Simultaneous UV-Spectrophotometric Estimation of Acetaminophen and Guaiphenesin by Second Order derivative Method in Combined Pharmaceutical Dosage Form

 

Rajan V. Rele.*

Central Research Laboratory, D.G. Ruparel College, Matunga, Mumbai 400016.

*Corresponding Author E-mail: drvinraj@gmail.com

The objective of the study was to develop a simple, accurate, precise and rapid a UV spectrophotometric i.e. second order derivative method for the determination of acetaminophen and guaiphenesin in combined dosage form i.e. tablets by using methanol as a solvent. The method was further validated by ICH guidelines. The proposed second order derivative method involves the measurement of absorbance of one drug at zero crossing point of other; hence wavelengths 215 nm and 234 nm were selected for the estimation of acetaminophen and guaiphenesin respectively. The linearity of the proposed method was found in the concentration range of 1 to 10 µg /ml (r²= 0.9993) for acetaminophen and 5 to 100 µg /ml (r²= 0.9998) for guaiphenesin respectively. The percentage mean recovery was found to be 99.945 % for acetaminophen and 99.955 % for guaiphenesin respectively. The method was also statistically validated for its linearity, accuracy and precision. Both intra and inter day variations showed less percentage (%) RSD values indicating high grade of precision of this method.

 

 

 

INTRODUCTION:

Acetaminophen is chemically known as N-(4- hydroxyphenyl) acetamide. It inhibits prostaglandin biosynthesis by blocking the enzyme cyclo-oxygenase 2-5. Acetaminophen is an analgesic-antipyretic agent. It is effective in treating mild-to-moderate pain such as headache, neuralgia and pain of neuro-muscular origin 6. Owing to widespread use of acetaminophen in different kinds of pharmaceutical preparations, therefore rapid and sensitive methods for the determination of acetaminophen are being investigated.

 

Guaiphenesin is, 3-(2-Methoxyphenoxy)-1,2-propanediol. It shows molecular formula as C₁₀H₁₀O₄ with molecular weight as 198.2.

 

It is used to increase the volume and reduce the viscosity of tenacious sputum and is used as expectorant for productive cough.

 

In literature survey reveals HPLC methods^{1, 2} for simultaneous determination of acetaminophen and guaiphenesin in combined dosage form.

 

MATERIAL AND METHOD:

Instrument and reagents:

Spectral scan was made on a Shimadzu UV-spectrophotometer, model 1800 (Shimadzu, Japan) with spectral band width of 0.5 nm with automatic wavelength corrections by using a pair of 10 mm quartz cells. All spectral measurements were done by using UV-Probe 2.42 software.

 

Reference standard of acetaminophen and guaiphenesin were obtained from reputed firm with certificate of analysis.

 

Preparation of standard drug solutions:

A 100 mg standard acetaminophen was weighed accurately and transferred to a 100 ml volumetric flask and sonicated with 30 ml methanol for 15 minutes. The volume was made up to the mark with methanol to give a stock solution of acetaminophen of concentration 1000 μg /ml. From this solution, 10 ml of solution was pipetted out and transferred into 100 ml volumetric flask. The volume was made up to mark with methanol to give a working standard solution of concentration 100 μg/ml.

Similarly 100 mg standard guaiphenesin was weighed accurately and transferred to a 100 ml volumetric flask and sonicated with 30 ml of methanol for 15 minutes. The volume was made up to the mark with methanol to give a stock solution of methanol of concentration 1000 μg /ml. From this solution, 10 ml of solution was pipetted out and transferred into 100 ml volumetric flask. The volume was made up to mark with methanol to give a working standard solution of concentration 100 μg/ml.

 

Estimation from tablets:

Twenty tablets were weighed accurately and average weight of each tablet was determined. Powder equivalent to 325 mg of acetaminophen and 200 mg of guaiphenesin was weighed and transferred in 100 ml of volumetric flask. A 30 ml of methanol was added and sonicated for 15 minutes and filtered. The filtrate and washing were diluted up to the mark with methanol to give concentration as 3250 μg /ml of acetaminophen and 2000 μg/ml of guaiphenesin respectively. For working sample solution 10 ml of such solution was diluted to 100 ml and such solution was used for analysis.

 

Method: Second order derivative method:

(a) For acetaminophen:

For the selection of analytical wavelength, 100 μg/ml solution of acetaminophen was scanned in the spectrum mode from 350 nm to 200 nm by using methanol as blank. The second order derivative spectrum was obtained by using derivative mode by UV probe 2.42 software. From the spectrum, the amplitude of the derivative spectrum was measured at 215 nm.

