INTRODUCTION:

Chronic obstructive pulmonary disease (COPD) is a type of obstructive lung disease. It is characterized by long-term breathing problems and poor airflow. The main symptoms of COPD include shortness of breath and cough with sputum production. COPD is a progressive disease, meaning it typically worsens over time. The term "chronic bronchitis" is also used to define a productive cough that is present for at least three months each year for two to three years.

Formoterol Fumarate Dihydrate is a bronchodilator and Budesonide is anti-inflammatory drugs available in different combinations in multiple Aerosol dosage forms to treat COPD, Asthma and Chronic bronchitis. Both drugs are available in white to almost white powder form. A bronchodilator is a substance that dilates the bronchi and bronchioles, decreasing resistance in the respiratory airway and increasing airflow to the lungs while Anti Inflammatory drug is used for the treatment of inflammation occurred on respiratory tract.^1-7

Inhalation suspension dosage forms are utilised as life saving formulations and administered with the help of different types of nebulizers. These dosage forms are mainly anti asthmatics and requires very low dose ranging from 5mcg to 400mcg per dose. So method development for analysis of these drugs is very challenging job.

In the present study, a developed HPLC method was validated for the determination of a lower concentration of Fumarate and Budesonide drugs in Inhalation suspension pharmaceutical dosage form. The proposed analytical method was found to be precise, repeatable, linear, accurate, rugged, robust and specific. RP-HPLC method for the combined determination of Formoterol Fumarate and Budesonide is considered the main contribution of this study.^8-14

Figure 1 Structure of Formoterol Fumarate dihydrate and Budesonide Drug molecules

Materials and Methods:

Chemicals and Reagents:

Formoterol Fumarate Dihydrate working standard used for this is manufactured by Vamsi labs limited, Budesonide working standard used for this study is manufactured by Aarti industries limited and Formoterol Fumarate and Budesonide Inhalation suspension is manufactured by Zydus Cadila healthcare limited. These standards and samples are provided for method development and validation by Zydus Cadila Healthcare Limited, Ahmedabad, India and Study performed at pharmaceutical technology centre, Analytical development laboratory of Zydus Cadila healthcare limited. Reagents Sodium Dihydrogen Orthophosphate, 1-Decane sulphonic acid sodium salt, Orthophosphoric acid and Acetonitrile used is of make Merck limited.

General Procedure (Standard and Sample Preparation)

Standard solution of Formoterol Fumarate and Budesonide was prepared at concentration of 0.2µg per mL and 10.0µg per mL respectively, by dissolving appropriate amount of drug substance standard in diluent. Sample of same concentration as standard was prepared using a following procedure. Taken a respule containing Formoterol Fumarate and Budesonide Inhalation suspension. Weigh the initial weight (W1 in gm) of respule. Shake it well. Cut it from the upper side cut mark and Transfer the whole content accurately to 100ml volumetric flask. Wash the each inner part of respule repetitively with 35ml of Acetonitrile with the help of suitable syringe and needle and transfer it to same 100ml volumetric flask. Further added about 35ml of Buffer solution to it and sonicated with intermittent shaking for about 10 minutes. Kept at room temperature for about 10 minutes after sonication and made volume up to the mark of 100ml with buffer solution. Weigh the end weight (W2 in gm) of respule. Report different weight of respule (W1-W2) in gm. Same procedure to be repeated for further 9 more Respules. Wt/ml of suspension also to be analysed with suitable glassware.

Detection method (Instrument and chromatographic system)

Chromatographic separation and quantification of assay of Formoterol Fumarate and Budesonide, were performed using the Shimadzu and Agilent HPLC system. The systems have a quaternary solvent manager, a sample manager and with Ultraviolet/diode array detector. The output signal monitoring and processing were done using LC solution and Chromeleon Software. Separation was achieved on Hypersil ODS (125 × 4.0 mm, 5.0μm) column (Thermo scientific). The isocratic liquid chromatographic method employs mobile phase prepared by adding 2.0gm of Sodium dihydrogen orthophosphate dihydrate and 1.5gm of 1-Decane sulphonic acid sodium salt monohydrate to 1000ml Milli-Q water and dissolved with the help of shaking with glass rod and ultrasonication. Adjusted pH of solution to 3.0 with Diluted Orthophosphoric acid. Then resulting solution was filtered through 0.45µ Nylon filter and degas. This buffer solution is mixed with Acetonitrile in the ratio of 650ml: 350ml (65: 35 % v/v). The mobile phase was pumped with the flow rate of 1.0 mL per min. The column oven temperature was maintained at 40°C and injection volume was kept as 50 μL. The detection was monitored at a wavelength of 215 nm. The mobile phase is used also as a diluent.^15-21

