Scientific paper First-Order Derivative UV Spectrophotometric Method for Simultaneous Measurement of Delapril and Manidipine in Tablets Vítor Todeschini,* Amanda Thomas Barden, Leticia Lenz Sfair, Maximiliano da Silva Sangoi and Nadia Maria Volpato *Postgraduate Program in Pharmaceutical Sciences, Faculty of Pharmacy, Federal University of Rio Grande do Sul, 90610-000 Porto Alegre-RS, Brazil * Corresponding author: E-mail: vitor_todeschini@hotmail.com; Tel.: + 55 51 33085214 Received: 18-01-2013 Abstract A first-order derivative spectrophotometric ('D-UV) method was developed and validated for simultaneous determination of delapril (DEL) and manidipine (MAN) in tablets. The 'D-UV spectra were obtained using ax = 4.0 nm and wavelength set at 228 nm for DEL and 246 nm for MAN. The method was validated in accordance with the ICH requirements, involving the specificity, linearity, precision, accuracy, robustness and limits of detection and quantitation. The method showed high specificity in the presence of two drugs and formulation excipients and was linear over the concentration range of 18-54 |g mL-1 (r2 = 0.9994) for DEL and 6-18 |g mL-1 (r2 = 0.9981) for MAN with adequate results for the precision (< 1.47%) and accuracy (98.98% for DEL and 100.50% for MAN). Moreover, the method proved to be robust by a Plackett-Burman experimental design evaluation. The proposed 1D-UV method was successfully applied for simultaneous analysis of DEL and MAN in tablets and can be used as alternative green method to separation techniques. The results were compared with the validated liquid chromatography, capillary electrophoresis and liquid chromatography-tandem mass spectrometry methods, showing non-significant difference. Keywords: Delapril, derivative UV spectrophotometry, green method, manidipine, validation. 1. Introduction High blood pressure is the major cardiovascular risk factor and one of main cause of death around the world. Control of blood pressure reduces the high mortality associated with hypertension and is a necessary step to reduce global cardiovascular risk and for management of their complications.1'2 Combination therapy of the antihypertensive agents is frequently required to achieve blood pressure targets in a large proportion of patients.3 Due to their complementary mechanisms of action, delapril hydrochloride (DEL), an angiotensin-converting enzyme inhibitor, and manidipine dihydrochloride (MAN), a dihydropyridine calcium antagonist, can be considered a rational drugs combination and an optimal antihypertensive drug treatment for mild to moderate essential hypertension.2 4 The chemical structures of both drugs are shown in Figure 1. Figure 1. Chemical structures of delapril (a) and manidipine (b). The literature reveals a few number of publications related to DEL and MAN determination in biological matrices using liquid chromatography (LC) with differents detectors and applied to pharmacokinetic studies or enantiomeric analysis.5-11 However, for the analysis of both drugs in pharmaceutical formulation, some analytical methods have been developed by our research group. Recently, two stability-indicating methods performed by LC,12 and by capillary electrophoresis, using micellar electrokinetic chromatography (MEKC) mode,13 were validated for simultaneous analysis of DEL and MAN in commercial tablets. Additionally, a liquid chromato-graphy-tandem mass spectrometry (LC-MS/MS) method was also developed with the same purpose.14 Many of these methods have to use expensive and hazardous chemicals, require expertise, making the process a challenge for the environment, time consuming and complex. Spectrophotometric techniques provides practical and economic advantages over other methods and could be an useful alternative for quality control routine.15-17 Multi-component analysis has been proved to be useful for resolving analytes mixtures in drug analysis. The derivative technique in UV spectrophotometry tends to emphasize subtle spectral features by presenting them in a new and visually more accessible way, allowing the resolution of overlapping spectra, and reducing the effect of spectral background interferences.18 Besides that, compared with conventional spectrophotometric analysis, derivative spectrophotometry allows to eliminate the interference from excipients and co-formulated drugs.18 There is no publication concerning the UV spectrop-hotometric determinations of DEL and MAN as finished product in the current literature. Thus, the aim of this study was to develop and validate a simple, fast and cost-effective first-order derivative spectrophotometric (1D-UV) method for simultaneous measurements of DEL and MAN in their combined dosage form. Additionally, the responses obtained by the proposed method were compared to those LC, MEKC and LC-MS/MS methods, contributing to research of green and practical alternatives methods for quality control and assuring the therapeutic efficacy of products. 