Int J Curr Pharm Res, Vol 14, Issue 5, 13-19Original Article


DEVELOPMENT AND VALIDATION OF STABILITY INDICATING RP-HPLC METHOD FOR THE SIMULTANEOUS ESTIMATION OF ACLIDINIUM BROMIDE AND FORMOTEROL FUMARATE IN BULK AND INHALER FORMULATION

A. JAYA LAKSHMI, K. ABHISHEK, Y. LEELA PRIYANKA, P. V. BALAJI, N. ASHRITHA*

School of Pharmaceutical Sciences and Technologies, Jawaharlal Nehru Technological University Kakinada, Kakinada-533003, Andhra Pradesh, India
*Email: ashrithanarikimalli@yahoo.com

Received: 15 Jun 2022, Revised and Accepted: 22 Jul 2022

ABSTRACT

Objective: To develop a simple, accurate, precise, rapid and sensitive method for the simultaneous estimation of Formoterol fumarate and Aclidinium bromide in pharmaceutical dosage form.

Methods: The chromatogram was run through Ascentis C18 150 x 4.6 mm, 5µ. Mobile phase containing Water: Acetonitrile taken in the ratio 60:40 was pumped through the column at a flow rate of 1.0 ml/min. The temperature was maintained at 30 °C. The optimized wavelength selected was 220 nm.

Results: The retention times of Formoterol fumarate and Aclidinium bromide were found to be 2.953 min and 2.364 min. %RSD of the Aclidinium bromide and Formoterol fumarate was found to be 0.6 and 0.9, respectively. %Recovery was obtained as 99.81 % and 100.20% for Aclidinium bromide and Formoterol fumarate, respectively. LOD, LOQ values obtained from the Signal-to-noise ratio of Aclidinium bromide and Formoterol fumarate were 0.84 µg/ml, 2.56 µg/ml and 0.01 µg/ml, 0.03µg/ml respectively. Regression equation of Formoterol fumarate is y =9023x+268.67, and y = 4661.2x+1941.9 of Aclidinium bromide. Retention times were decreased and that run time was decreased, so the method developed was simple, rapid, sensitive and economical that can be adopted in regular quality control tests in Industries.

Conclusion: Developed and Validated Formoterol fumarate and Aclidinium bromide in pharmaceutical dosage form by using RP-HPLC method.

Keywords: Reverse phase-high-performance liquid chromatography, Validation, Formoterol fumarate, Aclidinium bromide

© 2022 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/)
DOI: https://dx.doi.org/10.22159/ijcpr.2022v14i5.1973 Journal homepage: https://innovareacademics.in/journals/index.php/ijcpr

INTRODUCTION

Aclidinium bromide with the IUPAC name [[(3R)-1-(3-phenoxypropyl)-1-azoniabicyclo[2.2.2]octan-3-yl]2-hydroxy-2,2-dithiophen-2-ylacetate; bromide] [1], is an anticholinergic drug used to control and prevent symptoms caused by chronic obstructive pulmonary diseases (COPD) like bronchitis and emphysema [2]. The structure of Aclidinium bromide is shown in fig. 1.

Formoterol fumarate with the IUPAC name [((E)-but-2-enedioic acid; N-[2-hydroxy-5-[(1S)-1-hydroxy-2-[[(2S)-1-(4-methoxyphenyl)Propan-2-yl]amino]ethyl] phenyl] formamide] [3], is a long-acting bronchodilator used as a long-term treatment to prevent or to decrease wheezing and trouble breathing caused by asthma or COPD. The structure of Formoterol fumarate is shown in fig. 2.

Both drugs work by relaxing the respiratory muscles [2, 4].

HPLC is an accurate and sensitive method used for the quantitative analysis of several drugs [5, 6]. Literature shows a few methods for simultaneous estimation of Aclidinium Bromide and Formoterol Fumarate [7-10]. The present study aims to develop and validate an economical and effective HPLC method for simultaneous determination with good linearity and sensitivity for both drugs, which could be used in quality control analysis.

