Levetiracetam In Human Plasma Research Articles

Determination of levetiracetam in human plasma by online heart-cutting liquid chromatography: Application to therapeutic drug monitoring.

Levetiracetam is an antiepileptic drug for the treatment of psychiatric patients. In this study, a selective, straightforward, and rapid online heart-cutting liquid chromatography method was developed for the therapeutic drug monitoring of levetiracetam. This method allows for the determination of levetiracetam in human plasma without complex sample preparation. The mobile phases consisted of 30mM aq. orthophosphoric acid solution/methanol (70:30) at a flow rate of 1mL/min for the first system and 10mM aq. orthophosphoric acid solution/methanol (55:45) at a flow rate of 1mL/min for the second system. The first separation was carried out on a GL Sciences Intersil ODS-3 column (4.6mm × 150mm, 3µm) and the second separation was carried out on a Restek Ultra PFPP column (4.6mm × 150mm, 5µm). The detection was carried out at 205nm for both systems. The method was validated for selectivity and linearity, which were in the 6-60µg/mL range. Intra- and interassay accuracies were<112.6%, and the intra- and interassay precisions were<6.4% for all quality control samples. The lower limit of quantitation was 6µg/mL. The developed method was successfully applied for therapeutic drug monitoring of plasma samples from patients.

Computer-aided design of magnetic molecularly imprinted polymer nanoparticles for solid-phase extraction and determination of levetiracetam in human plasma.

Analytical methods should be accurate and specific to measure plasma drug concentration. Nevertheless, current sample preparation techniques suffer from limitations, including matrix interference and intensive sample preparation. In this study, a novel technique was proposed for the synthesis of a molecularly imprinted polymer (MIP) on magnetic Fe3O4 nanoparticles (NPs) with uniform core–shell structure. The Fe3O4@MIPs NPs were then applied to separate and enrich an antiepileptic drug, levetiracetam, from human plasma. A computational approach was developed to screen the functional monomers and polymerization solvents to provide a suitable design for the synthesized MIP. Different analysis techniques and re-binding experiments were performed to characterize the Fe3O4@MIP NPs, as well as to identify optimal conditions for the extraction process. Adsorption isotherms were best fitted to the Langmuir model and adsorption kinetics were modeled with pseudo-second-order kinetics. The Fe3O4@MIP NPs showed reasonable adsorption capacity and improved imprinting efficiency. A validated colorimetric assay was introduced as a comparable method to a validated HPLC assay for the quantitation of levetiracetam in plasma in the range of 10–80 μg mL−1 after extraction. The results from the HPLC and colorimetric assays showed good precision (between 1.08% and 9.87%) and recoveries (between 94% and 106%) using the Fe3O4@MIP NPs. The limit of detection and limit of quantification were estimated to be 2.58 μg mL−1 and 7.81 μg mL−1, respectively for HPLC assay and 2.32 μg mL−1 and 7.02 μg mL−1, respectively for colorimetric assay. It is believed that synthesized Fe3O4@MIP NPs as a sample clean-up technique combined with the proposed assays can be used for determination of levetiracetam in plasma.

Novel contribution in online sample cleanup for quantitative determination of Levetiracetam in human plasma by LC-Tandem mass spectrometry

The aim of this work was to develop and validate a simple, sensitive and rapid method for the quantitation of levetiracetam (LEV) in plasma using LC-Tandem mass spectrometry. Plasma samples were prepared by simple protein precipitation using acetonitrile; atenolol was used as internal standard (IS). Chromatographic separation was done on Luna 5 µm C18(2) (Phenomenex) 50 × 2.0 mm using gradient flow using solvent A (0.1% formic acid) and solvent B (0.1% formic acid in acetonitrile: water, 95:5, v : v ). The positive-ion mass spectrometric detection method utilized electrospray ionization and the multiple reaction monitoring (MRM) mode. The MRM ion transitions were170.9 → 140.8 and 267.3 → 145.1 for levetiracetam and atenolol, respectively. The retention time of levetiracetam and atenolol is 3.24 and 2.96 min, respectively. The total run time was 5 min. The assay was validated over a concentration range from 1 to 100 µg/mL. The method was robust (minimal matrix effect), sensitive (LOQ, 1 µg/mL) metabolites and reproducible (The precision and accuracy for both intra- and inter-day were acceptable <15%). The method can be done on traditional LC-MS equipment. The method was effectively applied to single case study receiving toxic dose of LEV.

Determination of Levetiracetam in Human Plasma by Dispersive Liquid-Liquid Microextraction Followed by Gas Chromatography-Mass Spectrometry.

