INTRODUCTION:

Beclomethasone dipropionate [BD]:

Beclomethasone dipropionate (α-chloro-11β,17,21-trihydroxy-16β-methylpregna-1,4-diene-3,20-dione 17,21-dipropionate) is synthetic halogenated inhaled glucocorticoid. It is used for treating steroid-dependent asthma, for relieving symptoms associated with allergic or nonallergic (vasomotor) rhinitis or for preventing recurrent nasal polyps following surgical removal.

Formoterol fumarate [FF]:

Formoterol fumarate ((±)-N-{2-hydroxy-5-[(1RS)-1-hydroxy-2-{[(2RS)-1-(4-methoxyphenyl) propan-2-yl]amino}-ethyl]phenyl}formamide fumarate is a longer-acting ß₂ adrenoreceptor agonist which is administered via inhalation. Due to its prolong duration of action it should not be used for the relief of an acute asthma attacks. Formoterol fumarate use in management of asthma and chronic obstructive pulmonary disease (COPD).

Figure 1: (I) Chemical structure of Beclomethasone dipropionate (II) Formoterol fumarate

MATERIALS AND METHODS

Chromatographic condition

C₁₈[250mm x 4.6m, 5µm] ODS Hypersil column was used at ambient temperature. Mobile phase consisted of Acetonitrile, 10mM Ammonium acetate buffer (pH 4.5, adjusted with OPA) in the ratio of 70:30 V/V, was pumped at a flow rate of 1.5 ml/min. The mobile phase was filtered through 0.45 µm nylon membrane filter and degassed before used. The elution was monitored at 218 nm and run time was 10 min.

Preparation of Mobile phase

Accurately weighed 0.77gm of ammonium acetate dissolved and diluted with HPLC grade water. pH of buffer solution was adjusted to 4.5 with OPA. The mobile phase comprised of Acetonitrile 10mM ammonium acetate buffer 70:30 V/V. It was degassed for 15min before used.

Preparation of standard solution of BD and FF:

Accurately weighed 50 mg of BD and 50 mg FF were transferred in 50 ml volumetric flask separately. It was dissolved properly and diluted up to the mark with mobile phase to obtain stock solution of 1000 µg/ml BD and 1000 µg/ml FF.

Preparation of sample solution (Formulation):-

Twenty capsules (Fullform-200 containing 200 µg of BD and 6 µg of FF) were weighed. The powder equivalent to 200 µg of BD and 6 µg of FF was weighed and transferred into 100 ml volumetric flask. Wash the capsules with mobile phase, add 50 ml of mobile phase, and sonicated for about 10 min. The volume was then adjusted upto the mark with mobile phase (4000 µg/ml of BD and 120 µg/ml of FF).

The solution was filtered using Whatman filter paper (0.45µm) to remove excipients. Aliquot of 1 ml was transferred to 100 ml volumetric flask and volume was adjusted upto the mark with mobile phase.

Preparation of calibration range

From the standard working solution, aliquot of 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 ml for BD and 0.9, 1.2, 1.5, 1.8 and 2.1 ml were transferred in a separate 10ml volumetric flask and diluted upto the mark with mobile phase to get the concentration range of 30-70 µg /ml and 0.9-2.1 µg /ml for BD and FF respectively.

FORCED DEGRADATION^[11,15-17]

Forced degradation study was carried out by subjected standard BD and FF individually, standard mixture of BD and FF, and formulation to various stress conditions to conduct forced degradation studies. Stress studies were carried out under the conditions of acid/base hydrolysis, oxidation, thermal and UV light.

Standard Solution of mixture is used for Forced degradation study.

Acid degradation

Aliquots of 4.0 and 1.2 ml were transferred from standard working solution in separate 10 ml Volumetric flask and. To this sample solutions 1 ml 0.1 N HCl were added in all the sample drug solutions and kept for 1 hr. at room temperature. Solutions were neutralized by adding 1 ml 0.1 HCl to all the solutions and diluted upto 10 ml with mobile phase to get final concentration 40 µg/ml and 1.2 µg/ml of BD and FF respectively.