2. Experimental 2. 1. Chemicals and Reagents Delapril hydrochloride and manidipine dihydrochlo-ride reference substances were kindly donated by Chiesi Farmaceutici (Parma, Italy). Hipertil® (Chiesi Farmacéutica Ltda, SP, Brazil) tablets, containing 30 mg of DEL and 10 mg of MAN, were obtained from commercial source and used within their shelf life period. The excipients contained in the dosage form (lactose monohydrate, magnesium stearate, hydroxypropyl cellulose, low substituted hydroxypropyl cellulose, riboflavin and alumi- num lake) were all pharmaceutical grades and acquired from different suppliers. LC-grade methanol was obtained from Tedia Company Inc (Fairfield, OH, USA). For all the analyses, ultrapure water was used (Milli Q Gradient System, Millipore Corp, Bedford, MA, USA). 2. 2. Instrumentation 2. 2. 1. UV The UV-Visible spectrophotometer was a double-beam (Shimadzu, Kyoto, Japan), model UV-1800, equipped with 1 cm quartz cells, with a spectral band width (1 ± 0.2 nm) and wavelength accuracy of ± 0.1 nm (with automatic wavelength correction). UV Probe software version 2.33 (Shimadzu) was used for instrument control, data acquisition and analysis. 2. 2. 2. LC The LC apparatus was a Shimadzu LC system (Shi-madzu Corp, Kyoto, Japan) equipped with a SCL-10AVP system controller, a LC-10ADVP binary pump, a SIL-10ADVP autosampler, a CT0-10ACvp column oven, and a SPD-M10AVP photodiode array (PDA) detector. The peak areas were automatically integrated using the Class VP (v 6.12) computer software. 2. 2. 3. MEKC The MEKC experiments were performed on an Agilent 3DCE apparatus (Agilent Technologies, Waldbronn, Germany) equipped with an autosampler, a PDA detector and power supply able to deliver up to 30 kV. CE Chem-Station software was used for instrument control, data acquisition and analysis. 2. 2. 4. LC-MS/MS The LC-MS/MS method was performed on a Shimadzu LC system equipped with a SCL-10AVP system controller, LC-10ADVP quaternary pump, DGU-14A de-gasser, a SIL-10ADVP autosampler, and a CT0-10ADVP column oven. The triple quadrupole mass spectrometer (Micromass UK Ltd., Manchester, UK), model Quattro LC, was equipped with an ESI source in positive mode. Data acquisition and analysis were performed using the Masslynx (v 3.5) software. A syringe pump (Bioanalytical Systems Inc, West Lafayette, IN, USA) was used to infuse the solutions. 2. 3. Preparation of Reference Solutions The stock solutions were prepared by weighing accurately, 15 mg of DEL (purity 99.6%) and 5 mg of MAN (purity 99.7%) reference standards, with ± 0.01 mg of precision, and diluted to volume with methanol, obtaining the concentration of 1.5 mg mL-1 and 0.5 mg mL-1 for DEL and MAN, respectively. The stock solution was stored, for not more than seven days, at 2-8 °C, protected from light and daily diluted to an appropriate concentration in water. 2. 4. Preparation of Sample Solutions To prepare the extraction sample solutions, tablets containing 30 mg of DEL and 10 mg of MAN were daily weighed and crushed to fine powder. An appropriate amount was transferred into an individual 20 mL volumetric flask, diluted to volume with methanol, kept in vortex for 5 min, sonicated for 10 min and filtered through a 0.45 pm membrane filter (Millipore Corp), obtaining theoretical concentrations of 1.5 mg mL-1 for DEL and 0.5 mg m-L-1 for MAN. Working sample solutions were prepared by diluting the extraction solution to an appropriate concentration with water. 2. 5. UV Procedure The experiments on UV method were performed using the 1D-UV mode. The solutions were recorded at a fast scan speed with a fixed slit to lead to a spectral resolution of 1 nm. The spectra were obtained by instrumental electronic differentiation using a wavelength interval (AX) of 4 nm in the range of 200-300 nm. The analysis were carried out at 228 nm (1D228) for DEL and 246 nm (1D246) for MAN, with working sample containing 36 pg mL-1 and 12 pg mL-1 of DEL and MAN, respectively. The analytical responses obtained were multiplied by 10 (scaling factor of 10). The spectrophotometric measurements were recorded using water as blank solution. 