DEVELOPMENT AND VALIDATION OF STABILITY-INDICATING
RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF
FORMOTEROL FUMARATE AND BUDESONIDE IN METERED
DOSE INHALER FORMULATION
*Nanasaheb R. Kale1, Dr. Ashok P. Pingle1, Javed A. Mirza1, Govind N. Dhongade1 1
NDMVP Samaja's College of Pharmacy, Gangapur Road, Nashik , Maharashtra
India
ABSTRACT
A stability indicating RP-HPLC method was developed and validated
for the simultaneous determination of Formoterol Fumarate (FF) 6µg
and Budesonide (Bude) 400µg in metered dose inhaler formulation.
The chromatographic separations was carried out with Waters alliance
2695 separation module, 2996 PDA detector and empower software
and Shimadzu LC HCT 10 with UV detector and LC Solution
Software instruments, using Hypersil BDS C-18, 150 x 4.6mm, 5 µ
column and gradient elution with NaH2PO4.H2O buffer (pH 3.1 with
OPA) and acetonitrile as mobile phase at a flow rate of 1.5 ml/min.
The detection was carried out on dual wavelength detector at 214nm
and 247nm for FF and Bude respectively. The detector response is linear from 0.3-0.9 µg/ml
and 20-60 µg/ml concentrations for FF and Bude respectively. The linear regression equation
was found to be y = 56974 x-170.74 (r² = 0.9997) for FF and y = 28487 x – 44972 (r² =
0.9994) for Bude. The stability indicating capability was established by forced degradation
experiments. The method was satisfactorily validated as per the ICH guidelines.
KEY WORDS : Formoterol Fumarate, Budesonide, HPLC, Gradient elution.
INTRODUCTION
Formoterol is long acting selective beta-2-adrenergic receptor agonist used as bronchodilator
in treatment of asthma. Formeterol fumarate is chemically (RS)-2′-hydroxy-5′ [(RS)
-1-hydroxy-2-[[(RS)-p-methoxy- α -methylphenethyl] amino] ethyl] formanilide.
Volume 3, Issue 6, 1386-1399. Research Article ISSN 2277 – 7105
Article Received on 20 June 2014,
Revised on 15 July 2014, Accepted on 10 August 2014
*Correspondence for
Author
Nanasaheb R. Kale
NDMVP Samaja's College of
Pharmacy, Gangapur Road,
OH H
O H
N H
COH
H CH
3
OCH3
HOOC COOH.2H2O
2
Figure: 1 Chemical structure of Formoterol Fumarate
Budesonide is synthetic corticosteroid. It is administered by systemic, local/topical route. In
the form of inhalation dosage form, it is used in management of asthma as prophylactic
therapy while Nasal sprays are used for management of seasonal and perennial allergic
rhinitis. Topically it is used in treatment of skin disorders. Chemically it is C-22S (epimer A)
and the C-22R (epimer B) epimers of 16α, 17-[(1RS)-butylidenebis (oxy)]-11β, 21
-dihydroxypregna-1, 4-diene- 3, 20-dione.
Figure: 2 Chemical Structure of Budesonide
Literature survey reveals that few RP-HPLC [12-21] and UV [22] spectrophotometric methods
have been reported for the estimation of FF and Bude. No stability indicating RP-HPLC
method for estimation of FF and Bude in Metered dose inhaler form has been reported.
MATERIAL AND METHOD Instrumentation
HPLC System Waters alliance 2695 separation module, 2996 PDA Detector and Empower
software and Shimadzu LC HCT 10 with UV detector and LC Solution Software. Hypersil
BDS C-18, 150 x 4.6mm, 5 µ column. Mettler Toledo AG245 aanalytical balance. Lab India
Materials
Formoterol Fumarate and Budesonide obtained from Glenmark R&D Sinnar. Water (HPLC
grade) was obtained from a Milli –Q water purification system, Acetonitrile (HPLC grade),
Methanol (HPLC grade) and NaH2PO4.H2O were of analytical grade purchased from Merck.