Fig. 1: Structure of aclidinium bromide [1]

Fig. 2: Structure of formoterol fumarate [3]

MATERIALS AND METHODS

The API Aclidinium Bromide, Formoterol Fumarate was obtained from MSN Pharma Ltd, Hyderabad. The marketed formulation DuaklirPressair® (Formoterol fumarate and Aclidinium bromide inhaler), MSN Pharma Ltd, Hyderabad, India was used. Acetonitrile, Phosphate buffer, Methanol, Potassium dihydrogen orthophosphate, Ortho-phosphoric acid are from Rankem. Denver Electronic balance, BVK Enterprise pH meter and Ultrasonicator, Thermo Scientific Hot air oven and Refrigerator, Millipore BM2EA9672R, WATERS HPLC 2695 SYSTEM equipped with quaternary pumps, Photo Diode Array detector and autosampler integrated with Empower 2 Software. UV-VIS spectrophotometer PG Instruments T60 with special bandwidth of 2 mm and 10 mm and matched quartz cells integrated with UV win 6 Software was used for measuring absorbances of Formoterol fumarate and Aclidinium bromide solutions.

Methodology

Diluent

Based upon the solubility of the drugs, Acetonitrile and Water taken in the ratio of 50:50 was selected as diluent.

Preparation of solutions

Preparation of standard stock solutions

Accurately weighed 3 mg of Formoterol fumarate, 100 mg of Aclidinium bromide and transferred individually to 50 ml volumetric flasks and 3/4th of diluents was added to these flasks and sonicated for 10 min. Flasks were make up with diluents and labelled as Standard stock solutions. (60µg/ml of Formoterol fumarate and 2000µg/ml of Aclidinium bromide).

Preparation of standard working solutions (100% solution)

1 ml from each stock solution was pipetted out and taken into a 10 ml volumetric flask and made up with a diluent. (6µg/ml of Formoterol fumarate and 200µg/ml of Aclidinium bromide).

Preparation of sample stock solution and sample working solution (100% solution)

The contents of the inhaler were collected by 50 actuations (1.2µg Formoterol fumarate and 40µg Aclidinium bromide) into a 50 ml volumetric flask. 20 ml acetonitrile was added and sonicated for 25 min and volume is made up to mark to yield 12 and 400μg. Then the supernatant was collected and filtered using 0.45 μm filters using (Millipore, Milford, PVD).5 ml from this solution was pipetted out and taken into a 10 ml volumetric flask and made up with diluent. (6µg/ml of Formoterol fumarate and 200µg/ml of Aclidinium bromide).

Optimization of chromatographic conditions

Method development for the analysis of Aclidinium Bromide and Formoterol Fumarate was done by changing mobile phase ratios, buffers, flow rate, columns, and run time. Acceptable retention times, good resolution, tailing factor and theoretical plates were observed with optimized chromatographic conditions mentioned in table 1. The optimized chromatogram is shown in fig. 3. Validation and stability studies of the optimized method were performed according to the ICH guidelines [11].

Method validation

Validation was performed as per the ICH Q2B (R2) guidelines [11]. The method was validated for the parameters like system suitability, specificity, linearity, precision (system precision and repeatability), accuracy, the limit of detection and limit of quantification, robustness, and assay. Stability Studies like acid degradation, base degradation, oxidative degradation, thermal degradation, photostability degradation, and aqueous degradation were carried out as per ICH guidelines [12].

Table 1: Optimized chromatographic conditions

S. No. Parameter Condition
1 Mobile phase 60% Water: 40% Acetonitrile
2 Diluent Water: Acetonitrile (50:50)
3 Column Ascentis C18 (4.6 x 150 mm, 5 µm)
4 Wavelength 220 nm
5 Column temperature 30 °C
6 Injection volume 10 μl
7 Flow rate 1.0 ml/min
8 Run time 5 min
9 Retention time

2.364 min (Aclidinium Bromide)

2.953 min (Formoterol Fumarate)

Fig. 3: Optimized chromatogram

Fig. 4: Chromatogram for system suitability

System suitability

It is performed to verify that the analytical system is working properly and can give accurate and precise results. Standard solutions of Aclidinium Bromide (6 ppm) and Formoterol Fumarate (200 ppm) were injected six times and the parameters like resolution, peak tailing, and USP plate count were determined. The chromatogram was represented in fig. 4, and the results of system suitability were shown in table 2. According to ICH guidelines, plate count should be more than 2000, tailing factor should be less than 2 and resolution must be more than 2. All the system suitability parameters were passed and were within limits.

