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Spectrophotometric Determination of Levetiracetam by Developing Coloured Complexes with 2-Chlorophenylhydrazine and Anthranilic Acid
Abstract
Two simple, sensitive, reproducible, rapid and economical spectrophotometric methods are described for the determination of levetiracetam (LVC) in bulk and formulations. Both the methods are based on the formation of coloured complexes of levetiracetam with 2- chlorophenylhydrazine (method-A) and anthranilic acid (method-B) in alcoholic medium. Under the optimized conditions the complexes show an absorption maximum at 560 and 485 nm with molar absorptivities of 1.130 × 10 4 and 7.110 × 10 4 mol -1 cm-1 and sandells sensitivities of 0.03546 and 0.03708 per 0.001 absorbance unit for method-A and method-B, respectively. The result of analysis has been validated statistically and by recovery studies. Both methods have been successfully applied for the assay of the drug in pharmaceutical formulations.
... Several methods have been reported for determination of LEV; these methods include spectrophotometric methods [4][5][6][7], chromatographic methods [8][9][10][11][12][13][14][15], electrochemical methods [16][17] and capillary electrophoresis [18]. ...
... Proposed method Reported method [14] Taken ng mL -1 Found ng mL -1 %Recovery %Recovery 5 ...
... The proposed method is applied for determination of LEV in tablets and syrup. The results are shown in Tables (5)(6)(7)(8). The results of the proposed and reference methods are compared in accordance with the Student's t-test and variance ratio F-test. ...
A new, simple and sensitive spectrofluorimetric method is developed for the determination of levetiracetam (LEV). The proposed method is based upon its reaction with 9-Fluorenylmethyl chloroformate (FMOC-Cl) in borate buffer of pH 10 to produce a highly fluorescent derivative at λ emission 308 nm after excitation at 265 nm. All parameters required for the reaction conditions are investigated. Linearity is verified with a range of 5-50 ng mL-1. Validation of the procedure is evaluated according to ICH guidelines and shows good accuracy and reproducibility, with mean percentage recovery 99.89%. The Correlation Coefficient is 0.9998 (n = 6), with limit of detection 1.1 ng mL-1 and limit of quantification 3.3 ng mL-1. The proposed method is applied for analysis of LEV in its different dosage forms (tablets and syrup) with percentage assay (100.4 ± 1.2% RSD and 100.3 ± 0.71% RSD respectively) without interference from common additives. Statistical analysis of the results reveals no significant difference between the data of the proposed method with those of the reported method (t-test = 0.615 and F-test = 8.327). Please cite this article in press as Mohamed. Rizk et al. Spectrofluorimetric determination of Levetiracetam in pure and dosage forms through derivatization with 9-Fluorenylmethyl chloroformate using structural elucidation of the reaction product by LC-MS/MS. Indo American Journal of Pharm Research.2015:5(06).
... The authors have summarised the analytical methods developed for the quantification of Levetiracetam in the present review article. These analytical methods include spectrophotometry [3][4][5][6], HPLC [7][8][9][10][11][12], UPLC [13], UHPLC [14], HPTLC [15], LC-MS/MS Figure 1: Structure of Levetiracetam. [16,17] and UPLC-MS/MS [18] for the estimation of Levetiracetam in pharmaceutical formulations and biological fluids such as human plasma and human serum (Table 1). ...
