1. INTRODUCTION:

Pentoxifylline (3,7-dimethyl-1-(5-oxohexyl)-3,7-dihydro-1H-purine-2,6-dione) (Figure I) is used to treat intermittent claudication resulting from obstructed arteries in the limbs, and vascular dementia. Pentoxifylline improves blood flow through blood vessels and therefore helps with blood circulation in the arms and legs¹. Few methods have been reported for the quantification of pentoxifylline in plasma, which involves gas chromatography and high-performance liquid chromatography with ultraviolet detection^2-9. Recently, the use of high throughput bioanalytical procedures using LC-MS-MS has been reported¹⁰. Few HPTLC methods also have been reported for quantification of Pentoxifylline^11-12. Some UV and HPLC methods also have been reported¹³. However till now, no stability indicating method for estimation of Pentoxifylline has been reported. The present work describes the development of a simple, precise and accurate method for the estimation of pentoxifylline in presence of its degradation products in bulk drugs and marketed formulation. The method validation and forced degradation studies were carried out as per ICH guidelines^14-15.

2. MATERIALS AND METHODS:

Pentoxifylline (purity 99.8 %) was provided as a gift sample by Aventis Pharma Limited, Goa, India. and was used without further purification. The drug was received along with certificate of analysis.

All the other reagents used were of analytical grade. Chloroform (AR grade), Acetone (AR grade), Methanol (AR grade), Toluene (AR grade), Dioxane (AR grade) NaOH (AR grade), HCl (AR grade), H₂O₂ (AR Grade) were purchased from Thomas Baker (chemicals) Pvt Limited, India.

2.1 Instrumentation:

Chromatographic separation of drugs were performed on Merck TLC plates precoated with silica gel 60 F₂₅₄ (10 cm ×10 cm with 250 mm layer thickness) from E. Merck, Germany. The samples were applied onto the plates as a band with 4 mm width using Camag 100 µl sample syringe (Hamilton, Switzerland) with a Linomat 5 applicator (Camag, Switzerland). Linear ascending development was carried out in a twin trough glass chamber (for 10 x 10 cm). Densitometric scanning was performed using Camag TLC scanner 3 in the range of 200-2000 ng/spot and operated by winCATS software (V 1.4.2, Camag).

2.2 Selection of Detection Wavelength:

After chromatographic development bands were scanned over the range of 200-400 nm. It was observed that the drug showed considerable absorbance at 275 nm. So, 275 nm was selected as the wavelength for detection (Figure II).

Fig I: Structure of Pentoxifylline.

Table I : Validation Parameters

S.N.	Validation Parameter	Pentoxifylline
1	Linearity Equation (r²) Range	y = 8.6112x + 1700.5 (0.9947) 200 – 2000 ng/spot
2	Precision (% RSD) Intraday Interday	NMT 0.9375 % NMT 1.6186 %
3	Accuracy (% mean recovery)	101.34 %
4	LOD	43.91 ng/spot
5	LOQ	131.73 ng/spot
6	Specificity Peak purity	Specific r (s, m) 0.9889 r (m, e) 0.9861

2.3. Method validation:

2.3.1 Linearity:

A stock solution of Pentoxifylline (1000 ng/μL) was prepared in Water. Different volumes of stock solution, 0.2, 0.5, 1, 1.5, 2 μL were spotted on TLC plate to obtain concentration of 200, 500, 1000, 1500, 2000 ng per spot of Pentoxifylline, respectively. The data of peak area v/s. drug amount were treated by linear least-square regression analysis.

2.3.2. Precision:

The intra and inter-day variation for the determination of Pentoxifylline was carried out at three different concentration levels of 500, 1000 and 1500 ng per spot. The % RSD values were determined for intra-day and inter-day variation.

2.3.3. Recovery studies:

The analysed samples were spiked with extra 80, 100 and 120 % of the standard Pentoxifylline and the mixtures were reanalyzed by the proposed method. The experiment was conducted in triplicate. This was done to check for the recovery of the drug by standard addition method.

2.3.4. Limit of detection and limit of quantification:

The limit of detection (LOD) and limit of quantification (LOQ), were obtained by calculating, using the standard formula as per the ICH guidelines, where σ is the standard deviation of response.

2.3.5. Specificity:

The specificity of the method was ascertained by peak purity profiling studies. Purity of the drug peaks was ascertained by analyzing the spectrum at peak start, middle and at peak end. The peak purity was determined on winCATS software using statistical equation.

Fig. II: Spectrum of Pentoxifylline.

