Plasma Of Epileptic Patients Research Articles

Simultaneous Determination of Lamotrigine and Carbamazepine in Plasma Using Ultrasound-Assisted Emulsification Microextraction-High Performance Liquid Chromatography

A simple, fast and efficient method based on ultrasound-assisted emulsification microextraction (USAEME) followed by HPLC has been developed for simultaneous determination of Carbamazepine (CBZ) and Lamotrigine (LTG) in biological samples. The influence of different parameters affecting the USAEME procedure was evaluated to optimize the efficiency of the process. At these conditions, validation of the proposed method was performed based on European medicines agency guidelines. The proposed method showed good linearity in plasma samples ranging from 0.25 to 6 µg/mL and 0.13 to 6 µg/mL (R2 ≥ 0.99) for CBZ and LTG, respectively. The precision of the proposed method was evaluated for repeatability which was ≤12% (n = 5). The detection limits of 0.06 and 0.05 µg/mL were obtained for CBZ and LTG, respectively. The proposed method can be applied as an effective technique for therapeutic drug monitoring of CBZ and LTG in plasma of epileptic patients.

A three phase hollow fiber liquid-phase microextraction for quantification of lamotrigine in plasma of epileptic patients by capillary electrophoresis.

A three phase hollow fiber liquid-phase microextraction technique combined with capillary electrophoresis was developed to quantify lamotrigine (LTG) in plasma samples. The analyte was extracted from 4.0 mL of a basic donor phase (composed of 0.5 mL of plasma and 3.5 mL of sodium phosphate solution pH 9.0) through a supported liquid membrane composed of 1-octanol immobilized in the pores of the hollow fiber, and to an acidic acceptor phase (hydrochloric acid solution pH 4.0) placed in the lumen of the fiber. The extraction was carried out for 30 min at 500 rpm. The eletrophoretic analysis was carried out in 130 mmol/L MES buffer, pH 5.0 with a constant voltage of +15 kV and 20°C. Sample injections were performed for 10 s, at a pressure of 0.5 psi. The detection was performed at 214 nm for both LTG and the internal standard lidocaine. Under the optimized conditions, the method showed a limit of quantification of 1.0 μg/mL and was linear over the plasmatic concentration range of 1.0-20.0 μg/mL. Finally, the validated method was applied for the quantification of LTG in plasma samples of epileptic patients.

A meta-analysis of pro-inflammatory cytokines in the plasma of epileptic patients with recent seizure

This review was to assess the concentration changing trends of various cytokines in plasma of epilepsy patients after recent seizure, as well as to assess the differences between temporal lobe epilepsy (TLE) and extra-TLE (XLE) patients. We performed a meta-analysis of studies measuring cytokine concentration in plasma of patients suffering from epilepsy after recent seizure, by searches of the English literature in Pubmed and Embase databases (to July 1st 2010) and a manual search of references. A random-effects model was used to do accumulative analysis for the included studies by RevMan 5.0 software. Eight studies were included and analyzed. We found the plasma concentrations of interleukin-6 (IL-6) within 72h after seizure were significantly increased in epileptic patients compared with control subjects (211 epilepsy patients vs. 564 controls, overall weighted mean difference 1.27pg/ml, 95% confidence interval 0.72–1.82, P<0.0001). There were no significant differences for IL-1 beta and, IL-1 receptor antagonist (IL-1RA) between the two groups. The concentration changes of IL-6 and IL-1RA were not significantly different between TLE patients and XLE patients. The result of this meta-analysis revealed significantly higher concentrations of the pro-inflammatory cytokines IL-6 in epileptic seizure patients compared with control subjects. Further rigorous studies are needed to clarify the precise role of cytokines in epilepsy.

Liquid chromatographic determination of oxcarbazepine and its metabolites in plasma of epileptic patients after solid-phase extraction

