INTRODUCTION:

Aclidinium¹ is a long-acting, reversible antagonist at muscarinic receptors, with equal affinity to all five subtypes, but with a half-life dissociation of 29.2 hours from subtype M3, or six times longer than that from M2. Inhaled Formoterol works like other β2 agonists, which causes bronchodilation by relaxing the smooth muscle in the airway to treat asthma exacerbation. A literature review resulted some methods of analysis in inhalation and human serum by volatmmetry², in urine by gas chromatography mass spectrometry³, UV spectroscopy^4,5 for the estimation of formoterol either alone and in other combinations^6,7,8,9,10 and chromatographic methods were also developed for the determination of aclidinium and formoterol in their dosage form^11,12.

The main aim of the project work is to develop a novel RP-HPLC method which is able to separate and quantify the drug candidates selected for study viz., Aclidinium bromide and Formoterol fumarate present in its pure form as well as formulation and validate the method by ICH Q2 (R1)¹³ guidelines with demonstrable accuracy, linearity, precision and robustness.

Fig. 1: Chemical structure of Aclidinium bromide (A) and Formoterol (B)

MATERIAL AND METHODS:

Chemicals and reagents:

All the chemicals and reagents used in this study were of analytical grade. Aclidinium bromide was obtained from Malladi Drugs and Pharmaceuticals, Chennai, India. Dipotassium hydrogen phosphates and Potassium dihydrogen phosphate were obtained from Merck specialties private limited, Mumbai. HPLC grade acetonitrile and water were obtained from Qualigens.

Instrumentation:

A liquid chromatograph, Cyberlab^tm equipped with LC100 pump LC100 UV LC detector was used for separation. The column used in this study is Analytical packed with BDS, 5µm, size: 250 x 4.6mm and the column effluents were monitored and integrated using WS-100 Work station software. Elico SL 159 double beam UV visible spectrophotometer with 1cm quartz cells was used for spectral studies.

Preparation of reagents and standards:

Preparation of Buffer (KH₂PO₄ 0.1 M):

Weight 3.8954g of disodium hydrogen phosphate in a beaker containing 1000ml of distilled water and 3.4023 of potassiumdihydrogen phosphate dissolve fully. Ph is then balanced with orthophosphoric acid, and routed through a membrane filter of 0.45μm.

Preparation of solution for stocks:

Reference solution: The solution was prepared by dissolving 20.0mg of precisely weighed Aclidinium Bromide and 25.0mg of Formoterol Fumarate in mobile process, separately in two volumetric flasks of 100.0mL and sonicating for 20min. From the above solutions take 10.0mL of each solution into a 50.0mL volumetric flask and then make up for 10min with mobile phase.

Preparing Standard Working Solution:

In combination with Aclidinium Bromide and Formoterol Fumarate above, stock solutions equal to 20ppm to 80ppm in respect of both drugs were prepared, sonicated and filtered through 0.45μ membrane.

Standard Solution preparation for trials:

Weighing down 10mg of Aclidinium Bromide and Formoterol fumarate drugs and dissolving in 10ml of Mobile phase taken in two 10ml volumetric flasks separately and sonicated for 20 minutes to get 1000ppms and 1ml of each solution was taken into a 10ml volumetric flask and diluted to 10ml with mobile phase.

Method Validation:

System Suitability:

The system suitability test was performed using 20μL of standard solution for five replicate injections before analysis of samples and the chromatogram was recorded. Retention time (R_t), number of theoretical plates (N), tailing factor (T), and peak asymmetry (A_S), resolution (R_S) were evaluated for five replicate injections of the drug.

Specificity:

435mg placebo with 250mL ACN was taken in 500mL volumetric flask and the volume was adjusted with water. Prepared blank, placebo, standard solution with diluent are analyzed to check the interference.

LOD and LOQ:

3.3 σ 10 σ

LOD=------- LOQ= ------------

S S

Where, σ is the standard deviation in the intercept and S is the slope of the calibration curve

Linearity:

A series of solutions are being formulated using Aclidinium bromide and Formoterol fumarate working standards at 20ppm to 80ppm target concentration levels.

