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Simultaneous estimation of fluticasone propionate, azelastine
hydrochloride, phenylethyl alcohol and benzalkonium chloride by
RP-HPLC method in nasal sprays
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K. Lakshmi Narasimha Rao*1, K. Padmaja Reddy2, K. Sudheer Babu1,
K. Soloman Raju1, K. Visweswara Rao2, Jafer Vali Shaik2
Asterisk (*) for the corresponding
author (Should not be superscripted)

1Department of Branded Formulations, Analytical Research & Development, Dr.Reddy's La- boratories, IPDO, Bachupally, Hyderabad- 500 072 India 2Department of Active pharmaceutical Ingredient, Analytical Research & Development, Dr.Reddy's Laboratories, IPDO, Bachupally, Hyderabad- 500 072 India Superscript number for indicating
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* Corresponding Author Name: Dr. K. Lakshmi Narasimha Rao Email: klnrao@gmail.com All fields are mandatory
except Fax no.

Contact: +91-9824526321 Fax: +91-480-3103284 Date of Submission: 15-03-2011 Font: Times New Roman, Size: 12,
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The Abstract should be minimum 150
words, not more than 300 words and

ABSTRACT
unstructured. In abstract author has
A simple, reproducible and accurate efficient reverse phase thods, results and conclusion. But au-
tographic method was developed for simultaneous estim without subheadings. It should be like
(FLP), Azelastine Hydrochloride (AZH), Phenylethyl alcohol (PEA) and Benzalkonium chlo- self explanatory of entire work done
ride (BKC) in combined nasal spray preparations. Formulati u itnhing
used as antihistamine-corticosteroid combination available as a metered spray formulation for nasal or intranasal administration. Chromatography was performed on a 250 mm x 4.6 mm, 5-μm particle size, Waters Spherisorb CN column with a 55:45 (v/v) mixture of buffer and acetonitrile as a mobile phase. The detection of the combined dosage form was carried out at 215 nm and flow rate employed was 1.0 ml/min. The retention times were 3.380, 4.855, 6.647 & 10.175, 12.179 min for Phenylethyl alcohol, Fluticasone propionate, Azelastine Hy- drochloride and Benzalkonium chloride (C12,C14), respectively. Linearity was established in the concentration range 70.0 to 120.0 μg/ml for Phenyl ethyl alcohol, 14.0 to 24.0 μg/ml for Fluticasone propionate, 39.2 to 67.2 μg /ml for Azelastine Hydrochloride and in the range 2.8 to 4.8 μg/ml for Benzalkonium chloride, with a correlation coefficient of 0.9961, 0.9992, 0.997 and 0.9997, respectively. The results of the analysis were validated statistically and re- covery studies confirmed the accuracy and precision of the proposed method. Keywords: Azelastine Hydrochloride; Benzalkonium chloride; Fluticasone propionate; Nasal
preparations; Phenylethyl alcohol; Simultaneous estimation Four to eight keywords should be add-
ed at the end of Abstract. Keywords
should not reflect the titles. Individual
keywords should be separated by (;)
and ordered alphabetically.

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Fluticasone propionate and Azelastine Hydrochloride Nasal Spray is an antihistamine- corticosteroid combination available as a metered spray formulation for intranasal administra- tion. It contains azelastine hydrochloride, which is a second generation H1 receptor- drochloride. Azelastine hydrochloride occurs as a white, almost odorless, crystalline powder with a bitter taste. It has a molecular weight of 418.37. Fluticasone propionate, a synthetic corticosteroid with anti-inflammatory properties. Chemically it is known as (6α,11β-16α,17
carbothioic acid S-(fluoromethyl) ester. Fluticasone propionate is a white to off-white powder with a molecular weight of 500.58. Benzalkonium chloride is chemically known as alkyldimethyl (phenylmethyl) ammonium chloride. Three major homologues consist of C12, C14 and C16 straight chain alkyls. It acts as a Pharmaceutical aid (preservative), Algeside. (The Merck Index.3, 2006, British pharmacopoeia, United states pharmacopoeia, Jadwiga Dudkiewicz-Wilczynska., et al., 2004; Louis-Philippe et al., 2007). Phenylethyl alcohol is chemically known as 2 – phenyl ethanol. Its molecular weight is 122.16. It acts as a Pharma- ceutical aid (antimicrobial). (The Merck Index.3, 2006). In this formulation FLP and AZH References should be mentioned in the body
was drug substance where as PEA and BKC used as a preservatives. All four peaks to be
text in parenthesis for e.g. (Murnane, 2006)
quantified in the formulation. There e rature for the
than two authors (Murnane et al., 2006)
simultaneous estimation of Fluticasone propionate with other combinational drugs (J. L. Ber- nal et al., 1998; Murnane et al., 2006; Nichole L. Korpi-Steiner., 2010; Sriram Krishnaswa- mi., 2000). Since these methods were based on HPLC, Capillary electrophoresis, UV spec- trophotometer and LC-MS/MS. Azelastine hydrochloride also with other combination drugs was appearing in the literature in human plasma (Pivonka J et al., 1987). References should appear in the body
text and follow Harvard system.

