Ionics DOI 10.1007/s11581-015-1534-8

ORIGINAL PAPER

Simultaneous determination of amlodipine besylate and rosuvastatin calcium in binary mixtures by voltammetric and chromatographic techniques Nurgul Karadas-Bakirhan 1,2 & Mehmet Gumustas 1,2 & Bengi Uslu 1 & Sibel A. Ozkan 1

Received: 9 April 2015 / Revised: 30 July 2015 / Accepted: 10 August 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Voltammetric and liquid chromatographic (LC) methods have been developed for the simultaneous determination of amlodipine besylate (AML) and rosuvastatin calcium (ROS) for the first time. Detailed electrochemical behavior and simultaneous voltammetric determination of AML and ROS were investigated in detail using glassy carbon electrode (GCE). High-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography (UPLC) were also developed for the comparison. Voltammetric method exhibited linear dynamic responses for the simultaneous assay of AML and ROS in the concentration range between 0.006 and 2.85 μg/mL and between 0.01 and 5.00 μg/mL, with detection limits of 0.001 and 0.003 μg/mL, respectively. On the other hand, LC methods presented a wider linearity range than that of the SWV method between 0.5 and 100 μg/mL with the detection limits of 0.011 and 0.027 μg/mL for AML and 0.034 and 0.042 μg/mL for ROS by UPLC and HPLC techniques, respectively.

Keywords Amlodipine besylate . Rosuvastatin calcium . Voltammetry . HPLC . UPLC . Determination . Pharmaceuticals

* Sibel A. Ozkan [email protected] 1

Faculty of Pharmacy, Department of Analytical Chemistry, Ankara University, Ankara, Turkey

2

Faculty of Science and Arts, Department of Chemistry, Hitit University, Corum, Turkey

Introduction Amlodipine (AML),({R,S-2-[(2-aminoethoxy) methyl]-4-(2chlorophenyl)-3-ethoxy-carbonyl-5-methoxycarbonyl-6-methyl-1,4-dihydro pyridine}) (Scheme 1), is a dihydropyridine calcium channel blocker [1] and its chemical formula differs from other dihydropyridines in possessing a basic side-chain attached to the 2-position of the dihydropyridine ring. It shows the highest oral bioavailability and the longest half-life of elimination in humans among all the drugs of its class [2]. It is clinically used in the treatment of hypertension and angina [3]. Rosuvastatin calcium (ROS),(3R,5S,6E)-7-[4-(4fluorophenyl)-2-(N-methylmethane sulfonamido)-6-(propan2-yl) pyrimidin-5-yl]-3, 5-dihydroxyhept-6-enoic acid calcium salt (Scheme 1) is a member of the drug class of statins used to treat high cholesterol and related conditions, and to prevent cardiovascular disease. In clinical trials, ROS achieved a marked reduction in serum levels of LDL cholesterol, accompanied by modest increases in HDL cholesterol and a reduction in triglycerides [4–6]. ROS is a selective and competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase [7, 8]. Use of the tablets with AML and ROS combination may provide a more integrated approach to the treatment of cardiovascular risk. This drug is widely used as binary combination with different active drug compounds in pharmaceutical preparations, hence clinical and pharmacological studies require rapid, selective, and sensitive and fully validated analytical techniques for the investigation and determination of these drugs in the presence of each other and excipients in their tablets and from biological samples. Voltammetric methods are sensitive, reliable, rapid, selective, and easy techniques applicable to analysis in most areas of analytical chemistry, especially compared with the classical analytical methods. These methods can make the investigation of the mechanism

Ionics Scheme 1 Chemical structures of AML and ROS

AML

of redox reactions possible. The most commonly used voltammetric methods are cyclic (CV), linear sweep (LSV), differential pulse (DPV), square wave (SWV), normal pulse (NPV), and adsorptive stripping voltammetry [9–11]. To the best of our knowledge, no scientific papers regarding the simultaneous determination of AML-ROS by use of neither ultra-performance liquid chromatography (UPLC) nor voltammetric methods have been published. In literature, a few methods using high-performance liquid chromatography (HPLC) method applied for the simultaneous determination of these drugs have been reported [12, 13]. Due to the importance of the simultaneous, sensitive, and selective determination of AML-ROS, it is interesting to develop a rapid screening method from its pharmaceutical formulations. For this reason, three different methods including HPLC, UPLC, and voltammetric techniques were developed. For chromatographic analysis at the first step, a HPLC method was optimized and then this method was transferred to UPLC system using the same mobile phase conditions. All three methods have been found as highly sensitive, convenient, and effective tools for the analysis of important biomolecules including drugs in pharmaceutical formulations. Thus, the electrochemical behavior of AML-ROS has been examined by developing new voltammetric methods to study its content uniformity in the pharmaceutical preparation at GCE using cyclic voltammetry (CV) and square wave voltammetric techniques (SWV). The aim of the present paper is to investigate the electrooxidative and chromatographic behaviors of AMLROS and their sensitive and selective determination by square wave voltammetry, HPLC, and UPLC techniques. The proposed methods were extensively validated according to the United States of Pharmacopeia (USP 32) and International Conference on Harmonization (ICH) guideline Q2A [14, 15].

