Validated and Sensitive Liquid Chromatography – Spectrophotometric Method for the Estimation of Doxofylline in Bulk Drug Formulation

M.V. Kumudhavalli*, V. Nandhinipriya¹, D. Umamaheswari¹, M. Kumar¹, B.S.Venketeswarlu¹      JSK. Nagarajan²

¹Department of Pharmaceutical Analysis, Vinayaka Mission’s College of Pharmacy, Yercaud Main Road, Kondappanaikenpatty, Salem, Tamilnadu 636008

²Department of Pharmaceutical Analysis, JSS College of Pharmacy, Ooty,

 

ABSTRACT:

 

 

INTRODUCTION:

Doxofylline, 2-(7’-Theophyllinemethyl)-1,3-Dioxolan is a new generation long acting oral bronchodilators. Doxofylline is a methylxanthine derivatives used in the treatment of asthma and Chronic obstructive pulmonary disease^[1]. The empirical formula of Doxofylline is C₁₁H₁₄N₄O₄ and its molecular weight is 266.25. It works by relaxing the muscles of the air way and widens airway effectively^[2]. Available form of Doxofylline for oral administration is 400mg and 800mg. A literature survey revealed that different high-performance liquid chromatographic (HPLC) methods for determination of Doxofylline in combination have been reported^[3-12].

Accepted on 18.09.2019           © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(2):742-746.

There is no method reported for the determination of Doxofylline. Hence, the present work aim for the development of UV spectrophotometric and RPHPLC method and determination of Doxofylline with possible shortest retention time. According to the ICH guidelines,the proposed method has also been validated^[13]

 

 

MATERIALS AND METHODS:

Experimental:

Chemicals and reagents:

HPLC grade Methanol and AR grade trimethylamine were obtained from E.Merck (India) LTD, Mumbai. AR grade Disodium hydrogen phosphate were obtained from Nice chemicals Pvt. Ltd. O-Phosphoricacid were obtained from Universal laboratories ltd.

 

Pure Doxofylline IP was obtained as a gift sample from Dr. Reddy’s Lab Ltd. hydrabad. Triple distilled Water from In House Production was used to prepare the sample and standard solutions. Pure Doxofylline IP was obtained as a gift sample from Dr. Reddy’s Lab ltd, Hydrabad. The used pharmaceutical preparation Doxobid 400mg tablets were purchased from local market and it was manufactured by Ranbaxy company.

 

Methods:

Instrumentation:

Shimadzu (UV 1601) UV- visible spectrophotometer with quartz cells of 1cm optical length incorporated with a PC computer was used, HPLC instrument equipped with UV-detection chromatographic separation was achieved at ambient temperature on column symmetry C18 (4.6x250mm, 3.5µm). The flow rate and runtime was set 1ml/min and 12 minutes. The wavelength selected was 274/nm. The injection volume was 20µl.

 

Preparation of Mobile Phase and Buffer:

Prepared the mixture of Methanol: 25mM Na₂HPO₄ in the ratio of (30:70). Filter and degased the mobile phase. For preparation of buffer, Accurately weighed 890mg of Na₂HPO₄was dissolved in 250ml of distilled water, adjusted the pH 7.0 with Orthophosphoric acid.

 

Procedure:

1. Calibration for Spectrophotometric method:

(A) Preparation of Doxofylline Standard and Sample Solution.

(i) Preparation of standard solutions:

Accurately weighed 100mg of doxofylline was transferred to a clean dry 100ml calibrated volumetric flask and dissolved in methanol. It was shaken for few minutes and the solution was diluted to 100ml with the same. From this various dilution were prepared to get final concentration of 10µg/ml. The absorbance was taken at 274nm.

 

(ii) Preparation of Sample Solution:

For estimation of Doxofylline IP in tablet formulation, Twenty tablets were accurately weighed and average weight was taken, weight of tablet powder equivalent to 160mg of doxofylline was taken in 100ml standard flask and the sample was dissolved in methanol and made up to 100ml with the same. The solution was then filtered through what mann filter paper No.1 From this two more dilution were prepared to get the concentration of 10µg/ml.

 

 

Validation of UV-spectrophotometric method:

 

ACCURACY:

Accurately weighed formulation sample equivalent to 100mg of Doxofylline was mixed with 50%, 100% and 150% of pure drug Doxofylline. The quantity of mixture corresponding to 100mg Doxofylline was weighed and sufficient amount of methanol was added to dissolve and the volume was made up with the same. The solution was filtered using Whatmann filter paper and further analysis were carried out according to the procedure described under preparation of standard absorbance curve.

 

Table - 1: UV Accuracy data of doxofylline

S. No	Concentration µg/ml	Amount of Doxofylline present in each tablet (mg)	Amount of pure drug added in stock solution (mg)	Amount of drug recovered (mg)	Percentage of drug recovered (%)
1	2	396.2	400	412.0	101
2	4	396.2	400	408.0	100
3	6	396.0	400	400.0	98.5
4	8	396.3	400	403.2	99
5	10	395.2	400	406.4	100
AVERAGE					99.7
S.D					0.9747
R.S.D					0.9776

 

 

Precision:

Procedure:

Standard and sample solution was prepared as per procedure given under preparation of standard absorbance curve. This parameter was validated by assaying number of aliquots of homogenous samples of Doxofylline and estimating its validity using parameters such as standard deviation (S.D) and relative standard deviation (R.S.D).