Base degradation

Aliquots of 4.0 and 1.2 ml were transferred from standard working solution in separate 10 ml Volumetric flask and. To this sample solutions 1 ml 0.1 NaOH were added in all the sample drug solutions and kept for 1 hr. at room temperature. Solutions were neutralized by adding 1 ml 0.1 HCl to all the solutions and diluted upto 10 ml with mobile phase to get final concentration 40 µg/ml and 1.2 µg/ml of BD and FF respectively.

Oxidative degradation

Aliquots of 4.0 and 1.2 ml were transferred from standard working solution in separate 10 ml Volumetric flask and. To this sample solutions 1 ml 6% H₂O₂ were added and kept for 5 hr. at room temperature. All solutions were warm to remove access H₂O₂and diluted upto 10 ml with mobile phase to get final concentration 40 µgml and 1.2 µg/ml of BD and FF respectively.

Thermal degradation

Solid material was exposed to 50⁰C in Oven for 1hr. Powder was taken after 1hr. and solution was made with final concentration of 40 µg/ml and 1.2 µg/ml of BD and FF respectively.

Photo degradation

Solid material was exposed to UV light (254 nm) for 2 hrs in UV chamber. Powder was taken after 1hr. and solution was made with final concentration of 40 µg/ml and 1.2 µg/ml of BD and FF respectively.

VALIDATION OF RP-HPLC METHOD^[5]:

Linearity

It is the ability of the method within the given range to obtain test results which is directly proportional to concentration of analyte in the sample. Linear correlation was obtained between absorbance and concentrations of BD and FF in the ranges of 30-70 µg/ml and 0.9-2.1µg/ml for BD and FF respectively. The linearity of calibration curve was validated by the value of correlation coefficient of regression. Each reading was a mean of three determinations.

Precision

(a) Repeatability

The repeatability study was performed by analyzing BD and FF (40 µg/ml and1.2 µg/ml) for six times by developed RP-HPLC method. The %RSD was calculated.

(b) Intraday and interday precision

The intraday and interday precisions of the proposed method was determined by analyzing corresponding responses in triplicate on the same day and on 3 different days, different concentrations of standard solutions of BD (30, 50 and 70 µg/ml )and FF(0.9,1.5 and 2.1µg/ml) were taken. Results were reported in terms of %RSD.

LOD and LOQ

LOD and LOQ were calculated from calibration curve by using the following equations as per ICH guidelines.

LOD = 3.3 µ/ S and LOQ = 10 µ /S

where, is the standard deviation of intercept to regression line and S is the slope of the corresponding calibration curve.

Accuracy (Recovery)

The proposed method was applied to determine BD and FF in rotacap formulation. The recovery study was carried out by spiking standard BD (24, 30 and 36µg/ml) and FF (0.72,0.9and1.08µg/ml) into pre quantified sample solution of 40µg/ml and1.2 µg/ml BD and FF, respectively at 80,100 and 120% levels.

Robustness of method:

Robustness study was performed in following altered chromatographic condition:

Variation in mobile phase (±2%)

Variation in flow rate (± 0.1ml/min)

System suitability parameters:

system suitability tests were carried out on freshly prepared standard stock solution of Beclomethasone dipropionate and Formoterol fumarate of both drugs. 30µL solution was injected under optimized chromatographic condition and parameters such as retention time, theoretical plates, capacity factor, resolution, peak asymmetry etc. were studied to evaluate the suitability of the system.

Analysis of marketed formulation

Twenty capsules (Fullform-200 containing 200 µg of BD and 6 µg of FF) were accurately weighed. The powder equivalent to 200µg of BD and 6µg of FF was weighed and transferred into 100 ml volumetric flask. Wash the capsules with mobile phase, add 50ml of methanol and sonicated for about 10min.The volume was then adjusted upto the mark with mobile phase (4000 µg/ml of BD and 120 µg/ml of FF). The solution was filtered using whatman filter paper (0.45µm) to remove excipients. Aliquot of 1 ml was transferred to 100 ml volumetric flask and volume was adjusted upto the mark with mobile phase. The resultant sample was run in the HPLC system and% label claim was calculated for BD and FF.