2. 6. LC, MEKC and LC-MS/MS Procedures The LC, MEKC and LC-MS/MS methods applied for the simultaneous analysis of DEL and MAN in the pharmaceutical dosage form were previously developed and validated by our research group and the data were compared with obtained results by the proposed 1D-UV method. The LC experiments were performed on an analytical column Shim-pack C8 (Shimadzu, Tokyo, Japan) maintained at 35 °C. The mobile-phase consisted of aceto-nitrile and a solution of triethylamine 0.3% adjusted to pH 3.0 with phosphoric acid (55:45; v/v), run at a flow-rate of 1.2 mL min-1, with detection at 220 nm using PDA detector. The injection volume was 20 pL of the solutions containing 30 pg mL-1 and 10 pg mL-1 of DEL and MAN, respectively.12 For MEKC method, the experiments were carried out on an uncoated fused-silica capillary with 50 pm i.d. and 72 cm of effective length, thermostatized at 35 °C. The buffer solution consisted of 50 mM of borate and 5 mM of SDS at pH 9.0. A constant voltage of 25 kV (cur- rent about 15 pA) was applied during the analysis, with detection at 208 nm using a PDA detector. Samples containing 60 pg mL-1 of DEL and 20 pg mL-1 of MAN were injected using the hydrodinamic mode at 50 mbar for 5 s. Salicilic acid was used as internal standard (IS).13 The LC-MS/MS method was achieved on an analytical column Luna C8 (Phenomenex, Torrance, USA), maintained at 45 °C, with a mobile-phase consisted of methanol and 10 mM ammonium acetate (90 : 10, v/v), run at a flow rate of 0.25 mL min-1. The injection volume was 10 pL of the solutions containing 450 ng mL-1 of DEL and 150 ng mL-1 of MAN. The mass spectrometry method was performed employing positive electrospray ionization operating in multiple reaction mode, monitoring the transitions of 453.1>234.1 for DEL, 611.1>167.0 for MAN and 412.2>223.0 for fesoterodine (IS).14 2. 7. Validation of the 1D-UV Method The aim of an analytical procedure validation is to demonstrate that it is suitable for its intended purpose. The method was validated by determining the parameters such as: specificity, linearity, precision, accuracy, LoD, LoQ and robustness following the International Conference on Harmonisation (ICH) guidelines.19 2. 8. Sample Analysis For quantitative determination of DEL and MAN in the tablet formulation, the respective working sample solutions were diluted to appropriate concentrations (as described above for each analytical technique) and the assay (%) of the drugs was calculated against the reference substances. The results were converted to milligrams (mg) for statistical comparison. 3. Results and Discussion 3. 1. Optimization of the Spectrophotometric Conditions During method development, several tests were performed in order to establish the assay parameters. In this context, different solvents were investigated to develop a suitable spectrophotometric method for the simultaneous analysis of DEL and MAN in tablets. For diluents selection, the employed criteria were the drugs solubility, the sample preparation easiness and the method sensitivity. Due to great capability to dissolve both drugs, methanol was selected for the first step of sample preparation. However, aiming the reduction of the organic solvent consumption, water was used as final diluent. Water is the better solvent considering toxicological risks and the absence of demanding residue storage, maintaining the stability of the drugs. Moreover, the use of methanol as final diluent changes the drugs absorption spectra, making this method unfeasible. The overlay zero-order UV spectra of DEL, MAN and placebo (which was prepared with the tablet excipients) are represented in Figure 2a. The maximum absorption wavelengths in water were around 210 nm and 230 nm for DEL and MAN, respectively. However, significant overlapping of the three spectra can be observed, Figure 2. Overlay UV spectra (a), first-order derivative spectra (b) and second-order derivative spectra (c), of delapril reference standard (36 |g mL-1), manidipine reference standard (12 |g mL-1) and placebo. which prevents the simultaneous determination of the two compounds by direct absorbance