Human Serum Albumin-Occupying-Based Fluorescence Turn-On Analysis of Antiepileptic Drug Tiagabine Hydrochloride.

Tiagabine hydrochloride (TGB) is a clinically frequently used drug for anticonvulsion and reducing epileptic frequency. Over administration of TGB could bring about adverse effects, such as speech disorder, depression, and even suicidal tendencies. Therefore, accessible and sensitive assay for analysis of TGB becomes an urgent need toward guiding clinical medication. Here, we present the first report on fluorescence turn-on detection of TGB in urine testing. In this protocol, a fluorescent dye, perylene tetracarboxylic acid imide derivative (PTAI), is found specifically occupying the Sudlow site II of human serum albumin (HSA) and displays a new phenomenon of binding-induced quenching (BIQ). In presence of TGB, competitive binding of the TGB to the site II of HSA will trigger release of PTAI, thus successfully lighting up the fluorescence of PTAI. This label-free assay enjoys a broader working range (1-350 µM) and lower detection limit (0.218 µM) than the traditional liquid chromatography method and is uninterfered by the miscellaneous in the artificial urine. The BIQ probe highlights the merits of HSA as a quencher and a molecular recognition unit, and it opens up a way for studying drug-HSA interaction mechanism and noninvasive pharmaceutical testing.

Multidimensional Liquid Chromatography Employing a Graphene Oxide Capillary Column as the First Dimension: Determination of Antidepressant and Antiepileptic Drugs in Urine.

Human mental disorders can be currently classified as one of the most relevant health topics. Including in this are depression and anxiety, which can affect us at any stage of life, causing economic and social problems. The treatments involve cognitive psychotherapy, and mainly the oral intake of pharmaceutical antidepressants. Therefore, the development of analytical methods for monitoring the levels of these drugs in biological fluids is critical. Considering the current demand for sensitive and automated analytical methods, the coupling between liquid chromatography and mass spectrometry, combined with suitable sample preparation, becomes a useful way to improve the analytical results even more. Herein we present an automated multidimensional method based on high-performance liquid chromatography-tandem mass spectrometry using a lab-made, graphene-based capillary extraction column connected to a C8 analytical column to determined five pharmaceutical drugs in urine. A method enhancement was performed by considering the chromatographic separation and the variables of the loading phase, loading time, loading flow, and injection volume. Under optimized conditions, the study reports good linearity with R2 > 0.98, and limits of detection in the range of 0.5-20 µg L-1. Afterward, the method was applied to the direct analysis of ten untreated urine samples, reporting traces of citalopram in one of them. The results suggest that the proposed approach could be a promising alternative that provides direct and fully automated analysis of pharmaceutical drugs in complex biological matrices.

Highly sensitive UHPLC-DAD method for simultaneous determination of two synergistically acting antiepileptic drugs; levetiracetam and lacosamide: Application to pharmaceutical tablets and human urine.

A simple and highly sensitive ultra-high-performance liquid chromatographic-diode array (UHPLC-DAD) detection method was developed and validated for the simultaneous estimation of levetiracetam (LEV) and lacosamide (LAC). It was clinically proven that the combination of LEV and LAC exhibits a synergistic effect against refractory seizures in mice, which was the motivation for the analysis of this binary mixture both in bulk and in human urine samples. The binary mixture was resolved on a Hypersil BDS C18 analytical column, utilizing a mobile phase of 0.050 mol L-1 phosphate buffer (pH 5.60), methanol and acetonitrile in the ratio (80:10:10 v/v/v) using catechol as an internal standard. The mobile phase was pumped at a flow rate of 1.2 mL min-1 with diode array detection at 205 nm for both drugs and 270 nm for IS. Calibration curves were linear with correlation coefficient >0.9990 over the studied concentration range of 0.1-70.0 µg mL-1 for both drugs. The developed method was reproducible with low relative standard deviation values for intra- and inter-day precision (<2.0%). Both drugs were determined in bulk, pharmaceutical formulations and human urine samples without any interference from complex matrices.

Species differences in metabolism of a new antiepileptic drug candidate, DSP-0565 [2-(2'-fluoro[1,1'-biphenyl]-2-yl)acetamide].

