Flow Injection Analysis With Trimetazidine Dihydrochloride Ion-Selective Electrode

New Plastic membrane ion-selective electrode for trimetazidine dihydrochloride based on trimetazidinium-tetraphenylborate was prepared. The electrode exhibited mean calibration graph slope of 29.5 mV per decade of (TrimCl2) concentration at 25°C. The electrode can be used within the concentration range 3.2×10-5-10-2 M (TrimCl2) at pH ranges of 1.5-3.8 and 4.5-7.5. The standard electrode potentials were determined at different temperatures and used to calculate the isothermal coefficient of the electrode, which was (0.00088 V°C-1). The electrode showed a very good selectivity for (TrimCl2) with respect to a number of inorganic cations, sugars and amino acids. The electrode was applied to the potentiometric determination of the trimetazidine ion and its pharmaceutical preparation under batch and flow injection conditions. Graphite coated wire was prepared and characterized as sensor for the drug under investigation. Also, trimetazidine was determined by conductimetric titrations.

Trimetazidine dihydrochloride has been determined using a limited number of techniques including high performance thin layer chromatography [I], reversed phase liquid chromatography [2], liquid chromatography-mass spectrometry [3], voltammetry 141, spectrophotometry [5,6] and gas chromatography-mass spectrometry [7], Although there is an increased demand for chemical surveillance in different fields including, pharmaceutical and industries which has created the need for highly sensitive, selective, precise and inexpensive techniques; no ion-selective electrode (ISE) has been constructed for the determination of Trimetazidine dihydrochloride. In this study, plastic membrane electrode for Trim cation was constructed based on the incorporation of trimetazidinium tetraphenylborate (Trim-TPB) ion associate in poly (vinyl chloride) (PVC) membrane plasticized with dibutylphethalate (DBP). The electrode was fully characterized under batch conditions according to IUPAC recommendations [8] and then used to determine the drug both in batch and with a flow injection technique (FIA) in which calibration standards and samples are allowed to flow over the membrane surface of the sensing electrode coupled to a reference electrode. The FIA technique has been used in this work for assessing the possibility of using the method for measuring Trim cation. Based on the success of the conventional type Trim-TPB electrode, a fine graphite coated electrode for trimetazidine has also been prepared and investigated.

Reagents
All reagents used for the preparation of solutions were of analytical-reagent grade. Doubly distilled water was used for preparing solutions and as a flow stream in FIA measurements. The carrier and reagent solutions were degassed by means of vacuum suction. All sample solutions used for injections were freshly prepared prior to measurements. Pure grade trimetazidine dihydrochloride and its pharmaceutical preparation (Vastarel, 20 mg per tablet) were provided by the Servier Egypt Industries Limited, 6th of October City, Giza, Egypt. Sodium tetraphenylborate (NaTPB), and diobutyl phthalate (DBP) were obtained from Fluka. Poly (vinyl chloride) (PVC) of high molecular weight and tetrahydrofuran (THF) were obtained from Aldrich.

Apparatus
Potentiometric measurements were carried out with an LPH 230T-pH meter (Tacussel Electronique) (France). A Techne circulator thermostat, model C-100 (Cambridge Limited) (England), was used to control the temperature of the test solution. The electrochemical system was of a sequence: AgIAgCllfilling solutionlmembraneltest solution//KCI salt bridgel1SCE.
The flow injection set-up is composed of a four channel peristaltic pump (ISM 827) (Ismatec, Zurich, Switzerland) and a Model 5020 injection valve with exchangeable sample loop from Rheodyne (Cotati, CA, USA). The electrode was connected to a WTW pMX 2000 microprcessor pHlion meter and interfaced to a Model BD 11 1 strip chart recorder from Kipp and Zonn (Deflt, The Netherlands). A wall jet cell, providing a low dead volume, fast response, good wash characteristics, ease of construction and compatibility with electrodes of various shapes and sizes, was used in flow measurements, where a Perspex cup with axially positioned inlet polypropylene tubing was mounted at the sensing surface of the electrode body.
The optimized distance between the nozzle and the sensing surface of the electrode was 5 mm; this provides the minimum thickness of the diffusion layer and consequently a fast response [9]. The ISE with flow cup, reference electrode (SCE) and the outlet tube were placed in a beaker, where the level of the solution was kept I cm above the electrode surface.