Experimental Instrumentation Cyclic (CV), differential pulse (DPV), and square wave (SWV) voltammetric methods were performed using a computer-controlled Autolab potentiostat/galvanostat PGSTAT 302 with GPES 4.9 software. A standard cell with one compartment and three electrodes of 10-mL capacity,

ROS

incorporating a GCE (Bioanalytical Systems, West Lafayette, IN, USA; Ø, 3 mm, diameter) was used. A platinum wire and an Ag/AgCl (BAS; 3 M KCl) were used as the auxiliary and reference electrode, respectively. The GCE was polished manually with aqueous slurry of alumina powder (Φ, 0.01 μm) on a damp smooth polishing cloth (BAS velvet polishing pad) before each measurement. All measurements were performed at room temperature. The pH was measured using a pH meter Model 538 (WTW, Weilheim, Germany) using a combined electrode (glass electrode-reference electrode) with an accuracy of ±0.05 pH. Agilent 1100 series HPLC system (Wilmington, DE) was used for the method development and validation studies. This chromatographic system was equipped with a degasser (G1379A), quaternary pump (G1311A), auto injector (G1313), and diode array detector (DAD) (G1315B). The separations were performed at 25 °C using X Bridge® RP-18 (150×4.60 mm ID, 5μm, Waters, Milford, MA, USA) analytical column as the stationary phase. UPLC system consisted of Waters Acquity system equipped with DAD detector, thermostatically controlled column oven, solvent delivery pump, and auto sampler. UPLC separation was performed using a Waters Acquity HSS C18 (50×2.1 mm, 1.8 μm) stationary phase and the column oven adjusted to 25 °C. The control of the system and data processing were performed by Empower 2 software (Waters, Milford, MA, USA). Reagents and solutions The standard stock solutions of AML and ROS were prepared as 408.88 and 481.54 μg/mL respectively for voltammetric studies, and 1,000 μg/mL for both compounds were prepared for chromatographic experiments. The solutions were freshly prepared for all techniques through dissolution directly in methanol and kept in the refrigerator at about 4 °C in the dark. Working solutions for electroanalytical assay were prepared by dilution of the stock solution which contained 20 % methanol as constant amount. 0.1 M H2SO4, 0.5 M H2SO4, 0.2 M phosphate buffer at pH 2.0–8.0, 0.04 M Britton—Robinson buffer at pH 2.00–12.0 and 0.2 M acetate buffer at pH 3.5 and 5.5 were used for the supporting electrolyte for voltammetric measurements. Analytical curves were obtained by the addition of aliquots of the previously prepared AML and ROS standard solutions into the measurement cell containing

Ionics

Analytical procedure

stainless steel analytical column which used as stationary phase. For this reason, an isocratic mobile phase system consisting of acetonitrile: water with the addition of 0.1 % H3PO4 (43:57; v/v) was selected. pH of the buffer solution was adjusted to 3.0 using 1 M NaOH. The injection volume was 10 μL and the mobile phase flow rate was kept constant at 1 mL/min. The detection wavelength was 225 nm. Different conditions, for instance, analytical columns, pH of the buffer, mobile phase composition, flow rate, and column temperature were varied to obtain efficient separation between AML, ROS, and internal standard (IS) for HPLC experiments. After the optimization of HPLC conditions, this method was transferred to work under UPLC conditions with sub 2-μm columns. In order to enhance chromatographic performances in terms of environmentally friendliness and rapidity, liquid chromatographic separations have recently evolved in the development of short columns (packed with sub 2-μm particles) which allowed working at high pressures up to 1, 000 bar. The advantages of these particles’ working at high pressure are examined in terms of analysis time and waste solvents [16]. Separation was performed using a Waters Acquity HSS C18 (50×2.1 mm, 1.8 μm) stationary phase with the same mobile phase and detection conditions mentioned in HPLC investigation. The injection volume was 5 μL and the mobile phase flow rate was kept constant at 0.3 mL/min. The dead time (to) was measured by injecting KBr solution [0.01 % (v/w), in water].

Electroanalytical conditions

Pharmaceutical dosage forms and recovery assay procedure

The operating conditions were as follows: For DPV—pulse amplitude, 50 mV; pulse width, 50 ms; scan rate, 20 mV/s and for SWV—pulse amplitude, 55 mV; frequency, 150 Hz; potential step, 10 mV. Average baseline correction was defined using a “peak width” of 10 mV. For CV technique, range of −0.25 and +1.8 V and scan rate of 100 mV/s were applied. CV, DPV, and SWV were employed to investigate the electrochemical behavior of AML and ROS. However, for the simultaneous determination of AML and ROS, the best responses were obtained with SWV methods. That is why further experiments were realized using SWV methods and it was applied to the assay of AML and ROS from bulk forms and their pharmaceutical dosage form.

To prepare the solutions of the pharmaceutical dosage forms of AML and ROS, 10 tablets were weighed and homogenated as fine powder in a mortar. A suitable amount of this powder was weighed and transferred to 25-mL volumetric flasks and diluted with methanol, and then completed to volume with the same solvent. After that, the working flask was sonicated for about 10 min using ultrasonic bath. The next step of this procedure was to take suitable aliquots of the supernatant, transfer to 10-mL calibrated flasks and complete to volume with 0.5 M H2SO4 and mobile phase for voltammetric and chromatographic experiments, respectively. All prepared solutions were filtered with 0.45-μm syringe filter before chromatographic run. The AML and ROS concentrations in each sample solution were determined using the regression equation of the previously plotted analytical curves obtained with standard solutions. Thus, the analytical parameters were compared and the obtained results showed that the simultaneous determination of both compounds in commercial pharmaceutical dosage form might be achieved without any separation techniques. For the recovery studies, aliquots of the standard solution of AML and ROS were added to the solutions of the pharmaceutical dosage form. The percent recoveries of AML and ROS were calculated using the comparison of the