The metabolism and pharmacokinetics of DSP-0565 [2-(2'-fluoro[1,1'-biphenyl]-2-yl)acetamide], an antiepileptic drug candidate, was investigated in rats, dogs, and humans. In human hepatocytes, [14 C]DSP-0565 was primarily metabolized via amide bond hydrolysis to (2'-fluoro[1,1'-biphenyl]-2-yl)acetic acid (M8), while in rat and dog hepatocytes, it was primarily metabolized via both hydrolysis to M8 and hydroxylation at the benzene ring or the benzyl site to oxidized metabolites. After single oral administration of [14 C]DSP-0565 to rats and dogs, the major radioactivity fraction was recovered in the urine (71-72% of dose) with a much smaller fraction recovered in feces (23-25% of dose). As primary metabolites in their excreta, M8, oxidized metabolites, and glucuronide of DSP-0565 were detected. The contribution of metabolic pathways was estimated from metabolite profiles in their excreta: the major metabolic pathway was oxidation (57-62%) and the next highest was the hydrolysis pathway (23-33%). These results suggest that there are marked species differences in the metabolic pathways of DSP-0565 between humans and animals. Finally, DSP-0565 human oral clearance (CL/F) was predicted using in vitro-in vivo extrapolation (IVIVE) with/without animal scaling factors (SF, in vivo intrinsic clearance/in vitro intrinsic clearance). The SF improved the underestimation of IVIVE (fold error = 0.22), but the prediction was overestimated (fold error = 2.4-3.3). In contrast, the use of SF for hydrolysis pathway was the most accurate for the prediction (fold error = 1.0-1.4). Our findings suggest that understanding of species differences in metabolic pathways between humans and animals is important for predicting human metabolic clearance when using animal SF.

Modification of polydopamine-coated Fe3O4 nanoparticles with multi-walled carbon nanotubes for magnetic-µ-dispersive solid-phase extraction of antiepileptic drugs in biological matrices.

In this study, multi-walled carbon nanotubes were coated on the surface of magnetic nanoparticles modified by polydopamine. The synthesized composite was characterized and applied to magnetic-µ-dispersive solid-phase extraction of oxcarbazepine (OXC), phenytoin (PHT), and carbamazepine (CBZ) from human plasma, urine, and cerebrospinal fluid samples prior to analysis by a high-performance liquid chromatography-photodiode array detector. The extraction parameters were investigated and the optimum condition was obtained when the variables were set to the following: sorbent type, Fe3O4@polyDA-MWCNTs (length < 2 µm); sample pH, 6; amount of sorbent, 15 mg; sorption time, 1.5 min at room temperature; type and volume of the eluent, 2.5 mL methanol; and salt content, none added. Under the optimized conditions, the calibration curves are linear in the concentration range 2-2000 ng/mL, the limits of detection are in the range 0.4-3.1 ng/mL, and the relative standard deviations and relative recoveries of plasma (spiked at 200 ng/mL) and CSF (spiked at 50 ng/mL) are in the ranges 1.4-8.2% and 92.8-96.5%, respectively. The applicability of the method was successfully confirmed by extraction and determination of OXC, PHT, and CBZ in biological matrices. Graphical abstract Magnetic multi-walled carbon nanotube core-shell composites were applied as magnetic-µ-dispersive solid-phase extraction adsorbents for determination of antiepileptic drugs in biological matrices.

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.

Risk factors for sodium valproate-induced renal tubular dysfunction.

