Optimization of solid-phase microextraction procedures for the determination of tricyclic antidepressants and anticonvulsants in plasma samples by liquid chromatography.

Simple, sensitive, and reproducible off-line solid-phase microextraction and liquid chromatography (SPME/LC) methods are described for the determination of seven anticonvulsants and tricyclic antidepressants in human plasma. Factorial design and simplex methodology were applied in the optimization of the SPME procedure for tricyclic antidepressants analyses. Important factors in the SPME efficiency are discussed, such as the fiber coatings (both lab-made and commercial), extraction time, pH, ionic strength, influence of plasma proteins, and desorption conditions. The development of the lab-made fiber coatings, namely, octadecylsilane, aminosilane, and polyurethane, are further described and applied to anticonvulsants analyses. The investigated plasmatic range for the evaluated anticonvulsants, using CW-TPR fiber, were the following: phenylethylmalonamide (3.00-40.0 microg mL(-1)), phenobarbital (5.00-40.0 microg mL(-1)), primidone (3.00-40.0 microg mL(-1)), carbamazepine and carbamazepine-epoxide (2.00-24.0 microg mL(-1)), phenytoin (2.00-40.0 microg mL(-1)), and lamotrigine (0.50-12.0 microg mL(-1)). The antidepressants' linear plasmatic concentration ranged from 75.0 to 500 ng mL(-1) for imipramine, amitriptyline, and desipramine, and from 50.0 to 500 ng mL(-1) for nortriptyline, being in all cases, the limit of quantification represented by the lowest value. The precision (interassays) for all investigated drugs in plasma sample spiked with different concentrations of each analyte and submitted to the described procedures were lower than 15%. The off-line SPME/LC methodologies developed allow anticonvulsants and antidepressants analyses from therapeutic to toxic levels for therapeutic drug monitoring.

Simultaneous plasma lamotrigine analysis with carbamazepine, carbamazepine 10,11 epoxide, primidone, phenytoin, phenobarbital, and PEMA by micellar electrokinetic capillary chromatography (MECC).

The determination of lamotrigine (LTG) simultaneously with carbamazepine (CBZ), carbamazepine 10,11 epoxide (CBZ-E), primidone (PRM), phenytoin (PHT), phenobarbital (PB), and 2-phenyl-2-ethyl-malonamide (PEMA) in human plasma was developed using micellar electrokinetic capillary chromatography (MECC) with a diode-array detector. The reproducibility of both separation and quantitation with MECC analysis were appropriate for the intra- and interassay coefficients. The evaluated drugs concentration intervals of LTG, 0.5-10.0 micro g/mL; CBZ, 1.0-16.0 micro g/mL; PEMA, 1.0-20.0 micro g/mL; PB, 1.0-60.0 micro g/mL; PRM, 1.0-20.0 micro g/mL; PHT, 0.7-40.0 micro g/mL; and CBZ-E, 1.0-14.0 micro g/mL were linear with correlation coefficients higher than 0.987 and coefficients of the variation of the points of the calibration curve lower than 10%. The limit of quantitation of the investigated drugs in plasma varied from 0.5 to 1.0 micro g/mL, depending upon the drug. The MECC technique was sensitive enough to work with microsamples into the subtherapeutic, therapeutic, and toxic concentrations, as well as showed to be simple and efficient when applied to monitoring therapeutic drugs in patients treated with a combination of lamotrigine and other antiepileptic drugs such as hepatic enzyme-inducing agents.

Simultaneous determination of primidone and its active metabolites in rat plasma by high-performance liquid chromatography using a solid-phase extraction technique.

Primidone (PRM) and its active metabolites, phenylethylmalonamide (PEMA) and phenobarbital (PB), in rat plasma were simultaneously determined using a solid-phase extraction technique followed by high-performance liquid chromatography (HPLC). Twenty microliters of plasma was applied to a Bond-Elut C-18 cartridge column with 0.1 microgram of acetanilide (internal standard, IS). After the column was washed, PRM, PEMA, PB, and IS were eluted with methanol and injected into the HPLC system. Calibrations for these substances were linear in the range of 0-20 micrograms/mL. The coefficients of variation were 1.5-7.9% and 3.4-9.1% in the within-day and between-day assays, respectively. The recovery rates were 96.8-101.8%. The pharmacokinetics of these substances were examined after oral administration of PRM (50 mg/kg) to rats. The Tmax values for PRM, PEMA, and PB were 1.4, 5.7, and 6.6 h, respectively, and the Cmax values were 18.2, 8.1, and 9.6 micrograms/mL, respectively. This method is useful for pharmacokinetic studies of PRM and its active metabolites.