The commercial combination [Airtec-FB, Formoterol Fumarate (6µg) and Budesonide
(400µg)] was obtained from Glenmark R&D Sinnar.
Method
Chromatographic separation was achieved on hypersil BDS C-18, 150 x 4.6mm, 5µ column
and gradient elution with sodium dihydrogen orthophosphate monohydrate (pH 3.1) Buffer:
Acetonitrile. The detection wavelength was 214 nm for Formoterol Fumarate and 247nm for
Budesonide, flow rate of 1.5 ml / min, injection volume 20 µl, column temperature 30°C and
run time of 30 min. Order of Elution first principal peak due to Formoterol Fumarate, Second
peak due to Budesonide, (Budesonide Epimer-B and then Epimer - A)
Table: 1 Gradient program
Time (minute) Buffer % Acetonitrile
0.01 82 18
6.50 82 18
8.00 67 33
22.0 67 33
23.0 82 18
Figure: 3. HPLC Chromatogram for Formoterol Fumarate and Budesonide
Preparation Of Buffer For Mobile Phase
Dissolve 3.73 gram of sodium dihydrogen orthophosphate monohydrate in 1000 ml of water
adjusting pH 3.1± 0.1 with orthophosphoric acid. Filter through nylon 0.45µm membrane
filter.
Preparation Of Standard Solution Reference Solution a
Weigh accurately about 20.0 mg of Formoterol fumarate working standard in 200 ml flask.
Add 160 ml of diluent and sonicate to dissolve and then dilute up to the mark with diluent.
Dilute 5 ml of above solution to 50 ml with diluent.
Reference Solution b
Weigh about 80.0 mg of Budesonide working standard in 200 ml flask. Add 160 ml of
diluent, sonicate to dissolve and then dilute up to the mark with diluent.
Reference Solution c
Dilute 3ml of Reference solution (a) and 5 ml of Reference solution (b) to 50 ml with diluent.
Preparation Of Sample Solutions
Procedure for Metered Dose Assay Sample, (X)
Remove all labels and markings present on the container with acetonitrile. Prime it with 3
Actuation. Wash the valve with methanol and wipe with tissue paper. Shake the pressurized
container for 30 seconds and place it inverted in the 50 ml beaker containing 30 ml diluent.
Discharge one delivery below the surface of the diluent using baseplate. Shake for 30 seconds
in vertical direction and again actuate below the surface of diluent using baseplate. Repeat the
sequence for 8 times. Transfer the solution in 100 ml volumetric flask. Rinse the beaker with
diluent, add rinsate to 100 ml volumetric flask and make up to the mark with diluent, filter
through nylon 0.45µ filter, and inject. (Actuations to take for the test, Initial 4 to 13,
Middle76 to 85 and End 141 to 150 of the canister)
Procedure for actuator Retention, (Y)
Fit the washed and dried actuator to the pressurized container and actuate the valve for 10
times at intervals of not less than 5 seconds. Remove the actuator from the container wash it
with 25 ml of diluent. Dilute the combined washings to 50 ml with diluent filter and inject.
Calculate the amount of active ingredient per actuation of the valve. This amount of active
ingredient is referred as actuator retention (Y).
Forced Degradation Studies [8]
It is a process in which the natural degradation rate of a pharmaceutical formulation is
increased by applying the additional stress. HPLC method is used to separate, detect, and
quantify the various drug related degradation substances. The prepared sample (0.6 µg of FF
and 40 µg of Bude) was treated with various degradation conditions such as acidic (0.1M
HCL, heat 15 min at 70ºC), alkaline (0.1M NaOH, 70ºC for 1 min), oxidization (3% H2O2,
70ºC, 60 min), and sunlight (2 hours). Both FF and Bude were found to be highly sensitive to
acidic and alkaline degradation. The peak area and assay value were dropped in all the above
mentioned conditions except peroxide degradation. The results of forced degradation studies
were given in Table.2 and shown in Fig.4, Fig.5, Fig.6, and Fig.7.