OBJECTIVE: To explore the risk factors for the development of sodium valproate (VPA)-induced renal tubular dysfunction for early diagnosis and treatment. STUDY DESIGN: The subjects were selected from patients who were diagnosed with epilepsy and administered VPA. Blood and spot urine samples were collected and measured the concentration of VPA, the level of serum phosphorus, serum uric acid, serum free carnitine, serum cystatin-c, and urine ß2-microglobulin (BMG). Patients with urine BMG/creatinine levels above 219.2 were treated as renal proximal tubular dysfunction (RTD), with all others treated as non-RTD. RESULTS: Eighty-seven patients, 4-48 years, 53 men and 34 women, were studied. RTD group is 17 patients and non-RTD group is 70 patients. Univariate analyses revealed that the RTD patients were more likely to be bedridden, receiving enteral tube feeding, taking more anticonvulsants, and demonstrating significantly lower serum levels of free carnitine, uric acid, and phosphorus. Among them, bedridden, free serum carnitine, and phosphorus levels were associated with the development of RTD by multivariate analysis. CONCLUSIONS: Bedridden patients receiving VPA are susceptible to hypocarnitinemia, which can cause RTD and may lead to FS. Therefore, urinary BMG should be measured regularly in all patients receiving VPA to assess renal tubular function. An additional measurement of serum free carnitine level should be considered in patients who developed RTD. Supplementation of carnitine for those patients to prevent such complication deserves for further study.

A silica fiber coated with a ZnO-graphene oxide nanocomposite with high specific surface for use in solid phase microextraction of the antiepileptic drugs diazepam and oxazepam.

A novel ZnO-graphene oxide nanocomposite was prepared and is shown to be a viable coating on fused silica fibers for use in solid phase microextraction (SPME) of diazepam and oxazepam from urine, this followed by thermal desorption and gas chromatographic quantitation using a flame ionization detector. A central composite design was used to optimize extraction time, salt percentage, sample pH and desorption time. Limits of detection are 0.5 µg·L-1 for diazepam and 1.0 µg·L-1 for oxazepam. Repeatability and reproducibility for one fiber (n = 4), expressed as the relative standard deviation at a concentration of 50 µg·L-1, are 8.3 and 11.3% for diazepam, and 6.7 and 10.1% for oxazepam. The fiber-to-fiber reproducibility is <17.6%. The calibration plots are linear in the 5.0-1000 µg·L-1 diazepam concentration range, and from 1.0-1000 µg·L-1 in case of oxazepam. The fiber for SPME has high chemical and thermal stability (even at 280 °C) after 50 extractions, and does not suffer from a reduction in the sorption capacity. Graphical abstract A hydrothermal method was introduced for preparation of ZnO- GO nano composite on a fused silica fiber as solid phase microextraction with high mechanical, chemical stability and long service life.

Challenges for Detecting Valproic Acid in a Nontargeted Urine Drug Screening Method.

BACKGROUND: Valproic acid (VPA) is a widely prescribed medicine, and acute toxicity is possible. As such, it should be included in any nontargeted urine drug screening method. In many published liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) methods, VPA is usually measured using a pseudo-multiple reaction monitoring (MRM) transition. We investigate a simple ultra-high-performance liquid chromatography-quadrupole time-of-flight (QTof) approach to detect the presence of VPA with more confidence. METHODS: Three commercially sourced VPA metabolites were characterized and added to a nontargeted high-resolution MS urine drug screening method. All analyses were performed on a Waters Xevo G2-XS LC-QTof in negative electrospray ionization mode. The mass detector was operated in MS mode, and data were processed with UNIFI software. Sixty-eight patient urine samples, which were previously identified by a well-established gas chromatography-MS method as containing VPA, were analyzed on the Waters Xevo G2-XS LC-QTof, to validate this approach. RESULTS: VPA metabolite standards were characterized, and their detection data were added to the broad drug screening library. VPA metabolites were readily detectable in the urine of patients taking VPA. CONCLUSIONS: The inclusion of characterized VPA metabolites provides a simple and reliable method enabling the detection of VPA in nontargeted urine drug screening.

Correlating blood and urinary concentrations of clobazam doses in Japanese children and adolescents with intractable epilepsy.

Clobazam (CLB) is an antiepileptic drug that is metabolized to the major metabolite N-desmethylclobazam (N-CLB). Our aim was to evaluate the utility of corrected urinary concentrations of CLB and N-CLB in Japanese children and adolescents with epilepsy. Blood and urinary concentrations of CLB and N-CLB were evaluated in 42 patients. The urinary and peak blood concentrations were measured 2-3 h after the last dose. The ratio of the blood and urinary creatinine concentrations was used to calculate the corrected urinary concentrations. A moderate correlation was found between the CLB dose and the CLB serum concentration, but this correlation was not found for N-CLB. Patients were dichotomized based on two regression lines, which were detected by statistical analyses with a cumulative distribution function: the lower ratio group (CLB/N-CLB < 0.275) and the higher ratio group (≥0.275). Moderate correlations were observed between the CLB dose and the serum concentration or the corrected value of CLB for the lower ratio group, and moderate to strong correlations were observed for the higher ratio group. The corrected urinary concentration of CLB correlates to the CLB dose when stratified by the CLB/N-CLB ratio and may prove practical for clinical estimation of the CLB serum concentration.

