ЖУРНАЛ АНАЛИТИЧЕСКОЙ ХИМИИ, 2010, том 65, № 11, с. 1190-1195
ОРИГИНАЛЬНЫЕ СТАТЬИ
УДК 543
RAPID AND SENSITIVE RP-LC METHOD WITH AMPEROMETRIC DETECTION FOR PHARMACOKINETIC ASSESSMENT OF PROPAFENONE IN HUMAN SERUM OF HEALTHY VOLUNTEERS
© 2010 г. Aleksandra Chmielewska, Lucyna Konieczna, Alina Plenis, Tomasz Baczek, Henryk Lamparczyk
Department of Pharmaceutical Chemistry, Medical University of Gdansk Hallera 107, 80-416 Gdansk, Poland Received 19.11.2009; in final form 05.05.2010
A rapid and sensitive liquid chromatography method with amperometric detection has been developed for the determination of propafenone in serum. Sample preparation was based on single extraction with dichlo-romethane using bupivacaine hydrochloride as internal standard. The compounds were separated on C-18 re-versed-phase analytical column with the mobile phase composed of methanol—acetonitrile — 10mM K2HPO4 (45 : 25 : 30, v/v/v). Analytes were detected electrochemically with the use of amperometric detector. The quantification limit for propafenone in serum was 10 ng/mL. Linearity of the method was confirmed in the range of 10—500 ng/mL with correlation coefficient of 0.9998. Inter-day relative standard deviations (RSD) ranged from 0.27 to 11.9% and intra-day RSD equalled from 1.1 to 9.7%. The newly developed method was applied to the monitoring of the drug in blood levels with18 healthy volunteers taking tablet with pro-pafenone.
Keywords: amperometric detection, propafenone.
Propafenone (2-(2-hydroxy-3-propylamino-propoxy)-3-phenylpropiophenone hydrochloride) is a class Ic anti-arrhythmic drug with weak b-blocking action. Its weak calcium-blocking action has also been described [1—4]. Propafenone contains a chiral centre and is available only as a racemate in therapeutic formulations.
In recent literature, a number liquid chromatography (LC) methods have been developed for the determination ofpropafenone in body fluids. However, these methods employ time-consuming sample preparation, which complicates routine analysis. An analytical method for the determination of propafenone was also developed in author's laboratory. This method involved one-step sample preparation for analyzing serum and used liquid-liquid extraction with dichloromethane. Some of chromatographic methods including gas chromatography [5, 6] and gas chromatography-mass spectrometry [7] have been described for determination of propafenone. Furthermore, micellar electrokinetic capillary chromato-graphic separation of propafenone in pharmaceutical formulations has been also reported with modified borate buffer containing sodium dodecyl sulphate [8]. Finally, enantioselective capillary electrophoresis was applied in chiral determination ofpropafenone and its main metabolites in biological samples. Limit of quantitation for all compounds equaled 25 ng/mL [9]. The LC methods were usually performed using UV detection because propafenone possesses UV absorption and can be determined directly at 209—315 nm. However, the proposed
methods were characterized by quantitation limits of20— 100 ng/mL in human plasma or serum, what might be not sensitive enough [10—13]. Only one LC-UV method has been described for the determination of propafenone and its metabolites in human biological fluids with particularly low volume of initial sample of 250 ^L, where the low limit ofquantitation 10 ng/mL was obtained [14]. However, it was achieved for the loop volume ofas high as 200 ^L. Propafenone and 5-hydroxypropafenone were determined in plasma after derivatization with dansylhy-drazine applying LC method with fluorescence detection using a normal phase [15]. A recently developed RP-LC method for estimation of propafenone without derivatization also uses fluorescence detector [16].
Separately described LC-MS methods reported about significantly lower quantitation limit [17], but propafenone was always isolated from biological matrix using complicated and expensive solid-phase extraction. The other LC-MS method proposed a longer procedure of sample preparation compared to the proposed method in the case of quantitative extraction of propafenone from human plasma, as well as quantita-tion limit was unsatisfactory [18—20]. Hence, it must be emphasized that none of the reported methodologies involved the use of electrochemical detection (ED). LC with electrochemical detection was applied only for determination of 5-hydroxypropafenone in plasma [21]. On the other hand, the electrochemical detection is a very powerful detection method, which
can detect weak currents of less than nA generated from oxidative or reductive reactions of interest. It can be also noted that propafenone possesses an oxidative group and thus can be detected by ED.
The LC methods reported lately [10—13] often were not enough sensitive for determinations of the serum concentration of propafenone after administration of therapeutic doses. Therefore, in order to investigate serum pharmacokinetics of propafenone it was necessary to improve the efficiency of a method for the quantitation ofthat compound. The current method provides a simple sample preparation procedure and electrochemical detection as detection method. Among detectors used in HPLC, electrochemical detectors (including ampero-metric ones) belong to the most specific and sensitive.
