Anal Bioanal Chem (2004) 379 : 308–311 DOI 10.1007/s00216-004-2539-8
O R I G I N A L PA P E R
Ceren Yardımcı · I˙ncilay Süslü · Nuran Özaltın
Determination of piribedil in pharmaceutical formulations by micellar electrokinetic capillary chromatography
Received: 19 November 2003 / Revised: 27 January 2004 / Accepted: 2 February 2004 / Published online: 20 February 2004 © Springer-Verlag 2004
Abstract A fast and simple micellar electrokinetic capillary chromatographic method was developed for the analysis of piribedil in pharmaceutical formulations. The effects of buffer concentration, buffer pH, sodium dodecyl sulphate (SDS) concentration, organic modifier, applied voltage and injection time were investigated. Optimum results were obtained with a 50 mM borate buffer at pH 8.0 containing 50 mM SDS by using a fused silica capillary (50 µm internal diameter, 72 cm effective length). The sample was injected hydrodynamically for 4 s at 50 mbar pressure and the applied voltage was +30 kV. The detection wavelength was set at 205 nm. Diflunisal was used as an internal standard. The analysis was performed at 25 °C and the total run time was 14 min. The method was suitably validated with respect to linearity range, limit of detection and quantification, precision, accuracy, specificity and robustness. The linear calibration range was 5–100 µg mL–1 and the limit of detection was determined as 1 µg mL–1. The method developed was successfully applied to the determination of piribedil in pharmaceutical formulations. The results were compared with a spectrophotometric method reported in the literature and no significant difference was found statistically. Keywords Piribedil · Capillary electrophoresis · Micellar electrokinetic chromatography · Pharmaceuticals
Introduction Piribedil (P, Fig. 1a), 2-(4-piperonyl-1-piperazinyl) pyrimidine, is a non-ergot dopamine agonist and has been tried in the treatment of parkinsonism and in depression. In some countries it is used in the treatment of circulatory disorders
C. Yardımcı · ˙I. Süslü · N. Özaltın (✉) Department of Analytical Chemistry, Faculty of Pharmacy, University of Hacettepe, 06100 Sıhhıye, Ankara, Turkey e-mail:
[email protected]
Fig. 1 Chemical structure of a piribedil (P) and b diflunisal (internal standard, IS)
[1]. Recently, binding and efficacy studies have shown that P presents antagonist actions at the two main adrenergic receptor subtypes present in the central nervous system: α2A and α2B [2]. In recent years, capillary electrophoresis has become an important analytical technique. It offers several advantages, including highly efficient and fast separations, relatively inexpensive and long-lasting capillary columns, small sample size requirements and low reagent consumption. Micellar electrokinetic capillary chromatography (MECC) is one mode of capillary electrophoresis. It was initially conceived for the electrokinetic analysis of neutral compounds but it is also effective for the separation of ionic compounds that have similar electrophoretic mobilities.
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Several analytical methods for the determination of P have been reported, including spectrophotometry [3, 4], high-performance liquid chromatography [5, 6], gas chromatography [7], gas chromatography–mass spectrometry [8], ion-selective electrodes [9] and voltammetry [10]. There is no capillary electrophoresis method for the determination of P. This work describes a new MECC method for the determination of P in pharmaceutical formulations. An optimization study of the method variables, pH and buffer concentration (borate buffer), surfactant concentration (sodium dodecyl sulphate, SDS), organic modifier, applied voltage and injection time, was carried out. The proposed method was also validated according to the official validation guidelines [11]. The aim of this work is to provide an effective and economical alternative for routine quality control studies of P.
Fig. 2 The effect of buffer pH on the migration time (t) of P and the efficiency (N) (20 µg mL–1 P, 50 mM borate buffer, 50 mM sodium dodecyl sulphate, SDS, potential 20 kV, hydrodynamic injection 3 s, detection 205 nm)
Experimental Apparatus All capillary electrophoresis experiments were performed using an Agilent 3D CE (Waldbronn, Germany) system using ChemStation software, equipped with a diode-array UV detector, an automatic sample injector, Peltier temperature controller and 30-kV high-voltage power supply. A fused-silica capillary pf 80.5-cm total length (72 cm to the detector) and 50-µm internal diameter was used. An Agilent model 8453 UV–vis spectrophotometer was used for the comparison spectrophotometric method.
Fig. 3 The effect of buffer concentration on the migration time of P and the efficiency (20 µg mL–1 P, pH 8.0 borate buffer, 50 mM SDS, potential 20 kV, hydrodynamic injection 3 s, detection 205 nm)
Reagents P and its pharmaceutical formulations were kindly provided by Servier Pharm. Ind. (Istanbul, Turkey). All other chemicals were of analytical reagent grade. Milli-Q water was used throughout the study. Stock solutions of 1,000 µg mL–1 P and the internal standard (IS), diflunisal (Fig. 1b), were prepared in methanol and stored in a refrigerator at 4 °C. Working standard solutions were prepared daily by diluting the stock solutions with the selected background electrolyte (50 mM borate buffer at pH 8.0 containing 50 mM SDS). For tablet sample solutions, ten tablets of Trivastal Retard, each one containing 50 mg P, were crushed in a mortar. The powder equivalent to one average tablet was accurately weighed and dissolved in 50 mL methanol with ultrasonication for 10 min. After centrifugation for 10 min, appropriate solutions were prepared by taking suitable aliquots of the clear supernatant and diluting them with the selected background electrolyte. All solutions were filtered through a 0.45-µm syringe filter and degassed in an ultrasonic bath for 5 min before injection into the capillary electrophoresis system. Capillary electrophoresis procedure When a new capillary was used, the capillary was washed for 30 min with 1.0 M NaOH solution, followed by 20 min with deionised water. Before each injection, the capillary was preconditioned with 0.1 M NaOH for 2 min, deionised water for 2 min and background electrolyte for 2 min to maintain proper reproducibility of run-torun injections. The sample injection was carried out under hydrodynamic pressure at 50 mbar for 4 s. A diode-array UV detector was set at 205 nm with a bandwidth of 20 nm. The capillary temperature was kept constant at 25 °C and a voltage of +30 kV was applied.