Paracetamol decreases steady-state exposure to lamotrigine by induction of glucuronidation in healthy subjects.

AIM: Patients receiving lamotrigine therapy frequently use paracetamol concomitantly. While one study suggests a possible, clinically relevant drug-drug interaction, practical recommendations of the concomitant use are inconsistent. We performed a systematic pharmacokinetic study in healthy volunteers to quantify the effect of 4 day treatment with paracetamol on the metabolism of steady-state lamotrigine. METHODS: Twelve healthy, male volunteers participated in an open label, sequential interaction study. Lamotrigine was titrated to steady-state (100 mg daily) over 36 days, and blood and urine sampling was performed in a non-randomized order with and without paracetamol (1 g four times daily). The primary endpoint was change in steady-state area under the plasma concentration-time curve of lamotrigine. Secondary endpoints were changes in total apparent oral clearance, renal clearance, trough concentration of lamotrigine and formation clearance of lamotrigine glucuronide conjugates. RESULTS: Co-administration of lamotrigine and paracetamol decreased the steady-state area under the plasma concentration-time curve of lamotrigine by 20% (95% CI 10%, 25%; P < 0.001) from 166 to 127 µmol l(-1) . Concomitant administration of paracetamol increased the formation clearance of lamotrigine glucuronide conjugates by 45% (95% CI 18%, 79%, P = 0.005) from 1.7 to 2.8 l h(-1) , while the trough value of lamotrigine was reduced by 25% (95% CI 12%, 36%, P = 0.003) from 5.3 to 3.9 µmol l(-1) . CONCLUSION: Paracetamol statistically significantly induced steady-state lamotrigine glucuronidation, resulting in a 20% decrease in total systemic exposure and a 25% decrease in trough value of lamotrigine. This interaction may be of clinical relevance in some patients.

Application of a magnetic molecularly imprinted polymer for the selective extraction and trace detection of lamotrigine in urine and plasma samples.

Magnetic molecularly imprinted polymers have been synthesized for the selective preconcentration and trace determination of lamotrigine (LTG) in urine and plasma samples. The magnetic nanoparticles were modified by tetraethyl orthosilicate and 3-methacryloxypropyl trimethoxysilane before imprinting. The magnetic molecularly imprinted polymers were prepared via surface molecular imprinting technique, using Fe3 O4 as a magnetic component, LTG as template molecule, methacrylic acid as a functional monomer, ethylene glycol dimethacrylate as a cross-linker, and 2,2'-azobisisobutyronitrile as a radical initiator in methanol/acetonitrile (50:50, v/v) as the porogen. The obtained sorbent was characterized using scanning electron microscopy, Fourier-transform infrared spectroscopy, X-ray diffraction, and thermal analysis. Separation of the sorbent from the sample solution was simply achieved by applying an external magnetic field. Determination of the extracted drug was performed by high-performance liquid chromatography with UV detection. Under the optimum extraction conditions, the method detection limits were 0.7 µg/L (based on S/N of 3) for urine samples and 0.5 µg/L for plasma samples. A linear dynamic range of 1-1000 µg/L was obtained for LTF in plasma and urine samples. Finally, the applicability of the proposed method was evaluated by extraction and determination of LTG in urine and plasma samples.

Pharmacokinetics and tolerability of eslicarbazepine acetate and oxcarbazepine at steady state in healthy volunteers.