... LEV is analyzed in the British , European and United States Pharmacopeia (2016) by liquid chromatography. In addition, it has been determined by various reported methods including spectrophotometric (Indupriya, Chandan, Gurupadayya, & Sowjanya, 2011;Muralikrishna, Ramu, Bikshambabu, Venkata, & Rambabu, 2012;Ravisankar, Niharika, Anusha, Himaja, & Basha, 2015;Thanuja, Chandan, Gurupadayya, Prathyusha, & Indupriya, 2014), spectrofluorimetric (El-Yazbi, Wagih, Ibrahim, & Barary, 2016;Rizk, Elshahed, Attia, & Farag, 2015), spectrophotometric and spectrofluorimetric (Ibrahim, El-Yazbi, Wagih, & Barary, 2017), electrochemical (Alonso-Lomillo, Domínguez-Renedo, Matos, & Arcos-Martínez, 2009;Arkan, Shamsipur, Saber, Karimib, & Majnooni, 2014) and HPTLC methods with other antiepileptics (Antonilli, Brusadina, Filipponia, Guglielmib, & Nencini, 2011;Santosh, Kadam, & Karad, 2014;Siddiqui et al., 2014), and HPLC methods either in bulk or in biological fluid samples (Ola'h, Bacso'i, Fekete, & Sharma, 2012;Saikrishna, Bikshapathi, kumar, Sabitha, & Saikumar, 2011;Nagaraju, Indira, & Appaji, 2014;Ramprasad & Sundar, 2014;Hamdan, Alsous, & Masri, 2017;Raju, Ganapathy, Sankar, & Naidu, 2014;Hadad, Abdel Salam, & Abdel Hameed, 2013;Kalaria, Dahmane, Armahizer, McCarthy, & Gopalakrishnan, 2018;Fonseca, Rodrigues, & Alves, 2018), or stability-indicating studies (Prasad, Sagar, & Sudhakar, 2013;Shah, Vidyasagar, & Barot, 2012). Several analytical methods have been reported for the determination of LAC, including spectrophotometry (Rao et al., 2012;Patel, Bhanubhai, Bhumi, Arpan, & Arpit, 2014;Rajeshri, Choksi, Divan, Patel, & Shraddha, 2013;Valarmathi, Banu, Senthamarai, & Dhharshini, 2013), gas chromatography (Nikolaou, Papoutsis, Spiliopoulou, Voudris, & Athanaselis, 2015), HPTLC for bulk drug (Kamdar, Vaghela, & Desai, 2012) and for a stress degradation study (Patel, Suhagia, & Patwari, 2013), and HPLC in bulk and biological fluids (Chakravarthy & Shankar, 2011;Jayasimha, Krishna, Sandeep, & Goud, 2015;Nazma, Yasaswini, Supraja, Vijayalakshmi, & Nalluri, 2015;Sreedevi et al., 2015;Valarmathi, Senthamarai, Akilandeswari, Banu, & Saratha, 2015) or stability-indicating studies (Ramisetti, Kuntamukkala, Lakshetti, & Sripadi, 2014). ...
A simple and highly sensitive Ultra High‐performance Liquid Chromatographic ‐Diode array detection method (UHPLC‐DAD) was developed and validated for the simultaneous estimation of levetiracetam (LEV) and lacosamide (LAC). It was clinically proven that the combination of LEV and LAC exhibits a synergistic effect against refractory seizures in mice, which was the motivation for the analysis of this binary mixture both in bulk and human urine samples. The binary mixture was resolved on a Hypersil BDS C18 analytical column, utilizing a mobile phase of 0.050 mol L‐1 phosphate buffer (pH 5.60), methanol and acetonitrile in the ratio (80:10:10 v/v/v) using catechol as an internal standard (IS). The mobile phase was pumped at a flow rate of 1.2 mL min‐1 with diode array detection (DAD) at 205 nm for both drugs and 270 nm for IS. Calibration curves were linear with correlation coefficient (> 0.9990) over the studied concentration range of 0.1 – 70.0 μg mL‐1 for both drugs. The developed method was found to be reproducible with low relative standard deviation (RSD) values for intra‐ and inter‐day precision (< 2.0 %). Both drugs were determined in bulk, pharmaceutical formulations, and human urine samples without any interference from complex matrices.
... It is official in BP [3] and USP [4]. Various analytical methods were reported for the determination of levetiracetam in pure and pharmaceutical dosage forms including spectrophotometry [2,[5][6][7][8][9], spectrofluorimetry [10], HPLC [11][12][13][14][15], HPTLC [16], capillary electrophoresis [17] and LC-MS [18,19]. ...
A new simple, rapid and sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed by employing Quality by Design (QbD) approach for determination of levetiracetam and pyridoxine HCl in prepared tablets. Levetiracetam and pyridoxine HCl are co-administered because treatment with the antiepileptic levetiracetam leads to a deficiency in pyridoxine HCl. Fractional factorial design (FFD) was applied for screening of the four independent factors: pH of the aqueous part of the mobile phase, flow rate, injection volume and % of the organic modifier. Analysis of variance (ANOVA) confirmed that the four factors were significant. Optimization of the chromatographic conditions was performed using central composite design (CCD). The Analysis was achieved on BDS Hypersil C8 (250 × 4.6 mm, 5 μm) column applying an isocratic mobile phase containing MeOH and 25 mM KH2PO4 buffer pH 3 (38.4:61.6, v/v) at 0.8 mL/min flow rate with UV detection at 214 nm and 5 μL injection volume. The proposed method was validated according to ICH guidelines. Under optimized conditions, linear ranges of 1.56–100 μg/mL and 0.39–100 μg/mL were obtained for levetiracetam and pyridoxine HCl, respectively, with correlation coefficients (R2) of 0.999. The average recoveries ranged from 95.46 to 101.14%. The inter- and intra-day precisions were lower than 1%. The proposed method showed good predictability and robustness.