Figure 2 Chromatogram of Diluent

Figure 3 Chromatogram of Sample preparation

Method Validation:

The developed analytical method was validated for its acceptable performance to ensure suitability of indent purpose. The validation parameters executed include accuracy, precision, specificity, detection limit, quantification limit, linearity, range, ruggedness and robustness experiments, as per ICH guidelines.^22-32

System suitability and system precision:

System suitability and system precision is determined by injecting multiple injection of same standard preparation and calculating the %RSD of Formoterol Fumarate and Budesonide peak areas. The theoretical plates and Tailing factor also recorded for the Formoterol Fumarate and Budesonide Epimer-B and Budesonide Epimer-A peaks.

The acceptance criteria was set to % RSD of multiple injections should not be more than 2.0%.

The Theoretical plates for Formoterol, Budesonide Epimer-B and Budesonide Epimer-A peaks should be not less than 1500 and tailing factor for each peak should be less than 2.0

Limit of detection (LOD):

The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value. A specific calibration curve was studied using samples containing an analyte in the range of detection. A typical signal-to-noise ratio is 3:1.

Limit of quantitation (LOQ):

The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. This approach can only be applied to analytical procedures that exhibit baseline noise. A typical signal-to-noise ratio is 10:1. LOD and LOQ were calculated according to the linearity of the calibration curve and its standard deviation.

Linearity:

The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample. Linearity was studied by analyzing the mixed calibration standard solutions at ten concentration levels. The linearity solutions were prepared from 20% level to 400% level of target concentration for Budesonide and 10% level to 200% level of target concentration for Formoterol Fumarate. The y-intercept, slope and correlation coefficient were calculated for both analytes from linear regression equation. The specification was set to the value of co-relation co-efficient r should not be less than 0.998 for all analytes, and Y intercept bias should not be more than 2% of area of 100% linearity level.

Accuracy:

Accuracy and recovery, each of them is a face for the same coin. The accuracy can be defined as the closeness of the measured value (actual concentration) to the true value (Theoretical concentration), where recovery it is defined as how much drug was recovered from the added concentration using the purposed method.

Accuracy and recovery were evaluated in triplicate by addition of 3 sets of Formoterol Fumarate and Budesonide standard to the formula placebo to get concentration at 50, 100% and 150% level of target concentration. The specification was set for accuracy is 95% to 105% and %RSD should not be more than 5.0%.

Method Precision:

Method precision study was performed by preparing ten same homogeneous sample preparations using single batch. Assay of Formoterol Fumarate and Budesonide was calculated for all sample preparations. Mean of all results, Standard deviation and percentage related standard deviation was also determined.

The specification was set to the assay value for each individual set should be between 90% and 110% and the %RSD of all 10 results should be less than 6.0%.

Intermediate precision (Ruggedness):

Intermediate precision expresses within-laboratories variations: different days, different columns, different equipment, etc. The study was performed by preparing ten same homogeneous sample preparations using single batch. On different day, by using different lot of HPLC column and by using different HPLC equipment. Mean of assay, Standard deviation and %RSD were determined. The specification was set same as method precision study.

Solution stability:

Stability of analytical solution was determined by injecting standard and sample solutions at different time intervals up to 36 hours. The %RSD of peak area of Formoterol Fumarate and Budesonide was measured. The specification was set to % RSD should not be more than 2.0% for all both analytes.

Robustness:

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. It was studied by making following variations in method

1. pH of buffer solution was changed by ± 0.1 unit (pH 2.90 for –pH condition and pH 3.10 for +pH condition).

