15
solvent B (acetonitrile). The mobile phase was pumped through the column at a fl ow rate of 2 mL/min (Table 1). The samples were made up in two solutions, diluent A or diluent B. Diluent A was a mixture of 0.05% phosphoric acid in methanol and water (70:30, v/v) and diluent B was a mixture of acetonitrile and diluent A (50:50, v/v).
An injection volume of 20 µL was used and the selected detection wavelength was 228 nm which is the optimal wavelength to ensure simultaneous determination of related impurities of SX and FP in Cyplos 50/500 mcg Salmeterol/Fluticasone Propionate Sanohaler inhalation powder.
Table 1. Mobile phase program for gradient elution. Figure 2. Chemical Structures of all Related Impurities of FP
powder, pre-dispensed monograph. According to this monograph the method could separate only seven impurities from nine impurities of FP and fi ve impurities from seven impurities of SX [7].
Yan C et al. [8] proposed an HPLC method of salmeterol and FP inhalation powder related substances determination with a Phenomenex Luna C18 column (25 cm x 4.6 mm, 5 µm). However, this method is not ideal. The method uses a different mobile phase and a higher column temperature, 40ºC, than the BP method but still results in a tailing factor for salmeterol peak greater than 1.3. Reducing the column temperature, results in the tailing becoming more severe which causes interference with the FP and salmeterol peaks for related substances [9].
The purpose of this study is to develop an HPLC method which can detect and separate all 16 known related substances of both salmeterol and FP, simultaneously. The difference between the proposed method from the BP and the literature methods relates to the solubilities of SX, FP and their impurities. The fi nal method has been shown to be able to detect and separate all 16 known related substances (SX based impurities: A, B, C, D, E, F, G and FP based impurities A, B, C, D, E, F, G, H, I) with good linearity, accuracy and precision. The method was validated for 11 known related substances (SX based impurities D, G and FP based impurities A, B, C, D, E, F, G, H, I) because SX impurities are process based and as such were not required to be monitored. This proposed method has been successfully applied for routine analysis.
2. Experimental 2.1. Reagents
SX, FP and their related impurities were purchased from an API producer in Mumbai, India. Ammonium dihydrogen phosphate and phosphoric acid were purchased from Merck Ltd. HPLC grade acetonitrile and methanol were purchased from
J.T.Baker. Deionised water was obtained from a Millipore, Milli-Q (Bedford, USA) purifi cation system. Cyplos 50/500 mcg salmeterol/ fl uticasone propionate Sanohaler inhalation powder product (Sanovel, Turkey) was used as the fi nished product. All impurities are European pharmacopoeial impurities (Figure 1 and 2).
2.2. Instrumentation
An HPLC system (Agilent, USA) equipped with inbuilt autosampler and quaternary gradient pump with an on-line degasser was used. The column compartment was temperature controlled and a PDA detector was employed throughout the analysis. Chromatographic data was acquired using Empower software.
2.3. Chromatographic Conditions
A Hypersil BDS C8 (15 cm x 4.6 mm) 5 µm (Thermo Fisher Scientifi c, Runcorn, UK) column was used as the stationary phase and maintained at 30ºC in the thermostatically controlled oven. Mobile phase consisted of solvent A (0.1 M NH2
Time (min)
0
30 46 85
110 112 120
Flow rate (mL/min)
2.0 2.0 2.0 2.0 2.0 2.0 2.0
Solvent A (%)
90 73 71 52 26 90 90
2.4. Impurity Stock Solutions
Salmeterol related substances A, B, C, D, E, F and G were prepared separately by dissolving 2.0 mg of each impurity in diluent B and completing volume to 25 mL to yield a concentration of 0.08 mg/mL. Four mg of each FP related substances (A, B, C, D, E, F, G, H and I) were weighed into 5 mL volumetric fl asks and completed to volume with diluent B to yield the fi nal concentrations of FP impurities of 0.8 mg/mL.
2.5. Standard Solutions PO4
buffer which was prepared as 11.5 g ammonium dihydrogen phosphate dissolved in 1000 mL of water and adjusted to a pH of 2.9 with orthophosphoric acid) and
Stock standard solutions for SX and FP were separately prepared by dissolving 29 mg of SX (equivalent to 20 mg Salmeterol) and 20 mg of FP in 100 mL volumetric fl asks and diluting them to volume with diluent B to yield the fi nal concentrations of solutions of 0.2 mg/mL. To obtain a mix standard solution, 0.25 mL from SX and 2.5 mL from FP stock standard solutions were pipetted out into a 20 mL volumetric fl ask and diluted to volume with diluent B. (CSalmeterol mg/mL, CFP
: 0.0025 : 0.025 mg/mL).
2.6. Test solution 500 mg sample from Cyplos Sanohaler
Solvent B (%)
10 27 29 48 74 10 10
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