Preparation of the electrode
The electrode was constructed as previously described [ I 01. The membrane composition was studied by varying the percentages (w/w) of the ion exchanger, PVC and DBP until optimum composition that exhibits the Nernstian behavior and the best performance characteristics is reached. The membrane was prepared by dissolving the required amounts of PVC, ion-associate and the plasticizer (DBP) in a 6.0 cm diameter Petri-dish containing 8.0 ml of tetrahydrofuran (THF) (the total weight of the ingredients was 350 mg). The dissolved components being well mixed to ensure homogeneity of the membrane. The Petri dish was then covered with a filter paper and the solvent was left to evaporate in air. The prepared membrane was cut and glued, using PVC-THF paste, to the polished end of a plastic cap attached to a glass tube. The electrode body was filled with a solution that is rnol I-' with respect to both NaCl and Trim-C12 and preconditioned by soaking in rnol I-' Trim-C12 solution.
For the preparation of the wire coated electrode, a graphite pencil rod, (3 mm in diameter and 12 cm in length) is used. The rod is enclosed in a polyethylene tube of a proper diameter leaving 2.0 cm bared at both ends. One of these terminals was used for connection while the other was dipped in a solution made by dissolving the required amounts of the ion-associate, DBP and PVC in about 4 ml of THF contained in a test tube. The plastic layer formed on the surface of the rod is left to dry in air and the process was repeated several times (10-15) till a layer of proper thickness is formed covering the terminal of the rod. The electrode did not need to be soaked in the respective drug solution before use.
General procedure for conductimetric measurements Volumes containing 10-1 00 mg TrimC12 was transferred to a 50 ml volumetric flask and made to the mark with bidistilled water. The contents of the volumetric flask were transferred to a beaker and the conductivity cell was immersed. Then 10-M NaTPB solution was added from a microburette and the conductance was where n is the electrolytic conductivity, vl is the initial volume (50 ml) and v2 is the volume of the added reagent (corr., corrected; ob.s., observed). A graph of corrected conductivity versus the volume of the titrant added was constructed and the end point was determined. One millilitre of M NaTPB is theoretically equivalent to 1.7mg trimCI2.

Solubility Product of the Trim-TPB ion-associate
The determination of the solubility product of the precipitate is important, since its reciprocal value is approximately equal to the equilibrium constant of the precipitation reaction leading to the formation of the ion-associate. If the ionassociate is sparingly soluble (highly hydrophobic), it is expected that its leaching to the aqueous bathing solutions, which is the main determining factor in the life time of the electrode, is very slow. The solubility product of the Trim-TPB precipitate was determined conductimetrically, as previously described [I21 and was found to be 3.75~10-17. This value indicates that the solubility of the ion-associate is very low (2.1x10-~ molll). Consequently, the equilibrium constant of the reaction, TrimC12 + 2Na-TPB = TrimTPB + 2NaCI is 2.67x10I6, which reflects that the reaction is more than 99.9% complete. In the above equilibria, the solubility of the undissociated ion-associate in water (i.e. the intrinsic solubility) was omitted as it only has a negligible contribution to the total solubility

Optimization of the ISE in batch conditions
Composition of the membrane Several membrane compositions were investigated in which the content of the Trim-TPB ion-associate ranged from 5.0 to 15.0% (wlw), the electrode was repeatedly prepared four times. The preparation process was highly reproducible as revealed from the low standard deviation (RSD) values of the slopes obtained employing the prepared membranes (the mean RSD was about 0.86%).
The best performance was obtained using composition 5.0% Trim-TPB, 47.5% PVC and 47.5% DBP, resulting in a slope of 29.52 mV per concentration decade after minimum pre-soak time of 1 h. The usable concentration range was 3.2~10-~-10" mol I-'. The above composition was used to prepare membrane electrode for all further investigations.