10.0 mL of the 0.5 M H2SO4. SW voltammograms were obtained after each aliquot addition. For chromatographic analysis, all solutions were diluted with mobile phase before use as described in the “Chromatographic conditions” section. The calibration graph for chromatographic methods were constructed by plotting the ratio of the peak area of the compounds to that of IS against the drug concentration using five replicated analysis. The linearity plots were constructed and the acceptable fit to the linear regression was demonstrated and reported by the necessary parameters. AML, ROS, and their dosage form were kindly provided by Salutis Pharm. Ind. (Istanbul, Turkey). Film coated tablet (Rosucor®) contained 5 mg of both AML and ROS. Etoposide (ETP) is used as an internal standard (IS) for LC assay. It was kindly supplied by Deva Pharm. Ind. (Istanbul, Turkey). All chemicals and solvents were of analytical and chromatographic grade and they were employed without further purification. Chromatography grade, acetonitrile, methanol, and analytical grade aluminum powder, potassium bromide, phosphoric acid (%85), and sodium hydroxide were obtained from Sigma-Aldrich (Munich, Germany). Doubly distilled water with conductivity lower than 0.05 μS cm−1 was used for the preparation of the mobile phase solutions. The mobile phase was filtered through 0.45-μm PTFE membranes using a vacuum pump and degassed before the chromatographic run.

Chromatographic conditions An initial literature search revealed that some LC methods have been developed for the simultaneous determination of these compounds on either C8 or C18 columns, using different mobile phase and temperature conditions. So, attempts were made to develop an isocratically basic method on an advanced new generation fully porous X Bridge® RP18 (150×4.60 mm ID, 5μm, Waters, Milford, MA, USA)

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concentration obtained from spiked samples with the labeled concentration. The results obtained from the recovery experiments showed the reliability and suitability of these three methods. Validation of the methods System suitability test results for both chromatographic methods were evaluated. A system suitability test ensures that the method can generate results of acceptable accuracy and precision. The requirements for system suitability are usually designed after method development, and then, validation steps are completed. The selected criteria will be based on the performance of the method. These parameters include resolution factor, theoretical plate number, selectivity, etc. which are shown in Table 1. Typically, at least two of these acceptable criteria are required for the demonstration of system suitability of the proposed method. As mentioned above, all of the developed methods were validated according to USP and ICH guidelines [14, 15]. The linearity of the proposed procedures was evaluated by analyzing a series of varied concentrations for AML and ROS. The detection limit (LOD) of an individual analytical procedure is the lowest amount of the analyte in a sample which can be detected but not necessarily quantified as an exact value. This value is calculated from the equation of LOD=3.3×s/m. The quantification limit (LOQ) of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. This value is calculated from the equation of LOQ=10×s/m, where (m) is the slope of the calibration curve and (s) is the standard deviation of the blank [17–23]. The ruggedness and precision were checked in same-day and between-days experiments, and the results were

represented as percentage of relative standard deviations (RSD %). The precision and accuracy of analytical methods are described in a quantitative fashion by use of relative errors (Bias %) [17–23]. One example of relative error is the accuracy, which describes the deviation from the expected results. All solutions were kept in the dark in a refrigerator at about 4 °C. The recorded voltammograms and chromatograms of the sample solutions after a period of 1 week from the preparation did not show any appreciable change in assay values.

Results and discussion Electrochemical behavior of AML and ROS The electrochemical behavior of AML and ROS on GCE was investigated by use of CV. Figure 1 shows the cyclic voltammetric profile of the electrochemical oxidation of AML and ROS at 5.67- and 10.0-μg/mL concentration in a 0.5 M H2SO4 solution at the GCE respectively. It is clear that the electrochemical reactions of these compounds at the GCE are irreversible. As can be seen in Fig. 1., AML and ROS exhibit only one well-defined oxidation peak at 0.94 V (· · · · ) and 1.25 V (—), respectively, without the presence of any cathodic peak on the reverse scan. Figure 1 (− − −) also shows both AML and ROS behaviors in 0.5 M H2SO4 solution on the GCE. The influence of pH on the oxidation peak of AML and ROS was examined using GCE with CV method. The linear relationships between the peak potential (Ep) and pH at GCE can be expressed by the equations below. The influence of pH was investigated by cyclic voltammetry.

For AML; E p ðmVÞ ¼ 1059−46:00 pH ðr ¼ 0:991Þ GCE ðpH : 3:5−11:0Þ f or AML with CV For ROS; E p ðmVÞ ¼ 1351 – 16:69 pH ðr ¼ 0:988Þ GCE ðpH : 2:0−6:0Þ f or ROS with CV

It is observed that with increase in pH, the AML peak potential shifts towards less positive values, which indicates the participation of protons in the electrode process. The Ep– pH equation of AML was linear and the slopes were between 46.0 and 49.58 mV using all the techniques, and these values suggested that equal numbers of protons and electrons are involved in the electrode reaction at GCE [24, 25]. The slope results obtained from all working techniques for both compounds are in good agreement with each other. However, the peak potential of ROS decreased in small values while the buffer solution pH was increasing. According

to the Ep–pH equations, all methods are aproximately almost in agreement about redox behavior of AML and ROS. For AML, a higher peak current was obtained in 0.1 M H2SO4 and for ROS, a higher peak current was obtained in 0.1 M H2SO4 (RSD%=1.07, N=3). However, a larger separation of peak potentials between AML and ROS and a better repeatability of analytical signal were obtained in 0.5 M H2SO4 (RSD%=0.61, N=3) without any separation techniques. AML and ROS redox reactions can easily be occured in the acidic media such as H2SO4 than other electrolyte or buffer media and pH values on the glassy carbon electrode

