An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for the quantification of phenobarbital in human serum and plasma.

OBJECTIVES: Phenobarbital serves as an antiepileptic drug (AED) and finds application in the treatment of epilepsy either as monotherapy or adjunctive therapy. This drug exhibits various pharmacodynamic properties that account for its beneficial effects as well as potential side effects. Accurate measurement of its concentration is critical for optimizing AED therapy through appropriate dose adjustments. Therefore, our objective was to develop and validate a new reference measurement procedure (RMP) for the accurate quantification of phenobarbital levels in human serum and plasma. METHODS: A sample preparation protocol based on protein precipitation followed by a high dilution step was established in combination with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using a C8 column to separate target analytes from known and unknown interferences. Assay validation and determination of measurement uncertainty were performed based on current guidelines. Selectivity and Specificity were assessed using spiked serum and plasma samples; to investigate possible matrix effects (MEs) a post-column infusion experiment and a comparison of standard line slopes was performed. Precision and accuracy were determined within a multiday precision experiment. RESULTS: The RMP was shown to be highly selective and specific, with no evidence of matrix interferences. It can be used to quantify phenobarbital in the range of 1.92 to 72.0 µg/mL. Intermediate precision was less than 3.2 %, and repeatability coefficient of variation (CV) ranged from 1.3 to 2.0 % across all concentration levels. The relative mean bias ranged from -3.0 to -0.7 % for native serum levels, and from -2.8 to 0.8 % for Li-heparin plasma levels. The measurement uncertainties (k=1) for single measurements and target value assignment were 1.9 to 3.3 % and 0.9 to 1.6 %, respectively. CONCLUSIONS: A novel LC-MS/MS-based candidate RMP for the quantification of phenobarbital in human serum and plasma is presented which can be used for the standardization of routine assays and the evaluation of clinically relevant samples.

Evaluation of anti-seizure medications and their serum concentration with regard to cardiovascular risk parameters: A cross-sectional study.

OBJECTIVE: Epilepsy is a chronic disease that requires long-term monitoring and treatment. It is suspected that there is a interaction between the use of anti-seizure medications and the risk of cardiovascular disease. The aim of the study is to investigate the association between the intake of phenobarbital, carbamazepine and valproic acid and their serum drug concentrations (SDC) with various cardiovascular risk parameters (homocysteine, folic acid, vitamin B12, total cholesterol (TC), triglycerides, high- and low-density lipoprotein (LDL)). METHODS: This is a cross-sectional study. Data (demographic characteristics and laboratory results) of patients treated for epilepsy in a tertiary care hospital between January 2020 and February 2022 were analyzed retrospectively (n = 2014). Kruskal Wallis, Mann-Whitney U, correlation analysis was used, p < 0.05 was considered statistically significant. RESULTS: The median age of patients was 15 years (IQR:8-31) and 48.3 % were women. The highest homocysteine level was found in patients receiving valproic acid, but it was not statistically significant. Patients receiving phenobarbital had the highest levels of folic acid and B12 and the lowest levels of total cholesterol and low-density lipoprotein cholesterol, which was statistically significant. In patients receiving carbamazepine, a moderately negative significant association was found between serum drug concentration and folic acid levels and a moderately positive significant association was found between TC and LDL levels. CONCLUSION: In our study, the majority of patients were children and adolescents. Regular monitoring of drug serum concentrations and metabolic parameters may be useful to select the safest drug in terms of cardiovascular disease risk. Randomized controlled trials on the long-term effects of anti-seizure treatment are needed.

The effect of acidic beverage versus mineral water on the change in serum phenobarbital concentrations: a randomized clinical trial on children with seizure.

OBJECTIVE: To assess the effect of an acidic beverage (Orange juice) on the change in serum Phenobarbital concentrations in children with seizure who take Phenobarbital as the main treatment. METHODS: We did a parallel design and placebo controlled randomized clinical trial. Patients attending Heshmatiyeh Hospital (Iran) were recruited from October 2016 to December 2017. Forty patients randomly assigned to either experimental group or control group. Firstly, 5 mL blood sample was taken from both groups to measure serum Phenobarbital concentration before experiment. Then, one oral dose of Phenobarbital (2.5 mg/kg) with 100 mL of corporate Orange juice (pH = 3.5) (experiment group) or 100 mL of mineral water (neutral pH) (control group) was given to each group, respectively. After 2 h of administration, another blood sample was taken. The high-performance liquid chromatographic system was used for measurement of serum Phenobarbital concentration. RESULTS: There was significant increase in serum Phenobarbital concentrations after taking Phenobarbital in experiment group in comparison to control group. Statistical analysis revealed a significant increase in change of serum Phenobarbital concentrations in experiment group versus control group. CONCLUSION: The results of the current trial indicate that the level of serum Phenobarbital in the experiment group was higher than that of control group.