measurements, being required another tool or analytical instrument. A suitable technique for overcoming this problem is the derivative spectrophotometry.20 Due to the overlapping spectra, the 1D-UV method was considered appropriate for resolving mixtures of both elements and eliminates the interference from excipients absorption over DEL and MAN signal. This approach, based on zero-crossing measurements, involves analysis of the absolute value of the total derivative spectrum at an abscissa value corresponding to the zero-crossing wavelength of the derivative spectrum of another component.20 As observed in Figure 2b, the zero crossing wavelengths at 246 nm for DEL and 228 nm for MAN did not suffer any mutual interference of drugs and pharmaceutical excipients. Therefore, these points were selected as optimum regions to simultaneous determination of drugs (228 nm for DEL and 246 nm for MAN). On the other hand, the use of second derivative was also possible, but it was discarded because did not present analytical advantages. The second-order spectra of DEL, MAN and placebo are showed in Figure 2c. Additionally, beyond the different orders tested, various smoothing and scaling factors were analyzed in order to optimize the derivative method. The smoothing factor of A^ = 4 and a scaling factor of 10 showed an adequate signal-to-noise ratio, high spectral resolution and were suitable to enlarge the drugs signals, facilitating its measurement and decreasing the signal reading errors. With A^ increase, the signal-to-noise ratio also improves and the fluctuation in a derivative spectrum decreases. However, if the value of A^ is too large, the spectral intensity signal deteriorates. 3. 2. 1D-UV Method Validation 3. 2. 1. Specificity Specificity is a procedure to determine the analyte in presence of others components such as different analytes, sample matrix, degradation products and impurities. The method specificity was assessed by comparing the obtained spectra from the commercial formulations and the synthetic mixture of drugs standard and placebo solutions. On both determinations, the absorption spectra were similar (Figure 3a; Figure 3b). Moreover, in 1D-UV spectra analysis (Figure 2b), no spectral interference of two compounds and placebo were observed at the both selected wavelength (228 nm for DEL and 246 nm for MAN). Therefore, the proposed method is specific for the simultaneous analysis of DEL and MAN. 3. 2. 2. Linearity The linearity was determined by constructing three independent analytical curves, each one with seven con- a) 3.00 2.00 1.00 0.00 l I I I \(a) _ 4b) \ (a) DEL (b) MAN (c) Placebo i I I I 200.0 220.0 240.0 260.0 280.0 300.0 nm b) 0.00 -0.50 3 -1.00 -o -1.50 ! ! -1-1- "(c)'/ j -^-(b) y _t«)\ \ V 228 2' t i (a) DEL (b) MAN " Ig (c) Placebo i i 200.0 220.0 240.0 260.0 280.0 300.0 nm c) ■< S 3 < TI 2.00 0.00 -2.00 V" " i i i i (C) VV \ (a) DEL 1 (b) MAN (c) Placebo i i i i 200.0 220.0 240.0 260.0 280.0 300.0 nm a) 3.00 2.00 1,00 0.D0 ti» 0.05) for the response studied (assay (%)). So, there were no significant changes in the assay regarding the percentage of drugs contents under the modifications made in the experimental conditions, showing the robustness of the developed method. 3. 2. 7. Solution Stability In order to demonstrate the stability of both reference solution and tablet sample solution during analysis, they were placed at 2-8 °C and also maintained at room temperature for 48 h. The assay results for DEL and MAN remained almost unchanged and no significant alteration relative to freshly prepared samples was observed (data not shown), showing the stability of the solutions within the indicated period, which was sufficient for the whole analytical process. As previously mentioned, two stability-indicating methods performed by LC and MEKC for DEL and MAN evaluation were developed by our research group. The different stress conditions that the drugs were submitted during the stability studies demonstrated the susceptibility of DEL and MAN under alkaline and photolytic conditions, respectively. Some degradation products formed could be visualized by PDA detector for both drugs, and lots of them presented very similar spectrum. Therefore, an UV method, even with the derivative tool, would be difficult to evaluate both drugs and theirs degradations products simultaneously and, thus, its application in stability studies becomes limited when compared with separation techniques. 