Ionics Table 1 System suitability tests parameters

Parameters

HPLC

Compounds

IS

AML

ROS

IS

AML

ROS

Retention time

2.231

3.246

4.449

0.403

0.815

1.321

Capacity factor (k)

0.560

1.268

2.109

1.166

3.382

6.102

Selectivity factor (a) Resolution factor (Rs)

– –

2.267 7.848

1.662 7.446

– –

2.900 4.960

1.800 5.040

Tailing Theoretical plate numbers

1.195 6,054

1.106 8,187

1.145 9,883

1.990 1,830

1.860 4,140

1.310 10,200

RSD % of retention time

0.167

0.135

0.224

0.769

0.292

0.342

surface due to the easy electron transfer rate. The pH of an aqueous solution can also affect the solubility of the compound. By changing the pH of the solution, the charge state of the compound can be changed. Hence, the response of the AML and ROS can be increased in H2SO4. The most reproducibile, precise, and accurate results were obtained in the lowest pH value (Fig. 2). Also, the sensitivity, the lowest LOD, LOQ values could be obtained in this pH value and this supporting electrolyte. According to the obtained result, 0.5 M H2SO4 was chosen as the optimum value for the simultaneous determination of AML and ROS in electroanalytical study.

Mechanism of electrochemical process Aiming to understand the determination mechanism of AML and ROS on the GCE, the effect of scan rate (from 5 to 500 mVs−1) on the oxidative reaction of 5.67 μg/mL AML and 10.0 μg/mL ROS solution were studied using CV. The cyclic voltammograms revealed that peak currents increase and peak potential shifts as the scan rate increases for the two compounds, a typical characteristic of irreversible electrochemical reactions [26]. In this study, the anodic peak was shifted at 47 and 46 mV per tenfold change in scan rate for AML and ROS,

i /A

9.47u 6.97u

respectively. The linear responses for AML and ROS were observed with the square root of the scan rate in 0.5 M H2SO4 solution as follows: I p ðμAÞ ¼ 0:124υ1=2 ðmV=sÞ−0:421 ðr ¼ 0:982Þ f or AML I p ðμAÞ ¼ 0:122υ1=2 ðmV=sÞ−0:418ðr ¼ 0:976Þ f or ROS

A plot of the logarithm of the peak current versus the logarithm of the scan rate for AML and ROS gave a straight line with a slope of 0.823 and 0.741, respectively. If this curve is linear, diffusion or adsorption process can be expected due to the slope value. If the slope is nearly 0.5, diffusion process can be expected. If the slope is nearly 1, the adsorption process can be occurred. But the intermediate value of the slope is observed, suggesting a mixed diffusion–adsorption peak [27].
 log
I p  ¼ 0:823 log υ–1:830ðr ¼ 0:999Þ f or AML log I p ¼ 0:741 log υ–1:713ðr ¼ 0:992Þ f or ROS Although adsorption controlled process seemed according to the log Ip and log υ relation, there is no significant increase in current with stripping voltammetry. Hence, square wave voltammetry without stripping parameters was used after its optimized conditions.

3.07u 2.57u 2.07u 1.57u 1.07u 0.57u 0.07u -0.43u -0.93u -1.43u 0.53

0.78

1.03

4.47u i/ A

Fig. 1 Cyclic voltammograms (100 mVs−1) obtained with GCE for: dark solid line, 5.67 μg/mL AML; light solid line, 10.0 μg/ mL ROS; and dashed line, 5.67 μg/mL AML and 10.0 μg/ mL ROS in 0.5 M H2SO4 solution

UPLC

1.28

1.53

E/V

1.97u -0.53u -3.03u 0.39

0.64

0.89 E/V

1.14

1.39

Ionics

The relationship between Ep and log υ can be expressed by the following equation:


 E p ðV Þ ¼ 0:0502 log υ V:s−1 þ 0:9523ðr ¼ 0:996Þ f or 5:67 mg=mL AML in 0:5 M H2 SO4 solution. .
 E p ðV Þ ¼ 0:0508 log υ V:s−1 þ 1:173ðr ¼ 0:999Þ f or 10:0 μg mL ROS in 0:5 M H2 SO4 solution.

For a totally irreversible electrode process, Ep and logv are defined by the following equation [28]:

a 0

Ep ¼ E0 −

b



     2:303RT RT k 0 2:303RT þ log logν αn F αn F αnF

Where E 0 ’ is formal potential, k 0 is the standard heteregenous rate constant, α is the transfer coefficient of the oxidation of AML, ROS, and n is the number of the electron transfer in the rate-determination step. F, R, T symbols have their usual significance. Tafel plot was drawn to obtaine results of cyclic voltammetry at 5 mV/s. The tafel plot slope is equal to the 2.3 RT/n(1−α)F equation. According to this equation, nα was calculated as 0.49 for AML and 0.47 for ROS. If we assume n as 1, α can calculated as 0.49 and 0.48 for AML and ROS, respectively. Then, αnα value can calculated from the below equation; E p –E p=2 ¼ 48=αnα ½28

