RP-HPLC Method for Simultaneous Estimation of Formoterol Fumarate, Tiotropium Bromide and Ciclesonide in Pharmaceutical Metered Dose Inhalers

 

Ravi Pratap Pulla¹*, B.S. Sastry², Y. Rajendra Prasad³ and N. Appala Raju⁴

¹Department of Pharmaceutical Chemistry, SSJ College of Pharmacy, V.N.Pally, Gandipet, Hyderabad-500 075.

²Department of Pharmaceutical Chemistry, Medak Institute of Technology-Pharmacy, Kothapet (V), Near Narsapur, Shivampet, Medak District- 502 334.

³Department of Pharmaceutical Chemistry, Andhra University College of Pharmaceutical Sciences,  Peda Waltair, Visakhapatnam- 530 003.

⁴Department of Pharmaceutical Chemistry, Sultan-Ul-Uloom College of Pharmacy, Mount Pleasant, Road No# 3, Banjara Hills, Hyderabad-500 034.

*Corresponding Author E-mail: ravi_477@rediffmail.com

ABSTRACT:

An accurate, sensitive, precise, rapid and isocratic Reversed-Phase HPLC, (RP-HPLC) method for simultaneous estimation of Formoterol Fumarate, Tiotropium Bromide and Ciclesonide in the bulk drug and in Pharmaceutical metered dose inhalers has been developed and validated.  The best separation was achieved on a 250 mm × 4.6 mm i.d., 5-μm particle, Inertsil ODS 3V-RP C18 column with Acetonitrile as the organic modifier and Di-Potassium Hydrogen Phosphate [0.03M] in water with pH 3.2 adjusted with Ortho-Phosphoric Acid (0.1% v/v) in the proportion of [30:70 v/v] as mobile phase at a flow rate of 0.8 mL min⁻¹. UV detection was at 254 nm. Retention times were found to be 5.206 min. for Formoterol Fumarate, 7.016 min. for Tiotropium Bromide and 9.991 min for Ciclesonide. The response was a linear function of concentration over the range of 0.72-8.64 mcg/ml, 1.08-12.96 mcg/ml and 24-288 mcg/ml respectively with correlation coefficient of 0.9999. The percentage assay Formoterol Fumarate, Tiotropium bromide and Ciclesonide were found to be 98.87 %, 99.42 % and 98.91 % respectively. The Limit of Detection (LOD) for Formoterol Fumarate, Tiotropium bromide and Ciclesonide were found to be 1.44 µg/ml, 5.4µg/ml and 48µg/ml respectively. The Limit of Quantification (LOQ) for Formoterol Fumarate, Tiotropium bromide and Ciclesonide were found to be 4.32µg/ml, 16.2µg/ml and 144µg/ml respectively. The excipients present in the formulation were not interfered with the assay. The method is suitable for application in quality-control laboratories, because it is simple and rapid with good accuracy and precision.

 

 

INTRODUCTION:

Chronic Obstructive Pulmonary Disease (COPD) refers to chronic bronchitis and Emphysema, which is a pair of two commonly co-existing diseases of the lungs in which the airways become narrowed.¹ COPD, is also known as Chronic Obstructive Lung Disease (COLD), Chronic Obstructive Airway Disease (COAD), Chronic Airflow Limitation (CAL) and Chronic Obstructive Respiratory Disease (CORD).

 