 

Linearity:

Procedure:

Accurately weighed 100mg of doxofylline was transferred to a clean dry 100ml calibrated standard flask and dissolved in methanol. It was shaken for few minutes and the solution was diluted to 100ml with the same. From this various dilution were prepared to get the final concentration of 10µg/ml. The absorbance was taken at 274nm.

 

Table – 2: UV Calibration data of Doxofylline

S. No	Concentration (mcg/ml)	Absorbance at 274 nm			Average
		Trail -I	Trail –II	Trail –III
1	2	0.111	0.1115	0.1120	0.1115
2	4	0.184	0.191	0.1875	0.1875
3	6	0.254	0.252	0.250	0.2520
4	8	0.317	0.320	0.314	0.3170
5	10	0.383	0.3885	0.394	0.3885
6	12	0.515	0.4925	0.470	0.4925
7	14	0.577	0.5775	0.578	0.5775
8	16	0.6645	0.678	0.651	0.6645
9	18	0.745	0.758	0.7515	0.7515
10	20	0.8485	0.856	0.841	0.8485

 

 

Method-ii calibration for HPLC method.

(i) Preparation of standard solutions:

Weighed accurately about 50mg of Doxofylline as working standard into a 50ml volumetric flask, and dissolved separately in methanol and made up the volume with Methanol. Pipette out 1ml of both solution into a 10ml volumetric flask and made up the volume with mobile phase. And again pipette out 1ml of this solution into a 10ml volumetric flask and made up the volume with mobile phase.

 

(ii) Preparation of test solution:

Twenty tablets were weighed accurately and powdered. Powder equivalent to 50mg of doxofylline was weighed and transferred to 50ml volumetric flask and dissolved in methanol by shaking the flask for 15minutes. Filter the first 20ml of the filtrate through 0.25µ filter. Pipette out 1ml of the solution into a 10ml volumetric flask and made up the volume with mobile phase. And again pipette out 2ml of this solution into a 10ml volumetric flask and make up the volume with mobile phase.

 

Initialization of the Instrument:

Initially, Column was placed on the instrument and switch on the instrument and washed with Methanol: Water (20:80) for 30 min. Then run the mobile phase for 30 Min. for column saturation.

 

After several trials the following data is fixed for the optimized condition

 

Table - 3: Optimized condition for HPLC

Parameters	Description
Column name	C18(2);250x4.6mm, 5µ, Phenomenox Luna Column
Mobile phase	Methanol: 25 m M Na2HPo4 in the ratio of (30:70)
Flow rate	1.0ml/min
Detection	UV at 274nm
Temperature	Ambient
Injection volume	20ul

 

Validation of HPLC method:

Linearity:

Weigh accurately 80mg Doxofylline working standard into a 50ml volumetric flask, and dissolved separately in methanol and was made up to volume with Methanol. Pipetted out 1ml of both solution and transferred into a 10ml volumetric flask and make up the volume with mobile phase. Pipette out 2ml from the above solution into a 10ml volumetric flask and make up the volume with mobile phase.

 

Table - 4: Linearity of data of doxofylline

Concentration ug/ml	Peak area
2	22500
4	41100
6	62160
8	84120
10	107040

 

Each values is the mean of 3 Readings

 

Precision:

A) Repeatability:

The chromatogram was run for five different concentrations of the standard and sample as per the optimized method with 3 repeatation of each concentration.

 

B) Intermediate Precision:

Intermediate Precision study was carried out by repeating the complete experiment with different analysts (Analyst-1 and Analyst-2) on different days (Day-1 and Day-2) in sample and standard as per the optimized method with 3 repeatation of each concentration.

 

 

Table – 5: Precision data of Doxofylline

S. No	Dilution (ml/ml)	Tria-1	Trial-2	Trial-3	Average	Con. From calibration cure	Amount present in each tablet	Percentage (%)
1	200	6180	6145	6215	6180	2.01	5.01	100.2
2	400	12810	11821	13010	12547	4.07	5.04	101.2
3	600	18840	18550	17510	18300	5.93	4.94	98.83
4	800	24580	249400	24680	24720	8.01	4.99	99.82
5	1000	30700	30800	31020	30840	10.01	4.97	99.75
Average							4.99	99.96%
S.D							0.038	0.857
S.E							0.0169	0.3833
R.S.D							0.76	0.857

 

CONCLUSION:

Literature review revealed that no UV-Spectrophotometry and RP-HPLC method is reported for the estimation of Doxofylline in tablet dosage form. Therefore, it was thought worthwhile to develop simple, precise, accurate UV-Spectrophotometery and RP-HPLC method for the estimation of Doxofylline in tablet dosage form.