The disposition of primidone in elderly patients.

1. The pharmacokinetics and metabolism of primidone at steady-state were studied in 10 elderly patients aged 70-81 years and eight control subjects aged 18-26 years. 2. Primidone half-lives and clearance values (mean +/- s.d.) were similar in the elderly and in the young (12.1 +/- 4.6 vs 14.7 +/- 3.5 h and 34.8 +/- 9.0 vs 33.2 +/- 7.2 ml h-1 kg-1 respectively. 3. The serum concentrations of the metabolites phenylethylmalonamide (PEMA) and phenobarbitone relative to those of parent drug were higher in the elderly than in the young, the difference being significant (P less than 0.01) in the case of PEMA. 4. The renal clearances of primidone, phenobarbitone and PEMA were moderately decreased in the elderly but this reduction was statistically significant only for PEMA. Elderly patients excreted a reduced proportion of unchanged primidone and an increased proportion of PEMA in urine. 5. Ageing is associated with a greater accumulation of PEMA, which is unlikely to have a major clinical significance.

Clearance of phenylethylmalonamide during haemodialysis of a patient with renal failure.

Information is presented for the serum concentrations during haemodialysis of primidone, phenobarbitone, and phenylethylmalonamide (PEMA) in a patient with renal failure receiving chronic primidone therapy. The concentrations of drug and metabolites fell during haemodialysis, but PEMA concentrations were above normal at all times. The average renal clearance of PEMA during 6 h of dialysis was found to be 84.7 +/- 4.6 ml min-1.

Primidone and essential tremor.

To clarify whether primidone itself, and not only its metabolite phenobarbitone, suppresses essential tremor, the effect of a high single dose of primidone was tested. Of 11 patients, 8 showed a reduction of their tremor by 54%-69% for up to 28 h. The serum concentration of primidone was as expected, whereas those of the metabolites phenyl-ethyl-malonamide and phenobarbitone were very low. The tremor suppression can thus be considered to be an effect of primidone. Three of the 11 patients did not show a reduction of tremor.

Gas chromatographic analysis of phenylethylmalonamide in human plasma.

A method is described for the analysis of phenylethylmalonamide in human plasma. Analysis of plasma requires only 200 microliter of sample which is extracted with dichloroethane. After filtration and evaporation of the solvent the residue is reconstituted in 50 microliter of chloroform and 5 microliter are injected onto the gas chromatograph. The column used is a mixture of CDMS/WG11 coated on Chromosorb W HP 100-120 mesh. The method is suitable for use in single-dose pharmacokinetic studies.

Phenylethylmalonamide serum levels in patients treated with primidone and the effects of other antiepileptic drugs.

Data are presented for the serum levels of 2-ethyl-2-phenylmalonamide (PEMA) in patients receiving anticonvulsant medication. Statistical analysis of these data indicates that the serum level of PEMA, which is a metabolite of primidone, is affected not only by the dose of primidone but also by the serum levels of other prescribed anticonvulsant drugs. In particular, phenobarbitone is shown to be a major perturbation upon the PEMA serum level.

Anticonvulsant effect of primidone in the gerbil. Time course and significance of the active metabolites.

The anticonvulsant effect of primidone was determined in gerbils, in which seizures were elicited by a blast of compressed air, over the time range of 30 min to 18 h after oral administration. ED50s remained fairly constant from 1 to 12 h after administration: 46-73 mumol/kg with the minimal value at 6 h. Of the metabolites, phenobarbital was maximally effective at 2 h after administration (ED50 35 mumol/kg), whereas phenylethylmalondiamide (PEMA) only had a weak anticonvulsant effect (ED50 1.55 mmol/kg at 2 h). By determination of primidone and its active metabolites in plasma and brain at 1, 4 and 12 h after administration of the respective ED50s, it could be shown that unchanged primidone is mostly responsible for the anticonvulsant effect of the first hours, but, at 12 h, only phenobarbital could be detected in both tissues. PEMA could not be detected in brain. From the effective brain concentrations at different times it could be calculated that primidone and phenobarbital have the same anticonvulsant potency on a molar base in the gerbil. The concentrations necessary to control seizures in this model were considerably lower than those needed to suppress convulsions in maximal seizure models in mice and rats.