Important management strategies are smoking cessation, vaccinations, rehabilitation and drug therapy (often using inhalers). The combination of Formoterol Fumarate, Tiotropium bromide and Ciclesonide will help in targeting different aspects of COPD viz. bronchodilation through different mechanisms and the inflammations with inhaled steroids⁵. Formoterol Fumarate² is a long-acting β₂  agonist used in the management of asthma and/or COPD; chemically it is (RS, SR)-N-[2-hydroxy-5-[1-hydroxy-2-[1-(4-methoxyphenyl) propan-2-ylamino]ethyl] phenyl] formamide (Figure:1). Tiotropium³ is a long-acting, antimuscarinic agent, which is often referred to as an anticholinergic agent. In the airways, it exhibits pharmacological effects through inhibition of M3 - receptors at the smooth muscle leading to bronchodilation. It is chemically (1α, 2β, 4β, 7β)-7-[(hydroxidi-2-thienylacetyl)oxy]-9,9-dimethyl-3-oxa-azoniatricyclo [3.3.1.0^2,4]-nonane bromide monohydrate (Figure:2). Ciclesonide⁴ is a glucocorticoid used to treat obstructive airway disease, which is chemically, 2-[(1S, 2S, 4R, 8S, 9S, 11S, 12S, 13R)-6-cyclohexyl-11-hydroxy-9, 13-dimethyl-16-oxo-5, 7-dioxapentacyclo [10.8.0.0^{2, 9}. 0^{4, 8}.0^{13, 18}] icosa-14, 17-dien-8-yl] - 2-oxoethyl 2-methyl propanoate (Figure: 3). Literature survey reveals the determination of Formoterol Furmarate^{5, 6}, and Tiotropium^{7, 8} by using Liquid Chromatography in biological fluids like plasma and urine. Colorimetric methods, with laborious derivatization, have been reported for analysis of Formoterol Fumarate in rotacaps⁹. In this article, a simple, easily available and reliable Reversed Phase HPLC method with UV-detection has been developed and validated for the simultaneous determination of Formoterol Fumarate, Tiotropium Bromide and Ciclesonide concentrations in metered dose inhalers.

 

(Figure: 1)

Formoterol Fumerate

 

(Figure: 2)

 

Tiotropium Bromide

(Figure: 3)

 

Ciclesonide

EXPERIMENTAL:

Chemicals and Reagents:

·        Formoterol Fumarate, 99%, (Molecular Weight: 804.88 g/mol), Tiotropium Bromide of 99% (Molecular Weight: 472.41 g/mol) and Ciclesonide of 99% (Molecular Weight 540.69 g/mol) purity are acquired from Cipla Pharmaceuticals, Mumbai, India.

·        Acetonitrile HPLC Grade from Rankem Fine chemicals of HPLC Grade.

·        Potassium Phosphate (Dibasic, K₂HPO₄) [0.03M] from Rankem Fine Chemicals AR grade.

·        Ortho-Phosphoric Acid, 85%, Quligens Fine chemicals and HPLC Grade water.

 

Chromatography Instrument:

Quantitative HPLC was performed on liquid Chromatograph, Waters separation 2996, PDA detector module equipped with automatic injector with the injection volume 20 µl, and 2693 pump. A RP Inertsil ODS 3V C-18 column (250x4.6 mm i.d; particle size 5 μm) was used. The HPLC system was equipped with Empower Software. The column was maintained at 40^o C and eluted under isocratic conditions over 14.0 min at a flow rate of 0.8 mL/min. Mobile phase consisted of acetonitrile as the organic modifier and Di-Potassium Hydrogen Phosphate[0.03M] in water with pH 3.2 adjusted with Ortho-Phosphoric acid (0.1% v/v) in the proportion of [30:70 v/v.]. Before use, it was filtered through a 0.45 μm Nylon membrane filters and then degassed.  UV detection was performed at 254 nm.

 

Preparation of the Primary Standard Drug solution:

A standard stock solution of the drugs was prepared by dissolving 7.2 mg of Formoterol Fumarate, 13.5 mg of Tiotropium Bromide( equivalent to 10.8 mg of Tiotropium) and 240 mg of Ciclesonide in 10 ml volumetric flask containing 5 ml of diluent (50:50 v/v Acetonitrile: Water), sonicated for about 15 min and then made up to 10 ml with diluent to get the primary standard stock solution containing 720 mcg/ml of Formoterol Fumarate, 1350 mcg/ml of Tiotropium Bromide (equivalent to 1080mcg/ml of Tiotropium) and 24 mcg/ml of Ciclesonide.

 

Preparation of Working Standard Drug Solution:

1.0 ml of the above stock solution was taken in 100 ml volumetric flask and thereafter made up to 100 ml with diluent (50:50v/v Acetonitrile: Water) to get the working standard solution containing 7.2 mcg/ml of Formoterol Fumarate, 13.5 mcg/ml of Tiotropium Bromide (equivalent to 10.8 mcg/ml of Tiotropium) and 240 mcg/ml of Ciclesonide.