... Hence quantitative determination of levetiracetam in pharmaceutical formulations thus becomes important, since it's over dosage causes ill effects. In literature [8][9][10] it was found that earlier workers used UV spectroscopy, HPLC, RPHPLC for the determination of levetiracetam. ...
A simple, accurate, precise and easy spectrophotometric method for the determination of levetiracetam in pharmaceutical formulations using molybdenum blue method was developed by the authors. Aqueous solution of the drug sample on reaction with ammonium molybdate solution and hydrazine sulphate solution on heating for 5 minutes at a temperature of 60 0 C gave a clear, stable and intense blue colored molybdenum blue complex. This molybdenum blue showed a λ max at 823nm. Beer's law was found to be obeyed in the range 0.02-0.08mg/ml. correlation coefficient was found to be 0.998. Molar abosrptivity was found to be 240.2 lit/mole/cm. LOD and LOQ was found to be 0.12 mg/ml and 0.37mg/ml respectively. The method is important in view of the fact that the excess dosage causes clinical problems and optimum dosage has to be fixed in the use of the drug.
A comprehensive profile of cefpodoxime proxetil including the nomenclatures, formulae, elemental composition, appearance, uses, and applications. The methods which were developed for the preparation of the drug substance and their respective schemes are outlined. The physical characteristics of the drug including the ionization constant, solubility, X-ray powder diffraction pattern, differential scanning calorimetry, thermal behavior, and spectroscopic studies are included. The methods which were used for the analysis of the drug substance in bulk drug and/or in pharmaceutical formulations includes the compendial, spectrophotometric, electrochemical and the chromatographic methods. The other studies which was carried out on this drug substance are including the drug stability, pharmacokinetics, bioavailability, drug evaluation, comparison and several compiled reviews. Finally, more than two hundred references are listed at the end of this profile.
A comprehensive profile of levetiracetam is presented in this chapter which includes its description, formula, elemental analysis, appearance, uses and applications. Different earlier studies included for example methods of synthesis are described with its typical structural schemes. The profile also listed the drug's physical characteristics indicating its solubility, X-ray powder diffraction pattern, thermal methods of analysis as well as its spectroscopic characteristics. Different methods of analysis which includes compendial method of analysis, as well as reported method of analysis which include spectrophotometry, spectrofluorometry, electrochemical method, chromatographic method, and immunoassay method of analysis. The study was include drug stability, clinical pharmacology, e.g., mechanism of action, pharmacokinetic study. Around 70 references are recorded as a proof of this chapter.
Two highly sensitive, simple and selective spectrophotometric and spectrofluorimetric assays have been investigated for the analysis of ezogabine, levetiracetam and topiramate in their pure and in pharmaceutical dosage forms. The suggested methods depend on the condensation of the primary amino-groups in the three drugs with acetylacetone and formaldehyde according to Hantzsch reaction yielding highly fluorescent yellow colored dihydropyridine derivatives. The reaction products of ezogabine, levetiracetam and topiramate were measured spectrophotometrically at 418, 390 and 380 nm or spectrofluorimetrically at λem/ex of 495/425 nm, 490/415 nm and 488/410 nm, respectively. Various experimental conditions have been carefully studied to maximize the reaction yield. At the optimum reaction conditions, the calibration curves were rectilinear over the concentration ranges of 8–25, 60–180 and 80–200 μg/mL spectrophotometrically and 0.02–0.2, 0.2–1.2 and 0.2–1.5 μg/mL spectrofluorimetrically for ezogabine, levetiracetam and topiramate, respectively with good correlation coefficients. The suggested methods were applied successfully for the analysis of ezogabine, levetiracetam and topiramate in their commercial tablets with high percentage recoveries and negligible interference from various excipients in pharmaceutical dosage forms. The results were statistically analyzed and showed the absence of any significant difference between both developed and published methods. The procedures were validated and evaluated by the ICH guidelines revealing good reproducibility and accuracy. Therefore, the two proposed methods may be considered of high interest for practical and reliable analysis of ezogabine, levetiracetam and topiramate in pharmaceutical dosage forms.