RESULT AND DISCUSSION:

Method Development:

Chromatographic peak of BD and FF on optimised condition

Chromatogram of formulation on Optimized condition

Optimized Chromatographic conditions

Sr.No.	Parameters	Specifications
1	Stationary phase (column)	C₁₈[250mm x 4.6m,5μm] ODS Hypersil
2	Mobile phase	10mM Ammonium Acetate Buffer (pH 4.5 adjusted with ortho-phosphoric acid): Acetonitrile (30:70)
3	Flow rate(ml/min)	1.5 ml/min
4	Column temperature	Ambient
5	Volume of injection(μl)	20 μl
6	Detection wavelength

Forced Degradation study:-

In following section various chromatograms of BD and FF under unstressed and stressed conditions are presented.

Acid degradation:-

Chromatogram of standard mixture in acid degradation (0.1 N HCl,1 hr.) at 218 nm

Chromatogram of formulation in acid degradation (0.1 N HCl,1 hr.) at 218 nm

Alkali degradation

Chromatogram of Standard mixture in alkali degradation (0.01N NaOH,1 hr.) at 218 nm

Chromatogram of Formulation in alkali degradation(0.01N NaOH,1 hr.) at 218 nm

Oxidative degradation

Chromatogram of Standard mixture in Oxidative degradation (6% H₂O₂,5 hrs.) at 218 nm

Chromatogram of Formulation in Oxidative degradation (6% H₂O₂,5 hrs.) at 218 nm

Thermal degradation

Standard mixture-thermal degradation at 50^ºC for 1 hr

Formulation-thermal degradation at 50^ºC for 1 hr.

Photo degradation

Chromatogram of standard mixture in UV light exposure (254 nm, 2 hrs.)

Chromatogram of formulation in UV light exposure (254 nm,2 hrs.)

Summary of force degradation study

Type of degradation	Conditions	% Degradation		R_t(min.) of degradation product
Type of degradation	Conditions	BD	FF	R_t(min.) of degradation product
Acid degradation	0.1 N HCl 1 hr.	17.10%	12.11%	3 (1.7, 2.5, 4.9)
Alkali degradation	0.01 N NaOH 1 hr.	14.4%	17.60%	3 (2.4, 4.5, 5.2)
Oxidative degradation	6 % H₂O_{2 5 hrs.}	NIL	NIL	-
Thermal Degradation	50°C for 1 hr.	12.79%	15.32%	3 (3.6, 4.5, 5.2)
Photo degradation	UV light exposure 254 nm 2 hrs.	16.56%	19.44%	2 (2.6,3.6)

Method Validation

Linearity

Table 1 Calibration curve Data

Sr. No.	FF		BD
Sr. No.	Concentration of Drug (µg/ml)	Peak Area ±SD(n=3)	Concentration of Drug (µg/ml)	Peak Area ±SD(n=3)
1	0.9	397701± 950.43	30	505702±5830.941
2	1.2	580416± 5716.64	40	7084371±5773.503
3	1.5	787014± 5767.73	50	886227±5771.012
4	1.8	940978± 1102.78	60	1139841± 5505.152
5	2.1	1127037± 1527.52	70	1316797±2309.401


Figure 2 Calibration curve of BD	Figure 3 Calibration curve of FF

Table 2 Regression analysis data for the proposed method

Parameters	BD	FF
Wavelength (nm)	218 nm	218 nm
Linearity (µg/ml)	30-70 µg/ml	0.9-2.1 µg/ml
Regression equation (y= mx + c )	y = 20536x - 115395	y = 606411x - 142988
Slope (m)	20536	606411
Intercept (c)	115395	142988
Correlation coefficient (r²)	0.9971	0.9984

Precision

(a)Repeatability

Table 3 Repeatability data for BD and FF

Sr.No.	BD		FF
	Conc.(µg/ml)	Peak area ± SD (n=6)	Conc.(µg/ml)	Peak area ± SD (n=6)
1	40	7082275	1.2	581413
2	40	7071365	1.2	581311
3	40	7085276	1.2	580810
4	40	7062351	1.2	580988
5	40	7068272	1.2	581215
6	40	7083450	1.2	580518
Mean	7075498.16		581042.5
SD	9480.417		338.007
%RSD	0.133		0.058