 

Analysis of Pharmaceutical Metered Inhalers:

Remove the pressurized container (Triohale® MDI, Cipla; Each puff contains: Formoterol Fumarate Dihydrate Ph. Eur -6mcg, Tiotropium Bromide monohydrate equivalent to Tiotropium-9 mcg,  Ciclesonide -200 mcg, are suspended in propellant HFA 227-q.s in net weight of contents of  8.4 grams) from the actuator and remove all the labels and markings with suitable solvent. Dry the container, replace in its actuator, shake for about 30 seconds and prime the metered valve as follows. Discharge once for waste; wait for not less than 5 seconds and discharge again to waste. Remove the pressurized container from its actuator, clean the valve stem (internally and externally) and the valve ferrule by washing with a suitable solvent. Dry the complete valve assembly, using an air line fitted with an appropriate narrow jet to ensure that all solvent is removed from the inside of the valve stem. Place a tripod stainless steel base plate with a central circular indentation of 1.5 mm in diameter in a small vessel suitable for shaking and add 15 ml of diluent. The size of the vessel is such that when the pressurized inhalation is discharged in to 15 ml of diluent, discharge takes place not less than 25 mm below the surface of the solvent. Shake the pressurized container for about 30 seconds and place in inverted position in the vessel. Discharge 120 deliveries below the surface of the solvent actuating the valve at intervals of not less than 5 seconds, maintaining the pressurized container in vertical plane and discharging the pressurized inhalation through the aperture of the base plate. Shake the pressurized container between each actuation of the valve. Shaking should be carried out without removing the pressurized container from inverted position in the vessel. Remove the pressurized container, wash it with mobile phase and combined solution and washings to 25 ml volumetric flask, and dilute up 100 ml to volume with diluent to get the concentration of 7.2 mcg/ml of Formoterol Fumerate, 13.5 mcg/ml of Tiotropium Bromide (equivalent to 10.8 mcg/mL of Tiotropium) and 240 mcg/ml of Ciclesonide.

 

Linearity: Aliquots of working standard solution contains Formoterol Fumarate, Tiotropium Bromide and Ciclesonide stock solution were taken in different 10 ml volumetric flasks and diluted up to the mark with the mobile phase such that the final concentrations of Formoterol Fumarate, Tiotropium Bromide and Ciclesonide are in the range of 0.72-8.64 mcg/ml, 1.08-12.96 mcg/ml and 24-288 mcg/ml respectively (Table: 4). Each of these drug solutions (20μL) was injected three times into the column, and the peak areas and retention times were recorded. Evaluation was performed with PDA detector at 254 nm and a calibration graph was obtained by plotting peak areas versus concentration of Formoterol Fumarate, Tiotropium Bromide and Ciclesonide.

 

Accuracy: Accuracy was evaluated in triplicate by addition of three different amounts of Formoterol Fumarate, Tiotropium Bromide and Ciclesonide, to a previously analyzed sample and comparing the amounts of analytes recovered with the amounts added. The amounts added were equivalent to 80, 100, and 120% of the amount originally present. %Recovery and RSD (%) were calculated for amount added. From these data, it is obvious that the method is remarkably accurate, which ensures that this method produces reliable results (Table: 2).

 

 

Precision:  Intra-day and Inter-day precision were evaluated by analyzing quality-control samples containing low, medium, and high concentrations of 80%, 100% and 120%  For Intra-day variation, sets of five replicates of the three concentrations were analyzed on the same day; for Inter- day variation, five replicates were analyzed on three different days. The low value (≤1%) of RSD indicates the repeatability of the method (Table: 3).

 

Limits of Detection and Quantification: Limit of Detection (LOD) of the method was determined as the lowest concentrations of active pharmaceutical ingredients producing a signal-to-noise (S/N) ratio of about 3. The Limit of Quantification (LOQ) was determined as the lowest concentrations of active pharmaceutical ingredients capable of being quantified with acceptable accuracy and precision producing signal-to-noise (S/N) ratio of about 10.

 

Method Applicability: The present developed method was evaluated by applying to pharma- ceutical metered dose aerosols for the estimation of Formoterol Fumarate, Tiotropium Bromide and Ciclesonide by our research group.

