Saliva Phenytoin Research Articles

Phenytoin concentration in people with epilepsy: a comparative study in serum and saliva

In clinical practice, therapeutic drug monitoring (TDM) makes it possible to measure the concentration of drugs in serum or saliva, the purpose of which is to reduce adverse effects and optimize pharmacological therapy. The objective was to determine the concentrations of Phenytoin in saliva and serum of people with epilepsy. Cross-sectional, descriptive study with dynamic recruitment of 30 people with epilepsy (n = 30; 17 men, 56.7% and 13 women, 43.3%; mean age 33.9 ± 11.83 years). Serum and saliva samples were collected at trough levels from patients, who were under phenytoin treatment for at least three months. Drug levels were assessed by the Cloned Donor Enzyme Immunoassay method. Phenytoin levels were found in saliva between 0.01 to 3.56 mg/L and in serum between 0.09 to 36.60 mg/L. Pearson’s analysis showed an association between the estimated serum and saliva phenytoin concentrations (R2 0.7026; 95% CI 0.685-0.921), with a significant statistical correlation (p < 0.05). The Bland-Altman test broke concordance, the difference between the two saliva/serum methods is within 95% confidence. It is concluded that there is an association and concordance between the concentrations of phenytoin in serum and saliva, therefore, this technique can be useful in the clinical monitoring of phenytoin.

Electromembrane extraction‐high‐performance liquid chromatography‐ultraviolet detection of phenobarbital and phenytoin in human plasma, saliva, and urine

AbstractThe current article is focused on introduction and validation of electro‐membrane extraction along with a high‐performance liquid chromatography combined with UV detector for the identification of the antiepileptic drugs phenytoin (PT) and phenobarbital (PB) in plasma, saliva, and urine. In addition, 3D‐printing was utilized to fabricate the designed caps required for fixing the electrodes in the prepared setup, giving us a high potential in device fabrication with various dimensions. To arrive at the highest extraction of PB and PT, the parameters impacting the extraction efficiency of electromembrane extraction for example, supported liquid membrane composition, sample solution pH, applied voltage, extraction time along with effect of graphene oxide are examined in detail. In optimized experimental conditions, the calibration plots were obtained to be linear on the concentration range of 25–500 ng/L with the detection limit and quantitation limit of 7.5 and 25 ng/L for both of PB and PT. Relative standard deviations of <9.9% and <9.1% were found for PB and PT, respectively and the enrichment factor for the presented method was computed to be 25.4 and 44.7 for PB and PT, respectively. The proposed method was satisfactorily employed in determination of PB and PT in plasma, urine and saliva.

Development and Validation of an LC-MS/MS Method and Comparison with a GC-MS Method to Measure Phenytoin in Human Brain Dialysate, Blood, and Saliva.

Phenytoin (PHT) is one of the most often used critical dose drugs, where insufficient or excessive dosing can have severe consequences such as seizures or toxicity. Thus, the monitoring and precise measuring of PHT concentrations in patients is crucial. This study develops and validates an LC-MS/MS method for the measurement of phenytoin concentrations in different body compartments (i.e., human brain dialysate, blood, and saliva) and compares it with a formerly developed GC-MS method that measures PHT in the same biological matrices. The two methods are evaluated and compared based on their analytical performance, appropriateness to analyze human biological samples, including corresponding extraction and cleanup procedures, and their validation according to ISO 17025/FDA Guidance for Industry. The LC-MS/MS method showed a higher performance compared with the GC-MS method. The LC-MS/MS was more sensitive, needed a smaller sample volume (25 µL) and less chemicals, was less time consuming (cleaning up, sample preparation, and analysis), and resulted in a better LOD (<1 ng/mL)/LOQ (10 ng/mL). The calibration curve of the LC-MS/MS method (10–2000 ng/mL) showed linearity over a larger range with correlation coefficients r2 > 0.995 for all tested matrices (blood, saliva, and dialysate). For larger sample numbers as in pharmacokinetic/pharmacodynamic studies and for bedside as well as routine analyses, the LC-MS/MS method offers significant advantages over the GC-MS method.