Since December 1999, 3 drugs have been cleared for marketing by the US Food and Drug Administration for the treatment of partial-onset seizures in adults with epilepsy — levetiracetam, oxcarbazepine and zonisamide. All are approved as adjunctive therapy; oxcarbazepine is also approved as monotherapy. Levetiracetam appears to have a novel mechanism of action, while the others block voltagesensitive sodium channels (oxcarbazepine and zonisamide) and T-type calcium channels (zonisamide). Levetiracetam and oxcarbazepine have short serum elimination half-lives and can be started at therapeutic dosages. All 3 drugs exhibit linear pharmacokinetics and have a low propensity for drug-drug interactions. There is extensive worldwide experience with oxcarbazepine and zonisamide, whereas exposure to levetiracetam has been limited to a relatively small number of patients in clinical trials.
These 3 drugs are important additions to the armamentarium for the treatment of seizures and offer patients whose lives are compromised by epilepsy the potential to achieve a better quality of life.
The anticonvulsant activity of ucb L059 ((S)-alpha-ethyl-2-oxo-pyrrolidine acetamide) was evaluated in a range of animal models. ucb L059 was active after oral and intraperitoneal administration in both rats and mice, with a unique profile of action incorporating features in common with several different types of antiepileptic drugs. The compound was active, with ED50 values generally within the range of 5.0-30.0 mg/kg, in inhibiting audiogenic seizures, electrically induced convulsions and convulsions induced chemically by pentylenetetrazole (PTZ), bicuculline, picrotoxin and N-methyl-D-aspartate (NMDA). ucb L059 retarded the development of PTZ-induced kindling in mice and reduced PTZ-induced EEG spike wave discharge in rats. The R enantiomer, ucb L060, had low intrinsic anticonvulsant activity, showing the stereospecificity of action of the molecule although the actual mechanism of action remains unknown. Neurotoxicity, evaluated with an Irwin-type observation test, the rotarod test and open-field exploration, was minimal, with only mild sedation being observed, even at doses 50-100 times higher than the anticonvulsant doses; at pharmacologically active doses, the animals appeared calm but slightly more active. ucb L059 thus presents as an orally active, safe, broad-spectrum anticonvulsant agent, with potential antiepileptogenic and anti-absence actions.
We have reviewed the pharmacokinetics of six antiepileptic drugs that are marketed (felbamate, gabapentin, lamotrigine, oxcarbazepine, vigabatrin, and zonisamide) and six drugs that are undergoing evaluation (levetiracetam, ralitoline, remacemide, stiripentol, tiagabine, and topiramate). In addition, we have compared the prodrugs eterobarb and fosphenytoin and the controlled-release formulations of valproic acid and carbamazepine with their parent compounds. Finally, we have devised a scoring system to compare the pharmacokinetics of new antiepileptic drugs. Using this system, vigabatrin, levetiracetam, gabapentin, and topiramate appea to have the most favourable pharmacokinetic profiles, whilst ralitoline and stiripentol have the least favourable.
The Fourth Eilat Conference on New Antiepileptic Drugs (AEDs) was held at the Royal Beach Hotel, Eilat, Israel, from 6th to 10th September 1998. Epileptologists and scientists from 20 countries attended the conference, which was held to discuss a number of issues in drug development, including outcome assessment in epilepsy (long-term efficacy, quality of life, safety), cost-effectiveness, an update on drugs in development, a progress report on recently marketed AEDs, and controversies in strategies for drug development. This review focuses on drugs in development and recently marketed AEDs. Drugs in development include ADCI, AWD 131-138, DP16, ganaxolone (CCD 1042), levetiracetam (ucb L059), losigamone, pregabalin (isobutyl GABA [CI-1008]), remacemide hydrochloride, retigabine (D-23129), rufinamide (CGP 33101), soretolide (D2916), TV1901, and 534U87. New information on the safety and efficacy of recently marketed drugs (felbamate, fosphenytoin, gabapentin, lamotrigine, oxcarbazepine, tiagabine, topiramate, vigabatrin, zonisamide) and of a new antiepileptic device, the neurocybernetic prosthesis (NCP), has become available. This paper summarizes the presentations made at the conference.
A simple and rapid method for determination of the new antiepileptic drug keppra (levetiracetam) by capillary electrophoresis in borate buffer containing sodium dodecyl sulfate is described. The serum was injected without any treatment. The method compared well to high performance liquid chromatography. The mean of keppra in the serum of 35 patients was 25 mg/l (range 7-77 mg/l).