 

 

RESULTS AND DISCUSSION:    

HPLC Method Development and Optimization:

In response to lack of simple, reliable and easy-to-use method for the determination of Formoterol Fumarate, Tiotropium Bromide and Ciclesonide concentrations in pharmaceutical matrices, an isocratic Reversed-Phase HPLC method was developed for quantification of above mentioned, active pharmaceutical ingredients. We examined several HPLC method variables with respect to their corresponding effects on the result of analysis. To optimize the chromatographic conditions, different combinations of Methanol-Water and Acetonitrile-Water and Acetonitrile- Di-Potassium Phosphate buffer were tested. Acetonitrile with Phosphate buffer system [pH 3.2] was preferred because it resulted in greater resolution of active pharmaceutical ingredients after several preliminary investigatory runs, compared with other mobile phases. The other parameters in this factorial design were temperature, flow rate, detection wavelength and volume of injection. Buffer molarity was changed and optimum buffer strength was selected as 0.03M on the basis of theoretical plate number. At 254 nm, UV responses of all three active pharmaceutical analytes were good and free form interferences. Under these conditions, the analyte peaks were well defined and free from tailing. Considering the whole body of the data obtained from this extensive study, the set of conditions indicated earlier in this article was selected for further validation.  Typical chromatogram of Formoterol Fumarate, Tiotropium Bromide and Ciclesonide has been shown in Figure: 4.

 

Figure: 4: Typical HPLC Chromatogram of Formoterol Fumarate, Tiotropium Bromide and Ciclesonide

 

 

Method Validation Tests:

Recommended method validation characteristics including Method precision (RSD, %), Method accuracy (Recovery % and RSD, %), Linear range (Correlation Coefficient), and LOD & LOQ, were investigated.

­

Linearity:  The plot of peak areas of each sample against respective concentrations were found to be linear, in the range of 0.72-8.64 mcg/ml for Formoterol Fumarate, 1.08-12.96 mcg/ml for Tiotropium Bromide and 24-288 mcg/ml for Ciclesonide with Correlation Coefficient of 1.0 (Table: 4). Linear regression least square fit data obtained from the measurements are given in Table: 1. The respective linear regression equation being Y= 766602x+29930.11 for Formoterol Fumarate, Y= 5309731.4x+184384.4 for Tiotropium Bromide and Y=36346.5x+ 40111.67 for Ciclesonide. The regression characteristics, such as slope, intercept, and %RSD were calculated for this method and given in Table: 1.These results show that there was an excellent correlation between peak areas and analyte concentration.

 

Accuracy: Recovery of the individual substances at 80%, 100%, and 120% of specified concentrations were between 97.1% and 104.25%, which proves the accuracy of the method. From these data, RSD was always less than 1%, which indicates it is obvious that the method is remarkably accurate, produces reliable results (Table: 2)

 

Precision: The Intra-day and Inter-day variability or precision data are summarized in Table: 2. The low value (<1%) of RSD indicates the repeatability of the method. These data indicate a considerable degree of precision and reproducibility for the method both during one analytical run and between different runs (Table: 3).

 

Robustness:  Robustness was studied out to evaluate the effect of small but deliberate variations in the chromatographic conditions at three different levels, i.e. –2, 0, +2. To determine the robustness of this method, the experimental conditions were deliberately altered at three different levels and retention time and chromatographic response were evaluated. One factor at a time was changed to study the effect. Variation of the detection wavelength by ±2 nm (252 nm and 256 nm), mobile phase buffer to Acetonitrile ratio (68:32 and 72:28, v/v), mobile phase pH by ±0.2 units (pH 3.0 and 3.4), and mobile phase flow rate by 0.8 mL min−1 (0.6 and 1.0 mL min−1) had no significant effect on the retention time and chromatographic response of the method, indicating that the method was robust. The results are shown in Table: 5.

 

 

Table 1: Results from regression analysis and System Suitability data:

Parameter	Formoterol Fumarate	Tiotropium Bromide	Ciclesonide
Retention Time (min)	5.206	7.016	9.991
Tailing Factor	1.53	0.99	1.19
Theoretical Plates	6629.29	9952.81	7018.80
Resolution	0.64	1.71	10.51
Linear range in (µg/mL)	0.72-8.64	1.08-12.96	24-288
Limit of Detection (LOD) (ng/mL).	1.44	5.4	48
Limit of Quantification (LOD) (ng/mL)	4.32	16.2	144
Slope(m)	766602	5309731.4	36346.5
Intercept©	29930.11	184384.4	40111.67
Correlation Coefficient (r)	0.9999	0.9999	0.9999
Method Precision (RSD, %, n=6)	0.23	0.40	0.33
%  of Assay	98.87	99.42	98.91

