INTRODUCTION:

ETO is almost white crystalline powder and is mainly used as bronchodilator. The chemical name is 7-(2-hydroxyethyl)-1, 3-dimethyl-3, 7dihydro-1H-purine-2, 6-dione¹. THEO indicated for the chronic obstructive diseases of the air ways, chronic obstructive pulmonary diseases (COPD) bronchial asthma, infant apnea². Chemically THEO is 1, 3-dimethyl-7H-purine-2, 6-Dione. Many methods^3-6 were described in the literature for the determination of ETO individually and in combination with other drugs. The techniques include serum RP-HPLC⁷, TLC methods^8-9 and derivative spectrophotometric method¹⁰. However there is no RP-HPLC method reported for the simultaneous estimation of these drugs in combined dosage forms. The aim of this work was to develope and validates RP-HPLC method with ultraviolet detection for the simultaneous determination of ETO and THEO in pharmaceutical dosage forms.

MATERIALS AND METHODS:

Chemicals and solvents:

A HPLC grade methanol, Potassium dihydrogen phosphate were procured from Merck, India. High pure water was prepared by using Millipore Milli Q plus purification system for the buffer preparation.

Chromatographic Conditions:

A High Performance Liquid Chromatograph system, with LC solutions data handling system (Shimadzu-LC 2010) was used for the analysis. The purity determination performed on a stainless steel column 150 mm long, 4.6 mm internal diameter filled with Octadecyl silane chemically bonded to porous silica particles of 5 mm diameter (Inertsil C18, 5 mm, 150 mm x 4.6 mm, make: Shimadzu ltd, Japan) with the mobile phase containing methanol and phosphate buffer in the ratio of 75: 25 v/v (pH 3.0 ± 0.05) at ambient temperature. Flow rate was kept at 1.0 ml/min and the eluents were monitored at 241 nm.

Standard solution and calibration curve:

A standard stock solution of ETO (100 mg/ml) and THEO (100 mg/ml) was prepared in methanol. Subsequent dilutions were made in mobile phase to give the concentrations 50, 100, 150, 200 and 250 µg/ml for ETO and 15, 30, 45, 60, 75 µg/ml for THEO. The calibration curve was obtained by plotting the ratio of peak area of drug versus concentration.

Assay:

Twenty tablets were weighed accurately and finely powdered. The powder equivalent to 25 mg of ETO 8.5 mg of THEO were weighed accurately and dissolved in 50 ml methanol (HPLC).The resulting solution was sonicated for 15 minutes. The solution was filtered through 0.45 μ membrane filter.

After setting the chromatographic conditions and stabilizing the instrument to obtain a steady baseline. The solution was injected and a chromatogram was recorded. The injections were repeated five times and the peaks were recorded. A representative chromatogram has been given in Fig.1. The peak areas of each of the drugs were calculated and the amount of each drug present per tablet was estimated from the calibration curve. The results of analysis are presented in Table 1.

Linearity:

The linearity of the method was determined at five concentration levels ranging from 50 to 250 μg/ml for ETO and 15 to 75 μg/ml for THEO. The calibration curve was constructed by plotting response factor against concentration of drugs. The results show that an excellent correlation exists between response factor and concentration of drugs within the concentration range indicated above.

Accuracy (Recovery Test):

The accuracy of the method was determined by recovery experiments. The recovery studies were carried out six times and the percentage recovery were calculated and presented in Table 2. From the data obtained, added recoveries of standard drugs were found to be accurate.

Precision:

The precision of the method was demonstrated by inter day and intraday variation studies. In the intraday studies, six repeated injections of standard and sample solutions were made and the response factor of drug peaks and percentage RSD were calculated. In the inter day variation studies, six repeated injections of standard and sample solutions were made for three consecutive days and response factor of drug peaks and percentage RSD were calculated. From the data obtained, the developed HPLC method was found to be precise.

Limit of Detection (LOD) and Limit of Quantification (LOQ):

The LOD and LOQ of ETO and THEO were determined by using standard deviation of the response and slope approach as defined in International Conference on Harmonization (ICH) guidelines .The LOD and LOQ were calculated and represented in Table no.2.

Table 2: summary and validation

Parameter	Observation
Parameter	ETO	THEO
Label claim (mg/tab)	77	23
% Label claim	100.19	101.95
% RSD	0.2821	0.1464
Linearity range (µg/ml)	50-250	15-75
Correlation coefficient	0.9997	0.9999
Asymmetry factor	1.20	1.12
Number of Theoretical Plates	2400	3200
System precision	0.6404	0.2092
Method precision	0.2814	0.1464
% Recovery	100.16	100.17
Limit of Detection (µg)	2.3	1.98
Limit of Quantification (µg)	7.2	6.0

RESULTS AND DISCUSSION:

Our method is simultaneous determination of ETO and THEO by RP-HPLC using a UV detector. Elution was carried out using mobile phase consist of methanol and phosphate buffer (pH 3.0 ± 0.05) in the ratio of (75: 25) v/v and flow rate was set on 1 ml/min at 241 nm. The retention time for ETO and THEO were 2.78 and 5.08 min, respectively, run time was 10 min. Overall summary and validation parameters were presented in Table2.

CONCLUSION:

A simple, specific, linear, precise and accurate RP-HPLC method has been developed and validated for quantitative determination of ETO and THEO in tablet formulation. The method is very simple and specific as both peaks are well separated with total runtime was 10 min, which makes it especially suitable for routine analysis work.

ACKNOWLEDGEMENTS:

The authors thank management of Chalapathi Institute of Pharmaceutical Sciences for providing the facilities.

REFERENCES:

1. United States of pharmacopoeia United Kingdom. 1988; 1, 238.

2. Indian pharmacopoeia government of India. 1996; 2, 752.

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9. Schlemmer W. Application of quantitative thin-layer chromatography in drug assay and stability testing: determination of codeine phosphate, noscapine, diphenhydramine hydrochloride, phenylephrine hydrochloride, caffeine, etofyllin, Phenobarbital and thiamine hydrochloride by in situ reflectance spectroscopy. Journal of chromatography A. 1973; 82, 143.

10. Dave HN, Mashru RC and Thakkar AR. Simultaneous determination of salbutamol sulphate, bromhexine hydrochloride and etofylline in pharmaceutical formulations with the use of four rapid derivative spectrophotometric methods. Analytica chimica Acta. 2007; 597, 113.

Received on 21.06.2010 Modified on 03.07.2010

Research J. Pharm. and Tech. 4 (1): January 2011; Page 128-130

Tablet Sample	Label Claim (mg)	Amount present (mg/tablet)	%Label Claim	%RSD
ETO	77	77.152	100.19	± 0.152
THEO	23	23.45	100.95	±0.45