2. Mobile phase ratio was changed to ± 2units absolute (67:33% v/v for –organic condition and 63:37% v/v for + organic condition).

3. Column oven temperature was changed to ± 5°C (35°C for –temperature condition and 45°C for +temperature condition).

4. Flow rate was changed by ± 10% (0.9ml per min for –Flow condition and 1.1ml per min for +Flow condition).

The system suitability criteria should be achieved for each changed condition during robustness study.

Specificity

Specificity is the ability to assess unequivocally the analytes in the presence of components which may be expected to be present. Typically these might include placebo components, impurities etc. Specificity study was performed by preparing Placebo, Formoterol Fumarate standard, Budesonide standard, Placebo spiked with Formoterol and Budesonide standards and sample preparation. These samples were analyzed with diode array detector and purity of analyte peaks were determined. The acceptance criteria for specificity study was set to peak purity should be greater than 990 for each peak. The Degradation in assay should be achieved between 10% to 30%.

Following solutions were prepared and injected in to HPLC system with Diode Array Detector to establish specificity of method.

1. Mobile phase / Diluent preparation

2. Standard preparation

3. Placebo preparation

4. Placebo spiked with API preparation

5. As such sample preparation (Test preparation)

6. Acid hydrolysis sample

7. Alkali hydrolysis sample

8. Peroxide oxidation sample

9. Humidity Degradation sample

10. UV Degradation sample

Results and discussion:

System Suitability and System Precision:

Table 1 System Suitability and System Precision

Sr. No.	Parameters (n= 5)	Formoterol Fumarate	Budesonide Epimer-B	Budesonide Epimer-A
1	Retention Time (min)	4.66	8.43	9.00
2	Theoretical Plates	4144	6900	7085
3	Tailing factor	1.3	1.0	1.0
4	% RSD	0.3	0.1	0.0

%RSD, Theoretical plates and Tailing Factor evaluated as mentioned in table 1. The %RSD of 5 replicate standard injections for each analyte peak was found less than 2.0.

LOD and LOQ:

LOD and LOQ values were calculated from the prediction linearity plot from 2.5% to 75% of target concentration. Based on linearity calibration curve data of Formoterol Fumarate and Budesonide analytes the LOD and LOQ concentration found is tabulated below. These values have been indicated on the method sensitivity at lower concentrations.

Table 2a Prediction linearity table

Linearity to decide LOD and LOQ
Linearity level	µg/ml of Formoterol	Area of Formoterol	µg/ml of Budesonide	Area of Budesonide	Linearity % with respect to target concentration
1	0.005	1.061	0.25	9.281	2.5%
2	0.01	2.342	0.50	17.161	5%
3	0.02	4.554	1.00	36.337	10%
4	0.04	8.400	2.00	68.684	20%
5	0.06	11.598	3.00	101.903	30%
6	0.08	16.899	4.00	136.716	40%
7	0.10	21.677	5.00	171.603	50%
8	0.12	24.337	6.00	199.748	60%
9	0.15	29.463	7.50	240.510	75%

Based on above data LOD and LOQ precision done by injecting LOD solution in duplicate and LOQ solution in six replicate injections.

Table 2b LOD and LOQ precision

Area of Analyte peaks
Set No.	LOD Formoterol	LOQ Formoterol	LOD Budesonide	LOQ Budesonide
1	1.061	3.895	11.696	33.217
2	1.141	3.950	10.475	37.203
3		3.971		37.851
4		3.848		38.676
5		4.206		37.178
6		4.364		34.874
Average	1.10	4.04	11.09	36.50
SD	0.1	0.2	0.9	2.0
RSD	5.1	5.0	7.8	5.6
concentration	0.005µg/ml	0.02µg/ml	0.25µg/ml	1.00µg/ml

Linearity and Range

Table 3a Linearity Data of Formoterol Fumarate and Budesonide

Linearity Level	Final concentration in µg/mL (Formoterol Fumarate)	Area of (Formoterol Fumarate)	Linearity Level	Final concentration in µg/mL (Budesonide)	Area of Budesonide
20%	0.0438	8793	10%	0.9830	40230
50%	0.1096	21625	25%	2.4575	95440
100%	0.2192	43957	50%	4.9150	192822
200%	0.4384	89030	100%	9.8300	380854
400%	0.8768	178328	200%	19.6600	761709

