Trileptal (300 mg)® Tablets Simultaneous Micellar HPLC determination of carbamazepine, oxcarbazepine and eslicarbazepine acetate in pharmaceutical preparations and biological fluids. Application to dissolution testing. F. Belal, F. Ibrahim, Z.A. Sheribah, H. Alaa* Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Mansoura University, Egypt. INTRODUCTION RESULTS and DISCUSSION ABSTRACT Assay results for the determination of the studied drugs in their tablets. Fig. (1): A typical chromatogram for a synthetic mixture under described chromatographic conditions: (S): solvent front; (A): OXZ (8.0 µg mL-1); (B): ESZ (18.0 µg mL-1); (C): CBM (8.0 µg mL-1). One of the most common serious neurological disorders is epilepsy; it seemed to affect more than 50 million persons all over the world [1]. Different forms of epilepsy are treated by a wide variety of pharmacological treatments as to provide a better life for most patients. Several new antiepileptic drugs (AEDs) were introduced into clinical practice, considerably the therapeutic options beyond the traditional first-line medications such as carbamazepine is one of the choices for partial Seizures and hemifacial spasm treatment [2]. New AEDs, which include oxcarbazepine, enhanced the therapeutic effect, decreased epilepsy treatment side effects and used as monotherapy or adjunctive therapy in the treatment of partial seizures [2, 3]. Because epileptic patients are still suffering from seizures, extensive research has been carried but for ideal AED such as, eslicarbazepine acetate [4]. ESZ was approved for use in Europe, the United States and Canada as an adjunctive therapy for partial-onset seizures [5]. This paper emphasizes on CBM, OXZ, and ESZ as they represent three generations of structurally related AEDs. Parameter Proposed method Reference methods [6-8] Tegretol 200® Tablets Mean  S. D 100.54 1.97 99.84  1.56 Student’s t-test 0.52 F-test 1.59 Trileptal (300 mg)® Tablets 100.29 1.81 100.37  0.91 0.07 3.96 Esilzepine® Tablets 100.47 0.77 98.98  0.58 2.38 1.76 A simple, sensitive and selective micellar liquid chromatography method was developed and validated to quantify simultaneously three structurally related antiepileptic drugs; namely carbamazepine (CBM), oxcarbazepine (OXZ) and eslicarbazepine acetate (ESZ). The analysis was achieved using Phenyl column (250 mm x 4.6 mm i.d., 5 µm particle size). A mobile phase consisting of a mixture of 0.3% triethylamine (TEA) and 10% n-butanol in a solution of 0.05 M sodium dodecyl sulphate (SDS) was adjusted to pH 7.0 using 0.02 M orthophosphoric acid was pumped at a flow rate of 1.5 mL min-1 with UV detection 215 nm. The method showed good selectivity and linearity (r2>0.998) over the concentration ranges of 0.1-10 for CBM and OXZ and 0.2-20 µg min-1 for ESZ. The limits of detection are 0.04, 0.06 and 0.14 µg min-1 and limits of quantification are 0.13, 0.21 and 0.47 µg min-1 for CBM, OXZ and ESZ, respectively. The suggested method was successfully applied for the analysis of the studied drugs in their different dosage forms. Statistical evaluation and comparison of the data obtained by both the proposed and reference methods revealed good accuracy and precision of the proposed method. Due to the high sensitivity of the method, it was further applied for the determination of CBM and OXZ in spiked human urine and plasma without prior extraction. The proposed method was successfully applied to real plasma sample of CBM and real urine samples of CBM and OXZ. It is a suitable tool for routine therapeutic drug monitoring and supports other clinical pharmacokinetic-based studies. Furthermore, the applications were extended to include drug dissolution testing, and the results were satisfactory. APPLICATIONS Fig. (2): Representative chromatograms showing CBM, OXZ and ESZ in different dosage forms: (I): For CBM: Tegretol 200® Tablets: CBM (4.0 µg mL-1). (II): For OXZ: Trileptal (300 mg) ® Tablets: OXZ (6.0 µg mL-1). (III): For ESZ: Esilzepine® Tablets: ESZ (12.0 µg mL-1). The values of tabulated t and F tests are 2.78 and 19.00 respectively at p =0.05, respectively. CONCLUSIONS The determination of carbamazepine, oxcarbazepine and eslicarbazepine acetate in pharmaceuticals preparations and to real human urine and plasma based on Micellar HPLC method was applied for the first time. Good recoveries indicated excipients do not interfere with the analysis. The main advantage of the proposed method over other method is that it can be utilized as a suitable tool for routine therapeutic drug monitoring and supports other clinical pharmacokinetic-based studies, as it based on direct analysis of biological samples without the need for prior sample extraction. Another advantage is that the developed method was extended to in-vitro dissolution tests as an important criteria for the pharmaceutical dosage formulations quality because it evaluates the suggested formulation suitability to release the required active substance correctly to the patient. Fig. (3): The proposed method is applied for the determination of OXZ and CBM in spiked human plasma and urine. (I): For plasma 1- Blank plasma under the described chromatographic conditions. 2- (A): CBM (2.0 µg mL-1). 3- (A): OXZ (0.28 µg mL-1). (II): For urine 1- Blank urine under the described chromatographic conditions. 2- (A): CBM (0.8 µg mL-1). 3- (A): OXZ (0.9 µg mL-1). METHODS Chromatographic condition Column: Nucleosil 100-5 Phenyl column (250 mm x 4.6 mm i.d., 5 µm particle size), Macherey-Nagel, USA. Mobile phase: a mixture of 0.3% triethylamine, 10% n-butanol in a solution of 0.05 M sodium dodecyl sulphate adjusted to pH 7.0 using 0.02 M orthophosphoric acid Injection volume: 20µL. Flow rate:1.5 mL min-1 UV delectation: at 215 nm Construction of calibration graphs Accurately measured volumes of each of CBM, OXZ and ESZ standard solutions were transferred separately into separate sets of 10 mL volumetric flasks to give concentrations in the range of 0.1-10.0 µg mL-1 for CBM, and OXZ, respectively, and 0.2-10.0 µg mL-1 for ESZ. The solutions were completed with the mobile phase to the mark and mixed well. 20 µL aliquots were injected (triplicate) and eluted with the mobile phase under the optimum chromatographic conditions. The average peak area was plotted against the final concentrations of the drugs in µg mL-1 to get the calibration graphs. Alternatively, the corresponding regression equations were derived. 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