Fig. 2 Effect of pH on a 5.67 μg/mL AML and b 10.0 μg/mL ROS peak currents at the GCE in (black circle) 0.5 M H2SO4, 0.1 M H2SO4, (black triangle) 0.2 M phosphate buffer pH 2.0; 3.0; 6.0; 7.0; 8.0, (white circle) acetate buffer (1.0 M) pH 3.5; 4.5; 5.5, (white triangle) Britton— Robinson buffer (0.04 M) at pH 2.00–10.0 SWV

where, Ep/2 is the potential corresponding to Ip/2. The values for αnα were found to be 1.09 and 1.04 for AML and ROS, respectively. The numbers of transferred electrons are equal to 2.22 and 2.17 for AML and ROS, respectively. From the slope of the Ep versus logv plot, the number of the electrons (n) were calculated as 2.36 for AML and 2.04 for ROS. Therefore, the oxidation of AML and ROS is found as a twoelectron transfer process. In literature, the oxidation mechanism of AML was believed to occur in the 1,4-dihydropyridine ring (Scheme 1), involving a two-electron two-proton mechanism [29]. However, up to now, there has not been any mechanism investigation about the oxidation of ROS. The value of E0 =0.865 and 1.21 V were obtained from the intercepts of a plot of Ep versus v for AML and ROS,

Ionics

Optimization of chromatographic conditions

14.66u 12.16u i/A

9.66u 7.16u 4.66u 2.16u -0.34u 0.620 0.720 0.820 0.920 1.020 1.120 1.220 1.320 1.420 E/V

Fig. 3 SW voltammograms with dashed line, blank solution; line of white circles, 5.67 μg/mL AML and 10.0 μg/mL ROS under unoptimized conditions; solid line, 5.67 μg/mL AML and 10.0 μg/mL ROS under optimized conditions as f=150 s−1, amplitude=55 mV, step potential=10 mV

respectively. From this, ko was calculated to be 0.84 s−1 for AML and 1.02 s−1 for ROS. Additionally, the number of electrons transferred (n) in the redox process was determined by the following equation [30]:
 Eap αn F ¼ ðlog f Þ2:3RT where α, n, and F have the meanings previously assigned above, f is the frequency parameter and the other symbols have their usual meanings. The slopes obtained from the Eap (anodic peak potential) vs. log f plots were 0.058 and 0.057 for AML and ROS, respectively. Thus, by means of the equation above, the numbers of transferred electrons are equal to 1.92 and 2.09 for AML and ROS, respectively. If α is again assumed as 0.5, n is estimated as equal to 2 by AML and ROS, in agreement with those results obtained at scan rate studies by cyclic voltammetry.

Optimization of SWV parameters Frequency, pulse amplitude, step potential Before recording the analytical curves for the simultaneous determination of AML and ROS using the GCE, the effects of experimental parameters of the SWV on the peak potential and peak current were studied in the following ranges: square-wave frequency (8–200 Hz), pulse amplitude (25–80 mV), and step potential (4– 10 mV). The values of the optimized SWV parameters used for the subsequent simultaneous determination of these analytes were: frequency ( f ), 150 Hz; pulse amplitude, 55 mV; and step potential: 10 mV. According to the optimized conditions of SWV, the responses of AML and ROS increased approximately 19 and 24 times, respectively, with optimized conditions (Fig. 3).

An isocratic HPLC method coupled with diode array detection (DAD) was developed to provide a suitable procedure for the routine quality control analysis of binary mixture of AML and ROS. The development of a method in liquid chromatography is the key point for analysis. To reach this goal, upmost important parameter is the achievement of sufficient resolution of the pharmaceutical target compounds from all other excipients and matrix effects. On the other hand, the developed method should be completed within an applicable and short analysis time with acceptable efficiency. For this reason, there are some analytical conditions which have been formed out in this step such as stationary phase type with the dimensions and particle sizes, composition of the mobile phase, column oven temperature, pH, and type of the buffer. For the optimization of the stationary phase, varied reversed columns such as Waters X-Bridge RP-18 (150 × 4.6 mm ID ×5μm), Waters X-Select C-18 (250 ×4.6 mm ID×5μm) and Waters X-Terra RP-18 (250×4.6 mm ID× 5μm) were tested. The best separation with the symmetrical and sharp peak shapes for these three compounds, including AML, ROS, and IS with the shortest retention time was achieved using X Bridge® RP-18 (150×4.60 mm ID, 5μm) column. Another step of optimization is to find the best organic solvent and arrange its composition with an aqueous buffer phase. For this reason, methanol and acetonitrile were evaluated using different percentages of buffer solutions. Firstly, methanol was chosen because of its economical advantage and toxicological hazard; in this trial, analysis time got longer with broader peak shapes and poor resolution. For the simultaneous determination of AML and ROS with the constant amount of IS, acetonitrile was choosen as an organic solvent because it showed efficient separation and resolution with a short analysis time. Different acetonitrile ratios, such as 35 to 50, were tested and 43 % (v/v) acetonitrile was approved for all studies. No interferences with other compounds that originated from excipients were observed even with this percentage. Different acidic and basic additives were added for the preparation of buffer. After the addition of 0.1 % H3PO4 and pH was adjusted to 3.0 with NaOH, peak shapes of the target compounds improved. In order to arrange the total separation time, flow rate was altered between 0.75 and 1.25 mL/min. The best resolution was achieved at 1.0 mL/min. The temperature of the column oven and detection wavelength were adjusted to 25 °C and to 225 nm, respectively, for all compounds. Final optimized conditions were as follows: mobile phase composition consisting of a mixture of acetonitrile/water (43:57; v/v), containing 0.1 % H 3 PO 4, (pH 3.0) at the

Ionics

i/A

Fig. 4 SW voltammograms (a) obtained for the oxidation of AML and ROS in 0.5 M H2SO4 solution, using GCE. The concentrations of AML and ROS were changed simultaneously as, blank (short-dashed line), 0.285 μg/mL AML and 0.500 μg/ mL ROS (solid line), 0.567 μg/ mL AML and 1.00 μg/mL ROS (wide-dashed line), 2.85 μg/mL AML and 5.00 μg/mL ROS (dotted line). The concentrations of HPLC (b) and UPLC (c) chromatograms; IS (5 μg/mL), AML (5 μg/mL), and ROS (5 μg/ mL), respectively