Accuracy:

Table 4a Accuracy Data of Formoterol Fumarate and Budesonide

For Formoterol Fumarate
Accuracy Level	Amount of Drug added (mg)	Amount of Drug recovered (mg)	Recovery (%)	Mean (%)	% RSD
50 %	0.0055	0.0056	101.8	101.2	1.0
	0.0055	0.0055	100.0
	0.0055	0.0056	101.8
100 %	0.0110	0.0110	100.0	99.1	0.9
	0.0110	0.0109	99.1
	0.0110	0.0108	98.2
150 %	0.0164	0.0162	98.8	99.0	0.3
	0.0164	0.0162	98.8
	0.0164	0.0163	99.4

Table 4b Accuracy Data of Budesonide

For Budesonide
Accuracy Level	Amount of Drug added (mg)	Amount of Drug recovered (mg)	Recovery (%)	Mean (%)	% RSD
50 %	0.2458	0.2477	100.8	100.6	0.3
	0.2458	0.2477	100.8
	0.2458	0.2466	100.3
100 %	0.4915	0.4907	99.8	99.6	0.3
	0.4915	0.4905	99.8
	0.4915	0.4879	99.3
150 %	0.7373	0.7316	99.2	99.4	0.3
	0.7373	0.7352	99.7
	0.7373	0.7324	99.3

Accuracy at each individual level was evaluated and found between 95% to 105% and % RSD was found less than 5.0%. Which indicates the method is accurate.

Method precision (Repeatability) and Intermediate Precision

Table 5 Method Precision and Intermediate precision data for Formoterol Fumarate and Budesonide

Assay Set No.	Method Precision		Intermediate Precision
	Day-1 / HPLC-1/ Column-1		Day-2 / HPLC-2/ Column-2
	Formoterol Fumarate	Budesonide	Formoterol Fumarate	Budesonide
1	99.1	98.9	101.5	102.6
2	98.4	99.5	100.1	101.7
3	98.6	100.4	99.4	99.8
4	99.2	99.3	99.2	97.3
5	97.5	98.8	99.7	99.5
6	97.3	97.8	98.3	97.7
7	98.0	98.6	101.7	98.7
8	98.2	99.1	99.4	99.2
9	97.7	99.0	100.1	100.2
10	97.9	98.3	99.6	98.1
Mean (% Assay)	98.2	99.0	99.9	99.5
% RSD	0.7	0.7	1.0	1.7
Absolute Difference between Assay results of Method Precision and Intermediate Precision			1.7	0.5

Stability of Analytical Solution:

Table 6a Results of Solution Stability for standard preparation

Time (hour)	Area		% Difference
Time (hour)	Formoterol Fumarate	Budesonide (Epimer-B+A)	Formoterol Fumarate	Budesonide
0 (Initial)	41931	349373	==	==
6	42033	350331	0.2	0.3
12	42151	349214	0.5	0.0
18	42474	350978	1.3	0.5
24	42259	351406	0.8	0.6
30	42163	353447	0.6	1.2
36	42172	352495	0.6	0.9

Table 6b Results of Solution Stability for sample preparation

Time (hour)	Area		% Difference
Time (hour)	Formoterol Fumarate	Budesonide	Formoterol Fumarate	Budesonide
0 (Initial)	42422	395082	==	==
5	42551	395075	0.3	0.0
11	42475	397016	0.1	0.5
17	43000	394708	1.4	-0.1
23	42165	395502	-0.6	0.1
29	42565	397222	0.3	0.5
35	42745	398514	0.8	0.9

The % difference in area at each level is less than 2.0%. The standard and sample solutions are stable at room temperature for 36 and 35 hours respectively.

