ЖУРНАЛ АНАЛИТИЧЕСКОЙ ХИМИИ, 2014, том 69, № 6, с. 626-635
ОРИГИНАЛЬНЫЕ СТАТЬИ
УДК 543
A VALIDATED RP-LC METHOD FOR SALMETEROL AND FLUTICASONE IN THEIR BINARY MIXTURES AND THEIR STRESS DEGRADATION BEHAVIOR UNDER ICH-RECOMMENDED STRESS CONDITIONS © 2014 Bediha Akmese*, **, Senem Sanli***, Nurullah Sanli***, Adem Asan **
*Department of Chemistry, Faculty of Science and Arts, Hitit University
£orum, Turkey
**Department of Chemistry, Faculty of Science and Arts, Ondokuz Mayis University
Samsun, Turkey
***Department of Chemistry, Faculty of Science and Arts, Usak University
Usak, Turkey Received 29.02.2012; in final form 06.03.2013
Simple, accurate, precise and fully validated analytical methods for the simultaneous determination of sal-meterol xinafoate and fluticasone propionate in combined dosage forms have been developed. These drugs were exposed to thermal, photolytic, hydrolytic and oxidative stress conditions, and the stressed samples were detected by the proposed method. Additionally, pKa values of three ionizable drugs (salmeterol xinafoate, fluticasone propionate and thioridazine) were determined using by the dependence of the retention factor on pH of the mobile phase. The effect of the mobile phase composition on the ionization constant was studied by measuring the pKa in different acetonitrile—water mixtures, ranging between 50 and 65% (v/v) using LC—UV method.
Keywords: salmeterol xinafoate, fluticasone propionate, liquid chromatography, pKa.
DOI: 10.7868/S0044450214060024
Salmeterol xinafoate (SX), 2-(hydroxymethyl)-4-(1-hydroxy-2-{[6-(4-phenylbutoxy) hexyl]amino}ethyl)-phenol— 1-hydroxynaphthalene-2-carboxylic acid (1 : 1), is a new long-acting p2-adrenoceptor agonist recently introduced for the treatment of asthma, which possesses both bronchodilator and antiinflammatory activity. In human medicine salmeterol is used for conditions such as asthma and chronic obstructive pulmonary disease [1—3]. Salmeterol is a weak base (a secondary amine) with an ionizable phenol group. The retention behavior of an ionizable compound in reversed phase liquid chromatography (RP—LC) is different from the retention of a neutral one. Fluticasone propionate (FP) is a glucocorticoid with potent antiinflammatory activity used in the prevention of asthma [4]. These two drugs are formulated both as dry powder inhalers and pressurized metered dose inhalers, both individually and as a combination formulation.
The degree of ionization of the drug strongly affects solubility, permeability and drug disposition properties (ADME) [5]. Besides, the knowledge on dissociation constant of ionisable compounds at different solvent composition is also significant to determine the optimal separation conditions in RP—LC.
Additionally, there are a wide range of techniques for determining pKa values. Many compounds of pharmaceutical interest tend to be poorly soluble in water and the classical potentiometric techniques for
studying protolytic equilibria are not practical. The advantage of the LC-method is that only a small amount of sample, which does not need to be of high purity, is required. The method of choice is pKa estimation by RP—LC. The protonation of the weak base in LC is regarded as a secondary chemical equilibrium, the first being the distribution equilibrium of the solute between the mobile and stationary phases. The secondary equilibrium is controlled by both the pH of the mobile phase and the content of the organic modifier [6, 7].
Stability testing provides evidence on how the quality of a drug substance or drug product varies with time under the influence of temperature, humidity, and light, and enables recommendation of storage conditions, retest periods, and shelf lives to be established. Till date no detailed report on the forced degradation study on SX and FP under the conditions of hydrolysis (across wide pH range), oxidation, dry heat and photolysis is available. An ideal stability indicating chro-matographic method should be able to resolve the drug from its potential impurities and degradation products. It also requires that analytical test procedures for stability samples should be stability indicating and fully validated [8-10].
LC-methods are useful to determine SX and FP in pharmaceutical dosage form and biological samples each dry individually. There is just one article on the
determination of SX and FP in their binary mixtures [11], whereas there is no publications for their stress degradation behavior. Although acetonitrile—water mobile phases have been used in RP—LC separation procedures, pKa values of drugs have not yet been determined and the reported hydro-organic pKa values of drugs have not been determined in water—acetonitrile binary mixtures.