8.38u 7.38u 6.38u 5.38u 4.38u 3.38u 2.38u 1.38u 0.38u -0.62u 0.745

a

0.845

0.945

1.045

1.145

1.245

1.345

1.445

E/V

b IS ROS

Detector Response

AML

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

c

IS AML

0.50

ROS

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

Time (min) stabilized room temperature (25 °C). This mobile phase composition was found to be optimal for symmetrical peaks as well as to achieve minimal background noise. Table 2

Regression data of the calibration lines for quantitative determination of AML and ROS by all techniques HPLC

Linearity range (μg/mL) Slope Intercept Correlation coefficient SE of slope SE of intercept Limit of detection (μg/mL) Limit of quantification (μg/mL) Within-day precisiona (RSD%) Between-day precisiona (RSD%) a

For better precision and accuracy, constant amount of internal standard (5 μg/mL) was added to all prepared solutions. In this work, Etoposide (ETP) was selected as the internal

UPLC

SWV

AML

ROS

AML

ROS

AML

ROS

0.5–100 0.113 −0.003 0.999 6.02×10−3 2.62×10−2 0.027 0.082 0.204 0.393

0.5–100 0.145 −0.007 0.999 1.11×10−3 4.84×10−2 0.042 0.128 0.184 0.790

0.5–100 0.149 −0.029 0.999 3.42×10−4 1.49×10−2 0.011 0.016 0.350 0.722

0.5–100 0.156 0.007 0.999 1.67×10−3 2.04×10−2 0.034 0.049 0.520 0.580

0.006–2.85 19.2×105 −0.123 0.998 5.78×104 12.1×10−2 0.001 0.004 1.87 2.00

0.01–5.00 15.1×105 0.045 0.998 4.18×104 8.75×10−2 0.003 0.009 1.92 1.95

Each value is the mean of five experiments

0.22 0.58 100.21 101.40 0.99 2.29 1 0.07 100.31 100.72 2.27 0.048 99.99 100.58 0.07 0.04 5.14 5.03 10.03 24.97

5.04 5.07 5 5 10 25

0.30 0.24 100.34 100.73 10.03 0.38 0.42 99.89 101.42 25.00

0.94 0.63 100.44 101.12 0.87 0.76 99.28 100.62 0.18 0.72 100.34 99.75 1.01 1.09 1.03 1 1 1 0.057 0.567 1.13

0.055 0.563 1.14

0.66 0.53 100.87 99.87 4.03 0.569 4 0.567

100.23 100.41 100.57 100.44 100.45 100.65 100.91 100.20 100.32 100.41 0.02 0.20 0.14 0.06 0.05 0.06 0.08 0.552 0.26 0.04 10.02 25.10 5.029 5.02 5.02 5.03 5.05 0.50 1.00 5.02 99.12 102.14 100.57 99.62 101.20 101.24 100.79 101.60 100.40 100.68 9.99 25.53 5.03 4.98 5.06 5.06 5.04 0.51 1.00 5.03

A 5.01 5.07 5.03

0.50 0.28 0.99 0.45 0.42 10 25 5 5 5 5 5 0.5 1 5

0.77 0.36 0.86 0.13 0.21

R 99.82 101.65 99.24 A 101.23 99.26 100.44 R 0.83 0.95 0.99 A 0.90 0.77 0.99 R 0.11 1.02 2.05 A 0.563 0.565 0.567 R 0.1 1 2 A 0.567 0.567 0.567 R 99.22 98.98 100.49 A 0.25 0.31 0.26 R 0.50 0.99 5.03 A 5.02 5.03 5.05 R 101.50 101.00 99.44 A 100.22 101.35 100.58 R 0.69 0.42 0.55 A 0.25 0.08 0.36 R 0.5 1 5 A 5 5 5

R 0.51 1.01 4.97

RSD %

Recovery %

Found* (μg/mL)

RSD %

R 0.78 0.51 0.32

A 100.46 100.52 100.92

RSD % Found* (μg/mL) Recovery %

Compounds (μg/mL)

Voltammetry UPLC HPLC

Compounds (μg/mL) Found* (μg/mL)

When using electroanalytical techniques; SWV and DPV established a good separation, which clearly allows the simultaneous determination of AML and ROS. Hovewer, SWV gave more sensitive responses than DPV for these

Assay results and mean recovery studies of AML (A) and ROS (R) in laboratory made mixtures