Precision:

Repetition (Repeatability):

Six sample preparations were prepared individually using single as per test process, and each solution was injected.

Intermediate precision (analyst to analyst variability):

Day-to-day variation was studied by taking six samples.

Robustness:

The prepared proposed system suitability solution was injected into the chromatographic system at finalized condition and variable conditions like flow variation, pH variation of buffer, column oven temperature variation, wavelength variation.

Accuracy (% Recovery):

A research was performed on accuracy. Drug Assay was performed in triplicate as per test method with equal amounts of Aclidinium bromide and Formoterol fumarate in each volumetric flask for each spike stage to get the concentration of Aclidinium bromide and Formoterol fumarate equivalent to 50percent, 100 percent, and 150percent of the labeled quantity as per test method. The average recuperation rate of Aclidinium bromide and Formoterol fumarate was estimated.

Robustness studies:

Effect of Flow Variation (Effect of variation of flow rate):

A research was carried out to determine the impact of flux rate variability. Using flow speeds, 1.0ml/min and 1.2mL/min, standard solution prepared according to the test method was injected into HPLC network. The parameters of suitability for the device were tested and found to be within the 1.0ml/min and 1.2ml/min flow limits. Aclidinium bromide and Formoterol fumarate were resolved from all other peaks and the retention times were comparable to those obtained for the 1.0 ml/min mobile process.

RESULTS AND DISCUSSION:

HPLC Method optimization:

The extent of the organic and aqueous phases were adjusted to attain a specific and reliable assay method for the determination of Aclidinium bromide and Formoterol fumarate with less run time, short retention time and the sharp peak. The UV absorption of the selected drug candidates shown good response at 240 (Fig. 2) nm for detection of analytes. Keeping in view all the trials, a mobile phase composed of phosphate buffer pH 3.4 and methanol was used in different proportions until to get the best separation conditions, and a ratio of 25:75 v/v, has been found suitable for the study. The optimized chromatographic conditions was given in table 1.

Fig. 2: UV absorption spectra of Aclidinium bromide and Formoterol fumarate

Method validation:

The method was validated with particular parameters like specificity, linearity, precision, LOD and LOQ, accuracy and robustness.

System suitability studies:

It was observed from the data tabulated above, that system suitability parameters i.e., tailing factor < 2.0 and theoretical plates are more than 2000. Both parameters were found to be within the acceptance criteria. Hence it can be concluded that the preferred system is suitable. The results were presented in table 2.

Table 1: optimised chromatographic conditions

Parameters	Method
Stationary phase (column)	Inertsil -ODS C₁₈(250 x 4.6 mm, 5 µ)
Mobile Phase	Methanol: Buffer (75:25)
Flow rate (ml/min)	1.0 ml/min
Run time (minutes)	10 min
Column temperature (°C)	Ambient
Volume of injection loop (ml)	20
Detection wavelength (nm)	240 nm
Drug RT (min)	4.713min for Aclidinium Bromideand 6.691min for Formoterol Fumarate.

Specificity:

Specificity part (A) includes the interference from blank and placebo. No interference was observed at the retention time of selected drug candidates. Therefore it can be concluded that no interference due to diluent, placebo and standard for the quantification of Aclidinium bromide and Formoterol fumarate in formulations. Hence, the method is specific and selective. The chromatograms were shown in fig. 3 and 4 and table 3.

Table 2: Data of System Suitability for Aclidinium bromide

Injection	RT	Peak Area	USP Plate count	USP Tailing	RT	Peak Area	USP Plate count	USP Tailing
	Aclidinium bromide				Formoterol fumarate
1	4.713	298654	21023.84	1.094	6.691	1588766	8325.87	1.056
2	4.708	298479	21010.54	1.101	6.684	1586897	8384.54	1.078
3	4.707	298364	21036.87	1.076	6.681	1584876	8314.87	1.058
4	4.708	298841	21027.25	1.059	6.680	1586075	8372.78	1.055
5	4.709	298699	21084.65	1.107	6.684	1582466	8392.08	1.088
Mean	4.746872	298610	21084.65	1.100	6.6367422	1585794	8358.87	1.060
SD	3.271948	298607	12036.82	1.089	0.0024183	2014.808	-------	-------
% RSD	0.002754	167.849	-------	-------	0.037625	0.132	-------	-------