A method based on high-performance liquid chromatography with UV detection in combination with solid-phase extraction for sample pretreatment has been developed for the simultaneous analysis of the antiepileptic drug oxcarbazepine and its main metabolites in human plasma. The extraction of the analytes from plasma samples was carried out by means of a selective solid-phase extraction procedure using hydrophilic–lipophilic balance cartridges. The separation was obtained on a reversed-phase column (C 18, 150×4.6 mm I.D., 5 μm) using a phosphate buffer–acetonitrile–methanol–triethylamine mixture (final apparent pH* 3.5) as the mobile phase. Under these chromatographic conditions, oxcarbazepine and its metabolites 10,11-dihydro-10-hydroxycarbamazepine, 10,11-dihydro-10,11-dihydroxycarbamazepine and the internal standard are baseline separated in less than 9 min. The extraction yield values were >94% for all analytes and the precision, expressed by the RSD%, was in the low percentage range. For the entire method, including sample pre-treatment and HPLC determination, the linearity of the calibration lines, expressed by the linear correlation coefficient, was better than 0.995; the limit of quantitation was 15 ng ml −1. The method was applied to plasma samples from patients undergoing chronic treatment with oxcarbazepine, both in monotherapy and in polytherapy. Based on the analytical parameters precision, accuracy, limit of quantitation and analysis time the method is suitable for routine application in therapeutic drug monitoring.

Simultaneous high-performance liquid chromatography determination of carbamazepine and five of its metabolites in plasma of epileptic patients

A high-performance liquid chromatographic method with UV detection for the simultaneous analysis of the antiepileptic drug carbamazepine and five of its metabolites in human plasma has been developed. The analysis was carried out on a reversed-phase column (C 8, 150×4.6 mm I.D., 5 μm) using acetonitrile, methanol and a pH 1.9 phosphate buffer as the mobile phase. Under these chromatographic conditions, carbamazepine and its metabolites 10,11-dihydro-10,11-epoxycarbamazepine, 10,11-dihydro-10,11-dihydroxycarbamazepine, 2-hydroxycarbamazepine, 3-hydroxycarbamazepine and 10,11-dihydro-10-hydroxycarbamazepine are baseline separated in less than 18 min. The extraction of the analytes from plasma samples was performed by means of an original solid-phase extraction procedure using Oasis HLB cartridges. The method requires only 250 μl of plasma for one complete analysis. The repeatability (RSD%<2.4), intermediate precision (RSD%<3.5) and extraction yield (84.8–103.0%) were very good for all analytes. The method is suitable for reliable therapeutic drug monitoring of patients undergoing chronic treatment with carbamazepine and for kinetic–metabolic studies of this drug.

Improved selectivity for high-performance liquid chromatographic determination of clonazepam in plasma of epileptic patients

We report a high-performance liquid chromatography method for clonazepam determination in plasma. The use of a synthetic silica-based stationary phase markedly improved clonazepam resolution compared to standard reversed-phase columns. A liquid–liquid extraction was used, associated with reversed-phase chromatography, gradient elution and ultraviolet detection. Accuracy and precision were satisfactory at therapeutic concentrations. Selectivity was studied for benzodiazepines or other antiepileptic drugs, with particular attention to newly marketed drugs i.e., gabapentine and vigabatrin. No interfering substance was evidenced. Under the conditions described, it was possible to quantify clonazepam at nanogram level even when carbamazepine was present at therapeutic concentrations.

Studies on the reactivity of acyl glucuronides—III: Glucuronide-derived adducts of valproic acid and plasma protein and anti-adduct antibodies in humans

The major metabolite of the anti-epileptic agent valproic acid (VPA) is its acyi glucuronide conjugate (VPA-G), which undergoes non-enzymic, pH-dependent rearrangement via acyi migration to a mixture of β-glucuronidase-resistant forms (collectively VPA-G-R). We have compared the reactivity of VPA-G and VPA-G-R towards covalent VPA-protein adduct formation by incubation in buffer, human serum albumin (HSA) and fresh human plasma at pH 7.4 and 37°. In all three media, the predominant reaction of VPA-G over 30 hr was rearrangement to VPA-G-R ( ca. 24%). Hydrolysis was quite minor ( ca. 2%) and covalent adduct formation negligible (when protein was present). On the other hand, both hydrolysis ( ca. 27%) and adduct formation ( ca. 7%) were extensive when VPA-G-R was incubated with HSA or plasma. These data do not support a transacylation mechanism for VPA-protein adduct formation, since this pathway should be much more highly favoured by VPA-G (an acyl-substituted acetal) than VPA-G-R (simple esters). VPA-protein adducts were found in the plasma of epileptic patients taking VPA chronically (mean 0.77 ± SD 0.63 μg VPA equivalents/mL, N = 17). An enzyme linked immunosorbent assay was developed, using HSA modified by incubation with VPA-G-R, to test the immunoreactivity of the patients' plasma. Of 57 patients tested, nine showed measurable levels of antibodies to these adducts, but the litres were very low, with no difference in response to modified and unmodified protein detectable at plasma dilutions of 1:16 or greater. These results suggest that the VPA-protein adducts have little immunogenicity, and are in agreement with clinical observations that drug hypersensitivity responses have not been associated with VPA therapy. Thus, although the in vitro data show that VPA-G is an example of a relatively unreactive acyi glucuronide, covalent VPA-plasma protein adducts and anti-adduct antibodies are nonetheless formed in vivo, at least in some patients on chronic therapy with the drug.