Simultaneous determination of AML and ROS

Table 3

standard. However, the peak shape of ETP was very sharp and this compound did not increase the analysis time when compared to other potential internal standards. Using the optimized operating conditions, the retention times were obtained as 2.23 min for IS, 3.25 min for AML and 4.45 for ROS, being extremely stable among injections. After the optimization of HPLC parameters, this method was transferred to develop a new UPLC method. The method transfer process decreased the total analysis time and reduced the suffered solvent cost. The proposed UPLC method would serve as a versatile analytical tool suitable for the analysis of this mixture and would be of interest for quality control and clinical monitoring laboratories. Since most pharmaceutical manufacturers try to reduce the research and development costs and time, faster and higher UPLC separation can decrease the time for method development in research and development laboratories, too. As an advantage of this technique, analysts achieved a dramatic decrease in solvent consumption with the decrease in column conditioning time. For this reason, it can be said that UPLC is a more environmentally friendly technique [31]. UPLC experiments were performed using the same mobile phase composition as mentioned above with the 225-nm detection wavelength for a better comparison with HPLC method. Several stationary phases such as Waters Acquity HSS C18 (50×2.1 mm, 1.8 μm), Waters BEH C18 (50×2.1 mm, 1.7 μm) and Waters BEH C18 (100×2.1 mm, 1.7 μm) were also tested for UPLC system. However, before selecting the composition of the mobile phase, varied acetonitrile, buffer mixtures have also been tested using Waters Acquity HSS C18 (50×2.1 mm, 1.8 μm) stainless steel column because of its separation capacity. Different flow rates (0.1– 0.75 mL/min) were evaluated for the efficient separation with the injection volume of 5 μL. Flow rate of 0.3 mL/min was selected as optimum for further analysis after several preliminary investigatory chromatographic runs. At the end of these investigations; when 0.1 mL/min was used as flow rate, total analysis time (3.50 min) was shorter than the HPLC run time with the broad peaks and if 0.75 mL/min was applied for this system, the analysis was completed in 0.70 min with sharp peaks without efficient resolution. By using optimized conditions, retention times for IS, AML and ROS were 0.40, 0.82 and 1.32 min, respectively. Under the described chromatographic conditions, peaks were well separated and free from tailing.

Recovery %

Ionics

Ionics

constant amount of IS (5 μg/mL). Later, ROS was fixed at 5 μg/mL and AML was changed within the same concentration range. Electroanalytical interference affects of these two compounds on each other were also investigated in the 0.5 M H2SO4 by SWV (Fig. 5). The determination of AML in the concentration range between 0.006 and 2.27 μg/mL was accomplished in solutions containing ROS at the fixed concentration of 1.00 μg/mL. On the other hand, the determination of ROS in the concentration range of 0.01 to 4.00 μg/mL was accomplished in solutions containing AML at the fixed concentration of 0.567 μg/mL. The tolerance limits were taken as the maximum concentration of the other compound, which caused a relative error of lower than 2 % in the determination of AML or ROS. The repeatability of the magnitude of peak areas, currents and potentials were determined by successive measurements (n=5) of AML and ROS at different concentrations. The reproducibility of magnitude of these values were evaluated by measuring the peak currents and areas for similar fresh solutions over a period of 5 days. As can

compounds. Hence, SWV conditions were optimized and determination procedure was carried out with this method. After this optimization, AML and ROS were determined by simultaneously changing their equal concentrations (Fig. 4a). For chromatographic separations, Fig. 4b, c illustrated that the separation of AML, ROS and constant amount of IS using HPLC and UPLC methods, respectively. Table 2 also presents the analytical characteristics and calibration data obtained for simultaneous determination of AML and ROS by all methods. In order to show the accuracy, validity and applicability of the proposed methods, recovery tests have been performed by analyzing laboratory-made mixtures of AML and ROS presented in five different ratios (Table 3). The interference of each analyte for the simultaneous determination of its pairs was performed by increasing the concentration of one compound linearly in the presence of unchanging concentrations of the other compound for each three methods. Chromatographic analysis was performed with 5 μg/mL fixed concentration of AML and varied concentrations of ROS (0.5–25 μg/mL) using the Fig. 5 SWV curves obtained with binary mixtures of a 0.10, 1.00, 2.00, 4.00 μg/mL ROS and constant 0.567 μg/mL AML, b 0.057, 0.567, 1.13, 2.27 μg/mL AML and constant 1.00 μg/mL ROS, in 0.5 M H2SO4 solutions. HPLC c and UPLC d chromatograms obtained from Rosucor ® tablets with the concentrations of IS (5 μg/mL), AML (12 μg/mL), and ROS (6 μg/mL), respectively

5.06u

a

4.06u

i/A

i/A

3.06u 2.06u 1.06u 0.06u -0.94u 0.809

0.909

1.009

1.109

1.209

1.309

1.409

8.84u 7.84u 6.84u 5.84u 4.84u 3.84u 2.84u 1.84u 0.84u -0.16u 0.769

b

0.869

0.969

1.069

1.169

E/V

E/V

c AML

IS

Detector Response

ROS

0

0.5

d

1

1.5

2

2.5

3

3.5

4

4.5

AML

IS ROS

0.50

1.00

1.50

2.00

2.50

3.00

Time (min)

3.50

4.00

4.50

1.269

1.369

Ionics Table 4 Assay results and mean recovery studies of AML and ROS in pharmaceutical dosage forms HPLC

UPLC

Voltammetry

AML ROS

AML ROS

AML

ROS

Labeled claim (mg)

5.00

5.00

5.00

5.00

5.00

Amount found (mg)a

5.01 0.34

4.98 0.22

5.02 0.08

5.03 0.05

5.06 0.81

5.00

5.04 1.24 RSD (%)a Bias (%) −0.20 0.40 −0.40 −0.60 −1.20 −0.80 ttheo: 2.31 0.19 0.40 0.23 0.16 tvalue Ftheo: 6.39 0.96 0.11 0.13 0.17 Fvalue Added (mg) 5.00 5.00 5.00 5.00 5.00 5.00 4.96 5.03 4.99 5.06 5.09 5.01 Found (mg)a Recovery (%) 99.23 100.64 99.80 101.20 101.80 100.30 0.14 0.14 0.06 0.13 1.39 0.73 RSD% of recoverya Bias (%) 0.77 −0.64 0.20 −1.20 −1.80 −0.30

a

Each value is the mean of five experiments

be seen in Table 2, good relative standard deviation (RSD %) values were obtained. The mean percentage recoveries obtained after five repeated experiments were between 99.89 and 101.60, between 99.99 and 100.92 for AML, and between 99.12 and 101.50 and between 98.98 and 100.72 for ROS using HPLC and UPLC, respectively, with low level of RSD values (Table 3). Related to these results, when working on synthetic mixture, results encourage the use of the proposed methods described for the simultaneous assay of AML and ROS in commercial tablet dosage forms.