Fig. 3: Chromatogram of placebo for specificity

Fig. 4: Chromatogram showing for specificity in separation of two drugs

Table 4: Results of linearity

Aclidinium bromide		Formoterol fumarate
*Concentration, µg/mL*	*Area*	*Concentration, µg/mL*	*Area*
0	0	0	0
20	149024	20	791282
30	223539	30	1186923
40	298048	40	1582564
50	368512	50	1955684
60	447072	60	2373864
70	521584	70	2769487
80	596096	80	3165128
*Regression analysis*
Slope	7446	Slope	39536
y-Intercept	-284	y-Intercept	-1580
Correlation Coefficient	0.999	Correlation Coefficient	0.999

Fig. 5: Calibration curve for Aclidinium bromide

Fig. 6: Calibration curve for Formoterol fumarate

Fig. 7: Chromatogram for linearity solution of 20 ppm

Fig. 8: Chromatogram for linearity solution of 30 ppm

Fig. 9: Chromatogram for linearity solution of 40 ppm

Fig. 10: Chromatogram for linearity solution of 50 ppm

Fig. 11: Chromatogram for linearity solution of 60 ppm

Limit of detection and limit of quantification:

The limit of detection and quantitation was determined from the calibration curve and it was found satisfactory since the lowest amount that can be detected by this method was 0.757 µg/mL and the minimum concentration of the analyte that can be quantified was found as 2.295 µg/mL. From the linearity plot the LOD and LOQ are calculated by the following formula: The LOQ was found as 0.411 µg/ml for Aclidinium bromide in this study. The LOD and LOQ was found as 0.072 µg/ml and 0.218 µg/ml for Formoterol fumarate.

3.3 σ 3.3X 1839.51

LOD=------- = --------------- = 0.135

S 44690

Precision:

Three different sample concentrations and triplicate of each concentration in linearity range were taken for intra and inter day precision studies. The % RSD for intra-day precision of the sample (n=6) was found as 0.068 and 0.17 for Aclidinium bromide and Formoterol fumarate respectively. The interday precision was achieved by performing the method in between the days and the % RSD was found as less than 0.1 for Aclidinium bromide and Formoterol fumarate respectively. In all the cases studied, the %RSD has been found by the proposed method was within 2.0% that has indicated a consistency in its precision and can be seen in the results of precision studies in table 5 and 6 for intra-day and interday precisions respectively. Chromatograms representing the precision studies were given in fig. 12 and 13.

Table1 5: Intra-assay precision of Aclidinium bromide

Injection	Peak Area	% Assay	Peak Area	% Assay
Injection	Aclidinium Bromide		Formoterol Fumarate
1	298598	100.34	1586845	100.44
2	298348	100.26	1583485	100.22
3	298168	100.20	1584697	100.30
4	298264	100.23	1585509	100.25
5	298433	100.27	1587946	99.51
6	298680	100.39	1583965	100.35
Mean	298415	100.28	1585407	100.25
SD	196.130	0.065	1720.599	0.108
% RSD	0.065	0.065	0.108	0.108

Fig. 12: Repeatability chromatogram

Table 6: Inter-day precision of Aclidinium bromide and Formoterol fumarate

Injection	Peak Area	%Assay	Peak Area	%Assay
	Aclidinium bromide		Formoterol fumarate
1	298450	100.30	1586423	100.41
2	298631	100.36	1585684	100.36
3	298187	100.21	1588793	100.56
4	298586	100.34	1587640	100.49
5	298298	100.24	1587790	100.50
6	298330	100.25	1586565	100.42
Mean	298413	100.28	1587149	100.46
SD	173.210	0.058	1128.63	0.071
% RSD	0.058	0.057	0.071	0.071

Fig. 13: Intermediate-precision chromatogram

Accuracy:

Recovery studies were performed on three different levels at 50, 100, and 150 in three replicates in each level in the present study. Standard drug was spiked to the pre-analyzed sample and injected into an HPLC system to determine the amount recovered by the proposed method. The % recovery values were observed to be in the range of 98 % - 102 % with % RSD NMT 2.0. The recovery results indicated that the method had an acceptable level of accuracy. Results for the accuracy study were shown in table 7 and the chromatograms were given in fig. 14-16.