Coma and crystalluria: a massive primidone intoxication treated with haemoperfusion.

The present paper describes a patient who was in danger of dying from a massive primidone overdose. She was comatose, hypotensive and in acute renal failure with crystalluria. Because of her clinical condition and high plasma primidone level (209 mg/l) haemoperfusion was instituted. Both the calculated drug clearances and the remarkable improvement in the patient's clinical condition suggest that haemoperfusion was very effective.

Simultaneous high performance liquid-chromatographic determination of carbamazepine, carbamazepine-10,11-epoxide, ethosuximide, phenobarbital, phenytoin, primidone and phenylethylmalonamide in plasma.

A common methodology is reported for the determination of five major anticonvulsants (carbamazepine, ethosuximide, phenobarbital, phenytoin, primidone) and their active metabolites (carbamazepine-10,11-epoxide, phenylethylmalonamide) in 30 microliters of plasma. After a single step of deproteinisation and extraction with acetonitrile, leading to an almost complete recovery of all the analytes, 5 microliters is injected on a reversed-phase column (Lichrosorb RP-18, 5 microns). The anticonvulsants are eluted isocratically at a column temperature of 50 degrees C with a mobile phase consisting of acetonitrile/phosphate buffer p' 6.9 (40/60 by vol), and monitored at 208 nm. Quantitation, using peak height or peak area, is based on the ratio of analyte to internal standard (allylisobutylbarbital) referenced to a serum-based multiple drug standard. The composition and pH of the mobile phase, temperature of the column, choice of wavelength of detection and size of the column material are crucial for the optimal separation of these five drugs and their two active metabolites in a chromatographic time of only 12 min, without sacrificing high sensitivity and column life.

Therapeutic serum concentrations of primidone and its metabolites, phenobarbital and phenylethylmalonamide in epileptic dogs.

Fifteen dogs with idiopathic epilepsy were included in a 9-month clinical trial to determine the therapeutic serum concentrations of primidone and its active metabolites, phenobarbital and phenylethylmalonamide. Dogs with a seizure frequency greater than 1/mo or with a record of multiple seizures greater than 1/day were chosen for the study. Each dog was given primidone 3 times daily at dosages intended to maximize seizure control and to minimize undesired side effects. Maintenance period blood samples were taken from fasted dogs 7 hours after dosing in the 3rd, 5th, 7th, and 9th months of the trial to determine therapeutic serum concentrations of primidone and its metabolites. Two blood samples also were taken from all dogs 7 hours after dosing, during an enforced drowsy period, to establish upper limits of desirable serum concentrations of the drug. Seizure frequencies during the trial were controlled in 13 dogs, 7 of which had no seizures during the 9-month trial. The mean percentage reduction in seizure frequency from pretrial frequency was 85%. Two dogs appeared refractory to primidone therapy. Serum phenobarbital was the best metabolite of primidone to use to assess therapeutic serum concentrations. The therapeutic antiepileptic serum concentration of phenobarbital was found to be between 25 and 40 micrograms/ml of serum. Serum phenobarbital concentrations greater than 40 micrograms/ml resulted in side effects in most dogs.

Pharmacokinetics of phenylethylmalonamide (PEMA) in normal subjects and in patients treated with antiepileptic drugs.

The pharmacokinetics of phenylethylmalonamide (PEMA), a major metabolite of primidone, were investigated following administration of single oral doses (400 mg) to six normal subjects and six patients receiving chronic treatment with antiepileptic drugs. Peak serum PEMA levels were usually attained with 2-4 h after intake. The oral bioavailability estimated on the basis of the recovery of unchanged drug in the urine of normal subjects was at least 80%. Half-life values ranged from 17 to 25 h in normal subjects and from 10 to 23 h in the patients. No statistically significant difference in any of the calculated kinetic parameters could be found between the two groups. The data indicate that PEMA is readily absorbed from the gastrointestinal tract and that it is eliminated predominantly unchanged in the urine of man.

