INTRODUCTION:

Subjecting the API or drug product to common stress conditions provides insight into the stability of the analytes under different conditions¹. The common stress conditions include acidic pH, basic pH, neutral pH, different temperature and humidity conditions, oxidation, reduction and photo-degradation^2-3. These studies help to determine the significant related substances to be used in method development and sample solvent that gives the best sample solution stability^4-8. In addition, the structures of the analytes will indicate the potential active sites degradation. Knowledge from basic organic chemistry will help to predict the reactivity of the functional groups⁹. The ICH guidelines have been incorporated as law in the EU, Japan and in the US, but in reality, besides these other countries are also using them. As these guidelines reflect the current inspectional tendencies, they carry the de facto force of regulation. The ICH guideline Q1A on Stability Testing of New Drug Substances and Products emphalrsizes that the testing of those features which are susceptible to change during storage and are likely to influence quality, safety and/or efficacy must be done by validated stability-indicating testing methods.

It is also mentioned that forced decomposition studies (stress testing) at temperatures in 10°C increments above the accelerated temperatures, extremes of pH and under oxidative and photolytic conditions should be carried out on the drug substance so as to establish the inherent stability characteristics and degradation pathways to support the suitability of the proposed analytical procedures^10-11.

Doxofylline is methyl xanthine derivatives¹²; it is a bronchodilator and plays a direct role in bronchial relaxation of bronchial smooth muscle. Doxofylline by inhibiting the phosphodiesterase within the smooth muscle cells and cause smooth muscle relaxation, thus achieving suppression of asthma. Doxofylline is a novel bronchodilator xanthine that differs from theophylline because the presence of a dioxalane group in position C-7^13-14. Like theophylline, mechanism of action of doxofyllines is related to the inhibition of phosphodiesterase activities. However, differently from theophylline, doxofylline appears to have decreased affinities toward adenosine A1 and A2 receptors which may account for the better safety profile of the drug^15-16.

Material and Method:

Pure samples of doxofylline were gifts from Ranbaxy Pharmaceutical, Gudgaon, India. Acetonitrile and methanol (HPLC grade) were obtained from Rankem, India. Formic Acid (AR grade) was obtained from Hi Media, India. Water from Milli Q water (Young Lin Basic 370 series was used throughout the HPLC procedure.

Selection of Wavelength:

The wavelengths were selected, to study the linearity of doxofyllin by preparing 100mg/ml solution of drug separately in various solvent systems and at the end of these study 274 nm is selected in methanol as the maximum absorbance maxima (λ_max) (fig.1).

Fig. – 01 UV Spectra of 100µg/ml solution of Doxofylline in Methanol.

Selection of Mobile Phase:

The scanning of doxofylline was done by preparing 100mg/ml solution of drug separately in combination of various solvent systems (varying the ratio and/or nature of organic modifier), at the end of these studies acetonitrile: formic Acid (90: 10); pH-3.0 was selected as the best mobile phase because in that drug was showing good elution (fig.2).

Fig. – 02 Chromatogram of 100µg/ml solution of Doxofylline using Acetonitrile: Formic Acid (90: 10) as mobile phase.

Linearity and Range:

Different dilutions of doxofylline between 1.0-200 µg/ml were scanned at their respective 274nm (λmax) in HPLC and found that doxofylline follow linearity between 1.0-200µg/ml.

METHOD VALIDATION:

Working Calibration Curve:

Accurately weighed 100mg doxofylline was transferred into 100 ml volumetric flasks and dissolved and volume was made up to 100 ml with methanol to get a concentration of 1000µg/ml (Stock-A) and sonicate for 3 min, filtered through whatmann filter paper (no 41). 10 ml of stock-A of doxofylline was taken in 100 ml volumetric flasks and diluted up to 100ml to give concentration of 100µg/ml (Stock-B). Finally from stock solution-B of doxofylline different of 20, 30,40,60,80 and 100µg/ml were prepared for analysis.

Analysis of Tablet Sample:

Twenty tablets were taken; average weight was determined and fine powdered. Amount equivalent 100 mg doxofylline was taken in 100 ml volumetric flask. This was dissolved in methanol and sonicate for 3 min. The volume was made up to mark with methanol and filtered through whatmann filter paper (no 41). Filtrate was further diluted with solvent get the final concentration of the drug on the working range. The responses of final dilutions were observed at selected wavelengths and the concentrations were obtained from regression equation. The procedure was repeated for three times.

