SIMULTANEOUS ESTIMATION METHOD DEVELOPMENT AND VALIDATION OF ACEBROPHYLLINE AND DOXOFYLLINE IN TABLET DOSAGE FORM BY RP HPLC METHOD

SIMULTANEOUS ESTIMATION METHOD DEVELOPMENT AND

VALIDATION OF ACEBROPHYLLINE AND DOXOFYLLINE IN

TABLET DOSAGE FORM BY RP-HPLC METHOD

Shinde Madhuri*, H. S. Bhawar and Prof. G. S. Shinde

Pravara Rural College of Pharmacy, Pravaranagar, Tal- Rahata, Dist. – Ahmednagar,

Maharashtra, India.

ABSTRACT

A simple, accurate, precise and reproducible Reverse Phase High

Performance Liquid Chromatography method was developed and

validated for simultaneous estimation of Acebrophylline and

Doxofyllinein tablet dosage form. Chromatographic separation was

achieved by Cosmosil C18 (250 mm x 4.6 ID, Particle size- 5 micron)

column and Methanol: 10mM KH2PO4 buffer (70:30)as mobile phase,

at a flow rate of 0.8 ml/min (millilitre per minute) using UV detection

at 243nm. The retention time for Acebrophylline and Doxofylline were

obtained as 4.076min and 5.193 min. respectively. The method has

been validated for linearity, accuracy, precision, LOD, and LOQ.

Linearity of Acebrophylline and Doxofyllinewere found to be 5-

25μg/ml.(R2=0.999) and 20- 100μg/ml.(R2=0.999) respectively. The accuracy of present

method was evaluated at 50%, 100%, 150%. Recovery was found to be in a range from

99.46%-99.98% for Acebrophylline and 99.91%-100.12% for Doxofylline. Intermediate

precision studies were carried out and the RSD values were less than 2%. Lower values of

LOD (0.150μg/ml) and LOQ (0.455μg/ml) for Acebrophylline and LOD (0.127μg/ml) and LOQ (0.385μg/ml) fordoxofylline indicated good sensitivity of the method. In this study, the

optimization of mobile phase, flow rate, injection volume and wavelength were achieved.

This demonstrate that the developed method is simple, precise, accurate and robust for

simultaneous estimation of Acebrophylline and Doxofyllinein tablet dosage form.

KEYWORDS: Acebrophylline and Doxofylline, RP-HPLC, Validation.

Volume 8, Issue 11, 644-657. Research Article ISSN 2277– 7105

Article Received on 05 August 2019,

Revised on 26 August 2019, Accepted on 14 Sept. 2019

DOI: 10.20959/wjpr201911-15741

*Corresponding Author

Shinde Madhuri

Pravara Rural College of

Pharmacy, Pravaranagar,

Tal- Rahata, Dist. -

Ahmednagar, Maharashtra,

1. INTRODUCTION

 Acebrophylline: It acts as a bronchodilator effect due to inhibition of the intracellular

phosphodiesterases followed by an increase of adenosine monophosphosphate cyclic

levels, which promote the relaxation of bronchial muscles. Ambroxol act as a mucolytic

agent by increases the mucociliary clearance by stimulating cilia motility.[1] ACEBRO

inhibit the synthesis and release of leukotrienes and tumoir necrosis factors and reduce

inflammation. Its chemical name is

(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purine_7yl) acetic acid- 4{[(2-amino-3,5-dibromophenyl)methyl]amino} cyclohexanol[2]

having molecular formula C22H28Br2N6O5 with molecular weight 616.311gm/mol. It

appears as white crystalline powder.

Figure no.1: structure of Acebrophylline.

 Doxofylline: One of the mechanisms of action of is thought to arise from the inhibition of

phosphodiesterase activity thus increasing the levels of cAMP and promoting muscle

relaxation. The interaction of DOXO with beta-2 adreno receptors was demonstrated by a

study using nonlinear chromatography, frontal analysis and molecular docking .serine 169

and serine 173 residues in the receptor are thought to be critical binding sites for DOXO

where hydrogen bonds are formed. Via mediating the actions of beta-2 adreno receptors,

DOXO induces blood vessel relaxation[3] and airway smooth muscle relaxation having

chemical name as:

7-(1,3-dioxolan-2-ylmethyl)-3,7-dihydro-1,3-dimethyl2,3,6,7-tetrahydro- 1H-purine-2,6-dione.

