Development and Validation of an Isocratic HPLC Method for Simultaneous Determination of Quaternary Mixtures of Antihypertensive Drugs in Pharmaceutical Formulations

Original Acta Chromatographica 29(201 7)1, 95 –110 Research Paper DOI: 10.1556/1326.2017.29.1.9 Development and Validation of an Isocratic HPLC Method for Simultaneous Determination of Quaternary Mixtures of Antihypertensive Drugs in Pharmaceutical Formulations J.F.F. A NDERSON 1, M.C.G. G ERLIN 1, R.A. S VERSUT 1, L.C.S. O LIVEIRA 2, A.K. S INGH 3, M.S. A MARAL 1,4 , AND N.M. K ASSAB 1,* 1Programa de Pós-Graduação em Farmácia, Centro de Ciências Biológicas e da Saúde, Universidade Federal de Mato Grosso do Sul, Brazil 2Instituto de Química, Universidade Federal de Mato Grosso do Sul, Brazil 3Departamento de Farmácia, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, 05508-900, São Paulo, SP, Brazil 4Instituto de Física, Universidade Federal de Mato Grosso do Sul, Brazil *E-mail: [email protected] Summary. The objective of this study was to develop and validate an assay method for simultaneous determination of atenolol, furosemide, losartan, and spironolactone in pharmaceutical formulations. A reverse-phase high-performance liquid chromatography procedure was developed, using a Kinetex ® C-18 column (100 mm × 4.6 mm, 2.6 µm). The mobile phase was composed of methanol–water (75:25 v/v, pH 3.0, adjusted with phosphoric acid), with a flow rate of 0.4 mL min −1. All drugs were separated in less than 5 min. The method was validated according to International Conference on Harmoniza- tion (ICH) and Association of Official Analytical Chemists (AOAC) guidelines. The method showed linearity in a concentration range of 0.75–12.0 µg mL −1 for atenolol (r = 0.9995), 0.30–12.00 µg mL −1 for furosemide ( r = 0.9997), 0.45–12.00 µg mL −1 for losartan ( r = 0.9995), and 0.45–12.0 µg mL −1 for spironolactone ( r = 0.9999). The method also showed repeatability and precision. The three-day average intra-day precisions were 101.35 ± 0.74% for atenolol, 95.84 ± 1.44% for furosemide, 98.90 ± 1.16% for losartan, and 97.19 ± 0.18% for spironolactone. Similarly, the inter-day precisions were 101.34 ± 0.72% for atenolol, 95.84 ± 0.1.50% for furosemide, 98.90 ± 1.17% for losartan, and 97.19 ± 0.83% for spironolactone. The method accuracy was also tested and validated — in this case, the average recovery values were 100.18 ± 1.20% for atenolol, 99.83 ± 1.54% for furosemide, 100.07 ± 0.95% for losartan, and 99.94 ± 0.93% for spironolactone. Finally, the method was successfully applied in the simultaneous determination of atenolol, furosemide, losartan, and spironolactone in magisterial formulas, as well as in commercial pharmaceutical formulations. Key Words: HPLC–DAD, antihypertensive, quality control, simultaneous determination, quantitative This is an open -access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium for non - commercial purposes, provided the original author and source are credited. First published online: April 10, 2016 ISSN 2083-5736 © The Author(s) Unauthenticated | Downloaded 10/02/21 06:31 PM UTC 96 J.F.F. Anderson et al. Introduction Hypertension is a main risk factor for cardiovascular and kidney diseases and is among the leading causes of mortality in Brazil and worldwide [1]. A very common disorder, hypertension, if neglected, can lead to coronary thrombosis, stroke, and kidney failure [2, 3]. Atenolol (Fig. 1) is a β-adrenergic receptor antagonist with cardioselec- tive activity and is widely used by hypertensive patients, especially in sus- pected angina or heart failure [3]. Its clinical use can be attributed to its car- dioprotective effect, and its effectiveness as an antihypertensive is well described when used in combination with a diuretic for elderly patients with isolated hypertension [2]. Fig. 1 . Chemical structures of hypertensive drugs used in the method validation Furosemide (Fig. 1) is a loop diuretic, able to cause elimination of 15–25% of filtered Na +[3], and is widely used in the treatment of hyperten- Unauthenticated | Downloaded 10/02/21 06:31 PM UTC Isocratic HPLC Method 97 sion [2]. Compared to other diuretics such as the thiazides, furosemide causes smaller disturbances in the lipid profile [2, 4]. Losartan (Fig. 1) is an angiotensin II receptor antagonist and has a smooth-muscle relaxing action. The vasodilation activity of losartan increases renal excretion of water and salt, reduces plasma volume, and de- creases cellular hypertrophy [2]. It has a satisfactory hypotensive effect and is indicated mainly for young persons, diabetics, or patients with heart failure [2, 3]. Spironolactone (Fig. 1) is a diuretic, although it has very limited effectiveness when used alone, being responsible for reabsorption of only 2% of the Na + filtered [2, 3]. However, spironolactone