Accuracy:

To test accuracy, recovery studies were performed. To a preanalyzed sample solution, a definite concentration of standard drug was added and then its recovery was studied. Different concentration of pure drug (Doxofylline) was added to preanalysed tablet sample, and then the solution was analyzed in the same manner. It was repeated for three times to emphasize validation.

Precision:

Standard stock solutions of Doxofylline were prepared in same manner and repeatability was performed for three times for all concentration. The intermediate precision was performed by doing day-to-day variation, analyst- to- analyst variation.

Robustness:

The robustness of the method was established by making deliberate minor variations in the flow rate and mobile phase composition.

ACID DECOMPOSITION:

Different molar concentrations of HCl were refluxed with doxofylline at varied temperature and time period. All samples were subjected to HPLC analysis. The initial analysis of different stressed samples was performed on HPLC system using a C-18 column and mobile phase composed of acetonitrile: formic Acid (90: 10); pH-3.0. It was filtered through 0.45 μm nylon filter and sonicated before use.

Fig. – 03 Chromatogram of pure Doxofylline and Acid Degradation product in Acetonitrile : Formic Acid (90: 10)

The injection volume was 20μl and the flow rate was set at 1ml/min. The detection was carried out at 274nm. Each time four samples were generated. At the end of these studies 5M HCl was used and refluxed for 02 hrs at 70⁰C in dark in order to exclude the degradative effect of light (fig. 3 and 4).

RESULT AND DISCUSSION:

A sensitive, selective, precise and accurate high performance liquid chromatographic method of analysis of doxofylline in both as bulk drug and in formulation was developed and validated. The mobile phase consisted of acetonitril: 0.05M formic Acid (90: 10v/v); pH-3.0. The detection wavelength was 274nm. This system was found to give the sharp peak for Doxofylline (RT-2.9). The method was validated as per ICH guideline (Table 1). Stability indicating assay method in which acid stress condition was used for quantitative estimation of doxofylline in tablet formulation and identification of acid degradation product The separation of drug from its degradation product were optimized by varying the ratio and/or nature of organic modifier. Finally method was developed using same mobile phase composed of acetonitrile: formic Acid (90: 10); pH-3.0, in that both drug and degradation product showing good elution RT-2.9 (Doxofylline) and RT-4.8 (Acid degradation product) and m/e-149. The acid degradation product and pure drug were identified by LC-MS/MS in order to establish acid degradation pathway (fig 5 and 6).

Fig.-05 Acid degradation pathway of doxofylline

Fig.-06 Acid degradation product of doxofylline

Table – 01 Result of method development and validation

Parameter		Condition/ Value
Mobile Phase (HPLC)		Acetonitril : 0.05 M Formic Acid
Diluent		Methanol
Flow Rate		1.0/ml
Column		Inertsil, C8, 250 mm X 4.6 mm, 5µ.
Injection Volume		20mL
Mobile Phase (LC-MS/MS)		Acetonitril : 0.05 M Formic Acid
lMAX		274nm
Correlation Coefficient (r²)		0.9991
Slope (m)		49.232
Y-Intercept		0.0465
Linearity and Range		1-200mg/ml
LOD		0.106
LOQ		0.301
Robustness		Robust
Theoretical plates		2982.60
Tailing factor		1.127
Repeatability	S.D.	0.179
	R.S.D	0.532
Day-to-Day	S.D.	0.134
	R.S.D	0.295
Analyst-to-Analyst	S.D.	0.127
	R.S.D	0.261
Accuracy	MEAN	99.32
	S.D.	0.3111
	R.S.D.	0.630
Synasma (Tablet formulation	Mean	98.75
	S.D.	1.489
	RSD	1.51

CONCLUSION:

Stress testing (or forced degradation studies) is an important part of the drug development process and the pharmaceutical industries have considerable interest in this topic. Although the concept of stress testing is not new to the pharmaceutical industry, the procedure was not clearly defined until the International Conference on Harmonization (ICH) provided a definition in its guidance on stability. The ICH guideline indicates that stress testing is designed to help “determine the intrinsic stability of the molecule by establishing degradation pathways in order to identify the likely degradation products and to validate the stability indicating power of the analytical procedures used.” In present work, validated stability indicating assay method was developed for determination of Doxofylline in presence of its degradation products. Doxofylline undergo degradation in acid stressed condition to give one degradation product. The LC-MS/MS analysis and further fragmentation and characterization suggest that doxofylline undergoes hydrolytic decomposition to give one degradation product at RT 4.8 and found that acid hydrolysis gives degradation product of m/e-149. The structure of the aforementioned products is yet to be identified and its merits future studies.