Robustness

Table 7 Robustness data

For Formoterol peak
Condition	As such	+Flow	-Flow	+Temp	-Temp	+Org	-Org	+pH	-pH
%RSD of 5 injection	0.3	0.4	0.5	0.5	1.0	0.3	1.1	0.4	0.2
Retention time	4.66	4.22	5.16	4.46	4.83	4.37	4.89	3.69	4.12
Theoretical plates	4144	3987	4426	4267	4039	4201	4219	3844	3988
Tailing factor	1.3	1.3	1.3	1.3	1.3	1.3	1.2	1.3	1.3
For Budesonide Epimer-B Peak
Condition	As such	+Flow	-Flow	+Temp	-Temp	+Org	-Org	+pH	-pH
%RSD of 5 injection	0.1	0.7	0.2	0.4	0.3	0.7	0.2	0.1	0.6
Retention time	8.43	7.67	9.31	8.23	8.59	7.59	9.97	7.01	8.23
Theoretical plates	6900	6723	7463	7282	6909	6862	7438	6869	7073
Tailing factor	1.0	1.0	1.0	1.0	1.0	1.0	1.0	1.0	1.0
For Budesonide Epimer-A Peak
Condition	As such	+Flow	-Flow	+Temp	-Temp	+Org	-Org	+pH	-pH
%RSD of 5 injection	0.0	0.7	0.2	0.4	0.6	0.5	0.3	0.3	1.0
Retention time	9.1	8.28	10.05	8.86	9.30	8.17	10.81	7.53	8.88
Theoretical plates	7085	6904	7560	7501	7075	7160	7541	7060	7273
Tailing factor	1.0	1.0	1.0	1.0	1.0	1.0	1.0	1.0	1.0

The RSD of five replicate of standard solution is less than 2.0% for all conditions. The theoretical plates for each analyte peak is higher than 1500 and Tailing factor for each analyte peak is less than 2.0. This indicates that the method is Robust.

Specificity:

Specificity of developed method was established by determining peak purity of active component in standard preparation and test preparation using PDA detector.

Table 8 Forced degradation data

For Formoterol Fumarate
Condition	As such	Acid deg.	Alkali Deg.	Peroxide Deg.	Humidity Deg.	UV Deg.
%Assay	96.9	78.7	87.2	92.5	98.8	94.3
% Degradation	0.0	18.8	10.0	4.5	0.0	2.7
For Budesonide
Condition	As such	Acid deg.	Alkali Deg.	Peroxide Deg.	Humidity Deg.	UV Deg.
%Assay	98.7	101.7	85.3	97.65	100.2	100.6
% Degradation	0.0	0.0	13.6	1.1	0.0	0.0

There is no any interference found at the retention time of analyte peaks in blank and placebo preparation. The Purity of analyte peaks are greater than 990 for all degradation conditions. Data represents that Formoterol is degraded in Acid and alkali hydrolysis condition and Budesonide degrades in Alkali hydrolysis condition. The above observations indicate that the method is specific for its intended purpose.

The above observations indicate that the method is specific for its intended purpose.

Conclusion:

A RP-HPLC method was developed and validated as per present ICH guidelines for quantitative determination of Assay of Formoterol Fumarate and Budesonide in Inhalation suspension pharmaceutical dosage form. The result of the study shows the suitability of the method as a stability indicating. The developed method is simple, robust, specific, selective and accurate, Linear within the performed range and is very much suitable for its intended purpose.

Acknowledgements:

The authors of the current work wish to acknowledge the management of Zydus Cadila Healthcare Limited for supporting this work and are grateful to our colleagues from Analytical Research and Development Laboratory.

Conflict of interest:

The authors declare that there is no conflict regarding publication of this paper.

References:

1. Snyder LR, Kirkland JJ, Dolan JW. Introduction to Modern Liquid Chromatography. John Wiley and Sons; 2011 Sep 20.

2. Ravi Sankar S. Text Book of Pharmaceutical Analysis, Rx publication, Tirunelveli. 2005; 4th ed: pp. 012-028

3. Sharma BK. Instrumental methods of chemical analysis. Goel publishing house, meerut. 2004; 23rd ed: pp. 081-189

4. ICH I. Q2 (R1): Validation of analytical procedures: text and methodology. In International Conference on Harmonization, Geneva 2005 Nov.

5. Elder D. ICH Q6A Specifications: test procedures and acceptance criteria for new drug substances and new drug products: chemical substances. ICH Quality Guidelines: An Implementation Guide. 2017 Sep 27: 433-66.