The chemical structures of Phenylethyl alcohol, Benzalkonium chloride, Fluticasone propio- nate and Azelastine Hydrochloride are shown in Fig. 1. MATERIALS & METHODS
Reagents and chemicals
HPLC-grade acetonitrile and potassium dihydrogen ortho phosphate analytical grade were procured from Rankem (Mumbai, India) and pure standards of Phenylethyl alcohol (99.9%), Benzalkonium chloride (95.76%), Fluticasone propionate (98.95%) and Azelastine Hydroch- loride (99.46%) was obtained from Dr. Reddy's Laboratories Ltd. HPLC grade water was prepared by using Millipore Milli Q plus purification system. The 0.45µm-nylon filter was obtained from Advanced Micro Devices Pvt. Ambala Cantt, India. Waters spherisorb CN column was procured from Waters Associates, Inc. Instrumentation and Chromatographic Conditions
Chromatography was performed with a Agilent technologies 1100 LC (Germany), gradient pump with inbuilt auto injector, variable wavelength detector and Waters Alliance HPLC sys- tem (Milford, USA) equipped with a 2695 separation module with inbuilt auto injector and 2996 photodiode array detector. Waters Spherisorb CN column (250 x 4.6nm, 5-μm particles) was used for chromatographic separation under suitable condition. Detection was carried out using a UV spectrophotometric detector at 215 nm and the software used was waters Em- power2. The mobile phase was a 55:45 (v/v) mixture of freshly prepared buffer (50mM of potassium dihydrogen ortho phosphate) and acetonitrile. The mobile phase was sonicated and degassed before use. The diluent was mixture of water and acetonitrile in the ratio of 40:60 (v/v). The flow rate of mobile phase was maintained at 1.0 ml/min. The column temperature was maintained at ambient conditions. The injection volume was 20 μL and total run time was 20 min. The peaks were identified by retention time; a typical chromatogram is shown in Mixed standard Preparation
Standard stock solution of AZH (1400 μg/ml), FLP (500 μg/ml) PEA (2500 μg/ml) and BKC (100 μg/ml) was prepared individually in diluent. 2.0ml of the each stock solution was pipet- ted out into 50ml volumetric flask and diluted to volume with diluent to achieve a final con- centration of AZH (56.6μg/ml), FLP (19.8 μg/ml) PEA (100 μg/ml) and BKC (4 μg/ml). A system suitability test was performed for five replicate standard injections. Sample preparation
Shaken and mixed the contents of 3 bottles (7ml each) in 100ml volumetric flask. Weighed accurately a quantity of nasal spray solution equivalent to 2ml into a 50ml volumetric flask. Added about 30ml diluent, sonicated for about 20minutes and then diluted to volume with It is recommended that results should be presented in figures and tables
xt. μg/m
e (19.8 μg/ml) PEA
landscape or long boring tables. Results should be structured.
(100 μg/ml) and BKC (4μg/ml). Filter through a 0.45µm nylon filter. A typical chromato- gram obtained from a sample solution of assay is shown in Fig. 2. RESULTS AND DISCUSSION
Method development
The objective of this s be ac omet
repeat results. Put your results into perspective with previously pub-
components AZH, FLP, PEA and BKC under isocratic conditions. The mobile phase used lished data. Include citation wherever applicable e.g. (Prakash, 2010)
was the mixture of acetonitrile with phosphate buffer in different ratios. Finally a mixture of acetonitrile-phosphate buffer in the ratio of 45:55 (v/v), proved to be effective mixture than the other mixture used for the separation. Then the flow rate tested includes 0.8, 1.0, 1.2 and 1.5 ml/min. Among the flow rates 1.0 ml/min was selected for the assay because of better vi- sibility and resolution of the peaks. The final chromatographic conditions revealed to provide better resolution among Phenylethyl alcohol, Fluticasone propionate, Azelastine Hydrochlo- ride and Benzalkonium chloride peaks ie greater than 2.0. Number of theoretical plates and tailing factor for each individual peak was more than 2000 and less than 2.0, respectively. The optimum wavelength for detection was 215 nm, no indigenous interfering compounds eluted at the retention times of the drugs. Labeled amount of the drugs present in nasal spray preparations are given in Table.1. Validation of the Method
The method was validated, in accordance with ICH guidelines (ICH Q2B), for precision, ac- curacy, linearity, specificity, ruggedness and robustness. Linearity