REFERENCES:

1. D.D. Hong, M. Shah (2000) Development and validation of HPLC stabilityindicating assays. Drug stability: principles and practices. J.T. Carstensen, C.T. Rhodes (Eds.) Marcel Dekker. New York, 329– 384.

2. S. Singh, M. Bakshi (2002) Development of validated stability -indicating assay methods-critical review, J. Pharm. Biomed. Anal. 28: 1011 –1040.

3. S. Ahuja, S. Seypinski (2001) Handbook of modern pharmaceutical analysis, academic press New York. 346-356, 415-441.

4. S.K. Baveja, S. Singh (1987) Thin-layer chromatographic examination of the degradation of centbucridine in aqueous solutions. J. Chromatogr. 396: 337–344.

5. R.Gannu, S.Bandari, S.G.Sudke, Y.M.Rao, B.P.Shankar (2007) Development and Validation of a Stability-Indicating RP-HPLC Method for Analysis of Doxifylline in Human Serum. Application of the Method to a Pharmacokinetic Study, Acta Chromatographica. 19: 149-156.

6. Badhe R.V., Ghulekar S.P. Thomas A.B. (2006) Stability indicating assay for the determination of Mizolastine in bulk and Pharmaceutical dosage form. Indian Drugs. 43: 199-204.

7. Mhaske Deepali Vijay, Dhaneshwar Sunil Rajaram (2007) Stability indicating HPTLC method for the determination of Irbesartan Pharmaceutical dosage form. IJPER. 41: 261-268.

8. Sundar Natarajan, Bhanu Raman (2007) Simultaneous Estimation of acetaminophen and aceclofenac from tablet using stability indicating isocratic HPLC method. Indian Drugs 44: 95-101.

9. S.K. Baveja, S. Singh, Thin-layer chromatographic examination of the degradation of centbucridine in aqueous solutions, J. Chromatogr., 1987,396, 337– 344.

10. S.P. Puthli, P.R. Vavia (2000) Stability indicating HPTLC determination of piroxicam. J. Pharm. Biomed. Anal. 22: 673 –677.

11. J.P. Salo, H. Salomies (1996) Two stability -indicating UV spectrophotometric methods for the analysis of hydrolyzed tinidazole. J. Pharm. Biomed. Anal.14: 1261–1266.

12. Nimmagadda Sreenivas, M Lakshmi Narasu, B Prabha Shankar, Ramesh Mullangi (2008) Development and validation of a sensitive LC-MS/MS method with electrospray ionization for quantitation of doxofylline in human serum: application to a clinical pharmacokinetic study. J. Biomed Chromatography. 6: 18254143-48.

13. F Tagliaro, R Dorizzi, A Frigerio and M Marigo (2001) Non-extraction HPLC method for simultaneous measurement of dyphylline and doxofylline in serum. Clinical Chemistry. 36: 113-115.

14. Franzone JS, Cirillo R, Barone D (1999) Doxofylline and theophylline are xanthines with partly different mechanisms of action in animals. Clinical Chemistry. 38: 128-132.

15. Franzone, J S: Cirillo, R: Biffignandi, P (1989) Doxofylline exerts a prophylactic effect against bronchoconstriction and pleurisy induced by PAF. Eur-J-Pharmacol.165: 269-77.

16. Frank Lloyd Dini, Unità Operativa di Cardiologia, Ospedale Villamarina, Piombino, Italy; and Roberto Cogo, Unità di Pneumologia Riabilitazione Respiratoria, Ospedale Zappatoni, Cassano d'Adda, Italy (2001) Doxofylline: A New Generation Xanthine Bronchodilator Devoid of Major Cardiovascular Adverse Effects. Current Medical Research Opinion. 16: 258-268.

Received on 21.02.2011 Accepted on 18.03.2011

Asian J. Pharm. Ana. 1(1): Jan.-Mar. 2011; Page 10-13