Fig. 4 The effect of SDS concentration on the migration time of P and the efficiency (20 µg mL–1 P, 50 mM pH 8.0 borate buffer, potential 20 kV, hydrodynamic injection 3 s, detection 205 nm)
Results and discussion Optimization of the electrophoretic conditions The neutral nature of P makes the development of the MECC method necessary for its determination. The effect of buffer pH (Fig. 2), borate buffer concentration (Fig. 3), SDS concentration (Fig. 4), organic modifier, applied voltage and injection time on the determination of P were investigated. Initially, the effect of pH was studied using an initial electrolyte consisting of 50 mM phosphate buffer (pH 7.0– 7.5) and 50 mM borate buffer (pH 8.0–9.0) with 50 mM SDS. In considering the efficiency, migration time and peak symmetry, borate buffer at pH 8.0 was selected as the optimum pH condition of the running buffer (Fig. 2).
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Fig. 5 The electropherograms of a standard solution of 50 µg mL–1 P and b commercial tablet solution of 50 µg mL–1 P in optimum conditions (20 µg mL–1 IS)
The effect of borate buffer concentration on the efficiency was examined by varying the concentration from 25 to 100 mM at fixed pH 8.0. The efficiency has a maximum value at 50 mM, so this concentration was chosen as the optimum concentration of the borate buffer (Fig. 3). The influence of the SDS concentration on the migration times and the efficiency can be observed in Fig. 4. The increment of the SDS concentration causes an increase in the migration time and the efficiency. A SDS concentration of 50 mM was chosen as optimum. In order to investigate the effect of organic modifiers, acetonitrile and methanol were added at various concentrations (5, 10, 15% v/v) to the background electrolyte of 50 mM borate buffer pH 8.0 including 50 mM SDS. The efficiency and the peak shapes did not change, but the migration times of P and IS increased significantly with the addition of organic modifiers, so no organic modifiers were added to the background electrolyte. In order to determine the optimal voltage to be applied, an Ohm’s plot (current versus voltage) was made. There was a linear increase up to 30 kV. At 30 kV, the analysis time was the shortest and the currents were not excessive (21.3 µA), so this voltage was selected as the optimum run voltage. The optimum conditions obtained by this method are 50 mM borate buffer at pH 8.0 containing 50 mM SDS, an applied voltage of 30 kV, a working temperature pf 25 °C, 4-s hydrodynamic injection (50 mbar) and detection at 205 nm. Under these conditions the migration times of P and IS are 13.16±0.03 and 7.65±0.02 min, respectively (Fig. 5a).
Validation Linearity By using the optimum conditions the MECC method was developed for the determination of P. A linear relationship was observed between 5.0 and 100.0 µg mL–1 using the peak area ratio method (n=6). The regression equation was y =0.0676x–0.0138 (r=0.9999), where y is the ratio of the peak areas and x is the concentrations of P (micrograms per millilitre). The detection limit was found as 1 µg mL–1 (signal-to-noise ratio of 3). The limit of quantification was found as 5 µg mL–1 with a relative standard deviation (RSD) of 1.85% (n=6). Accuracy and precision Intra-day accuracy and precision were evaluated by analysis of standard P solutions at levels of 10.0, 50.0 and 80.0 µg mL–1 (n=6 at each level) on the same day. These levels were chosen to demonstrate the accuracy and precision of the method at low, moderate and high concentrations. To assess the inter-day accuracy and precision, analysis of standard P solutions at the same levels was performed on six different days. Accuracy and precision were expressed as bias and RSD, respectively (Table 1). Recovery According to official validation guidelines [10], in cases where it is impossible to obtain samples of all drug product components, it may be acceptable to add known quantities of the analyte to the drug product for determining recovery. For this reason, in order to know whether the excipients in the tablets show any interference with the analy-
311 Table 1 Accuracy and precision data for piribedil (P) obtained by the micellar electrokinetic capillary chromatographic (MECC) method. Mean±standard error (x), relative standard deviation (RSD), accuracy=[(found–added)/ added]×100
Added (µg mL–1)
10.0 50.0 80.0
Intra-day
Inter-day
Found x (µg mL–1)
Precision RSD (%)
Accuracy bias (%)
Found x (µg mL–1)
Precision RSD (%)
Accuracy bias (%)
10.11±0.06 49.89±0.21 79.93±0.09
1.35 1.05 0.29
1.1 –0.22 –0.09
10.2 ±0.09 49.55±0.09 80.01±0.16
2.16 0.42 0.44
2.0 –0.9 0.01
Table 2 The robustness data for P obtained by the MECC method Standard pH 7.9 pH 8.1 Buffer molarity 40 mM Buffer molarity 60 mM Sodium dodecyl sulphate molarity 40 mM Sodium dodecyl sulphate molarity 60 mM Applied voltage 29 kV Applied voltage 29.5 kV Wavelength 203 nm Wavelength 207 nm
Table 3 The results for commercial tablets containing P (labelled amount 50 mg/tablet) P found by the MECC method (mg/tablet)
P found by the spectrophotometric method (mg/tablet)
x=49.59±0.1 SD=0.33 RSD=0.14% Ttabulated=0