 Molecular formula C11H14N4O4 with molecular weight 266.25gm/mol. It appears as

Figure no. 2: Structure of Doxofylline.

 Literature review revealed that UV- spectrophotometric[5] and HPTLC[6] methods are

reported for the estimation of Acebrophylline alone. It was determined by HPLC [7,8], and

RP- HPLC method with another drug in combination.

 RP-HPLC, HPLC, solvent extraction spectrophotometric method is reported for

Doxofylline with another drug.[9-11] A single work reported as stability indicating HPTLC

method for Acebrophylline and Doxofylline in combination.[12]

 The present STUDY DEVOLPMED AND VALIDATE a reliable, rapid, sensitive and

accurate method Acebrophylline and Doxofylline in tablet dosage form by RP-HPLC

method in accordance with ICH guidelines.

2. MATERIAL AND METHOD

2.1Equipment

HPLC SYSTEM: The method was performed on UV Spectrophotometer Shimadzu model

(UV245 UV probe v 2.3.3) and HPLC 3000 series. With column CosmosilC18 (250mm x

4.6ID, Particle size: 5µ). UV-3000-M (UV-Visible Detector) has been used for detection and

P-3000- M reciprocating (binary pump) is included in the system.

Balance: All Drug and chemical were weighed on Wensar High Precision Balance (model:

PGB 100).

Sonicator: Wensar Ultra-Sonicator (WUC-4L).

Calibrated glassware’s

2.2Materials

Pharmaceutically pure sample of Acebrophylline was obtained from Alkem Laboratories Ltd.

Doxofylline was taken from Zydus cadila pharmaceutical company. And Spirodin-AB®

2.3.Mobile phase

Mobile phase containing Methanol: 10mM KH2PO4 buffer (70:30).

2.4.Stock solution of Acebrophylline

Accurately weigh 10mg pure drug of ACEBRO and dissolved in sufficient quantity of

solvent and final volume was made upto 100ml of solvent (methanol: 10mM KH2PO4 Buffer

(70:30), this gives 100 ppm solution. From that dilutions was made as 5,10,15,20,25 µg/ml

respectively was taken in 10ml volumetric flask and diluted upto the mark using methanol.

2.5.Stock solution of Doxofylline

Accurately weigh 10mg pure drug of DOXO and dissolved in sufficient quantity of solvent

and final volume was made upto 100ml of solvent (methanol: 10mM KH2PO4 Buffer

(70:30), this gives 100 ppm solution. From that dilutions was made as 20,40,60,80,100 µg/ml

was taken in 10ml volumetric flask and diluted upto the mark using methanol.

2.6.Analysis of marketed formulation:

The marketed formulation, Spirodin-AB® was analyzed using the developed method. 20

tablets were weighed accurately and finely powdered. A quantity of powder equivalent to

10mg of ACEBRO and 40mg of DOXO was weighed and transferred into 10ml volumetric

flask and sonicated for 15min then make up the volume upto 10ml with methanol. The

resulting solution was filtered using 0.45µm filter.

i. Selection of analytical wavelength

Accurately weighed quantity of ACEBRO and DOXO 10mg pure drug dissolved in 10ml of

solvent ( methanol : 10mM KH2PO4 Buffer (70:30), this gives 1000ppm solution. From that

0.25ml was taken in 10ml volumetric flask and diluted upto the mark using methanol.

Solution was scanned using UV-Visible Spectrophotometer in the spectrum mode between

Figure no. 3: analytical wavelength graph.

ii. Selection of mobile phase

The pure drug of ACEBRO and DOXO was injected into the HPLC system and run in

different solvent systems. Mixture of different solvents were tried to determine optimum

chromatographic conditions for effective elution of relative drug. After several trial, it was

found that the methanol : 10mM KH2PO4 Buffer (70:30) give good result as compared to

other mobile phase.