Dried Blood Microsampling-Based Therapeutic Drug Monitoring of Antiepileptic Drugs in Children With Nodding Syndrome and Epilepsy in Uganda and the Democratic Republic of the Congo.

Nodding syndrome is a highly debilitating, generalized seizure disorder, affecting children in subregions of sub-Saharan Africa. Despite numerous efforts to uncover the etiology, the exact cause of this syndrome still remains obscure. Therefore, to date, patients only receive symptomatic care, including the administration of first-generation antiepileptic drugs for seizure control. As data on the efficacy of drugs within this population are completely lacking, the aim of this study was to explore how therapeutic drug monitoring could help to understand the differential response to therapy. Considering the challenging environment in which sampling had to be performed (remote areas, devoid of electricity, running water, etc), dried blood matrices [ie, dried blood spots (DBSs)] and volumetric absorptive microsampling (VAMS) were considered fit-for-purpose. In addition, owing to the similarities between the syndrome and other forms of epilepsy, samples originating from patients suffering from (onchocerciasis-associated) epilepsy were included. In total, 68 patients with Nodding syndrome from Uganda, 58 Ugandan patients with epilepsy, and 137 patients with onchocerciasis-associated epilepsy from the Democratic Republic of the Congo were included. VAMS samples and DBS were analyzed using validated methods, involving manual extraction or fully automated extraction, respectively, before quantification using liquid chromatography coupled with tandem mass spectrometry. Analysis revealed that serum concentrations (calculated from DBS) within the respective reference ranges were attained in only 52.9% of the 68 Nodding syndrome patients treated with valproic acid, in 21.4% of the 56 Ugandan epilepsy patients treated with carbamazepine, and in 65.7% of the 137 onchocerciasis-associated epilepsy patients from the Democratic Republic of the Congo treated with phenobarbital. In all other instances, concentrations were subtherapeutic. Furthermore, on comparing DBS with VAMS concentrations, an inexplicable overestimation was observed in the latter. Finally, no obvious link could be observed between the obtained drug concentrations and the number of seizures experienced during the last month before sampling, elaborating the fact that the level of improvement in some patients cannot simply be linked to reaching therapeutic concentrations.

Blood concentration, efficacy, and adverse events of phenobarbital: A prospective study in rural China.

OBJECTIVE: This study evaluated the relationship between blood concentration of phenobarbital (PB) and its efficacy as well as adverse events in people with epilepsy in rural China. METHODS: People with epilepsy being treated with PB monotherapy were recruited and followed up for averagely 2.5 years. Data of clinical characteristics were collected using a standardized questionnaire by face-to-face interviews both at baseline and follow-up. Plasma concentration of PB was detected by the high-performance liquid chromatography. RESULTS: Data on treatment response and PB blood concentration was obtained from 225 subjects. Among them, 119 (52.9%) were recognized as effective cases and 106 (47.1%) as ineffective cases. In the effective group, the blood concentration of 95% subjects ranged from 1.22 µg/ml to 41.36 µg/ml with a median at 13.18 µg/ml (IQR = 8.32-20.19 µg/ml). The PB concentration of 95% of the subjects in the ineffective group ranged from 2.73 µg/ml to 70.16 µg/ml with a median at 19.80 µg/ml (IQR = 11.30-30.40 µg/ml), which was significantly higher than that of the effective group (p < 0.001). Multivariate logistic regression analysis showed that PB concentration ≥26.38 µg/ml was related to a 4.5-fold (95% confidence interval [CI], 1.85-11.08) higher risk of inefficacy. A receiver operation characteristic curve was performed to determine the cutoff value of concentration for PB efficacy at 19.02 µg/ml. SIGNIFICANCE: Blood concentration may be an important indicator for clinical decision making when PB monotherapy cannot achieve a good efficacy and more attention should be paid on it in clinical practice especially in resource-poor settings.

Comparison of CYP3A4-Inducing Capacity of Enzyme-Inducing Antiepileptic Drugs Using 4ß-Hydroxycholesterol as Biomarker.