Table 2: System suitability parameters for Aclidinium bromide and Formoterol fumarate

S. No. Aclidinium bromide Formoterol fumarate
Injection Rt (min) USP plate count Tailing Rt (min) USP plate count Tailing Resolution
1 2.364 3518 1.31 2.953 5426 1.16 3.6
2 2.380 3554 1.33 2.975 5235 1.23 3.6
3 2.389 3461 1.34 2.993 5109 1.24 3.6
4 2.393 3467 1.37 3.000 5295 1.17 3.6
5 2.398 3574 1.33 3.000 5537 1.18 3.6
6 2.405 3580 1.31 3.009 5297 1.21 3.6

Specificity

The specificity of the method is performed by separately injecting the blank and placebo at sample solutions. The interference observed (if any) at the retention times of each analyte in all the chromatograms is evaluated. Chromatograms were as shown in fig. 5, fig. 6 and fig. 7. Retention times of Aclidinium Bromide and Formoterol Fumarate were 2.364 min and 2.953 min, respectively. The method is specified as no interfering peaks were observed in blank and place boat retention times of the drugs.

Fig. 5: Chromatogram of blank

Fig. 6: Chromatogram of placebo

Fig. 7: Typical chromatogram

Linearity

Standard solutions of 25%, 50%, 75%, 100%, 125%, and 150% concentrations were prepared by taking 0.25, 0.5, 0.75, 1.0, 1.25, 1.5 ml each from two standard stock solutions and make up to 10 ml. Six linear concentrations of Formoterol fumarate (1.5-9.0µg/ml) and Aclidinium bromide (50-300µg/ml) were injected in a duplicate manner. Peak areas were recorded for each injected concentration and the calibration curves-concentration vs. peak area were constructed fig. 8 and fig. 9. The results were given in table 3 and table 4. Linearity equations obtained for Formoterol fumarate was y = 9023x+268.67and of Aclidinium bromide was y =4661.2x+1941.9. Correlation coefficient obtained was 0.999.

Fig. 8: Calibration curve of Aclidinium bromide

Fig. 9: Calibration curve of Formoterol fumarate

Table 3: Results for linearity of Aclidinium bromide

Aclidinium bromide
Conc. (μg/ml) Peak area
50 233919
100 464647
150 694073
200 948755
250 1178371
300 1386179

Table 4: Results for linearity of Formoterol fumarate

Formoterol fumarate
Conc. (μg/ml) Peak area
1.5 13531
3 27459
4.5 40365
6 55279
7.5 68596
9 80608

Precision

System precision

System precision was determined by injecting 15 µl standard solution six times and the chromatograms were recorded. Average area, standard deviation, and %RSD were calculated for two drugs and the results are shown in table 5. %RSD was obtained as 0.6% and 0.1%, respectively for Aclidinium Bromide and Formoterol Fumarate. As the limit of precision was less than 2, the method is precise.

Table 5: Results for system precision of Aclidinium bromide and Formoterol fumarate

S. No. System precision
Area of aclidinium bromide Area of formoterol fumarate
1. 935674 49682
2. 934514 50660
3. 947330 50449
4. 943197 50965
5. 944125 50702
6. 944718 50360
Mean 941593 496031
SD 5230.9 440.1
%RSD 0.6 0.1

Repeatability

Repeatability (Method precision) was determined by multiple sampling from a sample stock solution and six working sample solutions of the same concentrations were prepared, 15 µl injection from each working sample solution was given, and obtained areas were mentioned in table 6. Average area, standard deviation, and % RSD were calculated for two drugs and obtained as 0.3% and 0.8%, respectively for Aclidinium Bromide and Formoterol Fumarate.

As the limit of Precision was less than 2 the method is repeatable.