(b) Intraday and Interday precision

Table 4 Intraday precision

Drug	Conc.(µg/ml)	Intraday Precision
		Peak area mean ± SD (n=3)	%RSD
BD	30	505805.3±82.87	0.016
	50	886693.7±1651.64	0.186
	70	1316426±384.98	0.029
FF	0.9	397407.3±355.62	0.089
	1.5	781260.3±249.39	0.031
	2.1	112139±201.51	0.017

Table 5 Intraday precision

Drug	Conc.(µg/ml)	Intraday Precision
		Peak area mean ± SD (n=3)	%RSD
BD	30	505637.7±123.56	0.024
	50	886559.3±1590.53	0.179
	70	1316187±358.75	0.027
FF	0.9	397706±165.34	0.041
	1.5	781427±165.41	0.021
	2.1	1127505±155.15	0.013

LOD and LOQ

Table 6 LOD and LOQ

Parameter	BD	FF
LOD(µg/ml)	1.763	0.030
LOQ(µg/ml)	5.343	0.091

Accuracy

Table 7 Accuracy data for BD

Amount of drug added (µg/ml)	Target conc. (µg/ml)	Total Amount of drug (µg/ml)	Amount of drug recover (µg/ml)
24	30	54	23.48
30	30	60	29.82
36	30	66	36.02

Table 8 Accuracy data for FF

Amount of drug added (µg/ml)	Target conc. (µg/ml)	Total Amount of drug (µg/ml)	Amount of drug recover (µg/ml)
0.72	0.9	1.62	0.73
0.9	0.9	1.8	0.88
1.08	0.9	1.98	1.071

Robustness

Table 9 Robustness data for BD and FF

Factor	Change Level	Retention time		Mean± SD(n=3)		%RSD
		BD	FF	BD	FF	BD	FF
Flow rate	1.4	6.12	3.12	0.058	0.024	0.97	0.79
	1.5	6.02	3.09
	1.6	5.98	3.06
Mobile phase ratio Buffer: ACN	28:72	6.09	3.09	0.035	0.030	0.58	0.99
	30:70	6.05	3.05
	32:68	6.02	3.11

Analysis of marketed formulation

Table 10 Analysis of marketed formulation

Sr.No.	Label claim		Amount of drug found		%Assay
	BD (µg)	FF (µg)	BD (µg)	FF (µg)	BD	FF
1.	200	6	197.9	5.97	98.9	99.5
2.	200	6	195.5	5.92	97.7	98.7
3.	200	6	197.7	6.06	98.8	101.5
Mean± SD(n=3)			197.03±1.32	5.98±0.070	98.43±0.678	99.9±1.42
%RSD			0.669	1.18	0.71	1.42

Table 11 System suitability Parameters

Parameters	Proposed method
	BD	FF
Retention time (R_t)	3.302	6.042
Capacity factor (k)	12.68	10.56
Plates number (n)	3467	2353
Tailing factor (T)	1.67	1.16
Resolution factor	11.10

Table 12 Summary of validation parameter

Parameters	Results
	BD	FF
Linearity (µg/ml)	30-70 (µg/ml)	0.9-2.1 (µg/ml)
Correlation coefficient(r²)	0.9971	0.9984
Precision(%RSD)
a) Repeatability	0.133	0.058
b) Intraday	0.016-0.029	0.013-0.089
c) Interday	0.024-0.179	0.013-0.041
Accuracy (%Recovery)	97.70-100.07	97.07-99.31
LOD (µg/ml)	1.763	0.030
LOQ (µg/ml)	5.343	0.091
Robustness	Robust

CONCLUSION:
Stability indicating RP-HPLC method has been developed for simultaneous estimation of Beclomethasone dipropionate and Formoterol fumarate. The chromatographic separation of BD and FF and were achieved on C₁₈(250mm x 4.6mm,5µm) ODS Hypersil column using mobile phase Buffer: ACN (30:70 v/v) at flow rate 1.5 ml/min. Detection was performed at wavelength 218 nm.