 

The developed UV-Spectrophotometric method utilizes Methanol as a solvent which is cheap as compared to Acetonitrile and the quality of the developed method is very good as evident from the analytical and statistical parameters calculated.The UV Spectra of Doxofylline are recorded. The absorption maxima (λ_max) were observed at 274nm. Doxofylline shows linearity in the concentration range of 2 to 20 µg/ml. . The validation of the proposed method was further confirmed by Recovery studies at 60%, 80%, 100%, and 120%. The percentage recovery values from 98%w/w,100.5%w/w, 99.7% w/w, 99.6% w/w. This serves as a good index of accuracy and reproducibility of the study.

 

Reverse phase HPLC method was developed using Methanol: Na2 H PO4 in the ratio of 30:70 as a mobile phase and Hypersil C18 (5 micron, 250 4.6 mm) column as a stationary phase. The recovery studies showed that the observed percentage recovery of doxofylline was found to be 98.4 to 98.9%. The retention time of Doxofylline was found to be 12.778 min. The developed method was accurate and precise which was evident from the analytical data and recovery studies. The repeatability of the method was confirmed by repeating the assay procedure with five different concentrations of three replicates in each. The assay percentage values in “Doxofylline” ranging from 99.7% to 100.1% W/W for respectively. The quantitative results obtained were subjected to statistical validation. The values of R.S.D are less than 2%, indicating the accuracy and precision of the method. The recovery studies at 50%, 100%, 150% were showed that the observed percentage recovery of Doxofylline was found to be from 100.04%, 99.44% and 99.69% w/w respectively. From the present study it is clear that both the methods of analysis are simple, accurate, specific and precise in operation and can be employed in the routine analysis..

 

REFERENCES:

1.      Vogel’s Text Book of Quantitative chemical Analysis. 5^th edition ELBS Longman, Londan,1997, pp.671-672

2.      Sharma. B.K., Instrumental Method of Chemical Analysis., 18^th edition. Krishna  prakashan media (p) ltd, Meerut, 1999, pp.39-133.

3.      Akhilesh Gupta, Swati Rawat, Mayuri Gandhi, Jaydeep Singh Yadav. Method Development and Acid Degradation Study of Doxofylline by RP-HPLC and LC-MS/MS. Asian J. Pharm. Ana. 1(1): Jan.-Mar. 2011; Page 10-13.

4.      Akhilesh Gupta, Jaydeep Singh Yadav, Swati Rawat ,Mayuri Gandhi. Method Development and Hydrolytic Degradation Study of Doxofylline by RP-HPLC and LC-MS/MS. Asian J. Pharm. Ana. 1(1): Jan.-Mar. 2011; Page 14-18

5.      Akhilesh Gupta, Swati Rawat, ArunPandey. Method Development and Photolytic Degradation Study of Doxofylline by RP-HPLC and LC-MS/MS. Asian J. Pharm. Ana. 1(2): April-June 2011; Page 29-33

6.      Patel Juhee J, HenalChorawala, Zarna R. Dedania, S.M. Vijendraswamy. Development and Validation of UV Spectroscopic Method for Simultaneous Estimation of Doxofylline and TerbutalineSulphate in Combined Dosage Form. Asian J. Pharm. Ana. 5(2): April-June 2015; Page 74-78.

7.      Akhilesh Gupta, VimalYadav, Jaydeep S. Yadav, Swati Rawat. An Analytical Approach of Doxofylline: A Review. Asian J. Pharm. Ana. 1(4): Oct. - Dec. 2011; Page 67-70.

8.      Shyamaladevi D., S. Suresh Kumar. Formulation and Evaluation of Bilayer Tablets of Montelukast Sodium Immediate Release and Doxofylline Sustained Release. Research J. Pharm. and Tech. 6(12): Dec. 2013; Page 1370-1374.

9.      Hamid Khan, Mushir Ali, AlkaAhuja, Javed Ali. Validated HPLC-UV Method for Simultaneous Determination of Some Anti-Inflammatory and Analgesic Drugs. Asian J. Pharm. Ana. 2016; 6(3): 183-187.

10.   K. Vijaya Sri, K. Prajawala, S. Deepthi, K. Niharika. Method Development and Validation of UV and RP-HPLC method for the estimation of Capecitabine in Bulk and Pharmaceutical Dosage Forms. Asian J. Research Chem. 2018; 11(4):731-738

11.   AmitRathi, Dinesh Dhamecha, Saifee Maria, Mohd Hassan G Dehghan. Development of UV Spectrophotometric method of Idebenone in Bulk and Pharmaceutical Formulation. Asian J. Research Chem. 2(2): April.-June, 2009 page 168-170

12.   Mitesh D Phale, Purnima D Hamrapurkar. A Validated and Simplified RP-HPLC of Metoprolol Succinate from Bulk Drugs. Asian J. Research Chem. 2(2): April.-June, 2009 page 119-122.

13.   ICH Harmonized Tripartite Guideline. Text on validation of analytical procedures. Recommend for adoption at step 4 of the ICH Process on 27^th Oct.1994, by the ICH Steering committee.

 

 

 

Accepted on 29.09.2019                                 © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(2):560-564.