Table 6: Results for repeatability of Aclidinium bromide and Formoterol fumarate

Repeatability
S. No. Area of aclidinium bromide Area of formoterol fumarate
1. 934899 50012
2. 930938 50196
3. 935516 50566
4. 938062 50747
5. 936682 50391
6. 935234 49663
Mean 935222 50263
SD 2397.5 392.4
%RSD 0.3 0.8

Accuracy

Three levels (50%, 100%, 150%) of Accuracy samples were prepared by the standard addition method. Triplicate injections were given for each level of accuracy. The results were shown in table 7 and table 8. Mean % Recovery was obtained as 99.83% and 100.20% for Aclidinium Bromide and Formoterol Fumarate, respectively.

Table 7: Accuracy results for Aclidinium bromide

% Level Amount spiked (μg/ml) Amount recovered (μg/ml) % Recovery Mean % recovery
50% 100 100.56 100.56 99.96%
100 100.04 100.04
100 99.26 99.29
100% 200 199.58 99.79 99.85%
200 200.45 100.23
200 199.07 99.54
150% 300 297.41 99.14 99.69%
300 295.81 98.60
300 304.02 101.34

Table 8: Accuracy results for Formoterol fumarate

% Level Amount spiked (μg/ml) Amount recovered (μg/ml) % Recovery Mean % recovery
50% 3 3.01 100.39 100.41%
3 3.05 101.66
3 2.98 99.20
100% 6 6.00 100.06 100.06%
6 6.00 100.07
6 6.00 100.05
150% 9 8.91 99.00 100.12%
9 9.14 101.51
9 8.99 99.87

Limit of detection and limit of quantification

The LOD and LOQ of the developed method were determined by injecting progressively low concentrations of the standard solutions using the developed RP-HPLC method. “LOD and LOQ is related to both the signal and the noise of the system and is usually defined as a peak whose signal-to-noise (S/N) ratio is at least 3:1 for LOD and 10:1 for LOQ” [13]. The results were shown in table 9.

Table 9: Results for LOD and LOQ of Aclidinium bromide and Formoterol fumarate

Drug LOD (µg/ml) LOQ (µg/ml)
Aclidinium Bromide 0.84 2.56
Formoterol Fumarate 0.01 0.03

Robustness

Robustness conditions like Flow rate minus (0.9 ml/min), Flow rate plus (1.1 ml/min), mobile phase minus (65W: 35A), mobile phase plus (55W: 45A), temperature minus (25 °C), and temperature plus (35 °C) were maintained and samples were injected in a duplicate manner. Results were given in table 10. System suitability parameters were not much affected and %RSD was within the limit. Hence the method was considered to be robust.

Assay

Assay was performed with (DuaklirPressair®) bearing the label claim Formoterol 12 µg, Aclidinium 400 µg. 20 μl of the Standard and Sample solutions were injected into Chromatographic System and areas for Aclidinium Bromide and Formoterol Fumarate were measured and results were shown in table 11. The average % Assay for Formoterol fumarate and Aclidinium bromide obtained was 99.39% and 99.12%, respectively.

Stability studies

Acid degradation studies

1 ml 2N Hydrochloric acid was added to 1 ml stock solution of Aclidinium Bromide and Formoterol Fumarate, refluxed for 30 min at 60 °C. The resultant solution was diluted to obtain 6µg/ml and 200µg/ml solution and 10 µl solution was injected into the system to assess the stability of the sample. The results were given in table 12.

Table 10: Results for the robustness of Aclidinium bromide and Formoterol fumarate

S. No. Condition %RSD of Aclidinium bromide %RSD of formoterol fumarate
1 Flow rate (-) 0.90 ml/min 0.9 0.9
2 Flow rate (+) 1.1 ml/min 0.5 0.3
3 Mobile phase (-) 65W: 35A 0.5 1.1
4 Mobile phase (+) 55W: 45A 0.5 0.7
5 Temperature (-) 25 °C 0.3 0.1
6 Temperature (+) 35 °C 0.7 0.2