Levetiracetam (LEV) is an antiepileptic drug that is clinically effective in generalized and partial epilepsy syndromes. The use of this drug has been increasing in clinical practice and intra- or -interindividual variability has been exhibited for special population. For this reason, bioanalytical methods are required for drug monitoring in biological matrices. So this work presents a dispersive liquid-liquid microextraction method followed by gas chromatography-mass spectrometry (DLLME-GC-MS) for LEV quantification in human plasma. However, due to the matrix complexity a previous purification step is required. Unlike other pretreatment techniques presented in the literature, for the first time, a procedure employing ultrafiltration tubes Amicon® (10 kDa porous size) without organic solvent consumption was developed. GC-MS analyses were carried out using a linear temperature program, capillary fused silica column, and helium as the carrier gas. DLLME optimized parameters were type and volume of extraction and dispersing solvents, salt addition, and vortex agitation time. Under chosen parameters (extraction solvent: chloroform, 130 μL; dispersing solvent: isopropyl alcohol, 400 μL; no salt addition and no vortex agitation time), the method was completely validated and all parameters were in agreement with the literature recommendations. LEV was quantified in patient's plasma sample using less than 550 μL of organic solvent.

A Simple Bioanalytical Method for the Quantification of Levetiracetam in Human Plasma and Saliva

A novel, sensitive and selective ultra-high-performance liquid chromatography - coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method was developed and validated for the quantification of levetiracetam (LEV), a broad-spectrum antiepileptic drug, in human plasma and saliva. A simple protein precipitation method with acetonitrile as precipitating solvent was used to extract LEV from human plasma and saliva. LEV and an internal standard (fluconazole, IS) were separated on a Kinetex analytical column (5 μm C18 100A, 100 × 2.1 mm) using an isocratic solvent system consisting of methanol, water and 100% formic acid at a ratio of 97:3:0.25 (v/v/v) and a flow rate of 0.2 ml/min over 2 min run time. Detection was performed using target ions of [M+H]+ at m/z 171.0 for LEV and m/z 307.0 for IS in selective ion mode. The method was validated in the calibration range of 1.0-50.0 μg/mL for plasma and 0.5-30.0 μg/mL for saliva. Thus the present HPLC-MS/MS method for determination of LEV in human plasma and saliva, is highly sensitive, rapid with a short run-time of 2 min, can be suitable for high sample throughput. The developed method required minimal sample preparation and less plasma sample volume compared to earlier published LC-MS/MS methods. The validation parameters were found to be well within the acceptance limit.

Development and validation of an analytical method based on high performance thin layer chromatography for the simultaneous determination of lamotrigine, zonisamide and levetiracetam in human plasma

Methods based on HPLC technology are the most frequently adopted for monitoring blood levels of novel antiepileptics. Here a rapid method based on HPTLC was developed for quantitative determination of lamotrigine (LTG), zonisamide (ZNS) and levetiracetam (LVT) in human plasma and compared with HPLC and LC–MS/MS methods. Chromatographic separation was achieved on silical gel 60F 254 plates using ethylacetate:methanol:ammonia (91:10:15 v/v/v) as mobile phase. Quantitative analysis was carried out by densitometry at a wavelength of 312, 240 and 210 nm for LTG, ZNS and LVT, respectively. Calibration curves were linear over range of 0–200 ng for LTG and ZNS and 0–400 ng for and LVT. The limit of quantification of LTG, ZNS and LTV was found to be 3.69, 3.7 and 6.85 μg/ml, respectively. Intra and inter-assay precision provided relative standard deviations lower than 10% for all three analytes. Correlation and Bland–Altman plot showed general agreement between HPTLC and LC–MS/MS quantification, with a mean bias of −0.25, −0.46 and 0.5 μg/ml for LTG ZNS and LVT, respectively. Likewise, comparison between HPLC–UV and LC–MS/MS showed good agreement for all the three compounds analyzed. In conclusion, the proposed HPTLC method is simple, rapid, precise and accurate. It therefore is appropriate for the routine quantification of therapeutic levels of LTG, ZNS and LVT in human plasma.

LC method for therapeutic drug monitoring of levetiracetam: Evaluation of the assay performance and validation of its application in the routine area

Objectives An accurate and precise high-performance liquid chromatographic method using diode array detection for the determination of levetiracetam in human plasma has been developed and validated for use in pharmacokinetic studies. Methods A harmonized validation strategy based on the accuracy profiles was used to select the most appropriate regression model and to determine the limits of quantitation as well as the concentration range of the developed analytical procedure. On the other hand, the present paper also shows this validation approach as a suitable tool to guaranty the quality of the results obtained by the use of the analytical validated methodology for plasma levetiracetam determination in a routine setting and to ensure the risk of obtaining the future measurements outside the previously fixed acceptance limits. Results As pointed recently, the FDA, a weighted 1/ x 2 quadratic regression model ranging from 0.53 to 107.00 mg/L was selected as the simplest calibration model that maximized the accuracy all over the range. Relative bias was < 5%, assay imprecision was always < 6% and mean extraction recovery from plasma was > 90%. So, accuracy did not exceed the acceptance limits settled at ± 20% according to the FDA or Washington conference regulatory requirements for bioanalytical methods. Internal quality control has been assessed over a 2 year time period. All controls were essentially found to provide levetiracetam concentrations within the target range according to the FDA. Conclusions The validated analytical procedure complies with strongest regulatory standards. The validated method has a sufficiently rapid turnaround time and their results are good enough to enable the laboratory to routinely provide useful and accurate pharmacokinetic data in time to adjust patient regimens.