 

Table 2: Results of Recovery Accuracy Studies:

Accuracy       parameter	Spiked with 80% of the Working Std. Solution contains analytes in mcg/mL			Spiked with 100% of the Working Std. Solution contains analytes in mcg/mL			Spiked with 120% of the Working Std. Solution contains analytes in mcg/mL
	Formoterol Fumerate	Tiotropium Bromide	Ciclesonide	Formoterol Fumarate	Tiotropium Bromide	Ciclesonide	Formoterol Fumarate	Tiotropium Bromide	Ciclesonide
Amount added	5.76µg/mL	8.64µg/mL	192 µg/mL	7.2µg/mL	10.8 µg/mL	240 µg/mL	8.64µg/mL	12.96µg/mL	288 µg/mL
Amount Found	5.673µg/mL	8.390µg/mL	188.4 µg/mL	7.29µg/mL	10.62µg/mL	238.2 µg/ mL	9.00µg/mL	12.99µg/mL	284.8 µg/mL
% Recovery	98.5	97.11	98.19	101.25	98.4	99.25	104.25	100.25	98.9
% RSD.	0.11	0.21	0.12	0.61	0.49	0.32	0.18	0.42	0.14

 

Table 3: Results of Recovery Precision Studies

Precision Parameter	80% of the Working Standard Solution contains analytes in µg/mL			100% of the Working Standard Solution contains analytes in µg/mL			120% of the Working Standard Solution contains analytes in µg/mL
	Formoterol Fumarate 5.76 µg/mL	Tiotropium Bromide 8.64 µg/mL	Ciclesonide 192 µg/mL	Formoterol Fumarate 7.2 µg/mL	Tiotropium Bromide 10.8  µg/mL	Ciclesonide 240 µg/mL	Formoterol Fumarate 8.64µg/mL	Tiotropium Bromide 12.96 µg/mL	Ciclesonide 288 µg/mL
Intra-day Mean area ± SD	4447089 ±12366.4	45976000 ± 103691	6987547 ±7070.3	5523121.8 ±24460	57453813 ±186637	8707779 ±39728.7	6578309 ± 2206.4	68157110 ±54908	10404861 ± 14628
%.RSD.	0.28	0.22	0.10	0.44	0.32	0.46	0.03	0.08	0.14
Inter-day Mean area ± SD	4462389 ±7663	46115095 ± 85835.7	6977783 ± 20291.1	5401844.5 ±12380.4	56214655 ±222989	8604594.6 ±28566	6574395 ±7830.6	68097823 ± 182120	11205941 ± 11650.8
% RSD.	0.15	0.17	0.26	0.23	0.40	0.33	0.11	0.25	0.10

 

Table 4: Summary of the Calibration data / Linearity studies

Concentration of Formoterol Fumarate (µg/mL)	Peak Area	Concentration of Tiotropium Bromide (µg/mL)	Peak Area	Concentration of Ciclesonide in µg/mL	Peak Area
0.72	573768	1.08	5912272	24	905068
1.44	1109095	2.16	11432329	48	1758687
2.88	2244280	4.32	23227236	96	3552253
4.32	3326159	6.48	34500237	144	5248052
5.76	4452161	8.64	46145492	192	7031169
7.2	5629409	10.8	58355601	240	8895375
8.64	6589528	12.96	68301451	288	10399790
Correlation Coefficient	0.9999	------	0.9999	----------	0.99999

 

Table 5: Results from testing of the Robustness of the method (n=3, 100% of the Working Standard Solution contains Formoterol Fumarate 7.2µg/mL, Tiotropium Bromide 10.8 µg/mL and Ciclesonide 240µg/mL)

