1. INTRODUCTION:

Theophylline¹ (TH) is chemically, 1, 3-dimethyl-3, 4, 5, 7-tetrahydro-1H-purine 2, 6-dione (Fig.1). TH is a bronchodilator drug commonly used for the asthmatic condition. It works by stimulation of β- adrenoreceptors which activates adenylate cyclase leading to an increase in intracellular cyclic AMP and decrease in intracellular concentration causing smooth muscle relaxation². Several chromatographic^3,4, spectrophotometric⁵ and titrimetric⁶ methods have been reported for determination of TH in raw material, tablets, plasma samples and pharmaceutical preparations. Etofylline⁷ (ETH) is chemically, 7-(2-hydroxyethyl)-1, 3-dimethyl-3, 4, 5, 7-tetrahydro-1H-purine-2, 6-dione (Fig.2).

It inhibits phosphodiesterase, which degrades cyclic nucleotides, hence increases amount of intra cellular cyclic AMP molecules causing smooth muscle relaxation. Etofylline is official in IP and BP, where titrimetric methods for Etofylline in bulk and tablet formulation were reported^8,9. Several analytical techniques like spectrophotometric methods^10,11 have been reported for Etofylline in combination with other drugs. In recent years pharmaceutical preparations containing both these drugs have been available commercially. Both of these molecules have great similarity in their structures and overlapping UV spectra which cause difficulty in estimating both the drugs in combination using UV spectrophotometric method. Only HPLC methods have been reported in the literature for estimation of TH and ETH in combination^12-14. Even though HPLC methods are highly sensitive and specific but the cost per analysis is high. In the present work an attempt has been made to develop UV spectrophotometric methods for the simultaneous determination of TH and ETH in tablet dosage form.

Fig 1

Fig 2

2. MATERIAL AND METHODS:

2.1 Instrument:

1. Shimadzu^®UV 1800 and UV 1700 double beam UV-visible spectrophotometer was used along with 1.0 cm path length matched pair of quartz cell for spectrophotometric method.

2. Shimadzu AUX 220 balance.

3. Calibrated glassware used for the study.

4. Sonicator (Enertech Electronics).

2.2 Materials:

Standard gift samples of TH and ETH were obtained from Zim Laboratories Ltd., Kalmeshwar, Nagpur. Tablets containing both Theophylline and Etofylline were purchased from local market. Sodium hydroxide A.R grade was procured from Loba Chem. Ltd., Mumbai. Double distilled water was used for making 5%w/v NaOH.

2.3 Selection of common solvent:

Absorbance maxima of both the drugs was checked in various solvents. Table 1 gives the list of solvents and the λmax obtained both for TH and ETH.

Table1: Absorbance maxima of TH and ETH in different solvents

Sr.No.	Solvents	Absorbance maxima (λmax)
Sr.No.	Solvents	TH	ETH
1	Distilled water	271.4nm	273.0nm
2	Methanol	271.0nm	273.0nm
3	Chloroform	272.0nm	273.1nm
4	0.1N HCL	270.6nm	273.2nm
5	0.1N NaOH	275.2nm	273.0nm
6	5% NaOH	275.0nm	243.5nm

2.4 Preparation of Stock solutions:

The stock solutions (500 µg/ml) of TH and ETH were prepared separately by dissolving accurately about 50 mg of drug in 100 ml of 5% w/v NaOH solution.

2.5 Selection of Wavelength:

The stock solutions of TH and ETH were appropriately diluted with 5% w/v NaOH to obtain the solutions of 20 g/ml concentration and the resulting solutions were scanned in the wavelength range (400-200 nm) in 1.0 cm cell against solvent blank. UV absorbance spectra of both the drugs is shown in figure 3.

Figure 3 : Overlain spectra of TH and ETH in 5%w/v NaOH

2.6 Study of Beer-Lambert’s law:

Aliquots of working stock solution of TH and ETH were diluted with 5%w/v NaOH to get a concentration in the range of 5-25 μg/ml for TH and ETH individually and in mixture. Absorbance of each of the resulting solution was measured at 243.4, 254.5 and 275.0 nm in 1.0 cm cell using solvent blank. The graphs were constructed as concentration vs. absorbance and correlation coefficient was calculated.