Analysis of pharmaceutical dosage form Rosucor ® tablet was analyzed to determine these substances in order to evaluate the validity of the developed methods. Each Rosucor® tablet contains 5 mg of AML and ROS with ingredients such as quinoline yellow aluminum lactate, lactose monohydrate, titanium dioxide, lecithin, black and yellow ferric oxides. Table 5

Comparison of our method and previous published methods

Compound

Linear range (μg/mL)

LOD (μg/mL)

Reference

AML AML ROS ROS ROS Simultaneous determination of AML and ROS

0.57–39.7 13.8–19.6 0.1–24.1 0.20–10.0 0.27–26.5 0.006–2.85 0.01–5.00

0.34 – 0.013 0.07 0.034 0.001– 0.003

[32] [33] [34] [35] [36] Present study

Adequate amount of Rosucor® was evaluated by quantifying AML and ROS in pharmaceutical dosage form as mentioned in the “Validation of the methods” section. The utility of all of the proposed methods was verified by means of replicate estimations of pharmaceutical preparations and the results obtained were evaluated statistically. Table 4 shows the results for pharmaceutical preparations. Table 4 also compares the results of the analysis of AML and ROS between voltammetric and chromatographic methods statistically with the 95 % confidence level using the aid of student t and F tests. The amounts of AML and ROS were found fairly close to the target amounts for all related techniques. The F and t tests were carried out on the data and statistically examined the validity of the obtained results by voltammetric and chromatographic methods. According to the values of these statistical tests, results were less than the theoretical values in both tests at the 95 % confidence level. This indicates that there are no significant differences between the performances of the voltammetric and chromatographic methods with regards to accuracy and precision. The accuracy of the proposed methods was determined by its recovery during standard addition experiments which are explained in the “Validation of the methods” section. The recovery of the procedure was carried out by spiking the already analyzed samples of tablet with the known amounts of standard solutions of AML and ROS. The results of the recovery analysis for the proposed techniques are tabulated in Table 4. It is concluded that the proposed methods are sufficiently accurate and precise in order to be applied to pharmaceutical dosage form of both compounds. High percentage recovery data also shows that the proposed methods are free from the interferences of the excipients used in the formulations. For electroanalytical technique, another assay was used to evaluate the interferences of foreign species on the determination of AML and ROS. Possible interferents such as ascorbic acid (17.6 μg/mL), dopamine (18.9 μg/mL), glucose (180 μg/mL), and sodium chloride (58.4 μg/mL) were individually added into the standard solution containing 5.67 μg/mL AML and 10.0 μg/mL ROS. K+, Ca2+, Mg2+, Cl─, SO42─ ions had no effect on signals at a concentration of about 100-fold of AML and ROS.

Conclusion In this work, both voltammetric and UPLC techniques were applied for the simultaneous determination of AML and ROS from their pharmaceutical dosage forms for the first time. Electrochemical behavior of AML and ROS was also investigated and the electrochemical process was found to be irreversible and controlled by diffusion–adsorption. By using

Ionics

SWV technique, simultaneous determination of both compounds was achieved. Under optimized conditions, current responses of both compounds significantly increased. The repeatability and reproducibility results of the voltammetric responses are in good agreement with the validation requirements (RSD< 2 %). Recovery experiments also showed that the proposed method was not affected from the matrix in the pharmaceutical dosage form. The result of AML and ROS was compared to published papers (Table 5). According to the results, the most sensitive reasponses were obtained at this study. From the analytical point of view, the developed isocratic HPLC method has advantages when compared to published papers. First of all, in the present study, three compounds including AML, ROS, and IS were well separated. The solvent consumption of the present work is less than those in the already published methods [12, 13]. Furthermore, the present work was fully validated according to the ICH guidelines. Banerjee et al. used 10-cm analytical column for the separation of two compounds using gradient conditions and the separation time was found to be very close to that of the present study; both of them were completed in 5 min [13]. Tajane et al. completed their analysis in 6 min for two compounds, too [12]. The resolution factor for the present study was higher than those for the published ones. From the sensitivity point of view, the proposed methods were found to be more sensitive than the reported ones. In this study, the LOD values were reported as 0.11 and 0.06 μg/mL for ROS and AML, respectively [12]. In our proposed method, LOD values were found as 0.042 and 0.027 μg/mL in HPLC method, and 0.034 and 0.011 μg/mL in UPLC method for ROS and AML, respectively. Also, the proposed UPLC method is newly developed as an environmentally friendly method. The UPLC method is found to be capable of giving faster retention times with high resolution values using less amount of sample when compared to other methods. It reduced the solvent consumption by 11-fold and the analysis time decreased three times than that of isocratic mode HPLC. All proposed methods are accurate, precise, reproducible, specific, sensitive, and robust. They have been found to be better than previously reported methods because of their wide range of linearity, use of an economical, readily available, and greener solutions and lack of extraction procedures. Both voltammetric and chromatographic methods involve a sensitive, simple, rapid, cost-effective process for the simultaneous quantification of AML and ROS in pharmaceutical dosage form without the necessity of sample pretreatment, and timeconsuming evaporation or extraction steps prior to analysis. All of the proposed methods are suitable for quality control laboratory, where economy and time are essential. High percentage recoveries showed that the proposed methods are free from interferences of the commonly used excipient and additives in pharmaceutical dosage forms.

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34. 35. 36.

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