Linearity was obtained in the concentration range 70.0 to 120.0 µg/mL for Phenylethyl alco- hol, 14.0 to 24.0 μg/ml for Fluticasone propionate, 39.2 to 67.2 μg/ml for Azelastine Hy- drochloride and in the range 2.8 to 4.8 μg/ml for Benzalkonium chloride. Linearity was per- formed with the aid of serially diluted calibration solutions from 70% to 120% of the target concentration. Calibration graphs were plotted on the basis of analysis of each calibration so- lutions. The coefficient of regression obtained was 0.9961, 0.9992, 0.997 and 0.9997, respec- tively for PEA, FLP, AZH and BKC. The slope obtained was 50988, 3883, 79373 and 854, respectively for PEA, FLP, AZH and BKC. Precision and intermediate precision
The method was found to be precise with six sample preparations for the quantification of PEA, FLP, AZH and BKC. The precision was also studied on different day. The precision and intermediate precision variation was calculated in terms of percentage relative standard deviation and the results of PEA, FLP, AZH and BKC were found to be less than 2.0% and the results are given in Table. 1. Accuracy
Accuracy was determined by the method of spiking the standard stock solution in placebo at six different levels, by multiple level recovery studies. Solution containing 2500 μg/ml PEA, 500 μg /ml FLP, 1400 μg/ml AZH and 100 μg/ml BKC standard stock solutions was prepared and spiked with amounts of the standard drugs equivalent to 70, 80, 90, 100, 110 and 120% of the amounts present in the original solution. These solutions were then analyzed for recov- ery studies and the average recoveries ranged from 97% to 106%. Results for determination of accuracy are presented in Table 2 and 3. Specificity
Placebo interference
Specificity was tested against standard compounds and against potential interferences in the presence of placebo. No interference was detected at the retention time of PEA, FLP, AZH and BKC (C12, C14) in placebo solution. Forced degradation
The Forced degradation of placebo and formulation was carried out as per ICH guidelines (ICH, Q2B) in acid, base, oxidation and water. The acid, base, oxidation and water stress stu- dies were carried out by refluxing sample & placebo into individual flasks at 60°C for half an hour with 10ml 0.1N HCl, 0.1N NaOH, 1% hydrogen peroxide and water respectively. The drug and the formulation were found to be stable under all the stress conditions. All the stress conditions with purity angle, purity threshold and purity flag results are reported in Table 4 to Table 8. Purity plots are shown in Fig. 3 to Fig. 7. Solution stability
To demonstrate the stability of both standard and sample solutions during analysis, both solu- tions were analyzed over a period of 24 h at room temperature. The results showed that for all the solutions, the retention times and peak areas of PEA, FLP, AZH and BKC remained al- most unchanged (RSD<2.0%) indicating that no significant degradation occurred within this period, i.e. both solutions were stable for at least 24 h, which was sufficient to complete the whole analytical process. Ruggedness and Robustness
The ruggedness of the method was determined by using different instruments (Waters 2489). The robustness of the method was determined by making slight changes in the chromato- graphic conditions ie organic phase composition ± 10%, flow rate ±0.2 ml/min and column oven temperature ± 5°C. These results indicated that the method was rugged and robust with regard to these conditions. However, proper resolution also been achieved; separation of the drugs was very robust to the mobile phase ratio, flow rate and column oven temperature. System suitability tests are an integral part of chromatographic method. They were used to verify that the reproducibility of the chromatographic system is adequate for the analysis. To ascertain its effectiveness, system suitability tests were carried out on freshly prepared stan- dard stock solution of Phenylethyl alcohol, Fluticasone propionate, Azelastine Hydrochloride and Benzalkonium chloride. In addition, relative standard deviation of PEA, FLP, AZH and BKC peaks were evaluated by injecting mixed standard of the PEA, FLP, AZH and BKC (100, 20, 56.6 and 4.0 μg/ml). All P rtohvei dpa
Do Table11.
not repeat the abstract. Indicate possible application
CONCLUSION
and extension.
The proposed RP-HPLC method for simultaneous assay of Phenylethyl alcohol, Fluticasone propionate, Azelastine Hydrochloride and Benzalkonium chloride in combined nasal spray preparations is simple, precise, specific, highly accurate and less time consumption for analy- sis could be recorded. So, it can definitely be employed for the routine and stability study analysis. Hence this RP-HPLC method is suitable for quality control of raw materials and Acknowledgement section may be included if author wants to
acknowledge to the research center, institution, organization,

