33 Compound Salmeterol EP Impurity A Fluticasone Propionate EP Impurity C Fluticasone Propionate EP Impurity D Chemical Name


(1RS)-1-[4-Hydroxy-3-(hydroxymethyl)phenyl]-2-[(4- phenylbutyl)amino]ethanol


6α,9-difluoro-17-[[(fluoromethyl)sulphanyl]carbonyl]-11β-hy- droxy-16α- methyl-3-oxoandrosta-1,4-dien-17α-yl acetate


6α,9-difluoro-17-[(methylsulphanyl)carbonyl]-11β-hydroxy- 16α-methyl-3-oxoandrosta-1,4-dien-17α-yl propanoate


6α,9-difluoro-17-[[(fluoromethyl)sulphanyl]carbonyl]- Fluticasone Propionate EP Impurity G


11β-hydroxy-16α-methyl-3-oxoandrosta-1,4-dien-17α-yl- 6α,9-difluoro-11β,17-dihydroxy-16α-methyl-3- oxoandro- sta-1,4-diene-17β-carboxylate


this detector is exhibited as an unknown peak in the chromatogram.


The new suggested method developed and proposed here utilises Potassium Dihydrogen Phosphate which is not classified as hazardous [7] instead of Sodium Dodecyl Sulphate. In addition, UV detection is used which is more common and shows fewer working condition contamination peaks compared to FLR detection. Furthermore, UV detection causes no selectivity difference in terms of the SX and FP peaks.


The purpose of this manuscript is to explain the assay method developed by Arven Pharmaceuticals for SX and FP fixed dose combination drug product, Cyplos 50/500 mcg Powder for Inhalation.


2. Materials and Methods 2.1. Reagents


SX, FP and their related impurities were purchased from a reputable API producer. Ortho-phosphoric Acid and Potasium Dihydrogen Phosphate Monohydrate were purchased from Merck Ltd. HPLC grade Acetonitrile was purchased from J.T.Baker. High purity deionised water was obtained from a Sartorius stedim, arium 611VF (Goettingen, Germany) purification system. Cyplos 50/500 mcg Salmeterol/Fluticasone Propionate Powder for Inhalation (Arven, Turkey) was used as the finished product. All impurities were European Pharmacopeia impurities as stated in Table 1.


2.2. Instrumentation


A Waters HPLC system consisting of inbuilt autosampler and quaternary gradient pump with an on-line degasser was used. The column compartment with temperature control and UV detector were engaged.


Empower software was used to obtain chromatographic data.


2.3. Chromatographic Conditions Equipment: Column:


HPLC System


Thermo BDS Hypersil C18


Flow Rate:


Column Temperature:


150 x 4.6 mm, 5 um 1.5 mL / min


Injection Volume: 40 µL 40ºC


Tray Temperature: 25ºC Run Time:


Wavelength: 10 mins UV, 210 nm


(Gradient Starting Wavelength)


Wavelength gradient:


Time (min) Wavelength (nm) 0.00 5.50 5.60 9.00 9.10


10.00


210 210 239 239 210 210


A Thermo Fisher Hypersil BDS C18 (15 cm x 4.6 mm) 5 µm column was used as stationary phase and maintained at 40ºC.


The Injection volume was 40 µL and the initial wavelength was 210 nm. In the analysis a wavelength gradient was applied representing the maximum wavelength response for SX and FP in Cyplos 50/500 mcg Salmeterol/Fluticasone Propionate Powder for Inhalation given above.


2.4. Preparation of Solutions


2.4.1. Mobile Phase


The mobile phase involved a fixed composition solvent A (pH 3.0 buffer) which was prepared by dissolving 1.0 g Potassium dihydrogen phosphate in a 1000 mL volumetric flask containing high purity deionised water and stirred at 1000 rpm for 20 minutes on a magnetic stirrer. The pH was adjusted to 3.00 ± 0.05 with 85% Ortho-phosphoric acid and stirred for a further 10 minutes.


Solvent A: Acetonitrile (520: 480 v / v) was prepared. Stirred for 15 minutes at 1000 rpm in a magnetic stirrer. The mixture was


filtered through a 0.2 µm membrane filter and degassed for 2 minutes. The mobile phase was pumped through the column with a flow rate of 1.5 mL/min.


A Diluent mixture of acetonitrile : high purity deionised water (50:50, v/v) was also prepared for standard solution preparation.


2.4.2. Standard Solutions


Salmeterol Stock Standard Solution: ~14.53 mg Salmeterol Xinafoate standard (equivalent to 10.0 mg Salmeterol) was weighed and transferred into a 100 mL amber coloured volumetric flask and then diluted to volume with diluent stated above and dissolved by sonication in an ultrasonic bath for 15 minutes. The stock solution was then permitted to come to room temperature. (CSalmeterol


= 0.1 mg/mL)


Fluticasone Propionate Stock Standard Solution: ~25.00 mg Fluticasone Propionate standard was weighed and transferred into a 100 mL amber coloured volumetric flask and then diluted to volume with aforementioned diluent. It was dissolved and dissolved by sonication in an ultrasonic bath for 15 minutes and allowed to come to room temperature. (C Fluticasone propionate


= 0.25 mg/mL)


Standard Solution: 1.25 mL of the standard stock solution of Salmeterol and 5.0 mL of the standard stock solution of Fluticasone Propionate, transferred into a 50 mL amber coloured volumetric flask. The solution was made up to volume with dilution solution and mixed by shaking. The solution was filtered through 0.45 µm filter to a sample vial. (CSalmeterol propionate


= 0.0025 mg/mL, C Fluticasone = 0.025 mg/mL)


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