System suitability

 The column was equilibrated with the mobile phase (indicatedby constant back pressure

at desired flow rate). Working standard solution of drug was injected into the system. The

Figure no.4. Chromatogram for System suitability standard.

Table no. 1: System suitability parameter for final optimized chromatographic

condition.

Time Area Resolution Th. plate Asymmetry

4.069 226780 4.70 9666 1.17

5.185 931707 0.00 9822 1.10

3. METHOD VALIDATION

3.1.Assay

By using the standard areas from linearity of reported concentration the % assay was

calculated. The data for assay of acebrophylline and Doxofylline os shown in table no. 2.

Table No. 2: Assay result of Tablet formulation.

Drug Concentration

(µg/ml)

Area of standard

Area of sample

% Assay(w/v)

ACEBRO 15 621862 617632 99.319

DOXO 60 2653366 2650653 99.89

3.2.Linearity

Linear relation was obtained between mean peak area and concentration of the drug in the

range of 5-25µg/ml for Acebrophylline and 20-100µg/ml for Doxofylline. The data of peak

areas obtained with the respective concentrations in µg/ml are shown in table no. 3 for

Acebrophylline and Doxofylline. The linearity curve for Acebrophylline is shown in figure

Table no 3: Data of linearity for ACEBRO and DOXO.

Conc. Of

ACEBRO (µg/ml) Area

Conc. Of DOXO

(µg/ml) Area

5 227462 20 938242

10 413613 40 1733012

15 621862 60 2653366

20 820878 80 3494726

25 1001396 100 4373448

Figure no. 5: Calibration curve for Acebrophylline.

Figure no. 6: Calibration curve for Doxofylline.

3.3.Accuracy

Recovery studies were carried out by addition of standard drug to the solution at 3 different

concentration level (50%, 100%, 150%) taking into consideration percentage purity of added

bulk drug samples. These was analysed and results were calculated the % recovery data is

Table no 4: % Recovery of ACEBRO. % composition Amount taken (µg/ml) Amount added (µg/ml) Area Amount recovered (µg/ml) % Recovery Mean % recovery 50%

10 5 618686 14.923 99.489

99.465

10 5 627581 14.914 99.432

10 5 617783 14.921 99.475

100%

10 10 816247 19.887 99.435

99.432

10 10 815358 19.884 99.423

10 10 816138 19.887 99.438

150%

10 15 1001335 24.998 99.993

99.984

10 15 1000425 24.992 99.971

10 15 1001345 24.997 99.989

Table no 5: % Recovery of DOXO.

% composition Amount taken (µg/ml) Amount added (µg/ml) Area Amount recovered (µg/ml) % Recovery Mean % recovery 50%

40 20 2651372 59.954 99.924 99.919

40 20 2647281 59.947 99.913

40 20 2654274 59.952 99.921

100%

40 40 3499318 80.104 100.131 100.125

40 40 3487244 80.092 100.116

40 40 3497584 80.103 100.129

150%

40 60 4378643 100.118 100.118 100.117

40 60 4367726 100.116 100.116

40 60 4384592 100.119 100.119

3.4. PRECISION

3.4.1. Repeatability

It was performed by 100% test concentration level and %RSD was calculated. The data for

repeatability (intraday) for Acebrophylline is given in table no.6 and for Doxofylline is given

in table no.7.

The data for repeatability (interday) for Acebrophylline is given in table no. 8 and for

Doxofylline is given in table no.9.

Table no. 6: Repeatability (intra-day) result of ACEBRO.

Conc. of Acebro

(µg/ml) Morning Evening Mean %RSD

15

621862 625010

623399.33 0.66%

627529 626488

Table no. 7: Repeatability (intra-day) result of DOXO.

Conc. Of DOXO (µg/ml) Morning Evening Mean %RSD

60

2653366 2644788

2651804.5 0.24% 2655358 2652699

2660396 2644220

Table no. 8: Intermediate (inter-day) result of ACEBRO.

Conc. Of ACEBRO (µg/ml) Day 1 Day 2 Mean %RSD

15

621862 618325

627922 0.58% 627529 623807

623394 627922

Table no. 9. Intermediate (inter-day) result of DOXO.