A Quantitative Phenytoin GC-MS Method and its Validation for Samples from Human ex situ Brain Microdialysis, Blood and Saliva Using Solid-Phase Extraction

This study describes the development and validation of a gas chromatography-mass spectrometry (GC-MS) method to identify and quantitate phenytoin in brain microdialysate, saliva and blood from human samples. A solid-phase extraction (SPE) was performed with a nonpolar C8-SCX column. The eluate was evaporated with nitrogen (50°C) and derivatized with trimethylsulfonium hydroxide before GC-MS analysis. As the internal standard, 5-(p-methylphenyl)-5-phenylhydantoin was used. The MS was run in scan mode and the identification was made with three ion fragment masses. All peaks were identified with MassLib. Spiked phenytoin samples showed recovery after SPE of ≥94%. The calibration curve (phenytoin 50 to 1,200 ng/mL, n = 6, at six concentration levels) showed good linearity and correlation (r² > 0.998). The limit of detection was 15 ng/mL; the limit of quantification was 50 ng/mL. Dried extracted samples were stable within a 15% deviation range for ≥4 weeks at room temperature. The method met International Organization for Standardization standards and was able to detect and quantify phenytoin in different biological matrices and patient samples. The GC-MS method with SPE is specific, sensitive, robust and well reproducible, and is therefore an appropriate candidate for the pharmacokinetic assessment of phenytoin concentrations in different human biological samples.

SPR Imaging‐Based Salivary Diagnostics System for the Detection of Small Molecule Analytes

Saliva is an underused fluid with considerable promise for biomedical testing. Its potential is particularly great for monitoring small-molecule analytes since these are often present in saliva at concentrations that correlate well with their free levels in blood. We describe the development of a prototype diagnostic device for the rapid detection of the antiepileptic drug (AED) phenytoin in saliva. The multicomponent system includes a hand-portable surface plasmon resonance (SPR) imaging instrument and a disposable microfluidic assay card.

Monitoring Phenytoin Therapy Using Citric Acid-Stimulated Saliva in Infants and Children

Two factors have limited the use of saliva in monitoring phenytoin therapy: availability of adequate volume of clear saliva and lack of a sensitive phenytoin assay. The applicability of citric acid-stimulated saliva and of a sensitive analytical assay (fluorescence polarization immunoassay, "TDx" Abbott) was evaluated in this study. Phenytoin was measured in paired plasma-saliva specimens from epileptic children during the long-term or the initial phase of phenytoin therapy. Analysis was carried out in plasma and in the clear supernatant of saliva (following centrifugation). Pooled-estimate SD of the analytical assay variability was 0.175 micrograms/ml for plasma total phenytoin, 0.063 for plasma free phenytoin, and 0.009 for saliva phenytoin. Recovery measurements of phenytoin spiked into saliva samples gave a coefficient of variation of less than 5%. Correlations between saliva and total plasma phenytoin levels and between saliva and free plasma phenytoin levels were strong and highly significant (r = 0.99, p less than 0.01). The percentage of temporal fluctuation (as determined by saliva phenytoin profiles) during 10-24 h ranged between 25.5-177 (mean, 58.3; SD, 47.3). Ratios of plasma total phenytoin/saliva phenytoin and of plasma free phenytoin/saliva phenytoin levels were 9.54 +/- 1.05 and 0.71 +/- 0.09, respectively. Dialysis experiments showed no binding of phenytoin to saliva supernatant. The greater saliva phenytoin concentrations as compared to plasma free phenytoin concentrations could be due to active transport of phenytoin from plasma to saliva. Measurement of phenytoin in citric acid-stimulated saliva by fluorescent polarization immunoassay is a reliable, noninvasive, and convenient method for monitoring phenytoin therapy in children.

