www.scielo.br/eq Volume 34, número 2, 2009 23 Potentiometric Determination of Minoxidil in Topical Use Pharmaceutical Samples Rogério Adelino de Sousa1, Éder Tadeu Gomes Cavalheiro2* 1 – Departamento de Química, Universidade Federal de São Carlos, Rod. Washington Luiz Km 235, São Carlos, São Paulo, Brasil, CEP 13565-905, Caixa Postal 676. 2 – Departamento de Química e Física Molecular, Instituto de Química de São Carlos, Universidade de São Paulo, Av Trabalhador São- carlense, 400, Centro, São Carlos, São Paulo, Brasil, CEP 13560-970, Caixa Postal 780. *cavalheiro@iqsc.usp.br Abstract: A potentiometric titration method for the determination of minoxidil based on its re- dox reaction with K2Cr2O7 is described. The best results were observed using 1.00 x 10-3 mol L-1 K2Cr2O7 and 1.00 x 10-2 mol L-1 minoxidil solutions, and the minoxidil as titrant in 2.00 mol L-1 H2SO4 medium. The method was applied to commercial samples and compared with the results from a chromatographic procedure. Recoveries from 97.4 to 98.7 % were observed depending on the sample. Comparison with the chromatographic procedure reveled agreement within 90% confidence level. Keywords: Minoxidil determination; titration; potenciometry. Introduction Minoxidil (2,4-diamino-6-piperidinopyri- midine-3-oxide) is an odorless white crystalline powder, insoluble in water, acetone or alkaline solutions, slightly soluble in alcohols, and freely soluble in acidic solutions [1]. It has been used as a peripheral vasodilator drug orally administrated, applied in the treatment of refractory hypertension patients [2] and liquid retention and hirsutism is being observed for excessive doses [3]. Minoxidil also shows new applications in dermatology, especially in the treatment of an- drogenic alopecia [3,4]. In this case, this drug has been topically applied in order to stimulate hair growth by inducing vasodilatation and increasing the local irrigation and blood flow [4]. Commercially available products typically contain 2% minoxidil (m/v), in topic use formu- lations, containing ethanol, and propylene glycol, or their mixture with 2-n-nonyl-1,3-dioxolane as a vehicle [5,6]. Concerning analytical methods, MnO4 - has been proposed for the oxidimetric titration of mi- noxidil [2,7-8]. In addition, several instrumental techniques have been described for minoxidil de- termination in matrixes such as serum and phar- maceutical products. Most of them are based in chromatographic methods as described in referen- ces [3, 9 – 12]. Potentiometric titration [13,14] using K2Cr2O7 is proposed in this paper. High purity K2Cr2O7 is available, it is thermally stable and standard solutions can be prepared, stable to the action of light. It is not reduced by hydrochloric acid (HCl), reacts with organic matter being redu- ced from Cr(VI), orange, to Cr(III), green, accor- ding to the half-reaction (Eq. 1) [15,16]. Cr2O7 2- + 14 H+ + 6 e- → 2Cr3+ + 7 H2O Eo = 1,33 V (Eq. 1) Ecl. Quím., São Paulo, 34(2): 23 - 27, 2009 Ecl. Quím., São Paulo, 34(2): 23 - 27, 200924 Experimental Reagents and Solutions All the reagents were of analytical grade and used as received. Solutions were prepared using water bi-distilled in quartz stiller. Potassium dichromate (Merck, Germany) solutions were pre- pared by direct dissolution of the salt in H2SO4 solution (2.00 mol L-1). Minoxidil was from Natu- ral Pharma (Brazil), and H2SO4 from Mallinckrodt (Mexico). Potentiometric methods Potentiometric titrations were performed within an glass cell containing a platinum electro- de (indicator) and a saturated calomel electrode (reference) and a pH-meter Corning M 425 (Cor- ning, USA) 10.00 mL of a 1.00 x 10-3 mol L-1 K2Cr2O7 in 2.00 mol L-1 H2SO4 were added to cell at (60±1) ºC controlled temperature. Aliquots of 0.500 mL of minoxidil solutions were added to the K2Cr2O7 solution in acid media from a 10.00 mL full capa- city burette. After each addition of titrant the sys- tem was let to stabilize during 3 minutes, before the potential measurement. Commercial Samples The commercial samples used in this work were Regaine® from Pharmacia (USA) and Neo- xidil® from Galderma (Brazil); both purchased in the local market. Samples were topic use formu- lations of minoxidil 2% (20 mg mL-1) in a mixture of ethanol and propylene glycol as vehicle. As observed in test titrations both propy- lene glycol and ethanol present in the commer- cial samples severely interfered in the minoxidil determinations. Thus they were removed first by evaporation of ethanol in a rotary evaporator at 70ºC under reduced pressure followed by addition of acetone to the resulting viscous liquid. At this point minoxidil precipitates as a white solid that was recovered by filtration in a glass plate fun- nel. The precipitate was dried at 60ºC in an oven during 30 minutes and dissolved in 2.00 mol L-1 H2SO4 and then titrated. Comparison Method In order to check the accuracy of the pro- posed procedure the results were compared with those from a chromatographic procedure describe by Zarghi et al. [8], using a HPLC LC-6AD Shi- madzu chromatographic system with a SPD–10A VP Shimadzu spectrophotometric detector, Supel- co