Table: 2. Result for Forced Degradation studies Sr.
No. Degradation Parameter
Peak area of Sample % of Recovery % Degradation
FF Bude FF Bude FF Bude
1 Control Sample 40761 1162830 103.8 111.9 - -
2 Acid Degradation, 0.1M HCl, 70°C, 15 min. 33461 1121598 83.4 107.9 20.4 4.0
4 Peroxide Degradation, 3%H2O2 70°C,60
min. 40964 1166524 104.4 112.3 - -
5 Photo degradation, Daylight, 2 hours. 40412 996537 102.9 95.9 1.0 16.0
Figure: 5. Chromatogram for Base Degradation
Figure: 7. Chromatogram for Photo Degradation
Method Validation
The developed method was validation as per ICH guidelines (ICH-Guidelines, Q2R1). The
validation parameters are linearity, accuracy, precision, and robustness.
System Suitability
The six replicated injections were made in the standards solution of both FF and Bude and
system suitability parameters such as theoretical plates (USP), resolution (USP) and tailing
Table: 3. System Suitability Parameter
Parameters Formoterol Fumarate Budesonide Epimer B
Budesonide Epimer A
Resolution Factor -- 1.82
Theoretical Plates 4448 20283 19612
Tailing 1.09 1.07 1.06
Retention time 5.75 18.84 19.85
RSD 0.326 0.260
Linearity
Linearity was demonstrated from five different concentration levels for both FF and Bude,
which were found to be linear in the range of 0.3µg/ml to 0.9µg/ml and 20µg/ml to 60µg/ml
respectively. The values were given in Table 4. Correlation coefficient for FF and was found
to be 0.9997 and 0.9994 respectively. The calibration curves were shown in the Fig.8 and
Fig.9.
Table: 4.Linearity of Formoterol Fumarate and Budesonide
Figure: 8. Linearity plot of Formeterol Fumarate
Concentration, (%) Formoterol Fumarate Budesonide
Concentration (µg /ml) Mean Area Concentration (µg /ml) Mean Area
Linearity-50% 0.300 16802 19.864 529834
Linearity-80% 0.480 27120 31.782 851944
Linearity-90% 0.539 30617 35.755 969918
Linearity-100% 0.599 34102 39.728 1072804
Linearity-110% 0.659 37682 43.700 1220050
Linearity-120% 0.719 40512 47.673 1306742
Linearity-150% 0.899 50974 59.591 1655930
Slope 56974 28487
Intercept -171 -44971
Figure: 9. Linearity plot of Budesonide
Accuracy (Recovery Studies)
To check the degree of accuracy of the method, the recovery studies were performed by
standard addition method at 80 %, 100 % and 120 %. Known amounts of standard mixture of
FF and Bude were added to pre-analyzed samples and were subjected to the proposed HPLC
method. Results of recovery studies were given in Table 5.
Table: 5. Accuracy (Recovery) Data
Parameters FF Bude
% Recovery % RSD % Recovery % RSD
80% 101.40 0.09 100.33 0.47
100% 101.53 0.15 100.73 1.02
120% 101.03 0.80 101.73 0.15
PRECISION
Intraday precision (Repeatability) and Inter day precision (Reproducibility) was evaluated by
carrying out six independent sample preparations (0.6µg/ml of FF and 40µg/ml of Bude)
from a single lot formulation. Percentage relative standard deviation (%RSD) was calculated.
The results for precision were given in Table 6.
Table: 6. Precision
Drug Intra-day assay Inter-day assay
% Obtained % RSD % Obtained % RSD
FF 102.7 0.710 102.6 1.688
Robustness
To evaluate the robustness of the developed RP-HPLC method, small deliberate variations in
the parameters of optimized method were done. The effect of ±0.2 unit change in buffer pH,
±50C change in temperature and ±0.15 ml change in flow rate the retention time and area
were studied. The results of robustness were tabulated in Table 7.