This method was also demonstrated to be stability-indicating as it can separate all the degradation Peaks and proved to be suitable for routine quality control used. The new stability indicating RP-HPLC method was successfully validated as per ICH guidelines.(Q2R1.)

REFERENCES:

1. Rang HP, Dale MM, Ritter JM, Flower RJ, Rang and Dale’S Pharmacology, Elsevier Limited, 2007, 6th ed: pp357-365.

2. Tripathi KD. “Essentials of Medical Pharmacology”, Jaypee Publication, New Delhi, 2008, 6th ed: pp216-218.

3. Sethi PD., Quantitative Analysis of Pharmaceutical Formulations, CBS Publication and Distributors, New Delhi, 2001, 5th ed: pp 7-10.

4. Snyder LR., Kirkland JL., and Glajch JL., Practical HPLC Method Development, Wiley Interscience, New York, 1977, 2nd ed: pp 1-57.

5. International conference on harmonization of technical requirements for registration of pharmaceutical for human use, validation of Analytical procedure: Text and Methodology ICH Q2 (R1), 2005.

6. Skoog DA., Holler FJ., and Crouch SR. Principle of Instrumentation Analysis, Thomas Asia Pvt. Ltd, Singapore, 2011, 8th ed: pp 13-14.

7. Kasture AV., Wadodkar SG., Mahadik KR., and More HM. Pharmaceutical Analysis, Vol. I, 2007, 17th ed: pp 1-3.

8. Singh S and Bakshi M, “Development of validated stability-indicating assay methods-critical review.” Journal of Pharmaceutical and Biomedical Analysis. Vol.28, 2002:1011-1040

9. ICH Q1A(R2): Stability testing of new drug substances and products.2003.

10. Alsante K, Ando A, Brown R, Ensing J, Hatajik T, Wei Kong and Tsuda Y, “The role of degradant profiling in active pharmaceutical ingredients and drug products.” Advanced Drug Delivery Reviews, ScienceDirect 2007, 59, 29–37.

11. “Systemic approaches of stability indicating methods” July 2014 available form http://www.pharmainfo.net/reviews/sensitive-analytical-methods-determination-stability-drugs-pharmaceutical-dosage-forms.

12. Indian Pharmacopoeia, Government of India, Ministry of Health and Welfare, Volume-II, Ghaziabad, 2010:873 and 1386.

13. US Pharmacopeia, 34 NF 29, The United State Pharmacopial Convention.Rockvill,2011:1487.

14. British Pharmacopeia, British Pharmacopeia Commission, 2009:590 and 2677.

15. Shah P.D, M. Pharm Thesis, “Development and Validation of analytical method for simultaneous estimation of Beclomethasone dipropionate and Formeterol fumarate” Gujarat Technological University, April 2014.

16. Singh S, Bakshi M, “Guidance on conduct of stress tests to determine inherent stability of drugs”, Pharmaceutical Technology On-Line, Vol.24, 2000:1-14.

17. Kim HB., Handbook of Stability Testing in Pharmaceutical Development, Springer publication, 2008, pp. 1-8, 21-42

18. Ahuja S., and Scypinski S. Handbook of Modern Pharmaceutical Analysis, Academic Press, United states of America, 2001, pp. 85-169.

19. Prasanna LB, Shetty SA, Nadh PT, “Development of New Spectrophotometric Methods for the Simultaneous Estimation of LevosalbutamolSulphate and Beclomethasone Dipropionate in Bulk Drug and Pharmaceutical Formulations (ROTACAP)”, International Journal of PharmTech Research, Vol.4, 2012:791-798,

20. Reddy UK, Reddy H, Bobbarala V, Penumajji S, “Determination of Beclomethasone Dipropionate, Clotrimazole , Chloramphenicol and Lidocaine In Pharmaceutical Formulations Using A Novel RP-HPLC Method”, International Journal of Pharmacy and Bio Sciences, Vol.2 , 2011:453-462.

Received on 18.02.2016 Accepted on 25.03.2016

Asian J. Pharm. Ana. 2016; 6(2): 83-90.

DOI: 10.5958/2231-5675.2016.00013.2

FORCED DEGRADATION[11,15-17]