This paper focuses on the determination of pKa values of salmeterol, xinafolic acid, fluticasone and thioridazine (internal standard, IS) in several aceto-nitrile—water mixtures, 55, 60 and 65% (v/v), in order to overcome the lack of information related with the acid—base equilibria of this type of compounds by means of chromatographic measurements. The determination of pKa values has been performed based on the relationships between the capacity factors and the pH values of the mobile phase. Also, the aim of the present work is the development and validation of a simple, accurate, precise, stability indicating and reliable RP—LC method for the simultaneous determination of the studied compounds and its application to the determination of these analytes in commercial dosage forms. The stability tests were also performed on all drug substances as per ICH norms [12].
<
F
Fluticasone propionate
O
HO I
Salmeterol xinafoate
OH
Thioridazine
O
Chemical structure of studied compounds.
EXPERIMENTAL
Chemicals and reagents. All chemicals were of analytical reagent grade and all solvents were of HPLC grade. The analytes (Scheme) were kindly provided by the following pharmaceutical companies: salmeterol xinafoate and fluticasone propionate by GlaxoSmith-Kline, and thioridazine by Novartis. HPLC grade acetonitrile (ACN) (Merck, Darmstadt, Germany) was used as organic modifier. Orthophosphoric acid (Riedel-de Haen, Germany; min. 85%) and sodium hydroxide (Merck, Darmstadt, Germany) were used for pH adjustment. All stock solutions of hydrochloric acid, potassium hydroxide and potassium hydrogen phthalate were prepared in water. Water with conductivity lower than 0.05 ^S/cm was obtained with a Zeneer Power I (Human Corp.).
Standard stock solutions of SX and FP and thior-idazine (TH) were prepared in ACN at concentrations of approximately 100 mg/L. Working solutions were diluted with the corresponding mobile phase to 10 mg/L. All stock and working solutions were protected from light and stored in a fridge at about 4°C.
Apparatus. The LC-analysis was carried out on a Shimadzu HPLC system with a pump (LC-20 AD), a DAD detector system (SPD-M 20A) and column oven (CTO 20 AC). This equipment has a degasser system (DGU 20 A). The system operates at 210 nm for salmeterol, 248 nm for xinafolic acid, 238 nm for FP and 260 nm for TH (IS). An X-Terra RP-18 (250 x 4.60 mm ID x 5^) column was used as stationary phase at 25°C. Mettler Toledo MA 235 pH/ion analyzer with Hanna HI 1332 Ag/AgCl combined glass electrode was used for pH measurements.
Thioridazine was chosen as the internal standard because it showed a shorter retention time with better peak shapes and better resolution, compared to other potential internal standards. Also, thioridazine is a basic compound like other studied compounds.
Chromatographic procedure. Throughout this study, the mobile phases assayed were ACN—water at 55, 60 and 65% (v/v), containing 25 mM o-phospho-ric acid. The pH of the mobile phase was adjusted between 3 and 12 by the addition of appropriate amount of sodium hydroxide. The flow rate was maintained at 1.0 mL/min, and injection volume was 20 ^L. For each drugs, the retention time values (tR) were determined from three separate injections for every mobile phase composition and pH values. Retention factors for each compound and mobile phase were calculated using the expression k = (tR — t0)/t0. The dead time (t0) was measured by injecting uracil solution (Sigma, USA, 0.1% in water) which was established for each mobile phase composition and pH studied.
The pKa values of the studied compounds were determined from k/pH data pairs by means of the nonlinear regression program NLREG [13]. This is a general purpose program, where the function to be mini-
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mized and the parameters to be estimated can be defined by means of the built-in program editor. NLREG refines these parameters according to equation (1) to give a minimum in the sum of square residuals ( Um) in order to obtain the dissociation constants of the compounds studied.
Um У (ki,exp ki',calc)
(1)
J=1
where ns indicates the number of solutions, £iexp — the experimental value of the retention factor for solution i, and ki calc — the calculated value.
Analysis of drugs. Twenty blisters of Seretide® (each containing 0.05 mg SX and 0.25 mg FP) were accurately weighed and powdered. The required amount of this powder, equivalent to a stock solution of 1 mg/mL FP and 0.2 mg/mL SX, was weighed and transferred into a 100 mL volumetric flask and diluted with ACN. The prepared solution was sonicated for 10 min to complete dissolution. After filtration, appropriate solutions were prepared by taking suitable aliquots of clear filtrate and adding the appropriate IS solution, diluting them with the mobile phase in order to obtain the final solution. The nominal contents of the tablet amounts were calculated from the corresponding regression equations of previously plotted calibration plots from the raw material.
To keep an additional check on the accuracy of the developed assay methods and to study the interference of formulation additives, recovery experiments were carr
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