PURPOSE: Investigate the pharmacokinetics of once-daily (QD; 900 mg) and twice-daily (BID; 450 mg) regimens of eslicarbazepine acetate (ESL) and BID (450 mg) regimen of oxcarbazepine (OXC) at steady state in healthy volunteers. METHODS: Single-center, open-label, randomized, three-way (n = 12) crossover studies in healthy volunteers. KEY FINDINGS: Mean eslicarbazepine Cmax,ss (in µm) following ESL QD (87.3) was 33.3% higher (p < 0.05) compared to ESL BID (65.5) and 82.1% higher (p < 0.05) compared to OXC BID (48.0). The mean area under the curve (AUC)ss,0-τ (in µmol h/L) following the last dose of an 8-day repeated dosing was 1156.3, 1117.6, and 968.4 for ESL QD, ESL BID, and OXC BID, respectively. The ratio eslicarbazepine plasma exposure (µmol h/L) to ESL daily-dose (µmol) was 0.381 (1156.3:3037.3), 0.368 (1117.6:3037.3), and 0.271 (968.4:3567.6) for ESL-QD, ESL-BID, and OXC-BID, respectively, which translates into a 40.6% increase in the ability of ESL-QD compared to OXC-BID to deliver into the plasma their major active entity eslicarbazepine. The extent of plasma exposure to ESL minor metabolites: (R)-licarbazepine and oxcarbazepine after ESL-QD was 71.5% and 61.1% lower, respectively, than after OXC-BID. Twenty, 24 and 38 treatment emergent adverse events were reported with ESL-QD, ESL-BID, and OXC-BID, respectively. SIGNIFICANCE: ESL-QD resulted in 33.3% higher peak plasma concentration (Cmax,ss ) of eslicarbazepine and similar extent of plasma exposure (AUCss,0-τ ) when compared to ESL-BID, which may contribute to the efficacy profile reported with once-daily ESL. In comparison to OXC-BID, administration of ESL-QD resulted in 40.6% increase in the delivery of eslicarbazepine into the plasma as well as a significantly lower systemic exposure to (R)-licarbazepine and oxcarbazepine.

Determination of carbamazepine in human urine and serum samples by high-performance liquid chromatography with post-column Ru(bipy)2(3+)-Ce(SO4)2 chemiluminescence detection.

A novel, sensitive and rapid CL method coupled with high-performance liquid chromatography separation for the determination of carbamazepine is described. The method was based on the fact that carbamazepine could significantly enhance the chemiluminescence of the reaction of cerium sulfate and tris(2,2-bipyridyl) ruthenium(II) in the presence of acid. The chromatographic separation was performed on a Kromasil® (Sigma-Aldrich) TM RP-C18 column (id: 150 mm × 4.6 mm, particle size: 5 µm, pore size: 100 Å) with a mobile phase consisting of methanol-water-glacial acetic acid (70:29:1, v/v/v) at a flowrate of 1.0 mL/min, the total analysis time was within 650 s. Under optimal conditions, CL intensity was linear for carbamazepine in the range 2.0 × 10(-8) ~ 4.0 × 10(-5) g/mL, with a detection limit of 6.0 × 10(-9) g/mL (S/N = 3) and the relative standard detection was 2.5% for 2.0 × 10(-6) g/mL (n = 11). This method was successfully applied to the analysis of carbamazepine in human urine and serum samples. The possible mechanism of the CL reaction is also discussed briefly.

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.

A novel microextraction by packed sorbent-gas chromatography procedure for the simultaneous analysis of antiepileptic drugs in human plasma and urine.

A simple, accurate, and sensitive microextraction by packed sorbent-gas chromatography-mass spectrometry method has been developed for the simultaneous quantification of four antiepileptic drugs; oxcarbazepine, carbamazepine, phenytoin, and alprazolam in human plasma and urine as a tool for drug monitoring. Caffeine was used as internal standards for the electron ionization mode. An original pretreatment procedure on biological samples, based on microextraction in packed syringe using C(18) as packing material gave high extraction yields (69.92-99.38%), satisfactory precision (RSD < 4.7%) and good selectivity. Linearity was found in the 0.1-500 ng/mL range for these drugs with limits of detection (LODs) between 0.0018 and 0.0036 ng/mL. Therefore, the method has been found to be suitable for the therapeutic drug monitoring of patients treated with oxcarbazepine, carbamazepine, phenytoin, and alprazolam. After validation, the method was successfully applied to some plasma samples from patients undergoing therapy with one or more of these drugs. A comparison of the detection limit with similar methods indicates high sensitivity of the present method over the earlier reported methods. The present method is applied for the analysis of these drugs in the real urine and plasma samples of the epileptic patients.