 

(b) For guaiphenesin:

For the selection of analytical wavelength, 100 μg/ml solution of guaiphenesin was scanned in the spectrum mode from 350 nm to 200 nm by using methanol as blank. The second order derivative spectrum was obtained by using derivative mode by UV probe 2.42 software. From the spectrum, the amplitude of the derivative spectrum was measured at 234 nm.

 

Fig. 1(a): Overlay spectra of second order derivative of Acetaminophen in the Concentration range of 2– 10 µg/ ml at 215nm.

 

Fig. 1(b): Overlay spectra of second order derivative of guaiphenesin in the concentration range of 5 – 100 µg/ ml at 234 nm.

 

 

Preparation of calibration curves:

Series of solutions containing 1 – 10 µg/ ml of acetaminophen and 5 -100 µg/ ml of guaiphenesin were used to determine linearity of the proposed method respectively. Solutions were scanned in the spectrum mode and absorbance spectra were converted to second order derivative spectra. The overlain spectrum of acetaminophen and guaiphenesin were given in Fig. 1(a), 1(b) respectively.

 

After observing the overlain second order derivative spectra of acetaminophen and guaiphenesin, the zero crossing points of both drugs were selected for analysis of other drug. The second wave length selected was 215 nm, the zero crossing point of guaiphenesin where acetaminophen showed considerable absorbance. The second wavelength was 234 nm, the zero crossing point of acetaminophen, where guaiphenesin showed considerable absorbance. The calibration curves were plotted of double derivative of amplitude against concentrations [Fig. 2 (a), 2(b)].

 

Fig.2 (a): Calibration curve of Acetaminophen in the concentration range of 2-10 µg/ml.

 

Fig.2 (b): Calibration curve of guaiphenesin in the concentration range of 5-100 µg/ml.

 

Results of the analysis are given in table 1.

 

Table 1: Values of results of optical and regression of drugs

Parameter	Acetaminophen	Guaiphenesin
Detection Wavelength (nm)	215	234
Beer Law Limits (µg/ml)	1-10	5-100
Correlation coefficient(r2)	0.9998	0.9993
Regression equation (y=b+ac) Slope (a)	0.001	0.0005
Intercept (b)	-0.00007	-0.0002

 

Estimation from tablets:

Powdered from twenty tablets were collected and weighed accurately and average weight of powder from each capsule was determined. Powder equivalent to 325 mg of acetaminophen and 200 mg of guaiphenesin was weighed and transferred in 100 ml of volumetric flask. A 30 ml of methanol was added and sonicated for 15 minutes and filtered. The filtrate and washing were diluted up to the mark with methanol to give concentration as 3250 μg /ml of acetaminophen and 2000 μg /ml of guaiphenesin respectively. A 10 ml of such solutions was diluted to 100 ml. It was scanned in the range of 200-350 nm against methanol as blank. The absorbance spectra were converted to second order derivative spectra. Calculations were done as per the equations. The concentrations of acetaminophen and guaiphenesin present in tablets were calculated by substituting the values of absorbance in linearity equations.

 

(a) For Acetaminophen Y = 0.001x + 0.00007

(b) For guaiphenesin Y = 0.0005x - 0.0002

 

Method Validation:

These methods were validated according to ICH guidelines.

 

Accuracy:

To ascertain the accuracy of proposed methods, recovery studies were carried out by standard addition method at three different levels (80%, 100% and 120%). Percentage recovery for acetaminophen and guaiphenesin was found in the range of 99.893 % to 100.076% and 99.91% to 100.09 % respectively. (Table2).

 

Linearity

The linearity of measurement was evaluated by analyzing different concentration of the standard solutions of acetaminophen and guaiphenesin. For both the drugs concentration range was found to be 1-10 µg/ml for acetaminophen and 5-100 µg/ml for guaiphenesin.