BACKGROUND: Enzyme-inducing antiepileptic drugs (EIAEDs) are among the clinically most important inducers of cytochrome P450 (CYP) 3A4, but there is limited evidence regarding the comparative potency of each EIAED in raising CYP3A4 activity. The aim of this study was to estimate CYP3A4-inductive potency of EIAEDs by comparing CYP3A4 activity in patients treated with carbamazepine, phenobarbital, or phenytoin. METHODS: Residual serum samples from patients treated with EIAEDs or levetiracetam were collected from a therapeutic drug monitoring service for analysis of 4ß-hydroxycholesterol (4ßOHC), which is an indicator of CYP3A4 activity. The samples were collected between January and September 2016 at Diakonhjemmet Hospital, Oslo, Norway. Concentration of 4ßOHC, EIAEDs, and levetiracetam was measured by ultra-performance liquid chromatography tandem mass spectrometry. Kruskal-Wallis and Mann-Whitney tests were used for comparison of 4ßOHC levels between the subgroups. RESULTS: In total, 4ßOHC measurements for 343 and 339 patients treated with EIAEDs and levetiracetam, respectively, were included in the study. Compared with levetiracetam-treated patients, the median 4ßOHC concentration was 3.3-fold, 5.8-fold, and 6.9-fold higher in patients using phenobarbital, phenytoin, or carbamazepine, respectively (P < 0.0001). Phenytoin users (n = 65) and carbamazepine users (n = 225) had 1.8- and 2.1-fold higher median 4ßOHC concentration than phenobarbital users (n = 28), respectively (P ≤ 0.0001). CONCLUSIONS: This study shows that phenytoin and carbamazepine have approximately twice the CYP3A4-inducing potency of phenobarbital. The results indicate that 2-fold higher doses of CYP3A4-metabolized drugs may generally be required during concurrent treatment with phenytoin or carbamazepine compared with phenobarbital.

Modified dispersive liquid-phase microextraction based on sequential injection solidified floating organic drop combined with HPLC for the determination of phenobarbital and phenytoin.

A modified dispersive liquid phase microextraction based on sequential injection solidified floating organic drop was developed for simultaneous separation/preconcentration of trace amounts of phenobarbital and phenytoin. The important factors affecting on the extraction recovery including pH, the volume of extraction solvent, ionic strength, and the number of injections were investigated and optimized by Box-Behnken design and desirability function. Under the optimum experimental conditions, the calibration graph was linear in the concentration range of 1.0-300.0 µg/L (r2  = 0.997) for phenobarbital and 2.0-400.0 µg/L (r2  = 0.996) for phenytoin. The limit of detection and limit of quantification were 0.35 and 1.2 µg/L for phenobarbital and 0.65 and 2.2 µg/L for phenytoin, respectively. The relative standard deviation for six replicate determinations at 10 µg/L was 3.3 and 4.1% for phenobarbital and phenytoin, respectively. The developed method was successfully applied to the determination of phenobarbital and phenytoin in urine and plasma samples.

A potential drug interaction between phenobarbital and dolutegravir: A case report.

In this report, we describe a human immunodeficiency virus (HIV)-infected patient in whom changes in phenobarbital (PB) dosage resulted in associated changes in plasma concentrations of dolutegravir (DTG). His plasma concentrations of DTG were 0.934, 0.584, 1.003 and 3.25 µg/mL, respectively, with concomitant daily PB doses of 40, 70, 30 and 0 mg, respectively. This case suggests that PB can lead to a remarkable reduction in the plasma concentration of DTG in a dose-dependent manner.

Concentration of antiepileptic drugs in persons with epilepsy: a comparative study in serum and saliva.

