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

OBJECTIVES: Primidone is an anticonvulsive drug used in the treatment of epilepsy and essential tremor. It offers beneficial effects in controlling seizures, but its usage is also associated with possible side effects. To ensure optimal therapy, it is crucial to measure its concentration through accurate quantification methods. Therefore, our main goal was to develop and validate a new reference measurement procedure (RMP) for accurately measuring primidone levels in human serum and plasma. METHODS: In our study, we focused on the separation of primidone from both known and unknown interferences using a C18 column. To achieve accurate sample preparation, we developed a protocol involving protein precipitation followed by a high dilution step. The validation of the assay and determination of measurement uncertainty were carried out following guidelines from organizations such as the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the Expression of Uncertainty in Measurement. These rigorous validation processes ensure the reliability and accuracy of our method for quantifying primidone levels in human serum and plasma samples. RESULTS: The RMP was shown to be highly selective and specific, with no evidence of matrix interference. It can be used to quantify primidone in the range of 0.150-30.0 µg/mL. Intermediate precision was less than 4.0 %, and repeatability CV ranged from 1.0 to 3.3 % across all concentration levels. The relative mean bias ranged from 0.1 to 3.9 % for native serum levels, and from -2.6 to 2.8 % for lithium-heparin plasma levels. The measurement uncertainties for single measurements and target value assignment were 1.5-4.1 % and 0.9-1.0 %, respectively. CONCLUSIONS: In this study, we introduce an innovative LC-MS/MS-based candidate RMP specifically designed for primidone in human serum and plasma. Our RMP offers a traceable platform, facilitating the standardization of routine assays and enabling the evaluation of clinically relevant samples. With this novel approach, we aim to enhance the accuracy and reliability of primidone measurements, ultimately benefiting the field of clinical research and patient care.

Therapeutic drug monitoring of anti-epileptic drugs - a clinical verification of volumetric absorptive micro sampling.

Background Therapeutic drug monitoring (TDM) of antiepileptic drugs (AEDs) can serve as a valuable tool in optimising and individualising epilepsy treatment, especially in vulnerable groups such as pregnant women, the elderly and children. Unfortunately, TDM is often performed suboptimally due to limitations in blood collection. Therefore, we investigated volumetric absorptive micro sampling (VAMS) - a new home-sampling technique. We aimed to evaluate VAMS to determine and quantify the different AEDs and concentrations of 16 different AEDs in whole blood collected by VAMS. Methods Patient blood samples (n = 138) were collected via venepunctures at the Academic Centre for Epileptology Kempenhaeghe. AED concentrations were determined, and these concentrations were used to compare the VAMS method (whole blood) with the conventional method (serum). In addition, the recovery was examined as well as the impact of haematocrit. Finally, AED-spiked blood was used to test the stability of the AEDs inside the micro-sampler devices over a period of time and whether temperature had an effect on the stability. Results VAMS allows for an accurate detection of 16 different AEDs within 2 days after sampling. Deviation in recovery was less than 10% and high correlations were found between VAMS and conventional sampling. Moreover, haematocrit does not have an effect with values between 0.3 and 0.5 (L/L). Finally, although storage temperature of VAMS does affect some AEDs, most are unaffected. Conclusions VAMS enables an accurate detection of a wide variety of AEDs within 2 days after sampling.

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.

Robust isocratic high performance liquid chromatographic method for simultaneous determination of seven antiepileptic drugs including lamotrigine, oxcarbazepine and zonisamide in serum after solid-phase extraction.

A rapid, simple and robust method is presented for the simultaneous determination of seven antiepileptic drugs (AEDs), including primidone, phenobarbital, phenytoin, carbamazepine with its two major metabolites carbamazepine-10,11-epoxide and carbamazepine-10,11-(trans)-dihydrodiol and the new AEDs lamotrigine, hydroxycarbazepine (active metabolite of oxcarbazepine) and zonisamide in serum by high performance liquid chromatography (HPLC)-diode array detector (DAD). After solid-phase extraction, separation is achieved on an Alltima 3C18 analytical column using isocratic elution with a mixture of acetonitrile, methanol and phosphate buffer at 45 degrees C. The method is exhaustively validated, including experimental design in combination with statistical evaluation (ANOVA) to study the robustness of chromatography and sample preparation. Commonly co-administered antiepileptic drugs do not interfere with the method. Intra-day precision (RSD<1.9%), linearity, lower limit of quantitation (LOQ<0.065 mg/l) and robustness make the method suitable for daily therapeutic drug monitoring and pharmacokinetic studies.