Table 11: Results for assay of Aclidinium bromide and Formoterol fumarate

S. No. Aclidinium bromide Formoterol fumarate
Standard area Sample area % Assay Standard area Sample area % Assay
1 935674 934899 99.09 49682 50012 98.89
2 934514 930938 98.67 50660 50196 99.26
3 947330 935516 99.16 50449 50566 99.99
4 943197 938062 99.43 50965 50747 100.35
5 944125 936682 99.28 50702 50391 99.64
6 944718 935234 99.13 50360 49663 98.20
Avg 941593 935222 99.12 50366 50263 99.39
SD 5230.9 2397.5 0.25 440.1 392.4 0.8
%RSD 0.6 0.3 0.3 0.9 0.8 0.8

Alkali degradation studies

1 ml 2N NaOH was added to 1 ml stock solution of Aclidinium Bromide and Formoterol Fumarate and refluxed for 30 min at 60 °C. The resultant solution was diluted to obtain 6µg/ml and 200µg/ml solution and 10 µl solution was injected into the system to assess the stability of the sample. The results were given in table 12.

Oxidative degradation studies

1 ml 20% H2O2 was added to 1 ml of stock solution of Aclidinium Bromide and Formoterol Fumarate. The solutions were kept for 30 min at 60 °C. The resultant solution was diluted to obtain 6µg/ml and 200µg/ml solution and 10 µl were injected into the system to assess the stability of the sample. The results were given in table 12.

Thermal degradation studies

The standard drug solution was placed in an oven at 105 °C for 1 h to study dry heat degradation. The resultant solution was diluted to 6µg/ml and 200µg/ml solution and 10 µl were injected into the system to assess the stability of the sample. The results were given in table 12.

Photostability studies

The photochemical stability of the drug was studied by exposing 60µg/ml Aclidinium Bromide and 2000µg/ml Formoterol Fumarate solution to UV light by keeping the beaker in UV Chamber for one day. The resultant solution was diluted to obtain 6µg/ml and 200µg/ml solution and 10 µl were injected into the system to assess the stability of the sample. The results were given in table 12.

Table 12: Degradation data of Aclidinium bromide and Formoterol fumarate

S. No. Degradation condition Aclidinium bromide Formoterol fumarate
Area % Recover % Drug degraded Area % Recover %Drug degraded
1 Acid 841207 89.16 10.84 45312 89.60 10.40
2 Alkali 922161 97.74 2.26 49724 98.33 1.67
3 Oxidation 814730 86.35 13.65 48637 96.18 3.82
4 Thermal 923257 97.86 2.14 49260 97.41 2.59
5 UV 926891 98.24 1.76 50002 98.88 1.12
6 Water 940657 98.24 1.76 50312 99.49 0.51

Aqueous degradation studies

Stress testing under neutral conditions was studied by refluxing the drug in water for 1h at 60 °C. The resultant solution was diluted to 6µg/ml and 200µg/ml and 10 µl were injected into the system to assess the stability of the sample. The results were given in table 12.

CONCLUSION

In the present study, an attempt was made to develop a simple, accurate, precise, rapid and sensitive method was developed for the simultaneous estimation of the Aclidinium bromide and Formoterol fumarate in bulk and dosage form. Retention time of Aclidinium and Formoterol was found to be 2.364 min and 2.953 min. %RSD of the Aclidinium and Formoterol were and found to be 0.6 and 0.9, respectively. % Recovery was obtained as 99.69%-99.96% and 100.12%-100.41% for Aclidinium bromide and Formoterol fumarate, respectively. LOD, LOQ values obtained from Signal-to-noise ratio of Aclidinium bromide and Formoterol fumarate were 0.8, 2.56, and 0.01, 0.03, respectively. Regression equation of Formoterol fumarate is y =9023x+268.67, and y = 4661.2x+1941.9 of Aclidinium bromide. Retention times were decreased, and that run time was decreased, so the method developed was simple and economical, which is useful in pharmaceutical industries.

ACKNOWLEDGMENT

The author wishes to thank MSN Pharma Ltd, Hyderabad, for providing the facility to complete the work.

FUNDING

Nil

AUTHORS CONTRIBUTIONS

All the authors have contributed equally.

CONFLICT OF INTERESTS

Declared none

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