AIM OF THE STUDY: The monitoring of antiepileptic drugs (AEDs) in clinical setting is important for measuring the efficacy of drugs and their safety and in personalizing drug therapy. We investigated the levels of AED, carbamazepine (CBZ), phenytoin (PHT) and phenobarbital (PHB), to understand their association in saliva compared with those in serum during the therapy. MATERIALS AND METHODS: In this study, we performed a prospective study of 116 persons with epilepsy (PWE; mean age 26.90 ± 11.83 years). Serum and saliva samples were collected at trough levels from the patients, who were under the treatment of CBZ, PHT and PHB either alone or in combination of these drugs for at least three months. The drug levels were assessed by high-performance liquid chromatography. RESULTS AND CONCLUSIONS: The number of males (n = 88; 75.86%) was higher than females (n = 28; 24.14%) among the recruited patients. The intake of CBZ, PHT and PHB was observed in 49.14%, 68.10% and 38.79% of PWE, respectively. The levels of these AEDs showed a significant correlation (p < 0.05) between serum and saliva. Interestingly, the levels of mono-therapy or bi-therapy showed a significant association (p < 0.05) between serum and saliva, however, there was no significant association in case of poly-therapy. This is the first report in the Indian population on simultaneous estimation of the three commonly used AEDs, such as CBZ, PHT and PHB in serum and saliva implicating their associations, either in mono-therapy or bi-therapy in PWE.

A randomised trial of a medium-chain TAG diet as treatment for dogs with idiopathic epilepsy.

Despite appropriate antiepileptic drug treatment, approximately one-third of humans and dogs with epilepsy continue experiencing seizures, emphasising the importance for new treatment strategies to improve the quality of life of people or dogs with epilepsy. A 6-month prospective, randomised, double-blinded, placebo-controlled cross-over dietary trial was designed to compare a ketogenic medium-chain TAG diet (MCTD) with a standardised placebo diet in chronically antiepileptic drug-treated dogs with idiopathic epilepsy. Dogs were fed either MCTD or placebo diet for 3 months followed by a subsequent respective switch of diet for a further 3 months. Seizure frequency, clinical and laboratory data were collected and evaluated for twenty-one dogs completing the study. Seizure frequency was significantly lower when dogs were fed the MCTD (2·31/month, 0-9·89/month) in comparison with the placebo diet (2·67/month, 0·33-22·92/month, P=0·020); three dogs achieved seizure freedom, seven additional dogs had ≥50 % reduction in seizure frequency, five had an overall <50 % reduction in seizures (38·87 %, 35·68-43·27 %) and six showed no response. Seizure day frequency were also significantly lower when dogs were fed the MCTD (1·63/month, 0-7·58/month) in comparison with the placebo diet (1·69/month, 0·33-13·82/month, P=0·022). Consumption of the MCTD also resulted in significant elevation of blood ß-hydroxybutyrate concentrations in comparison with placebo diet (0·071 (sd 0·035) v. 0·053 (sd 0·028) mmol/l, P=0·028). There were no significant changes in serum concentrations of glucose (P=0·903), phenobarbital (P=0·422), potassium bromide (P=0·404) and weight (P=0·300) between diet groups. In conclusion, the data show antiepileptic properties associated with ketogenic diets and provide evidence for the efficacy of the MCTD used in this study as a therapeutic option for epilepsy treatment.

Nonlinear mixed effects modelling approach in investigating phenobarbital pharmacokinetic interactions in epileptic patients.

PURPOSE: The present study aimed to establish population pharmacokinetic model for phenobarbital (PB), examining and quantifying the magnitude of PB interactions with other antiepileptic drugs concomitantly used and to demonstrate its use for individualization of PB dosing regimen in adult epileptic patients. METHODS: In total 205 PB concentrations were obtained during routine clinical monitoring of 136 adult epilepsy patients. PB steady state concentrations were measured by homogeneous enzyme immunoassay. Nonlinear mixed effects modelling (NONMEM) was applied for data analyses and evaluation of the final model. RESULTS: According to the final population model, significant determinant of apparent PB clearance (CL/F) was daily dose of concomitantly given valproic acid (VPA). Typical value of PB CL/F for final model was estimated at 0.314 l/h. Based on the final model, co-therapy with usual VPA dose of 1000 mg/day, resulted in PB CL/F average decrease of about 25 %, while 2000 mg/day leads to an average 50 % decrease in PB CL/F. CONCLUSIONS: Developed population PB model may be used in estimating individual CL/F for adult epileptic patients and could be applied for individualizing dosing regimen taking into account dose-dependent effect of concomitantly given VPA.

Simultaneous extraction and quantification of lamotrigine, phenobarbital, and phenytoin in human plasma and urine samples using solidified floating organic drop microextraction and high-performance liquid chromatography.