Therapeutic monitoring of antiepileptic drugs for epilepsy.

BACKGROUND: The aim of drug treatment for epilepsy is to prevent seizures without causing adverse effects. To achieve this, drug dosages need to be individualised. Measuring antiepileptic drug levels in body fluids (therapeutic drug monitoring) is frequently used to optimise drug dosage for individual patients. OBJECTIVES: To review the evidence regarding the effects of therapeutic drug monitoring upon outcomes in epilepsy. SEARCH STRATEGY: We searched the Cochrane Epilepsy Group Specialised Register (September 2006), the Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library 2005, Issue 4), MEDLINE (January 1966 to April 2005) and EMBASE (1974 to May 2005). No language restrictions were imposed. We checked the reference lists of retrieved articles for additional reports of relevant studies. SELECTION CRITERIA: Randomised controlled trials comparing the outcomes of antiepileptic drug monotherapy guided by therapeutic drug monitoring with drug treatment without the aid of therapeutic drug monitoring. DATA COLLECTION AND ANALYSIS: We based this review on published aggregate data. The main outcomes measured were the proportions of patients achieving a 12-month remission from seizures, reporting adverse effects, and being withdrawn from the treatment they had been randomised to receive. MAIN RESULTS: Only one study met the inclusion criteria for the review. In this open study, 180 patients with newly-diagnosed, untreated epilepsy were randomised to treatment with the antiepileptic drug selected by their physician either with or without therapeutic drug serum level monitoring as an aid to dosage adjustments. The antiepileptic drugs used were carbamazepine, valproate, phenytoin, phenobarbital and primidone. A 12-month remission from seizures was achieved by 60% of the patients randomised to therapeutic drug monitoring (intervention group) and by 61% in the control group. A total of 56% in the intervention group and 58% in the control group were seizure free during the last 12 months of follow up. Adverse effects were reported by 48% in the intervention group and 47% of the control group patients. Of those randomised to therapeutic drug monitoring, 62% completed the two-year follow up compared with 67% of the control group. AUTHORS' CONCLUSIONS: We found no clear evidence to support routine antiepileptic drug serum concentration measurement with the aim of reaching predefined target ranges for the optimisation of treatment of patients with newly-diagnosed epilepsy with antiepileptic drug monotherapy. However, this does not exclude the possible usefulness of therapeutic drug monitoring of specific antiepileptic drugs during polytherapy, in special situations or in selected patients, although evidence is lacking.

Characterization and application of restricted access carbon nanotubes in online extraction of anticonvulsant drugs from plasma samples followed by liquid chromatography analysis.

Anticonvulsant drugs are often used in the treatment of epilepsy. However, their therapeutic monitoring is often necessary in order to obtain an appropriate dose adjustment, due to the proximity between their therapeutic and toxic ranges. The aim of this study was to carry out the synthesis, characterization and use of restricted access carbon nanotubes (RACNTs) in an online method for the analyses of phenobarbital and carbamazepine and primidone from untreated human blood plasma by column switching liquid chromatography. Therefore, the synthesis of RACNTs was carried out through coating commercial Carbon nanotubes with bovine serum albumin (BSA) to subsequently use them as adsorbents in a column switching system operating in the backflush mode. This material was evaluated through the construction of the kinetic and isotherm curves. The experimental data for the interaction of primidone with RACNTs were adequately adjusted to the chemisorption and Sips models for the kinetic and adsorption studies, respectively. The analytical curves ranged from 2.0 to 40.0mgL-1, with correlation coefficients higher than 0.99, for all the analytes. The LODs of 0.1, 0.1 and 0.01µgmL-1 were defined for PHB, PRM and CBZ, respectively. The relative standard deviation values ranged from 1.0% to 8.4% for the intra assay precision and from 2.7% to 7.6% for inter assay precision. The relative error values ranged from -13.4% to 7.7% for the intra assay accuracy and from -8.6% to 2.5% for the inter assay accuracy. The method was adequately used in the therapeutic monitoring of anticonvulsant drugs in human plasma samples.