has an accentuated antihypertensive effect when combined with other diuretics, such as loop diuretics. This combination increases mobilization of body fluid, while causing less disturbance in K + homeostasis. When combined with conventional therapy, spironolactone substantially reduces morbidity and mortality in patients with heart failure [2, 5]. Combined-dose formulations have several advantages in the treatment of hypertensive patients, such as increased antihypertensive efficacy result- ing from the combination of drugs that act by different mechanisms, reduc- tion of adverse effects due to the use of lower doses, simplification, and im- provement of treatment adherence [1, 6]. In the case of antihypertensive therapy, adherence to the treatment is one of the major challenges in the treatment of hypertensive patients. Stud- ies show that patient compliance with the treatment is inversely propor- tional to the number of units taken daily and/or the number of daily doses. Nearly 2/3 of hypertensive patients require the combined dosage form for better compliance with the treatment [1]. The compounding pharmacy provides tailor-made capsules in a form that allows a custom drug dosage. In addition to the lower cost of the compounded dosage form for better adjustment of the dose, these formulations play an important role when no commercial combined dosage forms are available in the market [7]. Although compounding is often preferred, since it meets the needs of the individual patient, the Brazilian Society of Cardiol- ogy (SBC) does not recommend compounding, due to a lack of information on quality standards [8], bio-equivalence, and/or chemical interactions among the compounded drugs [9]. For these reasons, quality control of pharmaceutical products is indis- pensable, not only to ensure the effectiveness of production processes but also and primarily to ensure compliance with qualitative and quantitative standards that ensure the effectiveness and safety of pharmaceuticals. Unauthenticated | Downloaded 10/02/21 06:31 PM UTC 98 J.F.F. Anderson et al. Table I. Analytical methods described in the literature for the analysis of atenolol, furosemide, losartan, and spironolactone Method Reference Drug Type of sample Atenolol, atorvastatin, Commercial [10] acetylsalicylic acid, losartan formulation Atenolol, sotalol, diacetolol, carteolol, nadolol, pindolol, [11] acebutolol, metoprolol, celiprolol, Human plasma oxprenolol, labetalol, propranolol, tertatolol, betaxolol Raw material and [12] Spironolactone, torsemide tablet High-performance Combinations of liquid chromatography hydrochlorothiazide with: atenolol, Pharmaceutical [13] (HPLC) amlodipine, candesartan, moexipril, formulation valsartan [14] Carvedilol, losartan Plasma and urine Pharmaceutical [15] Hydrochlorothiazide, losartan formulation Atenolol, amlodipine, atorvastatin, Pharmaceutical [16] enalapril, hydrochlorothiazide, formulation lisinopril, losartan [17] Spironolactone, metolazone Tablet and urine Spironolactone, furosemide, Pharmaceutical Densitometry [18] hydrochlorothiazide, triamterene formulation Capillary Pharmaceutical [19] Atenolol, amiloride electrophoresis formulation Ultraviolet spectrophotometry [20] Hydrochlorothiazide, losartan Tablet (UV) Pharmaceutical Irbesartan, losartan, olmesartan, [21] formulation and valsartan Thin-layer human plasma chromatography Amlodipine, hydrochlorothiazide, Pharmaceutical (HPTLC) [22] losartan formulation [23] Atorvastatin, losartan Tablet Unauthenticated | Downloaded 10/02/21 06:31 PM UTC Isocratic HPLC Method 99 Table I. (continued) Method Reference Drug Type of sample Pharmaceutical Liquid chromatography [24] Tranexamic acid, losartan formulation and (LC) serum Acetylsalicylic acid, atenolol, Liquid chromatography bisoprolol, chlorthalidone, enalapril coupled to mass [25] and its active metabolites, Plasma spectrometry (LC– fluvastatin, hydrochlorothiazide, MS/MS) valsartan [26] Atenolol, nifedipine Plasma Several analytical methods have been described for the analysis of atenolol, furosemide, losartan, and spironolactone, in isolation or in combined dosage form (Table I). However, we are unaware of a method for simultaneous determination of all four drugs in a single pharmaceutical formulation. The objective of this study was to develop a single chromatographic method for simultaneous determination of all four drugs in compounded capsule form. Experimental Materials and Reagents The raw materials were purchased from distributors licensed by the Na- tional Health Surveillance Agency (ANVISA, Brazil). The stated purity lev- els were: atenolol 98.86%, furosemide 100.75%, losartan 99.12%, and spironolactone 99.4%. The placebo, consisting of 1% silicon dioxide, 59% micro- crystalline

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