A novel and simple method based on solidified floating organic drop microextraction followed by high-performance liquid chromatography with ultraviolet detection has been developed for simultaneous preconcentration and determination of phenobarbital, lamotrigine, and phenytoin in human plasma and urine samples. Factors affecting microextraction efficiency such as the type and volume of the extraction solvent, sample pH, extraction time, stirring rate, extraction temperature, ionic strength, and sample volume were optimized. Under the optimum conditions (i.e. extraction solvent, 1-undecanol (40 µL); sample pH, 8.0; temperature, 25°C; stirring rate, 500 rpm; sample volume, 7 mL; potassium chloride concentration, 5% and extraction time, 50 min), the limits of detection for phenobarbital, lamotrigine, and phenytoin were 1.0, 0.1, and 0.3 µg/L, respectively. Also, the calibration curves for phenobarbital, lamotrigine, and phenytoin were linear in the concentration range of 2.0-300.0, 0.3-200.0, and 1.0-200.0 µg/L, respectively. The relative standard deviations for six replicate extractions and determinations of phenobarbital, lamotrigine, and phenytoin at 50 µg/L level were less than 4.6%. The method was successfully applied to determine phenobarbital, lamotrigine, and phenytoin in plasma and urine samples.

[BIS values were useful on the evaluation of consciousness recovery in acute Vegetamin-A poisoning: report of a case].

A 37-year-old man was admitted to our hospital with acute phenobarbital poisoning. On arrival, he was in deep coma with respiro-circulatory depressions. The serum concentration of the agent was elevated to 149.04 µg/mL which was consistent with a lethal concentration level. He underwent a gastric lavage, administration of activated charcoal, urinary alkalinazation and bowel irrigation. Respiro-circulatory status was recovered rapidly, while the serum concentration of phenobarbital did not decrease smoothly. Although the concentration of the agent decreased to 77.07 µg/mL that should be a comatose level, BIS values were gradually elevated, and then eventually the patient regained his consciousness. Because he was a chronic user of Vegetamin-A containing phenobarbital, the serum level might not have been correlated with symptoms. BIS values were highly reflective of the consciousness level, so it could be a useful indicator for predicting the consciousness levels of patients in deep coma with acute poisoning from hypnotic agents.

The novel antiepileptic drug imepitoin compares favourably to other GABA-mimetic drugs in a seizure threshold model in mice and dogs.

Recently, the imidazolinone derivative imepitoin has been approved for treatment of canine epilepsy. Imepitoin acts as a low-affinity partial agonist at the benzodiazepine (BZD) site of the GABAA receptor and is the first compound with such mechanism that has been developed as an antiepileptic drug (AED). This mechanism offers several advantages compared to full agonists, including less severe adverse effects and a lack of tolerance and dependence liability, which has been demonstrated in rodents, dogs, and nonhuman primates. In clinical trials in epileptic dogs, imepitoin was shown to be an effective and safe AED. Recently, seizures in dogs have been proposed as a translational platform for human therapeutic trials on new epilepsy treatments. In the present study, we compared the anticonvulsant efficacy of imepitoin, phenobarbital and the high-affinity partial BZD agonist abecarnil in the timed i.v. pentylenetetrazole (PTZ) seizure threshold test in dogs and, for comparison, in mice. Furthermore, adverse effects of treatments were compared in both species. All drugs dose-dependently increased the PTZ threshold in both species, but anticonvulsant efficacy was higher in dogs than mice. At the doses selected for this study, imepitoin was slightly less potent than phenobarbital in increasing seizure threshold, but markedly more tolerable in both species. Effective doses of imepitoin in the PTZ seizure model were in the same range as those suppressing spontaneous recurrent seizures in epileptic dogs. The study demonstrates that low-affinity partial agonists at the benzodiazepine site of the GABAA receptor, such as imepitoin, offer advantages as a new category of AEDs.

Pharmacological and neuroethological studies of three antiepileptic drugs in the Genetic Audiogenic Seizure Hamster (GASH:Sal).

Epilepsy modeling is essential for understanding the basic mechanisms of the epileptic process. The Genetic Audiogenic Seizure Hamster (GASH:Sal) exhibits generalized tonic-clonic seizures of genetic origin in response to sound stimulation and is currently being validated as a reliable model of epilepsy. Here, we performed a pharmacological and neuroethological study using well-known and widely used antiepileptic drugs (AEDs), including phenobarbital (PB), valproic acid (VPA), and levetiracetam (LEV). The intraperitoneal administration of PB (5-20mg/kg) and VPA (100-300mg/kg) produced a dose-dependent decrease in GASH:Sal audiogenic seizure severity scores. The administration of LEV (30-100mg/kg) did not produce a clear effect. Phenobarbital showed a short plasmatic life and had a high antiepileptic effect starting at 10mg/kg that was accompanied by ataxia. Valproic acid acted only at high concentrations and was the AED with the most ataxic effects. Levetiracetam at all doses also produced sedation and ataxia side effects. We conclude that the GASH:Sal is a reliable genetic model of epilepsy suitable to evaluate AEDs.