[Rapid analysis of antiepileptic drugs in human plasma by micellar electrokinetic capillary chromatography].

AIM: To develop a rapid and feasible method based on micellar electrokinetic capillary chromatography (MECC) for the simultaneous determination of antiepileptic drugs (AEDs)--phenytoin (PHT), phenobarbital (PB), carbamazepine (CBZ), primidone (PRM) and clonazepam (CZP) in human plasma. METHODS: Several factors that impact the separation of AEDs with MECC were investigated, such as concentration of sodium dodecyl sulfate (SDS), buffer compositions, pH, organic modifier, internal diameter and temperature, and an optimized MECC running condition was obtained the running buffer consisted of 8 mmol x L(-1) phosphate, 3 mmol x L(-1) sodium tetraborate, and 50 mmol x L(-1) sodium dodecylsulfate (SDS) (pH 8.0), containing acetonitrile (ACN) (18%) as organic modifier. Detection at 210 nm, run at 25 kV at 30 degrees C in a untreated fused silica capillary (50/45.5 cm length, 50 microm ID). RESULTS: The reproducibility of both migration time and relative peak area with MECC analysis were appropriate for the intra- and inter-assay coefficients. The evaluated drugs concentration intervals of PRM 1.0-40.0 microg x mL(-1), PB 1.0-60.0 microg x mL(-1), PHT 1.0-40.0 microg x mL(-1), CBZ 1.0-40.0 microg x mL(-1), CZP 0.2-8.0 microg x mL(-1) were linear with correlation coefficients higher than 0.999 1, and coefficients of the variation of the points of the calibration curve lower than 10%. The recoveries of AEDs varied from 80.0% to 100.0%, depending on the drug, with coefficients of the variation lower than 10.0%. CONCLUSION: The MECC technique is showed to be rapid, simple, efficient and low cost when applied to monitoring therapeutic drugs in patient treated with a combination of PHT and other AEDs such as hepatic enzyme-inducing agents.

Pharmacokinetics of antiepileptic drugs.

The rational use of antiepileptic drugs requires the consideration of their pharmacokinetics, which may be influenced by the physiological and pathological factors. Pharmacokinetic interactions between antiepileptic drugs may lead to considerable fluctuation in plasma drug concentration, and monotherapy is often preferable. The absorption of phenytoin depends on pharmaceutical formulation. Phenytoin is highly bound to plasma proteins, thus the changes in the unbound fraction are of clinical significance. The saturation kinetics of its metabolism and drug interactions have further consequences. Carbamazepine is well absorbed and largely metabolized. Due to the autoinduction its half-life shortens in chronic administration. Valproate is highly, but variably bound to plasma proteins. It is eliminated mainly by metabolism. Due to the long half-life of phenobarbital its plasma concentrations change slowly, and time to the steady-state may be up to 30 days, if no loading dose is given. Primidone is partly metabolized to phenobarbital, and at steady-state plasma concentration of phenobarbital often exceeds that of primidone. Diazepam, clonazepam and nitrazepam are largely bound to plasma proteins and extensively metabolized with the half-lives of 20 to 60 hours.

Interlaboratory variability in determination of plasma antiepileptic drug concentrations.

The usefulness of plasma antiepileptic drug concentrations in treatment of epilepsy has been established, and many laboratories provide this service. A "blind" survey utilizing pooled patient plasma samples was conducted among 197 laboratories in the United States and Canada to establish the interlaboratory reproducibility. Three "patient specimens" containing different amounts of phenobarbital, phenytoin (diphenylhydantoin), primidone, and ethosuximide were employed; 112 laboratories reported results within five weeks. The average cost for analyzing four drugs in a single sample was $43.27. Half of the laboratories reported results outside +/- 1 standard deviation of the mean of five reference laboratories. Wide interlaboratory variability must be considered by the practicing physician. Until certified antiepileptic drug standards in a biologic matrix are available from the National Bureau of Standards, a volunteer quality control program among laboratories is needed.