An accurate and precise high-performance liquid chromatographic method using diode array detection for the determination of the novel antiepileptic, Levetiracetam, has been developed. Three clean-up procedures for the analysis of Levetiracetam in human plasma were implemented and evaluated, namely solid-phase extraction, deproteinization by addition of organic solvents and formation of insoluble salts. Adenosine was used as the internal standard for all three sample pretreatment procedures. Among the several cartridges used for solid-phase extraction, the hydrophilic-lypophilic balance (Oasis) HLB) phase provides the best extraction yield of Levetiracetam, together with high precision. With the two other clean-up procedures involving plasma deproteinization by addition of methanol or zinc sulphate, lower sensitivity and precision of the assays were obtained. However, they are cheaper and faster when compared with the solid-phase extraction procedure.
We here present a method for the routine quantification of the novel antiepileptic drug levetiracetam in human serum by HPLC-UV. The procedure is very easy, quick, inexpensive and rugged. The sample preparation consists only in the precipitation of serum proteins by perchloric acid and extraction of unpolar components by cyclohexane. The aqueous phase containing the analyte levetiracetam is injected onto a porous graphitic carbon analytical HPLC-column and separated by gradient elution with diluted phosphoric acid/acetonitrile. Detection is carried out at a wavelength of 205 nm. The calibration function is linear in the range of 1-75 microg/ml. The detection limit is 0.1 microg/ml. Using four quality control sample concentrations, the inter-day relative standard deviations (R.S.D.) are lower than 3% and the accuracies are better than 6%. The respective inter-day values are: R.S.D. < 4% and accuracies better than 2%. Frequently co-administered antiepileptic drugs do not interfere with the assay. The method has been successfully applied to patient samples.
The first liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the determination of levetiracetam, an antiepileptic drug, in human plasma is described. The plasma filtrate obtained after solid-phase extraction (SPE), using a polymer-based, hydrophilic-lipophilic balanced (HLB) cartridge, was submitted directly to a short column LC/MS/MS assay. There was no significant matrix effect on the analysis. For validation of the method, the recovery of the free analytes was compared to that from an optimized extraction method, and the analyte stability was examined under conditions mimicking sample storage, handling, and analytical procedures. The extraction procedure yielded extremely clean extracts with a recovery of 79.95% and 89.02% for levetiracetam and the internal standard (IS), respectively. The intra-assay and inter-assay precision for the samples at the lower limit of quantitation (LLOQ) were 6.33 and 6.82%, respectively. The calibration curves were linear for the dynamic range of 0.5 to 50 microg/mL with a correlation coefficient r >/= 0.9971. The intra-assay accuracy at LLOQ, LQC, MQC, and HQC levels ranged from 81.60 to 95.40, 93.00 to 103.47, 95.97 to 104.09, and 91.15 to 95.18%, respectively, while the inter-assay accuracy at LLOQ, LQC, MQC and HQC levels varied from 80.20 to 95.40, 88.53 to 107.53, 95.97 to 108.45, and 91.15 to 112.70%, respectively. The method is rugged and fast with a total instrumental run time of 2 min. The method was successfully applied for bioequivalence studies in human subject samples after oral administration of 1000 mg immediate release (IR) formulations.
Levetiracetam (Keppra) is a novel antiepileptic drug recently approved by the U.S. Food and Drug Administration as an add-on therapy in the treatment of partial onset seizures in patients. We developed and describe a simple and rapid high performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ESI-MS/MS) assay for the determination of levetiracetam in human matrix (plasma, serum, or saliva).
An API-3000 or API-4000 triple-quadrupole mass spectrometer (Sciex, Concord, Canada) coupled with the IonSpray source and Shimadzu HPLC system (Shimadzu Scientific Instruments, Columbia, MD) was used employing ritonavir as internal standard (IS) for levetiracetam. One hundred microliters of serum (or plasma, saliva) was deproteinized by adding 150 microl of acetonitrile containing internal standard. After centrifugation, 100 microl of supernatant was diluted with 300 microl of water and 10 microl aliquot was injected onto a C-18 column. After a 2.5 min wash the valve was activated to initiate the isocratic elution program which eluted the levetiracetam and internal standard into the MS/MS system. Quantitation by MRM analysis was performed in the positive ion mode. Within-day and between-day imprecision were evaluated for levetiracetam using three levels of in-house controls. Reliability and accuracy of this method were assessed by comparison of targets with external QC material (ChromSystems), between laboratory comparisons and by recovery studies.
Within-day coefficients of variation (CVs) were <6.1% and between-day CVs were <8.2%. The average spiked recoveries of levetiracetam added to the drug-free human plasma samples were 108% at low concentration level and 103% at high concentration level.
The method was found both specific and sensitive for the rapid and accurate measurement of levetiracetam in human matrices and correlated well with the Quest/Chantilly tandem mass spectrometric method (r=0.983).