HPLC method for the simultaneous analysis of valproic acid and other common anticonvulsant drugs in human plasma or serum

The derivatizing procedure of Moody et al. [20] for valproic acid has been simplified and applied to the simultaneous HPLC determination of valproic acid (VPA), barbital (B), primidone (PRM), phenobarbital (PB) and carbamazepine (CBZ) in serum or plasma of epileptic patients. The sample is deproteinized with acetonitrile containing esterification agents and an aliquot of the supernatant is heated to 70°C for 15 min with 4-bromophenacyl bromide. The reaction mixture is analysed on a C18 column at ambient temperature, with gradient elution and with detection at 205 nm. The time required for the chromatographic analysis is 13 min; identification is based on retention time and quantification is by peak area determination with an internal standard. The calibration curves show good linearity in the range 6.25 to 100 mg/L. The detection limits at a signal: noise ratio ≥3, ranged from 1 mg/L for B and CBZ to 2–3 mg/L for PRM, PB and VPA. The method described for the simultaneous determination of the five drugs in the same plasma pool, correlated well with isocratic HPLC methods specific for each drug. The simultaneous procedure described allows a reproducible (CVs≤6.5% within run) and rapid (25 min for sample preparation: 13 min for chromatographic run) therapeutic monitoring of patients treated with VPA and two or more antiepileptic drugs.

Analysis of Taurine in Blood Plasma of Epileptic Patients Using an Improved Isocratic HPLC Method for Amino Acids

Abstract An efficient, reversed-phase HPLC method is described in which 21 derivatized amino acids were isocratically separated in less than 90 minutes. An internal standard was used to improve precision and accuracy. The method, which separated taurine from α-, β-, and γ-aminobutyric acids, was used to quantify taurine levels in 83 blood plasma samples. A statistical comparison was made between taurine levels found in the plasma of epileptic and non-epileptic children.

Together, phenobarbital and carbamazepine lower the alpha 1-acid glycoprotein concentration in plasma of epileptic patients.

Together, phenobarbital and carbamazepine lower the alpha 1-acid glycoprotein concentration in plasma of epileptic patients.

Quantitative determination of n-dipropylacetamide in the plasma of epileptic patients by gas—liquid chromatography with nitrogen-selective detection

Quantitative determination of n-dipropylacetamide in the plasma of epileptic patients by gas—liquid chromatography with nitrogen-selective detection

Rapid and Simple GLC Determination of Valproic Acid and Ethosuximide in Plasma of Epileptic Patients

A GLC method for the determination of valproic acid and ethosuximide in plasma was developed. The procedure involved a single solvent extraction of drugs from acidified plasma samples, followed by a GLC injection of the unconcentrated organic phase. This rapid, sensitive, specific, and reproducible method has been used for 2 years for the routine determination of plasma levels of valproic acid and ethosuximide in epileptic patients who receive other antiepileptic drugs simultaneously.

Quantitative determination of clobazam in the plasma of epileptic patients by gas—liquid chromatography with electron-capture detection

Quantitative determination of clobazam in the plasma of epileptic patients by gas—liquid chromatography with electron-capture detection

Concentration of metabolites of valproic acid in plasma of epileptic patients.

With the help of synthetic reference substances, five metabolites of valproic acid (VPA) could be quantitated by gas chromatography in the plasma of 26 epileptic patients undergoing chronic therapy with sodium valproate. The products of beta-oxidation, i.e., 2-en-VPA, 3-hydroxy-VPA, and 3-keto-VPA were found to be the major metabolites of VPA in plasma, whereas the intermediates of omega-oxidation, 4-hydroxy-VPA and 5-hydroxy-VPA, were present only in markedly lower concentrations. It was thus confirmed that in addition to the excretion of VPA as the glucuronide, beta-oxidation is the preferred metabolic pathway of VPA in man. However, taking the anticonvulsant activity of the metabolites as derived from animal experiments into consideration, none of the metabolites found in human plasma seems to contribute markedly to the therapeutic effect of VPA. Thus, in most patients, VPA seems responsible for more than 90% of the antiepileptic activity during continued medication in man.