2.7 Determination of Absorptivity of TH and ETH:

Working standard solutions i.e. TH 10 μg/ml and ETH 10 μg/ml were used for the present study. The procedure for preparation of both the drugs were repeated five times and absorbance of each of the solutions were measured in triplicate against solvent blank at 243.4, 254.5 and 275.0 nm and the A(1 % 1 cm) values were calculated using the formula

2.8 Estimation in standard laboratory mixture:

Accurately measured aliquot portions of stock standard solutions of TH and ETH were mixed and diluted appropriately to get concentration in the ratio of 20:20, 20:5, 5:25, 7:23, 6.9:23.1, 6.9:23.1, 4:11, 3.5:11.5 of TH and ETH respectively in the mixture. The absorbance of each resulting solution was measured at 243.5, 254.5 and 275 nm in 1.0 cm cell using 5% w/v NaOH as blank. The contents of TH and ETH were calculated by substituting values in the formulae given below,

Method 1: Simultaneous equation method:

For estimation of TH

For estimation of ETH

Where, A1 and A2 are absorbances of mixture at 243.5 nm and 275.0 nm respectively, ax1 and ax2 are absorptivities of Theophylline at λ1 and λ2 respectively and ay1 and ay2 are absorptivities of Etofylline at λ1 and λ2 respectively. Cx and Cy are concentrations of Theophylline and Etofylline respectively.

Method II: Absorbance ratio or Q-analysis method:

For estimation of TH

For estimation of ETH

Where, Cx = Concentration of TH in g/100 ml, Cy = Concentration of ETH in g/100 ml, Qm = Ratio of absorbance of laboratory mixture at 275 nm and 254.5 nm, Qx = Ratio of absorptivity of TH at 275 nm and 254.5 nm, Qy = Ratio of absorptivity of ETH at 275 nm and 254.5 nm, a₂ = Absorptivity of TH and ETH at 254.5 nm, A₂ = Absorbance of mixture at isoabsorptive point.

2.9 Estimation of TH and ETH in marketed formulation:

Accurately weighed quantity of tablet powder equivalent to 50 mg of TH (~ 163.0 mg of ETH) was transferred to 100.0 ml volumetric flask and 25 ml of 5% w/v NaOH was added. The solution was sonicated for 10 min. and volume was made up to mark with the same solvent. The contents of flask were filtered through 0.45μ membrane filter paper. A 1.0 ml portion of the filtrate was further diluted to 100.0 ml with 5% w/v NaOH to get final concentration of about 5 μg/ml TH, 16.3 μg/ml ETH (on labeled claim basis). The absorbance of resulting solutions was measured at 243.5, 254.5 and 275 nm in 1.0 cm cell using solvent blank. Quantities of both TH and ETH were estimated by substituting the values in the equation used for standard laboratory mixture. Percent label claim was also determined and recorded in Table 2.

2.10 Recovery Studies:

It was performed by standard addition method. To the pre-analyzed powder, known quantities of TH and ETH were added at three different levels. The contents were dissolved in 5% w/v NaOH, filtered and the absorbances of each solution were measured at 243.5, 254.5 and 275.0 nm against blank. Percent recovery was calculated by using the formula, The results are recorded in Table 3.

2.11 VALIDATION:

Accuracy:

To check the accuracy of the developed methods and to study the interference of formulation additives, analytical recovery experiments were carried out by standard addition method. Total amount of drug found and percentage recovery was calculated and results are reported in Table 3.

Table 2: Linear regression analysis of calibration curves with their respective absorptivity values

Concentration (mcg/ml)	Theophylline			Etofylline
Concentration (mcg/ml)	243.5nm	254.5nm	275nm	243.5nm	254.5nm	275nm
5	0.084	0.108	0.276	0.148	0.119	0.047
10	0.187	0.232	0.533	0.275	0.23	0.08
15	0.278	0.347	0.822	0.421	0.322	0.115
20	0.373	0.464	1.145	0.551	0.425	0.159
25	0.458	0.573	1.425	0.689	0.533	0.198
Corr. Coeif.	0.999	0.999	0.998	0.999	0.998	0.997
Slope	0.018	0.023	0.057	0.027	0.021	0.007
Intercept	-0.002	-0.001	-0.015	0.004	0.009	0.002
LOD	0.146	0.0984	0.094	0.109	0.182	0.325
LOQ	0.442	0.298	0.287	0.331	0.553	0.985
Absorptivity	183.84	229.876	531.76	279.3	230.1	81.2

Table 3 : Summary of results of estimation of Theophylline and Etofylline and Recovery studies

Sr.No.	Sample	Statistical parameter	% label claim*				% Recovery**
			Method A		Method B		Method A		Method B
			TH	ETH	TH	ETH	TH	ETH	TH	ETH
1	Standard Lab. Mixture	Mean	100.3	100.3	99.75	100.3	-	-	-	-
		 SD	1.83	1.75	1.61	0.51
		% RSD	1.82	1.75	1.61	0.51
2	Marketed tablet formulation	Mean	101.9	101.7	101.2	100.6	99.3	99.6	99.2	99.5
		 SD	1.80	1.72	1.61	1.501	0.85	1.5	0.86	0.96
		% RSD	1.76	1.69	1.59	1.49	0.86	1.5	0.87	0.97

* mean of six determinations, ** mean of five determinations

Precision:

Precision of analytical method is expressed in terms of SD, %RSD of series of measurements. Study was carried out by replicate analysis of homogeneous samples of tablet powder. Analytical procedure was repeated after 0 hr, 3rd hr, 5th hr at selected wavelength for intraday study. Similarly estimations were also repeated on 1st, 3rd, and 5th day and % label claim was calculated. The results are given in Table 4.