6. USP E. United States Pharmacopoeia 30 and National Formulary 25. In The United States Pharmacopoeial Convention, CD ROM 2007.

7. Indian pharmacopeia, General chapter on Inhalation preparation. 2018.

8. Kotak RK, Pandya CV, Pandya AC, Thakur B, Laddha R. Solubility study Of Formoterol Fumarate Dihydrate, Glycopyrrolate And Budesonide Drugs As A practical Tool For Analytical Method Development. International Journal of Technical Innovation in Modern Engineering and Science (IJTIMES). 2019; 5-5: 386-388.

9. Ghany MF, Hussein LA, Magdy N, Yamani HZ. Simultaneous spectrophotometric determination of indacaterol and glycopyrronium in a newly approved pharmaceutical formulation using different signal processing techniques of ratio spectra. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 2016 Mar 15; 157: 251-7. https://doi.org/ 10.1016/j.saa.2016.01.002.

10. Wolthers OD. Budesonide+ formoterol fumarate dihydrate for the treatment of asthma. Expert Opinion on Pharmacotherapy. 2016 May 2;17(7):1023-30. https://doi.org/10.1517/14656566.2016.1165207

11. Lilla TS, Szilas M, Gabriella G. Clinical Effectiveness of Budesonide / Formoterol Fumarate Easyhaler for Patients with Poorly Controlled Obstructive Airway Disease: a Real-World Study of Patient-Reported Outcomes. Adv Ther. 2018. https://doi.org/10.1007/s12325-018-0753-6

12. Lu Y, Sun Z, Zhang Y, Chen X, Zhong D. Simultaneous quantification of 22R and 22S epimers of budesonide in human plasma by ultra-high-performance liquid chromatography–tandem mass spectrometry: application in a stereoselective pharmacokinetic study. Journal of Chromatography B. 2013 Mar 15; 921: 27-34.

13. Salem YA, Hammouda ME, El-Enin MA, El-Ashry SM. Multiple analytical methods for determination of formoterol and glycopyrronium simultaneously in their novel combined metered dose inhaler. BMC chemistry. 2019 Dec; 13(1): 75. https://doi.org/10.1186/s13065-019-0592-9

14. Moore CD, Roberts JK, Orton CR, Murai T, Fidler TP, Reilly CA, Ward RM, Yost GS. Metabolic pathways of inhaled glucocorticoids by the CYP3A enzymes. Drug Metabolism and Disposition. 2013 Feb 1; 41(2): 379-89.

15. Salem YA, Shaldam MA, El-Sherbiny DT, El-Wasseef DR, El-Ashry SM. Simultaneous Determination of Formoterol Fumarate and Budesonide Epimers in Metered Dose Inhaler Using Ion-Pair Chromatography. Journal of Chromatographic Science. 2017 Nov 1; 55(10): 1013-20. https://doi.org/10.1093/chromsci/ bmx067

16. A V S S Prasad. Simultaneous spectrometric determination of Formoterol Fumarate and Budesonide in their combined dosage form. Indian Journal of Chemical Technology. 2006; 13: 81-83.

17. Pai, N., Patil, S.S. Development and validation of RP-HPLC method for estimation of formoterol fumarate and budesonide in pressurised meter dose inhaler form. Der Pharmacia Sinica. 2013; 4: 15–26.

18. Farid NF, Abdelwahab NS. Development and Validation of Different Chromatographic Methods for Analysis of Cabergoline in the Presence of Its Degradation Products: Studying Degradation Profile. Chromatographia. 2019 Oct 1; 82(10): 1555-69. https://doi.org/10.1007/s10337-019-03763-4

19. Kale NR, Pingle AP, Mirza JA, Dhongade GN. Development and validation of stability-indicating RP-HPLC method for simultaneous estimation of formoterol fumarate and budesonide in metered dose inhaler formulation. World Journal of Pharmaceutical Research. 2014 Jun 20; 3(6): 1386-99.