Phenylethylmalonamide in essential tremor. A double-blind controlled study.

A randomised double-blind placebo-controlled trial of phenylethylmalonamide, the major metabolite of primidone was performed in eight patients with essential tremor. Phenylethylmalonamide was given in a daily dose of 400 mg for one week and 800 mg for a second week. The compound had no statistically significant effect on the amplitude of tremor assessed by an accelerometric method, tests of performance, clinical evaluation and patient self assessment. No side effects occurred. Serum levels of phenylethylmalonamide on a daily dose of 400 mg were 11-27 micrograms/ml and on 800 mg daily were 16-48.5 micrograms/ml.

The effects of phenytoin on phenobarbitone and primidone metabolism.

Serum concentrations of primidone and its metabolites-phenobarbitone and phenylethylmalonamide-were measured in 40 epileptic patients receiving treatment with primidone alone or primidone plus phenytoin. Serum phenobarbitone concentrations were also measured in 100 patients receiving phenobarbitone, alone or with phenytoin showed raised serum phenobarbitone concentrations. Phenytoin also caused raised phenylethylmalonamide concentrations in patients on primidone.

Benign familial tremor treated with primidone.

Primidone given to a patient for epilepsy produced an unexpected reduction in benign familial tremor. Over the next eight years the drug was therefore tried in a prospective study of 20 other patients with benign familial tremor alone. Of these, six could not tolerate the drug because of vertigo and nausea but 12 obtained a good response, which in some cases was dramatic. Investigations in two patients suggested that the effect was mediated predominantly by derived phenylethylmalonamide, though primidone had some effect, since tremor recurred slightly on withdrawing the drug despite a constant or rising blood phenylethylmalonamide concentration. Primidone is highly effective in benign familial tremor. More patients with the condition are intolerant of the drug than are usually found with epilepsy.

Monitoring 2-ethyl-2-phenylmalonamide in serum by gas-liquid chromatography: application to retrospective study in epilepsy patients dosed with primidone.

We describe a procedure for determining 2-ethyl-2-phenylmalonamide (I) in serum of epilepsy patients dosed with primidone (II) for seizure control, by extracting the sample with chloroform under basic conditions, with use of an internal standard, 2-ethyl-2-(p-tolyl)malonamide, and without derivative formation. The sensitivity limit is 1.0 mg/L. Within-run CVs for 5, 10, and 30 mg/L concentrations were 3.5, 2.5, and 0.7%, respectively. For a 1.0 mg/kg body wt per day dose of II in patients co-medicated with phenytoin (Group A), the mean steady-state concentrations of I, II, and phenobarbital (III) were 0.72, 0.62, and 2.24 mg/L, respectively. For patients co-medicated with carbamazepine and phenytoin (Group B), I, II, and III concentrations were 0.68, 0.44, and 2.12 mg/L, respectively. Between these groups, only for II were the concentrations statistically different (t = 2.762; p < 0.01). For Group A no correlation was found between II and III. For Groups A and B, the coefficients of correlation between I and III were 0.626 and 0.826, respectively.

Serum concentrations of primidone and its metabolites, phenylethylmalonamide and phenobarbital, in the dog.

The elimination of primidone, phenylethylmalonamide, and phenobarbital (administered IV) was studied in dogs. The elimination half-lives were primidone, 1.85 +/- 0.3 (SEM) hours; phenylethylmalonamide, 7.1 +/- 1.45 hours; and phenobarbital, 40.9 +/- 4.96 hours. Dogs given repeated oral doses of primidone for 14 or 21 days had smaller primidone serum concentrations after each dosing. Dogs given 1.0 g of primidone orally (59.2 decreasing to 50.5 mg/kg of body weight) for 21 days accumulated the phenobarbital metabolite with apparent steady-state concentrations of 10 to 20 micrograms/ml and phenylethylmalonamide in concentrations of 2 to 5 micrograms/ml. Serum primidone concentrations decreased after repeated dosing and were measurable in only 1 dog 24 hours after the 21st dose and peak concentrations of 4 to 7 micrograms/ml were measured at 4 hours after the 22nd dose.