Table 4 : Summary of validation parameters

Parameters	Method A		Method B
Parameters	TH	ETH	TH	ETH
Intra Day Precision (n=3)
Amount found ±	98.66	99.42	98.29	99.34
RSD (%)	1.07	0.62	0.88	0.59
Inter Day Precision (n=3)
Amount found ±	98.21	98.08	98.12	99.19
RSD (%)	2.01	2.1	1.56	1.42
Ruggedness (%RSD)
Different analyst	1.0318	1.5934	0.2078	0.7168
Different instrument	1.8517	1.0812	1.6789	1.0486

Method A = Simultaneous Equation Method, Method B = Absorbance Ratio Method

Linearity and Range:

The study was performed over the series of concentrations ranging from 0-25 µg/mL of TH, ETH and mixture of TH/ETH. The graph of concentration vs. absorbance were found to be straight line over the concentration range of 0-25 µg/mL of TH and ETH individually and also in mixture. The r² value is given in Table 2.

Ruggedness:

Different Analyst: The tablet sample was analyzed by proposed method by three different analysts and results are given in Table 4.

Specificity:

Accurately weighed six separate quantities of finely powdered tablets equivalent to about of 35 mg TH (also equivalent to 115 mg ETH), were transferred to 100.0 mL volumetric flasks and kept under the following conditions viz., Normal, Alkali (0.1 N NaOH), Acidic (0.1 N HCl), 3% H₂O₂ for 24 hrs (50^oC) for oxidation stress, Heat (60^oC) for 24 hrs and UV exposure at 254.0 nm for 24 hrs. After completion for different stress condition dilutions were made as described under marketed formulation. The absorbances of each of the above solutions were measured at 243.5, 254.5 and 275.0 nm against blank. Results were calculated as described under marketed preparation. Results are given in Table 5.

Table 5 : Results of Specificity Study

Sr.No.	Sample	%Label claim
		Simultaneous equation method		Absorbance ratio method
		ETH	TH	ETH	TH
1	Normal	100.79	98.28	101.59	97.71
2	HCL	104.88	98.28	104.44	98.57
3	NaOH	105.23	105.71	105.77	105.42
4	UV-chamber	118.11	71.42	118.73	71.14
5	60 heat	101.24	97.42	101.77	97.14
6	H2O2	109.41	59.14	107.99	59.71

3. RESULTS AND DISCUSSION:

The preliminary UV spectral studies have shown sharp and distinct absorption maxima for both the drugs in 5% w/v NaOH. An overlapping UV spectrum of TH and ETH with well separated absorption maxima prompted the development of simultaneous spectrophotometric estimation method adopting Vierodt’s method/ Simultaneous equation method and absorption ratio method. The overlain spectra exhibit TH shows well defined λmax at 275 nm, whereas ETH shows λmax at 243.5 nm. The two drugs also show an isoabsorptive wavelength at 254.5 nm where both the drugs have same absorptivity value. Of these, the wavelengths 243.5 and 275 nm were considered for development of Vierodt’s method/ simultaneous equation method whereas 254.5nm and 275 nm for absorbance ratio method. Standard calibration curves for ETH and TH were linear with correlation coefficients (r2) values in the range of 0.997- 0.999 at all the selected wavelengths. The method was repeated for three different days and average % RSD was found to be <2.1 for ETH and <2.01 for TH. The accuracy of the method was confirmed by recovery studies from synthetic mixtures at three different levels of standard additions; recovery in the range of 98 – 102% justifies the accuracy of method. LOD values of calibration curves indicates the lowest concentration of analyte(s) in a sample that can be detected under a stated experimental conditions and LOQ values of calibration curves indicates the lowest concentration of analyte(s) in a sample that can be determined with acceptable precision and accuracy under the stated experimental conditions. The specificity of the method concluded that the proposed spectrophotometric method is reasonably specific for determination of Theophylline and Etofylline.

CONCLUSION:

The proposed methods for simultaneous estimation of Theophylline and Etofylline in combined dosage forms were found to be simple, accurate, precise, economical and rapid. In both the methods, percentage recovery was close to 100% and % RSD was less than 2% for both the drugs. Hence, it can be employed for routine analysis in quality control laboratories.

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Received on 03.10.2014 Modified on 20.10.2014

Asian J. Research Chem. 7(12): December, 2014; Page 1030-1034