20. Patel VA, Pandya CV, Pandya AC, Patel D, Patel Z. Concurrent determination of Tenligliptin Hydrobromide Hydrate and Metformin Hydrochloride Development and Validation by UV-VIS Spectrophotometry in Bulk and Pharmaceutical Dosage form. International Journal of Technical Innovation in Modern Engineering and Science (IJTIMES). 2019; 5-3: 296-303.

21. Sridharan D, Thenmozhi UA, Kumar LP, Chintalapati AD, Ramanaiah MV, Phanikishore Y. Development and validation of UV spectrophotometric method of darifenacin hydrobromide in bulk and tablet dosage form. Asian Journal of Pharmaceutical Analysis. 2011;1(3): 43-5.

22. Roge AB, Tarte PS, Kumare MM, Shendarkar GR, Vadvalkar SM. Forced Degradation Study: An Important Tool in Drug Development. Asian Journal of Pharmaceutical Research. 2013; 3(4): 198-201.

23. Nagaraju P, Gopal NV, Srinivas VD, Padma SV. Spectrophotometric Methods for the Determination of Atorvastatin Calcium in Pure and It's Pharmaceutical Dosage Forms. Asian Journal of Research in Chemistry. 2008; 1(2): 64-6.

24. Chowdhury SR, Maleque M, Shihan MH. Development and validation of a simple RP-HPLC method for determination of caffeine in pharmaceutical dosage forms. Asian Journal of Pharmaceutical Analysis. 2012;2 (1): 1-4.

25. Pinaz AK, Muralikrishna KS. Method development and acid degradation study of rivaroxaban by RP-HPLC in bulk. Asian Journal of Pharmaceutical Analysis. 2013; 3(2): 62-5.

26. Kumar CH, Kannappan N. Development and validation of Bioanalytical Method for Quantification of phenytoin in rat brain tissues as per ICH guidelines. Asian Journal of Pharmaceutical Analysis. 2014; 4(4): 147-50.

27. Patel JJ, Chorawala H, Dedania ZR, Vijendraswamy SM. Development and Validation of UV Spectroscopic Method for Simultaneous Estimation of Doxofylline and Terbutaline Sulphate in Combined Dosage Form. Asian Journal of Pharmaceutical Analysis. 2015; 5(2): 74-8.

28. Reddy S, Nayak N, Ahmed I, Thomas L, Mukhopadhyay A, Thangam S. Development and validation of two LCMS/MS methods for simultaneous estimation of oseltamivir and its metabolite in human plasma and application in bioequivalence study. Asian Journal of Pharmaceutical Analysis. 2016; 6(2): 91-101.

29. Deepthi N, Rajendran SS, Gananadhamu S, Rao GD, Lohit S. Spectrophotometric Estimation of Ziprasidone in Bulk and In Pharmaceutical Formulations. Asian Journal of Pharmaceutical Analysis. 2011; 1(1): 8-9.

30. Kathirvel S, Raju R, Seethadevi B, Suneetha A, Pavani J. Stability Indicating RP-HPLC Method for the Determination of Process Related Impurities in Posaconazole API. Asian Journal of Pharmacy and Technology. 2014; 4(4): 167-78.

31. Mali AD. Simultaneous Determination of Carvedilol and Hydrochlorothiazide in Pharmaceutical Dosage Form by Second Order Derivative UV Spectrophotometry. Asian Journal of Pharmaceutical Analysis. 2015; 5(3): 133-8.

32. Koralla S, Konidala SK, Rao KG, Begum SM. Stability Indicating RP-HPLC Method For Simultaneous Estimation of Ramipril and Amlodipine Besylate in Pharmaceutical Dosage Form. Asian Journal of Pharmaceutical Research. 2016; 6(4): 242-9.

Received on 17.07.2020 Modified on 20.08.2020

Research J. Pharm. and Tech. 2021; 14(8):4383-4390.

DOI: 10.52711/0974-360X.2021.00761

Parameters	Formoterol Fumarate	Budesonide
Linearity Range	0.0438 – 0.8768 mg/mL	0.9830 – 19.6600 mg/mL
Correlation coefficient (R)	0.9998	0.9998
Slope	20392	38649
Y-intercept	-491.3	1671.0
Y-Intercept Bias	0.55%	0.44%