Conc. Of

DOXO (µg/ml) Day 1 Day 2 Mean %RSD

60

2653366 2646678

2659202 0.19% 2655358 2657469

2660396 2659202

3.5 Ruggedness

The data for ruggedness of Acebrophylline and Doxofylline is shown in table no 10.

Table no. 10: Data of ruggedness for ACEBRO and DOXO.

Conc. Of ACEBRO (µg/ml) Area Conc. Of DOXO (µg/ml) Area

5 226620 20 940468

10 416724 40 1736384

15 624260 60 2659736

20 822012 80 3497753

25 1004814 100 4376358

The ruggedness curve for Acebrophylline and Doxofylline is shown in figure 7 and 8

Figure no.8: Ruggedness curve for Doxofylline.

3.6. Robustness

It was calculated by using two methods, i.e. change in flow rate and change in wavelength.

3.6.1. The data for robustness of Acebrophylline and Doxofylline for change in flow rate is

shown in table no. 11 and 12 respectively.

Table No. 11: Result of change in flowrate for ACEBRO.

Conc. (µg/ml) Flowrate(ml/min) Area Mean SD %RSD

5 0.8 413613

413660.7 3002.784 0.725905073

5 0.9 410682

5 0.7 416687

Table No. 12: Result of change in flowrate for DOXO.

Conc. (µg/ml) Flowrate(ml/min) Area Mean SD %RSD

20 0.8 1733012

1735373 2422.157 0.139575566

20 0.9 1737852

20 0.7 1735255

3.6.2. The data for The data for robustness of Acebrophylline and Doxofylline for change

in wavelength is shown in table no. 13 and 14 respectively.

Table no. 13: Result of change in wavelength for ACEBRO.

Conc. (µg/ml)

wavelength

(nm) Area Mean SD %RSD

5 253 411689

411784.3 1782.913 0.432972421

5 257 410051

Table no. 14: Result of change in wavelength for DOXO.

Conc. (µg/ml)

wavelength

(nm) Area Mean SD %RSD

20 253 1735210

1732299 3325.332 0.191960627

20 257 1728675

20 243 1733012

3.7 Limit of Detection (LOD), Limit of Quantification (LOQ).

Limit of Detection: LOD=3.3(SD) S

Where,

SD=standard deviation. S= slope

3.7.1. Limit of quantitation

From the linearity data calculate the limit of quantitation, using the following formula

LOQ= 10(SD)

S

Where,

SD=standard deviation S=slope.

Limit of detection: LOD was found to be 0.1504 for ACEBRO and 0.1271 for DOXO. Limit

of quantitation: LOQ was found to be 0.4559 for ACEBRO and 0.3852 for DOXO.

4. RESULT AND DISCUSSION

 Optimized chromatographic condition

The optimize chromatographic condition figure 3. The best peak shape and maximum

separation was achieved with mobile phase Methanol: 10mM KH2PO4 (70:30) with the flow

rate of 0.8ml/min. Column used Cosmosil C18 (250mm x 4.6ID, Particle size: 5 micron) as

stationary phase. Run time was 8.01min and peak was observed at 243nm which was selected

as a wavelength for quantitative estimation. Chromatogram of acebrophylline and doxofylline

is shown in figure 4 and optimized chromatographic condition is shown in table 1.

 Assay

The result of assay were found to be 99.319% for ACEBRO and 99.89% for DOXO. Assay

result found close to 100%. The data was shown in table no. 2.

 Linearity

curve for ACEBRO and DOXO is shown in figure 5 and 6 respectively.

 Accuracy

The % recovery was found to be 99.432% for ACEBRO and 100.125% for DOXO that is

shown in table no. 4 and 5 respectively.

 Precision

4.5.1. Intraday

The data for intraday precision for ACEBRO and DOXO is present in table no. 6 and 7. The

% RSD for intraday was found to be 0.66% for ACEBRO and 0.24% for DOXO.

4.5.2. Interday

The data for interday precision for ACEBRO and DOXO is present in table no. 8 and 9. The

% RSD for interday was found to be 0.58% for ACEBRO and 0.19% for DOXO.