Is switching from brand name to generic formulations of phenobarbital associated with loss of antiepileptic efficacy?: a pharmacokinetic study with two oral formulations (Luminal(®) vet, Phenoleptil(®)) in dogs.

BACKGROUND: In human medicine, adverse outcomes associated with switching between bioequivalent brand name and generic antiepileptic drug products is a subject of concern among clinicians. In veterinary medicine, epilepsy in dogs is usually treated with phenobarbital, either with the standard brand name formulation Luminal(®) or the veterinary products Luminal(®) vet and the generic formulation Phenoleptil(®). Luminal(®) and Luminal(®) vet are identical 100 mg tablet formulations, while Phenoleptil(®) is available in the form of 12.5 and 50 mg tablets. Following approval of Phenoleptil(®) for treatment of canine epilepsy, it was repeatedly reported by clinicians and dog owners that switching from Luminal(®) (human tablets) to Phenoleptil(®) in epileptic dogs, which were controlled by treatment with Luminal(®), induced recurrence of seizures. In the present study, we compared bioavailability of phenobarbital after single dose administration of Luminal(®) vet vs. Phenoleptil(®) with a crossover design in 8 healthy Beagle dogs. Both drugs were administered at a dose of 100 mg/dog, resulting in 8 mg/kg phenobarbital on average. RESULTS: Peak plasma concentrations (Cmax) following Luminal(®) vet vs. Phenoleptil(®) were about the same in most dogs (10.9 ± 0.92 vs. 10.5 ± 0.77 µg/ml), and only one dog showed noticeable lower concentrations after Phenoleptil(®) vs. Luminal(®) vet. Elimination half-life was about 50 h (50.3 ± 3.1 vs. 52.9 ± 2.8 h) without differences between the formulations. The relative bioavailability of the two products (Phenoleptil(®) vs. Luminal(®) vet.) was 0.98 ± 0.031, indicating that both formulations resulted in about the same bioavailability. CONCLUSIONS: Overall, the two formulations did not differ significantly with respect to pharmacokinetic parameters when mean group parameters were compared. Thus, the reasons for the anecdotal reports, if true, that switching from the brand to the generic formulation of phenobarbital may lead to recurrence of seizures are obviously not related to a generally lower bioavailability of the generic formulation, although single dogs may exhibit lower plasma levels after the generic formulation that could be clinically meaningful.

Can we safely administer the recommended dose of phenobarbital in very low birth weight infants?

AIM: We investigated whether the recommended phenobarbital loading dose of 15-20 mg/kg with maintenance of 3-4 mg/kg/day can safely be administered to very low birth weight preterm newborns with seizures. METHODS: Twenty-four convulsive preterms of <1,500 g were enrolled in the study. Phenobarbital was administered intravenously with a loading dose of 15 mg/kg in approximately 10-15 min. After 24 h, the maintenance dose of 3 mg/kg/day was administered as a single injection. Blood samples were obtained 2, 24, 48, 72, and 96 h after the phenobarbital loading dose was administered, immediately before the next phenobarbital dose was injected. RESULTS: None of the cases had plasma phenobarbital concentrations above the therapeutic upper limit of 40 µg/mL on the 2nd hour; one case (4.7%), on the 24th; 11 cases (45.8%), on the 48th; 15 cases (62.5%), on the 72nd; and 17 cases (70.8%), on the 96th hour. A negative correlation was detected between the serum concentrations of phenobarbital and gestational age on the 72th (p, 0.036; r, -0.608) and 96th hour (p, 0.043; r, -0.769). CONCLUSIONS: We suggest that particular attention should be done while administering phenobarbital in preterms, as blood levels of phenobarbital are higher than the reference ranges that those are often reached with the recommended doses in these groups of babies.

Interaction profile of Zizyphus jujuba with phenytoin, phenobarbitone, and carbamazepine in maximal electroshock-induced seizures in rats.