C-18 (150 x 3.9 mm, 4 mm) column, detection at 254 nm, mobile phase methanol / water (80 /20) flowing at 1.3 mL min-1. Results and Discussion The redox reaction between the K2Cr2O7 and minoxidil was very slow at 25ºC. However significant stabilization of the potential was ob- served when the system was held for 3 minutes af- ter each addition of the titrant at 60ºC. The effect of temperature on the redox titration profile was investigated for 40, 50, 60 and 70°C, as shown in Figure 2. The profiles of potentiometric curves and the end points of each titration were compa- red Sousa et al. Fig.2 0 3 6 9 12 15 18 -975 -900 -825 -750 -675 -600 -525 -450 400C 500C 600C 700C E / m V v s S C E Volume of tritant / mL The best results for the potentiometric cur- ve shape and accuracy of the end point were rea- ched at 60ºC (Figure 2). Equation 2 gives a sug- Ecl. Quím., São Paulo, 34(2): 23 - 27, 2009 25 gestion for the reaction between the minoxidil and K2Cr2O7. N N N O NH2H2N + 2 Cr2O7 -2 + 8H2O H+ 4 Cr+316 - 3 N N N O NH2O2N + 3 1 2 34 5 6 (Eq.2) This suggestion is based on the stoichiome- try of the titration in which 2 mols of Cr2O7 2- are consumed by 3 mols of minoxidil. The oxidation of the amine group at position 5 of the aromatic ring is proposed considering the lower reactivity of the amine group in position 3. Sample Determinations The titrant addition resulted in a potential change of the platinum electrode. To better iden- tify the end point of potentiometric titrations the Gram method was used [17] which consists of a ΔE / ΔV vs volume of titrant added curve, in whi- ch the intersection between the two linear portions indicates the end point of the titration, see Figure 3 for an example. 0 30 60 90 120 0 2 4 6 8 10 12 14 0 30 60 90 120 A - Regaineend point = 5.76 mL ∆E / ∆ V m V / m L B - Neoxidilend point = 5.75 mL Titrant volume / mL Sousa et al. Fig.3 The values of minoxidil concentration (Table 1) contained in the commercial samples were consistent with the label values. The propo- sed method agrees in 90% confidence level, with the label value of commercial samples and 95% confidence level, with the values obtained by the comparative chromatographic method, according to the Student t-test [18]. Table 1: Results of potentiometric titrations Samples Label a Found b* Comparison b* E1 / % c E2 / % d Neoxidil ® 20.0 20.7 ± 0.2 19.4 ± 0.4 +3.72 +6.70 Regaine ® 20.0 21.0 ± 0.3 20.9 ± 0.5 +4.71 +0.48 * n = 3 a – Concentration in mg mL-1 b – Concentration in mg mL-1 ± SD c – Relative error to the label d – Relative error to the comparison methods In order to evaluate the accuracy of the proposed method recovery tests were performed with recoveries between 98.7% for Neoxidil® and 97.4% for Regaine®. Ecl. Quím., São Paulo, 34(2): 23 - 27, 200926 FIGURE CAPTIONS Figure 1: Structural formula of minoxidil. Sousa et al. Fig.1 N NO NH2 N NH2 Figure 2: Effect of the temperature in the potentio- metric titration. Conditions: [minoxidil] = 1,00 x 10-2 mol L-1 ; [K2Cr2O7] = 1,00 x 10-2 mol L-1; Volume of K2Cr2O7(aq) = 3,00 mL; [H2SO4] = 2,00 x 10-2 mol L-1; Volume of H2SO4(aq) = 5 mL; Stabilization time = 3 minutes. Figure 3: Identication of the end point of the poten- tiometric titration (Gram’s method) of the commer- cial samples; A – Regaine®; B – Neoxidil®. Condi- tions: [minoxidil] = 1,00 x 10-2 mol L-1 ; [K2Cr2O7] = 1,00 x 10-2 mol L-1; Volume of K2Cr2O7(aq) = 3,00 mL; [H2SO4] = 2,00 x 10-2 mol L-1; Volume of H2SO4(aq) = 5 mL; Stabilization time = 3 minutes; T = 60ºC. Conclusions The method proposed in this study was effective in the determination of minoxidil in commercial samples for topical applications, con- sidering the apparatus limits and errors inherent to the classical technique. Although time consuming, this procedure can be an alternative for quality control in the ma- gistral pharmacies, presenting low cost and good precision when compared with other more expen- sive and waste generating methods as chromato- graphy,. Acknowledgements The authors are grateful to Brazilian agen- cies CAPES and CNPq for RAS and ETGC fello- wships. Determinação Potenciométrica De Minoxi- dil Em Formulações Farmacêuticas De Uso Tó- pico Received November 03 2008 Accepted October 14 2009 Resumo: Um método potenciométrico para a determinação de minoxidil em formulações far- macêuticas baseado na reação redox entre K2Cr2O7 e o minoxidil, foi desenvolvido. Os melho- res resultados foram obtidos usando concentrações de 1,00 x 10-2 mol L-1 para o minoxidil e 1,00 x 10-3 mol L-1 para o K2Cr2O7 ambos diluídos em uma solução em de H2SO4 2,00 mol L-1, a 60°C. As recuperações para método proposto foram da ordem de 98,7 % a 97,4 % dependendo da amostra comercial. O método proposto foi aplicado à amostras comerciais contendo mino- xidil e, quando comparado com resultados obtidos a partir de procedimentos cromatográficos, apresentou concordância no nível de confiança de 95%, de acordo com o teste t-Student. Palavras chave: Determinação minoxidil; titulação; potenciometria. Ecl. Quím., São Paulo, 34(2): 23 - 27, 2009 27 References [1] S. S. 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