 Ruggedness

The result of ruggedness curve for ACEBRO and DOXO is shown in figure no. 7 and 8 and

in table no. 10.

 Robustness

The result of robustness is shown in table %RSD of change in wavelength was found 0.432%

for ACEBRO and 0.191% for DOXO it is shown in table no. 13 and 14 respectively. %RSD

of change in flow rate was found 0.725% for ACEBRO and 0.139% for DOXO, it is shown

in table no. 11 and 12 respectively.

 LOD and LOQ

LOD was found to be 0.150µg/ml for ACEBRO and 0.127µg/ml for DOXO. LOQ was found

to be 0.455µg/ml for ACEBRO and 0.385µg/ml for DOXO.

 Retention time

The retention time for Acebrophylline and Doxofylline were obtained as 4.076min and 5.193

min.

5. CONCLUSION

A simple, fast, accurate and precise RP-HPLC analytical method has been developed and

form. The result obtained were within the acceptance criteria as per the ICH guidelines.

REFERENCES

1. Sravani T, Thota S, Venisetty R, Venumadhav N. RP-HPLC analysis of acebrophylline in

API and capsule dosage form. Res J pharm., 2014; 5: 480-486.

2. Jadhav NS, Lalitha KG. Development and validation of spectroscopic method for

simultaneous estimation of acebrophylline and acetylcysteine in capsule dosage form. Int

J Phaem Phytopharmacol Res., 1998; 2: 2249-2265.

3. Tripathi KD. Essential of medical pharmacology. 6th edition, New Delhi : Jaypee Brothers

Medica Publishers Ltd, 2010.

4. Indian Pharmacopoeia, Govt. Of India, Published By Indian Pharmacopoiea Commission,

Gaziabad, 2014: I And II, 7th edition: 1625.

5. Aniket R. Aligave, Harshad S. Dhamne, Shubhangee S. Gaikwad, M.S.Kondawar.

Determination Of Acebrophylline In Bulk And Pharmaceutical Formulation By UV

Spectrophotometer. Current Pharma Research, ISSN: 2230-7842, CPR, 2011; 1(3):

267-270, 267-267-270, 2015.

6. Ajinkya A. Deosthali And Mrinalini C. Damle. Development And Validation Of

Stability- Indicating HPTLC Method For Determination Of Acebrophylline. Wjpmr,

2016; 2(5): 122- 127.

7. Mohini Mittal, Yogesh Upadhyay, Durgadas Anghore, Anshul Kumar, Ravindra K.

Rawal. Simultaneous Estimation of Acebrophylline, Montelukast, And Levocetrizine

Dihydrocloride In Marketed Formulation By High-Performance Liquid Chromatography

Method. Pharm Aspire, 2018; 10(1): 23-28.

8. A.Geetha Susmita, G.Aruna, S. Angalaparameswari, M. Padmavathamma, A

Simultaneous Estimation Of Acebrophylline And Acetylcysteine In Tablet Dosage Form

By RP-HPLC Method. Asian Journal Of Pharmaceutical Research, 2015; 5(3): 143-150.

9. M Bhavani, D Sireesha, M Akiful Haque, S Harshini, Vasudha Bakshi, A Padmanabha

Rao. Analytical Method Development And Validation Of Doxofylline And Terbutaline

Sulphate By RP-HPLC Method. CODEN (USA)-IJPRUR, E-ISSN: 2348-6465, 2014;

2(6): 502-506.

10.Deepali Nanaware, Vidhya Bhusari, Sunil Dhaneshwar. Validated Method For

Simultaneous Quantitation Of Doxofylline And Terbutalline Sulphate In Bulk Drug And

11.Maulik Oza, Jagdish Kakadiya, Chirag Oza. Development And Validation Of Solvent

Extraction Spectrophotometric Method For Simultaneous Estimation Of Doxofylline And

Terbutaline Sulphate In Their Combined Dosage Form. Am. J. Pharmtech Res., 2012;

2(4): 592-604.

12.Ram S. Sakhare, Sanjay S. Pekamwar. Development And Validation Of

Stability-Indicating High-Performance Thin-Layer Chromatography Method For The

Determination Of Acebrophylline And Doxofylline In Bulk And Combined Dosage