Table 7: Results of Robustness
RESULTS AND DISCUSSION
To develop a suitable HPLC method for analysis of the drugs in pharmaceutical formulation,
in the beginning various tests were carried out to select the optimum conditions. After many
number of trials various proportions of solvents including buffers and acetonitrile were used
for this study. The goal of this study was to develop a suitable HPLC method for the analysis
of FF and Bude in a finished combined metered dose inhaler form using a NaH2PO4.H2O
buffer and acetonitrile in the gradient program pH was adjusted to 3.1 with ortho phosphoric
acid at a flow rate of 1.5ml/min, hypersil BDS C18 150 x 4.6mm, 5 µ column particle size
with the PDA and UV detection at 214 and 247 nm. The retention time was found to be 5.01
min and 17.58 and 18.44 min for FF and Bude epimer B Bude and epimer A respectively.
The linear regression equations were y = 56974x-171 (r2 = 0.9997) for FF and y= 28487x
-44971 (r2 = 0.9994) for Bude. The developed method was found to be accurate and precise;
the RSD values are in the range. The stability indicating capability was established by forced
degradation experiments.
CONCLUSION
The developed method was simple, accurate, and precise for simultaneous determination of
Formoterol Fumarate and Budesonide in combined metered dose inhaler form. This method
is accurate, precise, sensitive and also economical. Thus, the proposed method can be easily
adopted for routine analysis of Formoterol Fumarate and Budesonide in combined metered
dose inhaler form.
Factor Level Retention time Area FF Area Bude A + Bude B
FF Bude Epimer B Bude Epimer A
Standard ---- 5.4 17.58 18.44 34150 1100441
Buffer pH 2.9 5.7 17.95 18.85 33254 1135697
3.3 5.8 18.12 18.96 36009 1158966
Column oven temperature
250 C 6.1 17.32 18.26 39746 1157924
350 C 5.3 18.52 19.45 40353 1163152
Flow rate 1.35ml/min 6.2 18.99 20.09 32619 1292618
ACKNOWLEDGEMENTS
The authors are very much thankful to Glenmark Pharmaceuticals (R&D) Sinnar Nashik,
India for providing all the facilities to perform the research work.
REFERENCES
1. G.D. Christen, Analytical Chemistry, 5th edition, John Wiley and Sons, 2003; pp 35-42:
pp131-132.
2. B.K. Sharma, Instrumental Methods Of Chemical Analysis, 25th edition, Goel
Publication Co., Meerut, 1983, pp3-6.
3. K.A. Connors, A textbook of Pharmaceutical Analysis, 3 rd edition, Jhon Wiley and sons,
1999: pp 373-390.
4. J.W.Munson, Pharmaceutical Analysis, Modern methods, International Medical book
Distributors, Mumbai, Part B; 2001: pp 51-54.
5. ICH-Q1A (R2): Stability Testing of New Drug Substances and Products (Second
Revision), FDA, Vol. 68; No. 225, 21 November 2003: 65717-18.
6. ICH-Q1B: Photo stability Testing of New Drug Substances and Products, FDA, Vol. 62,
No. 95; 16 May1997: 27115-27122.
7. S. Singh, M. Bakshi, Guidance on conduct of: Stress tests to determine Inherent Stability
of Drugs, April 2000.
8. Blessy M.et.al, Development of forced degradation and stability indicating studies of
drugs – A review, J. Pharm. Anal.(2013)http://dx.doi.org/10.1016/j.jpha.2013.09.003
9. USP-NF, Validation of Compendial Procedures, General Chapters, Volume-1, 2010,
734-736.
10.ICH-Q2 (R1): Validation of Analytical Procedures: Text and Methodology, FDA, Vol 60,
1March; 1995: 11260.