Acetazolamide-induced interference with primidone absorption. Case reports and metabolic studies.

Effects of acetazolamide on primidone plasma levels were studied in three patients. Apparent interaction occurred in two patients. Primidone was not detected in the plasma when given orally with acetazolamide in one patient. In another, peak serum concentration was delayed, with corresponding delays in urinary excretion of primidone and metabolites. Plasma and urine concentrations of the two metabolites, phenylethylmalonamide and phenobarbital, were also studied.

Fluorescent immunoassay for determining antiepileptic drug concentrations: clinical usefulness.

The need for rapid and accurate antiepileptic drug measurement in blood is well established. A substrate-labeled fluorescent immunoassay (FIA) has been developed that can measure phenobarbital, phenytoin, primidone, and carbamazepine in serum. To our knowledge, the primidone and carbamazepine assays have not previously been tested in a field trial. We compared FIA and the well-established antiepileptic drug immunoassay technique EMIT for the quantitation of both carbamazepine and primidone. In our hands, the FIA method compared favorably with the EMIT method for accuracy and reliability but is somewhat more time consuming. This method has the advantage of being more sensitive, however, and requires only a finger-stick blood sample. Because of this and the simplicity of the equipment required, the FIA system should also be relatively inexpensive to set up and to operate.

Seizure activity and anticonvulsant drug concentration.

Current methods permit frequent, accurate serum anticonvulsant drug concentration measurements and continuous, 24-hour electroencephalographic recording with minimal environmental restriction. These techniques were used to perform longitudinal, 24-hour recordings of electroencephalographic paroxysmal activity and sleep-wake state concurrently with frequent measurements of serum anticonvulsant drug concentrations in two patients with poorly controlled convulsions. Drug administration was designed with the intent of producing high serum concentrations at times of maximum electroencephalographic paroxysmal activity. The suppression of clinical seizures coincided with decreased numbers of paroxysmal bursts in the electroencephalogram and increased serum anticonvulsant drug concentration.

Efficacy of primidone in essential tremor.

Primidone, 50 to 1,000 mg/d, reduced the amplitude of essential tremor in both untreated and propranolol-treated patients. Low doses were as effective as high doses. Primidone decreased tremor more than propranolol. There was no correlation between therapeutic response and serum levels. Acute reactions to the initial dose and side effects of higher doses caused drug intolerance. A single oral dose (250 mg) decreased tremor by 60% 1 to 7 hours after ingestion, with stable serum primidone levels but no detectable phenobarbital levels. Tremor control was lost when phenobarbital was substituted for primidone. Primidone is an effective agent for the treatment of essential tremor.

Double-blind comparison of primidone and phenobarbital in essential tremor.

In a double-blind cross-over trial, primidone was superior to both placebo and phenobarbital in reducing essential tremor in 13 patients. Phenobarbital, at a dosage yielding serum barbiturate levels greater than those seen with primidone, was not better than placebo. Thus, primidone has an effect in essential tremor independent from that of its metabolite phenobarbital.

Isoniazid as an inhibitor of primidone metabolism.

Isoniazid inhibited the metabolism of primidone in a patient with focal seizures. The steady-state serum level of primidone rose when the patient received both drugs simultaneoulsy. The serum levels of the primidone metabolites, phenobarbital and phenylethylmalonamide, fell and the rate of metabolism of primidone decreased. The results are similar to those observed when isoniazid is adminstered with diphenylhydantoin.

Changes in primidone/phenobarbitone ratio during pregnancy and the puerperium.

Plasma concentrations of primidone and its metabolite phenobarbitone were monitored in 9 pregnant epileptic patients treated with primidone (and in 3 cases other antiepileptic drugs) given at constant doses throughout pregnancy and the puerperium. Phenobarbitone plasma concentrations were monitored in another 6 patients given phenobarbitone itself. A trend towards increasing primidone plasma concentrations during the second quarter of pregnancy was evident in all patients, with a concomitant significant decrease in primidone-derived phenobarbitone plasma concentrations. A trend towards a lowering of plasma concentrations of phenobarbitone administered as such was confirmed. These results suggest the usefulness of a careful monitoring of primidone and primadone-derived phenobarbitone during pregnancy and the puerperium. Discrepancies of findings with primidone and phenobarbitone are discussed in view of the possible mechanism involved.