Except of the methods based on derivatization [15, 22], sensitive quantification of propafenone can be performed using UV detectors that are operating at wavelengths between 209 and 210 nm [11, 14]. The main problem is that almost all LC mobile phases contain organic liquids, which are non-transparent for short-wavelength UV light. Hence, application of chromatographic methods which are based on liquid phases modified by organic solvents and use UV detection are strongly limited. Therefore, development of analytical methods using electrochemical detection seems to be crucial, because it leads to lower quantita-tion limit in comparison to earlier described methods. Electrochemical detection can be used for determination of electrochemically active compounds, which typically possess aromatic rings in their chemical structure as well contain hydroxyl, methoxyl or amine group. Propafenone is electrochemically active, probably due to the presence of aliphatic amine group as well a phenolic group, which can be oxidized under the influence of the current on glassy carbon electrode. Therefore, the main aim of this work was to develop a direct and more sensitive LC-ED method based on amperometric detection. In this study, the optimal conditions for the analysis by such a LC-ED method with simple sample preparation and high sensitivity for the determination of propafenone in serum were investigated. Finally, the applicability of the developed method was tested with 18 healthy volunteers taking 300 mg of propafenone in tablets.
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EXPERIMENTAL
Reagents. Propafenone hydrochloride was kindly provided by Pharmaceutical Enterprise Polpharma S.A. (Starogard Gdanski, Poland). Rytmonorm comprising tablets 300 mg of propafenone was supplied by Knoll (Ludwigshafen, Germany). Bupivacaine hydrochloride (internal standard) was a product ofSigma-Aldrich (Saint Louis, Missouri, USA). Potassium dihydrogen phosphate was purchased from POCh (Gliwice, Poland). Acetoni-trile, methanol and dichloromethane (HPLC grade) were supplied by Merck (Darmstadt, Germany). Control serum was obtained from healthy volunteers.
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Fig. 1. Hydrodynamic voltammogram of propafenone (P) and bupivacaine (I.S.) in a mixture of methanol—acetoni-trile - 10mM K2HPO4 (45 : 25 : 30, v/v/v) at a flow rate of 1mL/min.
Apparatus. The high-performance liquid chro-matographic system used in the study was purchased from Knauer (Berlin, Germany), and was equipped with a solvent pump (Mini-Star K-500), amperomet-ric detector (Merck, L-3500 A) and a computer system for data acquisition (Eurochrom 2000).
Chromatographic conditions. A LiChrospher-100 C-18 5 ^m, 125 x 4 mm I.D., column from Merck (Darmstadt, Germany) was used. As mobile phase a triple mixture of methanol—acetonitrile—10 mM K2HPO4 (45 : 25 : 30, v/v/v) was applied. Measurements were carried out at a flow-rate of 1 mL/min. Injections were achieved with a syringe loading injector valve Rheodyne 7725i fitted with a 20 mL loop. Detector working conditions were as follows: working electrode: glassy-carbon, reference electrode: silver/silver chloride refill and cell volume 1.5 ^L. The working potential was 1.05 V and the input current 0.1 nA (Fig. 1).
Sample preparation. To 0.5 mL of human serum, 50 ^L of internal standard (I.S.) solution (bupivacaine hydrochloride, 500 ng/mL in methanol) and 4 mL of dichloromethane were added. Resulting mixture was shaken mechanically 10 min. After centrifugation for 10 min at 900 g, the organic layer was transferred to the conical tube and evaporated to dryness in a water bath at 45°C under a stream ofair. Finally, the residue was reconstituted in 200 ^L of mobile phase, vortex-mixed for 1 min, and a 20 ^L aliquot was injected into LC-ED equipment for subsequent analysis. Standard samples were prepared by spiking blank serum with known amounts of propafenone and used for construction of calibration curves.
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Table 1. Intra-day precision and inaccuracy. AVG, SD, RSD and BIAS denote mean value, standard deviation, relative standard deviation and percentage bias, respectively
Substance Amount added, ng/mL
Propafenone 10 20 50 100 200 300 400 500
Internal standard 50 50 50 50 50 50 50 50
Series Back-calculated concentrations, ng/mL
A B C D E F AVG SD RSD, % Bias, % 9.93 11.0 9.93 11.36 12.43 8.86 10.58 1.26 11.90 5.8 18.50 17.78 20.28 18.50 19.93 19.20 19.03 0.95 4.99 -4.85 47.07 47.43 57.43 43.50 49.20 49.93 49.09 4.65 9.47 -1.82 100.28 95.64 96.71 106.36 98.86 98.50 99
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