Quantification of levetiracetam in human plasma by liquid chromatography–tandem mass spectrometry: Application to therapeutic drug monitoring

A rapid, selective, reliable, precise, accurate, and reproducible tandem mass spectrometric (MS-MS) method for the quantification of levetiracetam (LEV) in human plasma using adenosine as an internal standard (IS) has been developed and validated. The drug and IS were extracted by solid phase extraction (SPE) technique and analyzed on Symmetry ® C 18 column (5 μm, 3.9 mm × 50 mm) using a mobile phase of methanol–water–formic acid (97:03:0.25, v/v/v) at a flow rate of 0.2 ml/min. Quantitation was achieved using a positive electrospray ionization (ESI+) interface employing multiple reaction monitoring (MRM) mode at MRM transitions m/ z 171 > 126 and m/ z 268 > 136 for LEV and IS, respectively. The method was validated over the concentration range of 1.0–40 μg/ml ( r > 0.99) with a limit of quantification of 1.0 μg/ml (R.S.D.%; 4.1 and Bias%; −9.0 to + 11.0%). Intra- and inter-run precision of LEV assay at three concentrations ranged from 0.6 to 8.9% with accuracy (bias) varied from −4.0 to 8.6% indicating good precision and accuracy. Analytical recoveries of LEV and IS from spiked human plasma were in the range of 91.7–93.4% and 80.2–84.1%, respectively. Stability of LEV in human plasma samples at different conditions showed that the drug was stable under the studied conditions. Matrix effect study showed a lack of matrix effect on mass ions of LEV and IS. The described method compared well with the commercial HPLC-UV method of Chromsystem ( r 2 = 0.99). The suitability of the developed method for therapeutic drug monitoring was demonstrated by measuring LEV in human plasma samples of epileptic patients treated with LEV.

Therapeutic Drug Monitoring of Levetiracetam by High-Performance Liquid Chromatography With Photodiode Array Ultraviolet Detection: Preliminary Observations on Correlation Between Plasma Concentration and Clinical Response in Patients With Refractory Epilepsy

Levetiracetam is a new antiepileptic drug prescribed for the treatment of patients with refractory partial seizures with or without secondary generalization as well as for the treatment of juvenile myoclonic epilepsy. A rapid and specific method by high-performance liquid chromatography diode array detection was developed to measure the concentration of levetiracetam in human plasma. The trough plasma concentrations measured in 69 epileptic patients treated with 500 to 3000 mg/d of levetiracetam ranged from 1.1 to 33.5 microg/mL. The mean (range) levetiracetam plasma concentrations in responders and nonresponders were 12.9 microg/mL (4.6-21 microg/mL) and 9.5 microg/mL (1.1-20.9 microg/mL), respectively. A wide variability in concentration-response relationships was observed in patients. Using a receiver operating characteristic curve, the threshold levetiracetam concentration for a therapeutic response was 11 microg/mL. The sensitivity and specificity for this threshold levetiracetam concentration were 73% and 71%, respectively. According to chi analysis, this finding was not significant probably because of the small number of patients and because of their refractory seizure type. Nevertheless, the levetiracetam plasma concentration could be used to help clinicians detect severe intoxication or to verify compliance by repeating the measurement in patients.

Determination of levetiracetam in human plasma by liquid chromatography/electrospray tandem mass spectrometry and its application to bioequivalence studies

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.

Determination of levetiracetam in human plasma with minimal sample pretreatment

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 μg/ml. The detection limit is 0.1 μg/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.

Microemulsion electrokinetic chromatography applied for separation of levetiracetam from other antiepileptic drugs in polypharmacy.

Microemulsion electrokinetic chromatography was applied for the separation of levetiracetam from other antiepileptic drugs (primidone, phenobarbital, phenytoin, lamotrigine and carbamazepine) that are potentially coadministered in therapy of patients. The influence of the composition of the microemulsion system (with sodium dodecyl sulfate as charged surfactant) was investigated, modifying the kind of cosurfactant (lower alcohols from C3 to C5), the pH (and salinity) of the aqueous background electrolyte, and the ratio of aqueous phase to organic constituents forming the microdroplets of the oil-in-water emulsion. Separation selectivity was depending on all these parameters, resulting even in changes of the migration sequence of the analytes. Only moderate correlation was observed for the microemulsion system compared with a micellar system, both consisting of the aqueous borate buffer (pH 9.2) and SDS as micelle former (linear correlation coefficient for analyte mobilities is 0.974). The sample solvent plays an important role on the shape of the resulting chromatograms: methanol at concentrations higher than 35% impairs peak shape and separation efficiency. The microemulsion method (with 93.76% aqueous borate buffer (pH 9.2, 10 mM), 0.48% n-octane, 1.80% SDS, 3.96% 1-butanol, all w/w) is suitable for the determination of levetiracetam in human plasma (combined with a sample pretreatment based on solid-phase extraction).