funding agency or to any scientist(s).
The authors are grateful to the Dr.Reddy's Laboratories Ltd. (Hyderabad, India) for gift sam- ples (Phenylethyl alcohol, Fluticasone propionate, Azelastine Hydrochloride and Benzalko- nium chloride) for providing laboratory facility for this research work. Authors are responsible for the accuracy and completeness of the
content including the list of references. References should appear at
end of the article and should consist of the author name, surname

REFERENCES
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ref ,e rIz
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 Louis-Philippe Labranche, Suzanne N. Dumont, Suzanne Levesque, Alain Carrier. For Patent reference
Rapid determination of total benzalkonium chloride content in ophthalmic formula- H. Aviv, D. Friedman, A. Bar-Ilan, and M. Vered. Submicron emul-
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An improved method for the determination of fluticasone propionate in human plas- ma. J. of Pharm and Biomed Anal, 21(4), 1999, 749-758. cessed: 24 Aug 2001].
 Murnane D., G.P. Martin, C. Marriott. Validation of a reverse-phase high perfor- mance liquid chromatographic method for concurrent assay of a weak base (salmete- rol xinafoate) and a pharmacologically active steroid (fluticasone propionate). J. of Pharmaceutical and Biomedical Analysis, 40(5), 2006, 1149-1154.  Nichole L. Korpi-Steiner, Brian C. Netzel, Jesse C. Seegmiller, John B. Hagan, Ra- vinder J. Singh. Liquid chromatography–tandem mass spectrometry analysis of uri- nary fluticasone propionate-17beta-carboxylic acid for monitoring compliance with inhaled-fluticasone propionate therapy Steroids, 75(1), 2010, 77-82.  Pivonka J., F. H. Segelman, C. A. Hartman, W. E. Segl, N. Kucharczyk, R. D. Sofia. Determination of azelastine and desmethylazelastine in human plasma by high- performance liquid chromatography. J. of Chromatog: Biomed Applic, 420, 1987, 89-  Quantitation of active ingredients and excipients in nasal sprays by high-performance liquid chromatography, capillary electrophoresis and UV spectroscopy. J. of Chroma- tog A, 823(1-2), 1998, 423-431.  Ramón Vargas, Robert J. Dockhorn, Steven R. Findlay, Phillip E. Korenblat, Eliza- beth A. Field, Kenneth M. Kral. Effect of fluticasone propionate aqueous nasal spray versus oral prednisone on the hypothalamic-pituitary-adrenal axis, J. of Allergy and Clinical Immunology, 102(2), 1998, 191-197.  Sriram Krishnaswami, Helmut Möllmann, Hartmut Derendorf, Günther Hochhaus. A sensitive LC–MS/MS method for the quantification of fluticasone propionate in hu- man plasma. J. of Pharm and Biomed Anal, 22(1), 2000, 123-129.  The Merck Index – An Encyclopedia of chemicals, Drugs, and biologicals, 14 th edn, Merck Research Laboratories, Whitehouse station, NJ, 2006, pp 909, 1058, 4237,  United states Pharmacopeia 32–NF27 Page 4129, 30–NF25 Page 1543, 30–NF25  Ute Heinemann, Gottfried Blaschke, Norbert Knebel. Simultaneous enantioselective separation of azelastine and three of its metabolites for the investigation of the enant i- omeric metabolism in rats: I. Liquid chromatography–ionspray tandem mass spectro- metry and electrokinetic capillary chromatography. J. of Chromatog B, 793(2), 2003, Figures, Tables, images and other
illustrations should be given at

end of the manuscript with suita-
Figure caption should be at the
ble caption and number.
bottom. Not exceeding two lines
or 20 words. Should be small let-
ter starting from Figure 1:

Figure caption, Must be in the text
form and should not be a part of
the image

Figure 1: Chemical structures of Azelastine Hydrochloride
Images should be legible, sharp and
should be 200dpi or higher resolu-
tion to fit A4 portrait not for land-
scape size page, JPEG, TIFF formats

are preferred.
Figure 2: Typical chromatogram obtained from assay sample
Table caption should be on top. Not
exceeding two lines or 20 words.

Should be small letter starting from
Table 1:

Table 1: Results from validation and system suitability studies of the method
Fluticasone
Azelastine
ethyl al-
Propionate
chloride
chloride
Theoretical plates % RSD for six injec- Accuracy (RSD(%))* Precision (RSD(%)) Intermediate Preci- * Results are mean of three sample preparations with six different drug concentration levels. ** Results are mean of six sample preparations. ***Results are mean of total area of benzalkonium chloride (C12, C14). Place abbreviations and footnotes
immediately below the table

Table 2: Accuracy results of Phenylethyl alcohol and Fluticasone propionate in nasal
spray preparations
Phenylethyl alcohol
Fluticasone propionate
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size portrait page not to fit land-

scape page. Table must be in text
form not a part of image.

Table should be bordered in all
sides of the cells without shading.

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