Phenytoin kinetics during pregnancy and the puerperium

During pregnancy changes in maternal physiology and plasma composition may alter drug binding and dose requirements. We have measured plasma unbound and total phenytoin, and saliva concentrations at intervals in 11 pregnant epileptics. Plasma albumin concentrations were also measured in pregnant and non-pregnant women. Saliva phenytoin correlated closely with the plasma unbound concentrations (r = 0.98). The saliva:plasma (S:P) ratio, reflecting the free fraction, was variable during pregnancy but tended to increase to maximal values at delivery and return to non-pregnant values within 2-8 weeks thereafter. Plasma albumin concentrations correlated poorly with phenytoin binding. Binding in umbilical cord plasma appeared higher than that in maternal plasma and total fetal concentrations correlated closely with maternal plasma concentrations at delivery. No ill effects of phenytoin were detected in the newborn infant. During the third trimester phenytoin dose increments were necessary to maintain therapeutic concentrations. After delivery maternal saliva phenytoin concentrations rose, and dose reductions were necessary to avoid clinical symptoms of toxicity. It is therefore appropriate to monitor saliva phenytoin concentrations regularly both during pregnancy and the puerperium.

Plasma concentrations of unbound phenytoin in the management of epilepsy.

In 46 epileptic patients the range of the unbound fraction of phenytoin in plasma measured by ultrafiltration (at 37 degrees C) and tracer-labelling with [14C]-phenytoin was 6.7%-33.3% with a median of 11.9%. The total and unbound phenytoin plasma concentrations were significantly correlated (r = 0.93, P less than 0.001), but in six patients the unbound concentration fell on or outside the 90% predictability limits for a single value. In all patients the unbound concentration reflected the clinical status of the patient equally or better than the total concentration. An inverse relationship was found between the plasma albumin concentration (within the normal reference range) and the phenytoin unbound fraction (r = -0.83, P less than 0.001) indicating that plasma albumin concentration is one of the important overall determinants of phenytoin protein binding. Saliva and plasma unbound phenytoin concentrations were significantly correlated (r = 0.98, P less than 0.001) but both collection of plasma samples and preparation of plasma ultrafiltrate using the Amicon micropartition system are simpler than collection and processing saliva, and interpretation of plasma unbound concentration does not require allowance for potential contamination. The additional value of the unbound phenytoin concentration in a clinically significant number of individuals would justify routine measurement of unbound phenytoin concentration in monitoring therapy, once available simplified methodology has been adequately characterised.

Phenytoin induced gingival overgrowth in institutionalized epileptics.

In a cross-sectional, epidemiological study of phenytoin induced gingival overgrowth in 77 institutionalized persons with epilepsy, the severity of the gum lesions was quantified by means of a precise new technique. Lesion severity was then compared statistically to other clinical and laboratory parameters. Positive correlations were detected between overgrowth severity and gingival inflammation, probing depths, calculus accumulation, plaque score and the measurement gingival margin to mucogingival junction (GM-MGJ). No correlation was observed between lesion severity and patients age, daily drug dosage, plasma or saliva phenytoin level, or salivary concentration of the major phenytoin metabolite.

The place of saliva in antiepileptic drug monitoring.

It has previously been shown that saliva phenytoin concentration bears a constant relationship to plasma free concentration whether protein binding of phenytoin is normal or disturbed by other drugs, pregnancy, renal failure, or hypoalbuminaemia. The present work examines the relationship between saliva (S), plasma free (F), and plasma total (P) concentrations of other anticonvulsants in 100 epileptic patients. Mean S/P ratios were for phenobarbitone 0.37 (r = 0.95), primidone 0.95 (r = 0.87), and carbamazepine 0.27 (r = 0.94). A highly significant correlation of S with F was found for these drugs, more significant than the correlation of S with P for carbamazepine in patients receiving multiple anticonvulsant drugs. Saliva valproate, however, had no predictive value for P or F. No binding to saliva proteins was demonstrated for any drug. Data for in vitro binding to plasma proteins was in good agreement with ex vivo data. Saliva is therefore a valid medium for monitoring treatment with phenobarbitone, primidone, and carbamazepine, as well as phenytoin.

Salivary phenytoin levels in institutionalized epileptics

Measurement of phenytoin (PHT) levels in saliva has been advocated as a monitor of plasma levels, and therefore of effective dosage. All studies to date have been performed on outpatients. We have now analyzed whole saliva from 62 mentally retarded, institutionalized, PHT-treated persons with epilepsy. Both PHT and its major human metabolite, 5-( para-hydroxyphenyl)-5-phenylhydantoin ( pHPPH) were measured using gas-liquid chromatography. We detected large inter-individual variations in saliva PHT and pHPPH levels. Data were ranked and subjected to statistical analysis according to Spearman. Significant positive correlations were detected between daily PHT dose and plasma total PHT concentration, plasma PHT and salivary PHT, and daily dose and salivary PHT level. When data were categorized and analyzed according to the type of medication being administered in addition to PHT, different results were obtained. Polypharmacy can influence drug and metabolite levels and relationships in institutionalized persons with epilepsy.