 

Table 2: Statistical evaluation of the data subjected to accuracy

Level of % recovery	Amount present in µg/ml		Amount added in µg/ml		Amount found in µg/ml		% Recovery		Mean % recovery
	ACET	GUI	ACET	GUI	ACET	GUI	ACET	GUI	ACET	GUI
80%	32.5	20	26	16	58.453	35.906	99.92	99.74	100.076	99.91
	32.5	20	26	16	58.646	36.064	100.25	100.18
	32.5	20	26	16	58.535	35.021	100.06	99.81
100%	32.5	20	32.5	20	64.902	39.940	99.85	99.82	99.893	99.93
	32.5	20	32.5	20	65.045	40.044	100.07	100.11
	32.5	20	32.5	20	64.844	39.948	99.76	99.87
120%	32.5	20	39	24	71.585	44.070	100.12	100.16	100.07	100.09
	32.5	20	39	24	71.621	44.096	100.17	100.22
	32.5	20	39	24	71.442	43.960	99.92	99.91

ACET = Acetaminophen, GUI = Guaiphenesin

 

 

Precision:

The method precision was established by carrying out the analysis of powder blend from tablets containing 325 mg of acetaminophen and 200 mg of guaiphenesin. The assay was carried out for the drugs by using proposed analytical method in six replicates. The values of relative standard deviation were 0.1306 % for acetaminophen and 0.1019 % for guaiphenesin respectively indicating the sample repeatability of the method. The results obtained are tabulated in table 3.

 

Table 3: Statistical evaluation of the data subjected to method of precision

Sr. No.	Sample No.	% Assay
		Acetaminophen	Guaiphenesin
1	1	100.16	99.94
2	2	99.92	100.15
3	3	99.87	99.97
4	4	99.89	99.91
5	5	100.04	99.89
6	6	99.81	99.87
Mean % assay		99.945	99.955
%R.S.D.		0.1306	0.1019

 

Intra-day precision was estimated by assaying tablets powder blend containing 325 mg of acetaminophen and 200 mg of guaiphenesin. The assay was carried out for the drugs by using proposed analytical method in six replicates. The results were average for statistical evaluation.

 

Table 4: Summary of validation parameter for intra-day and inter-day

Sr. No.	Parameters	Acetaminophen	Guaiphenesin
1	Intra-day precision (N=3)amount found ± % R.S.D.	99.87%   0.1205	99.81%   0.1206
2	Inter-day precision (N=3)amount found ±  % R.S.D.	98.874   0.1286	98.899%   0.1368

 

Inter-day precision was estimated by assaying tablets powder blend containing 325 mg of Acetaminophen and 200 mg of guaiphenesin for three consecutive days (i.e. 1st, 3rd and 5th days). The statistical validation data for intra and inter day precision is summarized in table 4.

 

Both intra- day and inter-day precision variation found to be less in % RSD values. It indicates high degree of precision of the method.

 

RESULT AND DISCUSSION:

The developed second order derivative spectrophotometric method for simultaneous determination of acetaminophen and guaiphenesin in tablet formulation was found to be simple and convenient for the routine analysis of two drugs. The method is used to eliminate the spectral interference from one of the two drugs while estimating the other drug by selecting the zero crossing point on the derivative spectra of each drug as the selected wavelength. The proposed method is accurate, precise and reproducible. It is confirmed from validation data as given in tables 1 to 4. The % RSD was found to be less than 1, which indicates validity of method. Linearity was observed by linear regression equation method for acetaminophen and guaiphenesin in different concentration range. The correlation coefficient of these drugs was found to be close to 1.00, indicating good linearity figure 2 (a) and 2 (b).

 

The assay results obtained by proposed method is shown in table 2 are in good agreement. Hence proposed method can be used for routine analysis of these two drugs in combined dosage form. Method is simple, accurate, precise, reliable, rapid, sensitive, reproducible and economical. It is validate as per ICH guidelines.

 

CONCLUSION:

The proposed method is simple, precise, accurate and rapid for the determination of acetaminophen and guaiphenesin in combined dosage form. This method can be adopted as an alternative to the existing methods. It can be easily and conveniently adopted for routine quality control analysis.

 

ACKNOWLEDGEMENT:

Authors express sincere thanks to the principal of D.G. Ruparel College, Dr. Tushar Desai, for encouragement and providing laboratory facilities.

 

REFERENCES:

1.        Satyanarayana M.V., Satyadev TNVSS, Anuradha V., simultaneous determination of acetaminophen and guiafenesin in pharmaceutical dosage form by validated RP-HPLC method, Indo American Journal of Pharmaceutical Research, 4(2); 2014 :1140-1152.

 

2.        Mohammad Younus, T. Karunaker Reddy, Md. Mohiuddin, Md. Fasiuddin Arif, Method development and validation for simultaneous estimation of acetaminophen and guaiphenesin in tablets, International Journal of Pharmacy and Pharmaceutical Sciences, 4, (1); 2012 : 623-626,.

 

 

 

Received on 01.03.2016         Modified on 04.04.2016

Accepted on 30.04.2016         © AJRC All right reserved