Rapid centrifugal analyzer enzyme immunoassays for phenytoin, phenobarbital, and primidone.

The adaptation of the homogeneous enzyme immunoassays (EMIT) for phenytoin, phenobarbital, and primidone to a centrifugal analyzer is described. The sample volume required was 10 microliter, and the assay had the capacity to analyze sera from 28 patients within 180 sec. The assay temperature was 30 C, and absorbance was monitored at 340 nm. Coefficients of variation for within-day precision ranged from 2.1% to 3.7%, and analytic recovery was quantitative. The centrifugal analyzer EMIT assay results correlated well with those obtained using high-pressure liquid and gas-liquid chromatographic technics. A logit-log transformation of the absorbance rate versus concentration data was obtained using a modified Gauss-Newtonian nonlinear least-squares fit analysis. Severe hemolysis and lipemia caused interference.

Emit assays for five major anticonvulsant drugs. An evaluation of adaptations to two discrete kinetic analyzers.

Because of the complex reaction kinetics observed with the enzyme multiplied immunoassay technic (EMIT), each adaptation of these assays must be specifically evaluated. Evaluations were done of the totally automated adaptations of these immunoassays for diphenylhydantoin, phenobarbital, primidone, carbamazepine, and ethosuximide to two kinetic analyzers that use markedly different reaction conditions, the Gilford System 3500 and the Abbott Bichromatic Analyzer-100. Comparisons of data from EMIT assays on the Gilford 3500 with data from EMIT assays on the ABA-100 and with data from gas-liquid chromatographic analyses for the five anticonvulsant drugs showed good correlations (r greater than or equal to 0.96) and little or no additive or proportional biases, as determined by joint confidence intervals. Between-run precision for each EMIT anticonvulsant drug assay on both analyzers was less than 6%, better than that obtained with gas chromatography. No interference by more than 40 different drugs, by hemoglobin (6 g/l), or by bilirubin (200 mg/l), and no or minimal cross-reactivity with any of the five EMIT anticonvulsant drug assays on either analyzer were found.

Acute phenytoin and primidone intoxication: a pharmacokinetic analysis.

Serial plasma levels of phenytoin, primidone, and phenobarbital were determined in a patient following massive overdose of phenytoin and primidone. The patient's neurologic status improved slowly over a period of 10 days and correlated best with the rise and fall of phenytoin plasma concentrations. The pharmacokinetics of all three agents were characterized by nonlinear regression analysis of their respective plasma concentration-time profiles during the elimination phase, followed by analog computer simulations of their entire plasma concentration-time profile closely resembled the observed values. Average values of Km and Vmax obtained from patients undergoing chronic therapy were used in the simulation and appear to adequately describe phenytoin elimination in this overdose situation. The elimination half-lives of primidone and phenobarbital of 6.2 and 83.5 hours, respectively, were within the "normal range" for patients on chronic therapy. Two distinct absorption phases for primidone and three for phenytoin were noted. The marked decrease in the estimated absorption rate constant between phases 1 and 2 for each drug may have been due to slow dissolution of a large congealed mass of phenytoin and primidone in the gut. The analysis of serial plasma samples following a massive overdose is recommended to provide a reliable data base for therapeutic decisions.

A rapid gas liquid chromatography method for simultaneous determination of various anticonvulsants.

The purpose of this study was to develop a rapid GLC method for the simultaneous determination of the following anticonvulsants: Carbamazepine, Diphenylhydantoin, Phenobarbital, and Primidone. Using a metal column (OV-17) with flash heater methylation as well as high N2 flow-rate, a quantitative methylation of the above anticonvulsants was obtained. A selective temperature program proved to be the decisive factor in separating the various methylated anticonvulsants. The GLC program was completed within 13 min. This method permits a rapid, simultanous and quantitative determination of various important anticonvulsants.