Table 7: Data of Accuracy studies

% of spiked level	Amount added (ppm)	Amount found (ppm)	% Recovery	Amount added (ppm)	Amount found (ppm)	% Recovery
	Aclidinium bromide			Formoterol fumarate
50% Injection 1	20	20.14	100.70	20	20.05	100.27
50% Injection 2	20	20.11	100.56	20	20.05	100.28
50% Injection 3	20	20.12	100.62	20	20.06	100.30
100 % Injection 1	40	40.07	100.18	40	40.12	100.30
100 % Injection 2	40	40.10	100.26	40	40.15	100.38
100% Injection 3	40	40.12	100.31	40	40.18	100.47
150% Injection 1	60	60.08	100.13	60	60.08	100.13
150% Injection 2	60	60.13	100.22	60	60.12	100.20
150% Injection 3	60	60.16	100.27	60	60.16	100.27

Fig. 16: Chromatogram for Accuracy at 150 % level

Table 9: Data for Effect of variation in flow rate for Aclidinium bromide

Flow rate	0.8 mL/min		1.0 mL/min		1.2 mL/min
Injection	Std Area	Tailing factor	Std Area	Tailing factor	Std Area	Tailing factor
1	290845	1.065	298709	1.086	305864	1.048
2	290684	1.087	298568	1.048	305655	1.050
3	290168	1.048	298458	1.091	305540	1.078
4	290386	1.068	298165	1.056	305487	1.096
5	290564	1.075	298655	1.037	305904	1.046
Mean	290529.4	1.0686	298511	1.0636	305690	1.0636
SD	262.6191	0.014293	215.4727	0.023776	187.7538	0.022334
%RSD	0.090393	1.337578	0.072183	2.23543	0.06142	2.099834

Table 10: Data for Effect of variation in flow rate (Formoterol fumarate)

Flow rate	0.8 mL/min		1.0 mL/min		1.2mL/min
Injection	Std Area	Tailing factor	Std Area	Tailing factor	Std Area	Tailing factor
1	1554425	1.048	1585455	1.055	1614877	1.078
2	1558464	1.051	1588642	1.065	1617870	1.086
3	1556482	1.075	1586124	1.078	1616540	1.055
4	1553565	1.054	1583693	1.056	1618025	1.060
5	1554878	1.026	1584710	1.015	1615520	1.029
Mean	1555563	1.0508	1585725	1.0538	1616566	1.0616
SD	1937.565	0.017456	1864.151	0.023573	1394.329	0.02221
%RSD	0.124557	1.661178	0.117558	2.236979	0.086252	2.092159

Fig. 17: The robustness chromatogram for 0.8 ml flow rate

Fig. 18: The standard robustness chromatogram for 1.0 ml flow rate

Fig. 19: The standard robustness chromatogram for 1.2 ml flow rate

CONCLUSION:

Due to good peak area the flow rate was set at 1.0 ml / min, satisfactory retention time and good resolution was obtained for separation and simultaneous estimation of the selected drug candidates. The new recovery over the same range as 98.0-100.25 was found to be reliable. The detection limit was found to be 0.074 Aclidinium Bromide, and 0.175 for Formoterol Fumarate. The linearity analysis in the concentration range 20-80 µg/mL was found to exhibit excellent correlation coefficient (0.999) and best curve fitting. The analytical procedure has passed sturdiness as well as robustness tests. In both occasions the relative standard deviation was very satisfying. The method may serve to estimate the drugs of interest for quality control in pharmaceutical industry.