2.4. Analysis of the marketed formulation:

To determine the content of Pentoxifylline in tablets (label claim: 400 mg per tablet). Twenty tablets were powdered and powder equivalent to 25 mg was weighed and transferred to a 25 ml volumetric flask containing about 20 ml water, the solution was filtered through whatmann filter paper and finally volume was made upto 25 ml to get the stock solution of (1000 ng/μL) Appropriate volume of solution was applied on TLC plate followed by development and scanning.

2.5. Forced degradation of Pentoxifylline:

A stock solution containing 25 mg Pentoxifylline in 25 mL distilled water was prepared. This solution was used for forced degradation.

2.5.1. Degradation under acid and alkali catalysed hydrolytic condition:

To 2.5 mL stock solution, 2.5 mL of 0.5 N NaOH and 5 N HCl were added separately. The volume was made upto 25 ml with distilled water. These mixtures were refluxed for 3 h at 80 ⁰ C. Appropriate volume of resultant solution (1000 ng per spot) was applied on TLC plate and densitograms were developed.

2.5.2. Degradation under neutral hydrolytic condition:

To 2.5 mL stock solution, distilled water was added. The volume was made upto 25 ml. The mixture was refluxed for 3 h at 80 ⁰ C. Appropriate volume of resultant solution (1000 ng per spot) was applied on TLC plate and densitograms were developed.

2.5.3 Degradation under oxidative condition:

To 2.5 mL stock solution, 2.5 mL of 30 % H₂O₂ was added. The volume was made upto 25 ml with distilled water. The mixture was refluxed for 3 h at 80 ⁰ C. Appropriate volume of resultant solution (1000 ng per spot) was applied on TLC plate and densitograms were developed.

2.5.4 Degradation under dry heat:

Dry heat studies were performed by keeping drug sample in oven (80⁰ C) for a period of 8 hours. Accurately weighed 25 mg of drug was transferred to the 25 ml of volumetric flask and dissolved in water, the volume was made up with distilled water. Appropriate volume of resultant solution (1000 ng per spot) was applied on TLC plate and densitograms were developed.

Fig III: Representative densitogram of Pentoxifylline at R_f 0.48

2.5.5 Photo-degradation studies:

The photochemical stability of the drug was also studied by exposing the drug sample to UV light up to illumination of 200 watt hr/m² followed by visible light up to illumination of 1200 lux-hr. Accurately weighed 25 mg of drug was transferred to the 25 ml of volumetric flask, the volume was made up with distilled water. Appropriate volume of resultant solution (1000 ng per spot) was applied on TLC plate and densitograms were developed.

2.6 Stress degradation of Formulation:

Pentoxifylline tablets, each containing 400 mg pentoxifylline were purchased from local market, The tablets were weighed, crushed and sample powder was exposed to stress conditions as mentioned under study for bulk drugs. Then the sample was filtered and appropriate volume was spotted on to TLC plate.

3. RESULTS AND DISCUSSION:

3.1. Development of the optimum mobile phase:

TLC procedure was optimized with a view to develop a stability-indicating assay method. The drug reference standard was spotted on the TLC plates and developed in different solvent systems. Initially, Acetone : toluene and Acetone : Dioxane with varying concentration were tried, but finally, the mobile phase Acetone : Chloroform (5:5, v/v) gave sharp and symmetrical peak with Rf 0.48. Well-defined spots were obtained when the chamber was saturated with the mobile phase for 15 min at room temperature. The representative densitogram is given in Figure III.

3.2 Validation of the method:

3.2.1 Linearity:

The response for the drugs was found to be linear in the concentration range 200–2000 ng / spot with correlation co-efficient of 0.9947.

3.2.2 Precision :

The % RSD value for intra-day and inter-day variation study was found to be not more than 0.9375 % and 1.6186 % respectively, thus confirming precision of the method.

Fig. IV: Overlain Spectra of Pentoxifylline (R_f 0.48) and its degradation product after base catalysed hydrolysis (R_f 0.27).

3.2.3 Recovery:

Excellent recoveries were obtained at each level of added concentration. The results obtained (n = 3 for each 80 %, 100 %, 120 % level) indicated the mean recovery 101.34 %

3.2.4 Limit of Detection and limit of Quantitation:

The limit of detection and limit of quantitation as calculated by standard formula as given in ICH guidelines was found to be 43.91 and 131.73 ng/spot respectively.

3.2.5 Specificity:

The specificity of the method was ascertained by peak purity profiling studies. The peak purity values were found to be r(s,m) 0.9889 and r(m,e) 0.9861 for Pentoxifylline, indicating the non interference of matrix, degradants and matrix in the peak of Pentoxifylline. .

The results are listed in Table I.