11.US FDA, General principles of validation, Rockville, MD, Center for Drug Evaluation
and Research (CDER), May 1987.. US FDA, Guidelines for submitting samples and
analytical data for method validation, Rockville, MD, Center for Drugs and Biologics
Department of Health and Human Services, Feb. 1987.
12.Katari Srinivasaro, Vinayk Gorule, Ch.Venkata Reddiah And Venkata Krishna A,
Method Development for Estimation of Formoterol Fumarate And Mometasone Furoate
In Metered Dose Inhalation Form By HPLC, Pharmacophore 2012; Vol. 3 (6); 301-306
13.A.Sule, V.Vaidya, S, Simultaneous Determination of Impurities from a combination
corticosteroid by RP- HPLC, IJPWR Vol 3 Issue 3 (Jul-Sep) – 2012
14.Katari Srinivasaro, Vinayk Gorule , Venkata Krishna Akula, Development and Validation
for Simultaneous Estimation of Budesonide and Salmeterol Xinafoate in Metered Dose
Inhalation Form by RP-HPLC, Int.J.Pharm.Phytopharmacol.Res. 2012; 1(5): 271-275.
15.Hiral N. Davea, Ashlesha G. Makwanab, and Bhanubhai N. Suhagiac, Validated
Reversed Phase High Performance Liquid Chromatographic Method for Determination
of Three Novel Steroids in Bulk and Pressurized Metered – Dose Commercial
Preparations Using a Common Mobile Phase, International Journal of Applied Science
and Engineering 2013. 11; 2: 125-135
16.J. Varshosaz1, J. Emami, N. Tavakoli1, M. Minaiyan, N. Rahmani, F. Ahmadi, and F.
Dorkoosh Development and validation of a rapid HPLC method for simultaneous analysis
of budesonide and its novel synthesized hemi esters in colon specific formulations,
Research in Pharmaceutical Sciences, October 2011; 6(2): 107-116
17.L.R.Synder, J. J. Kirkland, L. J. Glajch, Practical HPLC Method Development,
2ndedition, John Wiley and sons, Inc, 2,10,20,22,42.
18.Nandini Pai and Swapnali Suhas Patil, Development and validation of RP-HPLC method
for estimation of Formoterol fumarate and budesonide in pressurised meter dose inhaler
form, Pelagia Research LibraryDer Pharmacia Sinica, 2013; 4(4):15-25
19.Malik K., Kumar D., Tomar V., Kaskhedikar S., Soni. L.; Simultaneous quantitative
determination Formoterol fumarate and fluticasone propionate by validated
reversed-phase HPLC method in metered dose inhaler; Pelagia Research Library Der Pharmacia
Sinica; 2011; 02(06): 77-84.
20.Patil A.T., Patil S.D., Shaikh K.A.; Sensitive LC method for simultaneous estimation of
ciclesonide and Formoterol fumarate in dry powder inhaler; Journal of Liquid
Chromatography & Related Technologies; 2011: 34(15).
21.Katari Srinivasaro, Vinayk Gorule, Ch.Venkata Reddiah and Venkata Krishna, Validated
method development for estimation of Formoterol fumarate and mometasone furoate in
metered dose inhalation form by high performance liquid chromatography,
Pharmacophore (An international research journal) 2012,;Vol. 3 (6): 301-306
22.Prasad A.V.S.S., Simultaneous spectrophotometric determination of Formoterol fumarate
and budesonide in their combined dosage form, Indian Journal of Chemical Technology,
Vol. 13, Jan 2006, pp 81-83.
23.R.Nash: Pharmaceutical Process Validation, 3rd edition, Marcel Dekker Publication, New
24.J.T.Carstensen, C.T.Rhodes, Drug Stability Principles and Practices, 3rd edition, Marcel
Dekker, New York, 107; 2002: 340-370.
25.S.W.PettersonTricks of the Trade in Pharmaceutical Analysis by HPLC. International
Conference on Analytical Process Hplc.2006.