ACKNOWLEDGEMENT:

The authors are grateful to the management, Koringa educational society, Korangi, for providing us the facilities.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

REFERENCES:

1. Jones P. Aclidinium bromide twice daily for the treatment of chronic obstructive pulmonary disease: a review. Advances in therapy. 2013; 30(4): 354-68.

2. Demircigil B, Yılmaz S. Electrochemical behavior of formoterol fumarate and its determination in capsules for inhalation and human serum using differential-pulse and square-wave volatmmetry. Electroanalysis. 2002; 14(2): 122–127.

3. Kamimura H, Sasaki H, Higuchi S, Shiobara Y. Quantitative determination of the β-adrenoceptor stimulant formoterol in urine by gas chromatography mass spectrometry. Journal of Chromatography B: Biomedical Sciences and Applications. 1982; 229(2): 337–345.

4. Gousuddin M, Raju SA, Sultanuddin MS. Development and validation of spectrophotometric methods for estimation of formoterol bulk drug and its pharmaceutical dosage forms. International Journal of Pharmacy and Pharmaceutical Sciences. 2011; 3: 300–309.

5. Hassib ST, El-Zaher AA, Fouad MA. Validated stability-indicating derivative and derivative ratio methods for the determination of some drugs used to alleviate respiratory tract disorders and their degradation products. Drug Testing and Analysis. 2011; 3(5): 306–318.

6. El-Bagary RI, Fouad MA, Manal A, Tolba EH. Forced degradation of mometasone furoate and development of two RP-HPLC methods for its determination with formoterol fumarate on salicylic acid. Arabian Journal of Chemistry. 2016; 9(3): 493–505.

7. Assi KH, Tarsin W, Chrystyn H. High performance liquid chromatography assay method for simultaneous quantitation of formoterol and budesonide in Symbicort Turbuhaler. Journal of Pharmaceutical and Biomedical Analysis. 2006; 41(1): 325–328.

8. Parmar VK, Patel HN, Patel BK. Sensitive, and robust methods, for simultaneous determination of beclomethasone dipropionate and formoterol fumarate dihydrate in rotacaps. Journal of Chromatographic Science. 2014; 52(10): 1255–1266.

9. Rizk M, Sultan M, Talaat N, Youssef N. A validated TLC—densitometric method for the simultaneous determination of formoterol fumarate and budesonide in pressurized metered-dose inhaler. JPC-Journal of Planar Chromatography-Modern TLC. 2017; 30(1): 63–67.

10. Merey HA, El-Mosallamy SS, Hassan NY, El-Zeany BA. Validated chromatographic methods for the simultaneous determination of Mometasone furoate and Formoterol fumarate dihydrate in a combined dosage form. Bulletin of Faculty of Pharmacy, Cairo University. 2016; 54(1): 99–106.

11. Gowda R. Simultaneous RP-HPLC method for determination of impurities in formoterol fumarate and aclidinium bromide in pharmaceutical dosage forms. International Journal of Chemical and Pharmaceutical Analysis. 2016; (3)3: 1005.

12. Shyamala S, Sai Kala G, Sravani A, Anusha T, Srikanth K. Validated RP-HPLC method for simultaneous estimation of aclidinium and formoterol in bulk drug and dosage form. The Pharma Innovation Journal. 2020; 9(4): 242-247.

13. International Conference on Harmonization, Q2 (R1), Harmonised tripartite guidelines, Validation of analytical procedures: text and methodology, Geneva, November, 2005.

Received on 09.09.2020 Revised on 18.01.2021

Asian Journal of Pharmaceutical Analysis. 2021; 11(2):63-69.

DOI: 10.52711/2231-5675.2021.00012

Name of sample	Retention time (min)		Area		Efficiency (N)		Tailing factor
Name of sample	ACB*	FRM*	ACB	FRM	ACB	FRM	ACB	FRM
Diluent	Not found	Not found	Not found	Not found	Not found	Not found	Not found	Not found
Standard	3.049	4.316	1786594	3684584	15036.81	8325.87	1.032	1.035
Placebo	Not found	Not found	Not found	Not found	Not found	Not found	Not found	Not found
% RSD*	0.21	0.20	0.81	0.15	0.35	0.43	0.31	0.35