Phenytoin-valproate interaction: importance of saliva monitoring in epilepsy.

Sodium valproate is often used with phenytoin when epilepsy cannot be controlled by a single drug. Sodium valproate depresses phenytoin protein binding and so invalidates plasma phenytoin monitoring as a means of determining precise phenytoin dosage requirements. Plasma and saliva phenytoin and plasma valproate concentrations were measured in 42 patients with epilepsy receiving both drugs. Phenytoin protein binding was also measured by ultrafiltration in 19 of these patients and 19 patients taking phenytoin alone. Saliva phenytoin concentration bore the same close correlation to unbound (therapeutically active) phenytoin in patients receiving both drugs as it did in patients receiving phenytoin alone, whereas plasma total phenytoin did not. The same therapeutic range for saliva phenytoin (4-9 mumol/1; 1-2 microgram/ml) was therefore valid in both groups. The depression of phenytoin binding was directly related to the plasma concentration of valproate both in random samples taken from the 42 patients and in samples taken throughout the day in two of these patients. This was confirmed in vitro. Even when the concentration of valproate is known the degree of binding cannot be predicted. Saliva rather than plasma monitoring of phenytoin treatment is therefore valuable in the presence of valproate and with reduced phenytoin binding from any cause.

Determination of Antiepileptic Drugs in Body Fluid by a Competitive Binding Enzyme Immunoassay and Clinical Application

A competitive binding enzyme immunoassay (MARKIT) procedure for determination of the concentration of antiepileptic drugs was assessed and compared with gas-liquid chromatographic (GLC) procedures. Furthermore the Possibility of antiepileptic drugs determination in serum ultrafiltrate and mixed saliva was investigated with MARKIT. The two methods of MARKIT and GLC gave essentially the same results in determination of the serum levels of phenobarbital. The correlation coefficient of MARKIT between GLC was as high as 0.922. The recovery of the phenytoin in serum, serum ultrafiltrate and saliva were 98.1±1.9, 97.5±2.9 and 72.5±3.6%, respectively. The correlation coefficients between serum and serum ultrafiltrate and serum and saliva were 0.926 and 0.654, respectively. These data suggest that MARKIT is a reliable method for the determination of the concentration of antiepileptic drugs in serum, serum ultrafiltrate and saliva.

Intravenous valproate

Plasma and saliva phenytoin was evaluated at fixed-time intervals after intravenous (IV) administration of 800 mg valproate in epileptics chronically treated with phenytoin monotherapy. A significant fall in plasma and a concurrent significant increase of the drug in saliva (which reflects the free amount in plasma) were observed, with maximal changes occurring at 30 to 90 minutes after valproate infusion. Present data indicate that valproic acid is able to displace phenytoin from plasma proteins, suggesting a critical evaluation of the routine phenytoin plasma proteins, suggesting a critical evaluation of the routine phenytoin plasma levels (which reflect free + bound fraction) when this drug is administered concurrently with sodium valproate.

Comparison of Concentrations between Antiepileptic Drugs in Saliva Determined by EMIT and MARKIT Methods

The concentrations of phenytoin, phenobarbital and prlmidone in plasma and saliva were determined. The result obtained by MARKIT method showed a good agreement with that by EMIT method, with the exception of the determination of low concentration of phenytoin in saliva. However, when the phenytoin concentration in plasma was above 2.0μg/ ml, it was possible to determine salivary phenytoin in low concentration by adding one of the following operations to EMIT method: 1) concentration determined by CP 1000 and then calculated from the half-dose standard curve of DPH-calibrators diluted by DPH negative human serum; 2) concentration determined by CP 5000 in similar test method of plasma sample. In both operations, the salivary dilution omitted from EMIT method.

Phenytoin and Phenobarbital Levels in Saliva Measured by Homogeneous Enzyme Immunoassay

Phenytoin and Phenobarbital Levels in Saliva Measured by Homogeneous Enzyme Immunoassay

Compliance with anticonvulsant therapy in a hospital clinic and in the community.