Condition Studied in Robustness	Modification In OFAT analysis.	Mean Peak Areas ± S.D			% RSD			Mean Retention Time (in min) ± S.D
		Formoterol Fumarate	Tiotropium Bromide	Ciclesonide	Formoterol Fumarate	Tiotropium  Bromide	Ciclesonide	Formoterol Fumarate	Tiotropium Bromide	Ciclesonide
Detector Wavelength in (nm)	252	5572554± 2445.9	57494262 ± 186637.6	8682630 ± 36445.6	0.56	1.18	0.68	5.221±0.84	7.059± 1.18	9.991±0.03
	254	5522311± 12380.4	57455798± 222989.5	8758883 ± 374366.1	0.821	1.28	0.21	5.233±0.43	7.056± 0.94	9.982±0.12
	256	5509629± 31681.8	57755569± 54908.3	8693069 ± 44809.6	0.789	0.921	0.38	5.217±0.42	7.057± 0.58	9.986±0.45
Mobile Phase pH	3.0	5514140± 7821.9	57361218± 182324.6	8757562 ± 9845.8	0.921	0.825	0.26	5.236±0.65	7.034± 0.32	9.976±0.74
	3.2	5502051± 5689.8	57250000± 239193.4	8684017 ± 4407.7	0.619	0.625	1.02	5.219±0.81	7.056± 1.18	9.993±0.93
	3.4	5512170± 6437.5	57551700± 94432.3	8670518 ± 12346.2	0.527	0.825	0.94	5.234±0.94	7.063± 1.27	9.995±1.24
Mobile Phase Composition in (v/v)	68:32	5510819± 18130.9	57241699± 96350.4	8747577 ± 16573.8	0.661	1.47	0.85	5.235±0.63	7.062± 0.54	9.782±0.83
	70:30	5527875± 9995.2	57390457± 89445.8	8734904 ± 33689	0.982	1.19	0.28	5.228±1.04	7.068± 0.76	9.989±0.15
	72:28	5519419± 2206.4	57318435± 268422.6	8748297 ± 38885.5	1.292	0.73	0.84	5.237±1.13	7.072± 0.54	9.957±0.65
Flow rate in      mL/minute.	0.6	5492663± 7830.6	56848412± 112360.2	8648468 ± 8089.5	0.654	0.58	0.52	5.229±0.76	7.083± 0.69	10.012±1.03
	0.8	5537009± 3745.3	57726939± 162129.1	8708839 ± 50582.6	0.563	1.26	1.02	5.234±0.78	7.054± 0.26	9.993±0.94
	1.0	5487356± 5689.7	56969661± 93318.2	8615604 ± 27269.3	0.76	1.58	1.05	5.228±0.65	6.877± 0.82	9.756±0.75

 

 

Limit of Detection and Limit of Quantification:  The Limit of Detection (LOD) for Formoterol Fumarate, Tiotropium Bromide and Ciclesonide were found to be 1.44 ng/ml, 5.4 ng/ml and 48 ng/ml respectively. The limit of Quantification (LOQ) for Formoterol Fumarate, Tiotropium Bromide and Ciclesonide were found to be 4.32 ng/ml, 16.2 ng/ml and 144 ng/ml respectively. These values reflect the high sensitivity of the method, which is of great importance in most studies and also indicating the method can be used for detection and quantification of analytes in a very wide concentration range.

 

Specificity:  No evidence of  signals, in the corresponding times of the chromatogram were monitored as a sign of potential interfering peaks, were found when the pharmaceutical metered dose inhalers were tested. Hence, this method can be used reliably for the estimation of respected active pharmaceutical ingredients in a variety of dosage forms.

 

CONCLUSION:

A simple and easily available HPLC method was developed in this study for the quantification of Formoterol Fumarate, Tiotropium bromide and Ciclesonide in pharmaceutical matrices. The main advantages of this method are its considerably shorter run times, easy-to-use and its simplicity. All of these properties are very important in practice, particularly when a large number of samples are to be analysed. The results of validation tests were, collectively, indicative for a method with a relatively wide linear range, acceptable precision and accuracy and practically reliable sensitivity. The method enables simple, selective, sensitive, and specific analysis of Formoterol Fumarate, Tiotropium Bromide and Ciclesonide and can be used for routine analysis in pharmaceutical quality control.

 

ACKNOWLEDGEMENTS:

The authors wish to express their gratitude to M/s Cipla, Mumbai for the supply of Formoterol Fumarate, Tiotropium Bromide and Ciclesonide as gift samples.

 

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Received on 16.11.2010        Modified on 02.12.2010

Accepted on 05.12.2010        © AJRC All right reserved