Effect of soaking
The continuous soaking of the electrode in mol I-' TrimC12 solution affects negatively its response to the trimetazidinium cation, which may be attributed to leaching of the active ingredients [ion-associate and solvent mediator] to the All graphs start from pTrim = 6 .
bathing solution [13]. It was observed that the slopes of the calibration graphs obtained using the pre-conditioned electrode decreased gradually starting from 29.5 mV per concentration decade reaching about to 21.7 mV per concentration decade within six days, Fig.1. It is noteworthy that the electrodes which have been kept dry in a dark closed vessel and stored in a refrigerator showed nearly constant slope value and the same response properties on daily use extending to about 10 days. Hence it is recommended that unused electrodes be kept dry in a closed vessel in a refrigerator in order to extend its life spans substantially.
For the coated graphite electrode, it has been found that the plastic coat of the ionassociate must be reformed prior to every use, this is accomplished by immersing the bared terminal of the electrode in the solution mixture containing the Trim-TPB  for Trim-TPB electrode. This reveals a fairly good thermal stability of the electrode within the investigated temperature range.

Optimization of FIA response
The flow injection measurements were carried out in a two-line configuration.
The sample was injected into a distilled water stream, which was then merged with another stream of distilled water. In both lines the same tubing size was used, offering the same flow rate. The connector of the two streams was connected to the detector by a 50 cm tube of 0.4 mm internal diameter. Fig.3. shows the configuration of the system used in measurements. The dispersion coefficient was found to be 1.22, i.e., limited dispersion coefficient that aids optimum sensitivity and fast response of the electrode [I 51.

5'
To Waste

Optimization of operational conditions of FIA
Optimization of flow rate and sample volume.
Samples of different volumes (5-340 pl) were injected. In general, the higher the sample volume, the higher is the peak height and the longer is the residence proportional to the residence time of the sample at the active membrane surface. It was found that, as the flow rate increased, the peaks becomes higher and narrower until a flow rate of 25.00 ml min-' was reached, after which the peaks obtained at higher flow rates were nearly the same. A flow rate of 9.7 ml min-' was adopted, providing about 99% of the maximum peak height obtained by higher flow rates, a short time to reach the baseline and less consumption of the carrier.

Optimization of pH
The effect of pH of the test solution on the electrode potential was studied in batch and FIA measurements. In batch measurements, the variation in potential interfere because of the differences in ionic size, mobility and permeability. Also, the smaller the energy of hydration of the cation, the greater is the response of the membrane. The electrode exhibits good tolerance towards amino acids and sugars.
Regarding the selectivity coefficients obtained for the investigated electrode under both batch and FIA conditions, Table 2, it is clear that in most cases, the electrode was selective in both FIA and batch.
Comparing the selectivity coefficients obtained for the investigated electrode under both batch and FIA conditions (  For batch method, the mean recoveries of the amounts taken (10-100 mg) ranging from 97.1-99.7% with RSD = 0.14-1.07% (Table 3).
The results of the determination of trimetazidine in its pure state or pharmaceutical preparation by conductimetric method are given in Table 3. The results showed good recovery of the amounts taken (10-100 mg) ranging from 96.9 to 101.2% with RSD = 0.25-1.4% (Table 3).
Under FIA conditions, a series of solutions of different concentrations was prepared from the tablets and the peak heights were measured at the selected flow rate (9.7 ml min-I), then compared with those obtained from injecting a standard solution of the same concentration prepared from pure trimetazidine dihydrochloride. The mean recoveries for the amounts taken (10-100 mg) ranged from 96.9 to 100.9%

Conclusion
The application of the proposed methods usingTrim-TPB electrode under batch and FIA conditions in addition of conductimetric titration for determination of trimetazidine dihydrochloride in pure Solution and its pharmaceutical preparation (Vastarel, 20 mgltablet) are characterized by high degree of precision and accuracy when compared with the official method (potentiometric titration of 0.01 M TrimCI* with 0.1 M silver nitrate) as revealed by one tailed F critical value, Table 3.
It is clear that the applied methods do not exhibit significant differences in comparison with the official method. The standard additions method and conductimetric titrations take about 15-20 min in each run. In addition, the FIA conditions shortened the time needed for the determination of the drug in pure state or its pharmaceutical preparation. Therefore, FIA is mostly recommended for determination of trimetazidine and its pharmaceutical preparations.