1 Seizure control, saliva anticonvulsant concentration, prescribing habits and compliance with anticonvulsant medication have been compared in 86 epileptic subjects attending either a specialist hospital clinic or general practice surgeries. 2 Of all subjects experiencing recurrent seizures 70% had saliva concentrations of phenytoin below the range equivalent to the 'therapeutic range' of plasma concentration. Mean saliva phenytoin concentrations did not differ significantly between the two treatment settings and were low largely because of the low mean dosage prescribed. 3 Eleven subjects in all had no detectable phenytoin in their and could clearly be identified as noncompliant. Freedom from seizures appeared to predispose to poor compliance in these subjects as well as among those admitting repeated omission of doses.

Phenytoin concentrations in mixed, parotid and submandibular saliva and serum measured by radioimmunoassay.

1 Concentrations of phenytoin in mixed, parotid and submandibular saliva and serum were determined in normal subjects after an oral dose, using a specific double antibody radioimmunoassay which requires only 20 micronl fluid. 2 Semi-log concentration-time plots of phenytoin concentration in mixed saliva and serum gave good parallelism after the initial 14 h post-administration period. 3 The mean ratio of the mixed saliva: serum phenytoin concentration was 10.3% +/- 1.5 (s.d.) in seven normal subjects. 4 Phenytoin concentrations found in separate parotid and submandibular salivary fractions did not differ but were significantly greater (P less than 0.001) than those found in mixed saliva. 5 Phenytoin concentrations in all salivary fractions were independent of the volume of fluid produced and the degree of stimulation. 6 The rate of phenytoin secretion in the parotid and submandibular fluid was proportional to the salivary flow rate. 7 These data suggest that mixed saliva may be a suitable medium for the monitoring of phenytoin concentrations and may provide a non-invasive alternative to the direct determination of phenytoin in serum.

Use of saliva in therapeutic drug monitoring.

We measured the concentrations of phenobarbital, phenytoin, primidone, ethosuximide, antipyrine, and caffeine in paired samples of saliva and plasma by gas chromatograph-mass spectrometer-computer (GC/MS/COM) and enzyme immunoassay. Mixed saliva was collected for the antipyrine and caffeine studies, parotid saliva for the phenobarbital, primidone, ethosuximide and phenytoin studies. The saliva/plasma (S/P) ratios (by weight) obtained by GC/MS/COM were: phenobarbital, 0.31-0.37; phenytoin, 0.11; ethosuximide, 1.04; antipyrine, 0.83-0.95; caffeine, 0.55. The S/P ratio obtained by enzyme immunoassay were: phenobarbital, 0.32; phenytoin, 0.12; primidone, 0.85. The concentrations of phenytoin, primidone, ethosuximide and antipyrine in saliva correspond to the free fraction of the drug in plasma. When we analyzed samples containing phenobarbital or phenytoin (plasma or saliva) by both techniques, we found that the enzyme immunoassay values were generally higher than GC/MS/COM values, suggesting that the metabolites as well as the parent drug were measured in the immunoassay.

Salivary phenytoin radioimmunoassay. A simple method of the assessment of non-protein bound drug concentrations.

A simple, specific and rapid radioimmunoassay (RIA) method for the assessment of non-protein bound ('free') phenytoin (DPH) concentrations in mixed saliva is described. Epileptic patients on maintenance phenytoin therapy have mixed saliva phenytoin concentrations similar to 'free' drug levels measured directly in serum or cerebrospinal fluid (CSF), Salivary phenytoin levels are approximately 10% of the total serum level in treated epileptic patients and in normal subjects after ingestion of a single oral dose. The half time of disappearance of phenytoin after 100 or 300 mg doses is 12.2 +/- 3.0 (SD) h in serum and 12.3 +/- 3.2 (SD) h in saliva. This method of assessing the biologically active fraction of the drug may be particularly valuable in situations where serum protein binding is abnormal or in drug interactions. It is also non-invasive and requires small sample volumes (20mul) and may therefore be valuable in paediatric practice and in pharmacokinetic studies in which multiple venepunctures would otherwise be required.