The antiepileptic effect of hydroalcoholic extract of Zizyphus jujuba (HEZJ) in experimental seizures was demonstrated earlier. The present study aimed to evaluate the pharmacokinetic and pharmacodynamic interactions of HEZJ with phenytoin (PHT), phenobarbitone (PB), and carbamazepine (CBZ) in maximal electroshock (MES)-induced seizures in male Wistar rats. Maximal electroshock (70 mA, 9 ms pulse width, 0.2 s) was used to induce seizures. Blood samples were collected at two time points for estimation of serum PHT, PB, and CBZ levels using high-pressure liquid chromatography (HPLC). Co-administration of HEZJ with the sub-therapeutic doses of PHT, PB, and CBZ exhibited 66.7, 66.7, and 50.0% protection against tonic hind limb extension as compared to 33.3, 33.3, and 50% protection respectively, in the groups treated with PHT, PB, and CBZ alone in their sub-therapeutic doses. Co-administration of HEZJ with the sub-therapeutic doses of these antiepileptic drugs (AEDs) showed significant improvement in cognitive functions as compared to MES group as well as these AEDs alone. A significant increase in glutathione levels and decrease in malondialdehyde levels were observed with pretreatment of HEZJ with the sub-therapeutic doses of these AEDs. Co-administration of HEZJ with PHT, PB, and CBZ did not cause any significant changes in the serum concentrations of these AEDs. The results of the present study indicate that the co-administration of HEZJ with sub-therapeutic doses of PHT and PB potentiated the antiepileptic effect of PHT and PB in MES-induced seizures with no change found in the antiepileptic effect of CBZ.

Novel micro-extraction by packed sorbent procedure for the liquid chromatographic analysis of antiepileptic drugs in human plasma and urine.

A method for the simultaneous determination of the antiepileptic drugs, phenobarbital (PHB), phenytoin (PTN), carbamazepine (CBZ), primidone (PRM) and oxcarbazepine (OXC) in human plasma and urine samples by using micro-extraction in a packed syringe as the sample preparation method connected with LC/UV (MEPS/LC/UV) is described. Micro-extraction in a packed syringe (MEPS) is a new miniaturized, solid-phase extraction technique that can be connected online to gas or liquid chromatography without any modifications. In MEPS approximately 1 mg of the solid packing material is inserted into a syringe (100-250 µL) as a plug. Sample preparation takes place on the packed bed. The bed can be coated to provide selective and suitable sampling conditions. The new method is very promising, easy to use, fully automated, inexpensive and quick. The standard curves were obtained within the concentration range 1-500 ng/mL in both plasma and urine samples. The results showed high correlation coefficients (R(2) >0.988) for all of the analytes within the calibration range. The extraction recovery was found to be between 88.56 and 99.38%. The limit of quantification was found to be between 0.132 and 1.956 ng/mL. The precision (RSD) values of quality control samples (QC) had a maximum deviation of 4.9%. A comparison of the detection limits with similar methods indicates high sensitivity of the present method. The method is applied for the analysis of these drugs in real urine and plasma samples of epileptic patients.

One-way multiplexed immunoassay strategy for simultaneous determination of multi-analytes by microchip electrophoresis.

An integrated microchip electrophoresis (MCE) system with online immunoreaction and laser induced fluorescence (LIF) detection has been developed for simultaneous determination of multi-analytes. In this system, the multiplexed immunoreactions between multiple antibody-immobilized glass beads with analytes and respective fluorescently labeled antigens were performed in a sample reservoir. After online incubation, the immunoreaction solution was injected into a one-way separation channel, and free fluorescently labeled antigens were separated and detected in the separation channel. With the help of glass beads, the immunocomplex can not move into the separation channel, which simplifies the separation of fluorescently labeled antigens. With the use of phenobarbital (PB), phenytoin (PHT), carbamazepine (CBZ) and theophylline (Th) as proof-of-principle analytes, the one-way multiplexed immunoassay could be completed within 20 min, resulting in a response curve over the range of 4.0-400 nM for each analyte. Detection limits (S/N = 3) for the drugs tested were in the range of 1.8 × 10(-9) to 2.5 × 10(-9) M. Compared with the conventional immunoassays, this assay is simple, rapid, sensitive and low cost, and provides an accurate procedure for a multiplex immunoassay. The present method has been applied for the simultaneous determination of PB, PHT, CBZ and Th in human serum, which showed a promise of automated clinical application.