3.3Analysis of marketed formulation:

A single spot at Rf 0.48 was observed in the chromatogram of the drug samples extracted from tablets. There was no interference from the excipients present in the tablets. The drug content was found to be 99.16 %.

3.4 Stress degradation:

Pentoxifylline was found to be degraded in base induced and neutral hydrolytic conditions. Under these conditions, the peak area showed considerable reduction. The densitogram of the base-degraded sample of Pentoxifylline

showed a well resolved spot for product of degradation at R_f 0.27. The spectrum of the degradation product and pentoxifylline is shown in figure IV. While the peak area was found to be reduced in neutral and oxidative conditions, no peaks for degradation products were detected.

However the non interference by the product of degradation can be confirmed by peak purity values for Pentoxifylline peak after degradation having correlation coefficient r(s,m) 0.9889 and r(m,e) 0.9861. Limit for peak purity values were set to > 0.950 for both r(s,m) and r(m,e). It confirms that degradation product of drug can be separated from that of drug by this method. No degradation was seen in acid induced hydrolytic, oxidative, dry heat and photodegradation conditions. There were no extra peaks seen and the peak areas of the drug also showed no significant change. The results for stress degradation studies for Pentoxifylline bulk drug and tablet formulation are listed in Table II.

4. CONCLUSION:

From the above study we can conclude that Pentoxifylline undergo degradation to different extent under different, above mentioned, stress conditions. From the peak purity profile studies, it was confirmed that the peak of the degradation product was not interfering with the peak of Pentoxifylline. It confirms that degradation product of drug can be separated from the drug by this method. Since different formulations of same drug are available in the market so the developed method can also be used for the stability study of marketed formulations also.

5. ACKNOWLEDGEMENT:

The authors wish to express their gratitude to Aventis Pharma Limited, Goa, (India), for providing working standard of Pentoxifylline. The authors are also thankful to The Principal, AISSMS College of Pharmacy for providing necessary facilities to carry out the research work.

6. REFERENCES:

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4. Mancinelli A. et al. Determination of pentoxifylline and its metabolites in human plasma by high-performance liquid chromatography with solid-phase extraction. J Chromatogr B, 1992; 575: 101–107.

5. Musch G. et al. Determination of pentoxifylline and its 5-hydroxy metabolite in human plasma by solid-phase extraction and high-performance liquid chromatography with ultraviolet detection. J Chromatogr B, 1989; 495: 215–226.

6. Burrows J.L. Determination of pentoxifylline and three metabolites in plasma by automated capillary gas chromatography using nitrogen-selective detection. J Chromatogr B, 1987; 423: 139–146.

7. Grasela D.M. and Rocci M.L. Jr. High-performance liquid chromatographic analysis of pentoxifylline and 1-(5'-hydroxyhexyl)-3,7-dimethylxanthine in whole blood. J Chromatogr B, 1981; 419: 368–374.

8. Bauza M.T. Gas chromatographic determination of pentoxifylline and its major metabolites in human breast milk. J Chromatogr B, 1984; 310: 61–69.

9. Chivers D.A. et al Simultaneous determination of pentoxifylline and its hydroxy metabolite in plasma by high-performance liquid chromatography. J Chromatogr B, 1981; 225: 261–265.

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13. Christova-Bagdassarian V. et al. UV spectrometric and high performance liquid chromatographic determination of Pentoxifylline in workplace air. Journal of the University of Chemical Technology and Metallurgy, 2007; 42: 223-227.

14. Validation of Analytical Procedures: Text And Methodology Q1A (R2), ICH Harmonized Tripartite Guideline, Feb 2003.

15. Stability Testing Of New Drug Substances And Products Q2 (R1), ICH Harmonized Tripartite Guideline, Nov 2005.

Received on 16.03.2009 Modified on 14.05.2009

Research J. Pharm. and Tech.2 (3): July-Sept. 2009,;Page 527-530

S. No.	Conditions	% of Pentoxifylline degraded
S. No.	Conditions	Bulk Drug	Formulation
1.	Acid (5 N HCl, reflux, 3 hours)	6.4 %	8.52 %
2.	Base (0.5 N NaOH, 3 hours)	58.6 %	56.89 %
3.	Water (reflux, 3 hours)	14.35 %	13.5 %
4.	Hydrogen peroxide 30% (reflux, 3 hours)	5.89 %	No degradation
5.	Heat dry (80 ⁰C, 8 hours)	9.1 %	5.8 %
6. (a)	Photostability UV 200 watt hours/square meter	No degradation	No degradation
(b)	Visible 1200 Lux.Hr	No degradation	No degradation