyderabad, India for providing necessary facilities to carry out the research work.

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Received on 10.09.2021 Modified on 29.01.2022

Research J. Pharm. and Tech 2023; 16(1):111-114.

DOI: 10.52711/0974-360X.2023.00020

MATERIALSANDMETHODS16