3. 3. DEL and MAN Assay in Commercial Tablets The validated 1D-UV method was applied for the simultaneous measurements of DEL and MAN in tablets, and the results compared to those obtained using the previously validated LC, LC-MS/MS and MEKC methods. The experimental values of the four methods (in milligrams per tablet) were compared statistically by ANOVA Table 3: Assay results obtained by 1D-UV, LC-UV, MEKC and LC-MS/MS methods for delapril (DEL) and manidipine (MAN) determination in the pharmaceutical formulation and P-value furnished by ANOVA analysis. Experimental amount" DEL MAN Methods Assayb (mg) RSDC (%) Assayb (mg) RSDC (%) 1D-UV 30.18 140 10.01 1.19 LC-UV 30.18 0.65 10.00 0.85 MEKC 30.13 1.17 9.99 1.17 LC-MS/MS 30.11 0.76 9.98 0.90 P-value (P>0.05) 0.978 0.979 a Tablets containing 30 mg of DEL and 10 mg of MAN b Mean of five replicates c RSD = Relative standard deviation a) Gwt of the Effects (Response is Assay (%) of DEL. alpha = 0.05) i/> v. O Scatling factor Stirring time Delta lambda a.a 0.5 1.0 1.5 Effect 2.306 2.0 2.5 b) § (Ï Chart of the Effects (Response is Assay (%)of MAN, alphas 0.05) 2.306 Stirring time Delta lambda Scatling factor 0.Û 0.5 1.0 1.5 Effect 2.0 2.5 Figure 5. Bars charts representing the effects of the factors on quantitative determination (assay) of delapril (a) and manidipine (b) in Placket-Burman experimental design (n = 12). The tcritical is represented by the vertical line. showing non-significant difference (P > 0.05) for content of DEL and MAN, as shown in Table 3. Hence, the :D-UV method can be applied to routine analysis of both drugs in pharmaceutical formulation in the specific wavelengths, with benefit of short analysis time and low organic solvents consumption when compared with the others. 4. Conclusions The first-order derivative spectrophotometric method was successfully validated and demonstrated to be simple, specific, reproducible, rapid and cost effective. Moreover, the proposed method can be used without any prior separation of drugs and tablet excipients and, when the assay results were compared, non-significant difference between the previously validated LC-UV, LC-MS/MS and CE methods was found. Considering that the chroma-tographic and electrophoretic methods are more expensive, time consuming, and need more steps, the proposed method is adequate for routine analysis. It is a safe alternative for the simultaneous analysis of DEL and MAN in their combination formulation and can be conveniently used for the routine quality control. 5. Acknowledgements The authors wish to thank CAPES (Coordenaçao de Aperfeiçoamento de Pessoal de Nivel Superior) for the financial support and Chiesi Group for the provision of de-lapril and manidipine reference standards. 6. References 1. A. D. Lopez, C. D. Mathers, M. Ezzati, D. T. Jamison, C. J. L. Murra, Lancet. 2006, 367, 1747-1757. 2. M. L. Otero, Vas. Health. Risk Manag. 2007, 3, 255-263. 3. K. K. Gaddam, A. Verma, M. Thompson, R. Amin, H. Ventura, Med. Clin. North Am. 2009, 93, 527-786. 4. P. L. McCormack, G. M. Keating, Drugs. 2006, 66, 961-969. 5. T. Miyabayashi, K. Yamashita, I. Aoki, M. Motohashi, T. Yashiki, K. Yatani, J. Chromatogr. 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Povzetek Razvili in validirali smo derivativno spektrofotometrično metodo prvega reda (1D-UV) za hkratno določitev delaprila (DEL) in manidipina (MAN) v tabletah. 1D-UV spektre smo pridobili pri AX = 4,0 nm in valovni dolžini 228 nm za DEL ter 246 nm za MAN. Metodo smo validirali v skladu z ICH zahtevami: specifičnost, linearnost, natančnost, točnost, robustnost, meje zaznave in določanja. Metoda je bila visoko specifična v prisotnosti dveh učinkovin in pomožnih snovi. Bila je linearna v koncentracijskem območju 18-54 |g mL-1 (r2 =0.9994) za DEL ter 6-18 |g mL-1 (r2 = 0.9981) za MAN. Dobili smo primerne rezultate za natančnost (< 1,47%) ter točnost (98,98 % za DEL in 100,50 % za MAN). Glede na Plackett-Burmanovo evaluacijo eksperimentalnega načrta se je metoda izkazala za robustno. Predlagano 1D-UV metodo smo uspešno uporabili za hkratno analizo DEL in MAN v tabletah in jo lahko uporabimo kot alternativno zeleno metodo separacijskim tehnikam. Rezultate smo primerjali z validirano tekočinsko kromatografsko, kapi-larno elektroforezno in tekočinsko kromatografsko - tandemsko masnospektrometrično metodo, razlike so bile nesigni-fikantne.