Pharmacokinetics of the monohydroxy derivative of oxcarbazepine and its enantiomers after a single intravenous dose given as racemate compared with a single oral dose of oxcarbazepine.

Oxcarbazepine (OXC) is an antiepileptic drug. In humans, OXC is metabolized via reduction and conjugation. Monohydroxy derivative of OXC (MHD) is the major pharmacologically active component after OXC ingestion. This study was performed to characterize the disposition of the two enantiomers of MHD after oral and intravenous administration and to estimate the bioavailability of MHD after a single oral dose administration of OXC compared to a single intravenous administration of MHD. The study was performed in two parts. In a first pilot study, three intravenous doses were given in an ascending manner (150, 200, and 250 mg of MHD; one subject per dose level) to assess the safety, tolerability, and basic pharmacokinetics. Part two was an open, single-center, randomized, two-way crossover, single-dose trial in 12 healthy adult subjects (n = 6 males and n = 6 females) given OXC orally (one film-coated 300-mg tablet of OXC) and MHD intravenously (250 mg infused over 30 min). Concentrations of OXC and its metabolites were measured by means of high-performance liquid chromatography methods. OXC given as a tablet is completely absorbed in man under fasting conditions. When MHD is given intravenously, (S)-MHD predominates as free compound in plasma. When OXC is administered orally, the ratio of the area-under-the-curve values of (S)-MHD over (R)-MHD equals 3.8, indicating an enantioselective reduction of the prochiral carbonyl group of OXC.

Excretion of the principal urinary metabolites of phenytoin and absolute oral bioavailability determined by use of a stable isotope in patients with epilepsy.

BACKGROUND: The anticonvulsant properties of phenytoin (PHT) were discovered in 1938. Since then, it has been one of the most widely used antiepileptic drugs. It is slowly absorbed, extensively plasma protein-bound, exhibits a nonlinear, concentration-dependent pharmacokinetic profile, and has a narrow therapeutic range. METHODS: We determined PHT bioavailability during steady-state therapy by 1) measurement of the two principal deconjugated PHT urinary metabolites, 5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) and 5-(3,4-dihydroxy-1,5-cyclohexadien-1-yl)-5-phenylhydantoin (DHD); and 2) direct determination of absolute bioavailability after simultaneous administration of an oral formulation and parenteral stable-labeled PHT (SL-PHT). Urinary metabolites were quantified by an isocratic HPLC-NI-APCI-MS method. The urinary dose recovery was calculated by dividing the molar recovery of the major PHT urinary metabolites by the molar dose received. RESULTS: Urinary metabolite recovery was surprisingly low, 35.4% ± 15.7% in younger patients (age 21-49 years old) and 32.9% ± 15.0% in patients aged 65 to 93 years. Absolute bioavailability was 86.4% ± 19.4% and 92.5% ± 25.2%, respectively. A weak, but significant, Spearman rank correlation was observed between urinary metabolite recovery and oral bioavailability (P = 0.00924, R = 0.166). CONCLUSION: This weak correlation may be the result of variability in urinary versus biliary-fecal excretion of p-HPPH glucuronide. This study demonstrates that daily PHT oral absorption exhibits wide interpatient variability, which may account for fluctuations in PHT concentration over time.

High performance liquid chromatography: A versatile tool for assaying antiepileptic drugs in biological matrices.

Epilepsy is a recurrent long-term illness occurring in approximately 1.0% of the world's population. There are currently about 29 approved antiepileptic drugs for the management of epilepsy. Due to narrow therapeutic indices of most antiepileptic drugs, clinical pharmacokinetic characteristics and therapeutic drug monitoring of these drugs are imperative. The objectives of this review were to identify common chromatographic principles, requirements and/or conditions for high-performance liquid chromatography as applied to assay of antiepileptic drugs in biological matrices. The review was conducted using 66 peer reviewed articles (1990 to 2020) from 29 journals that were sought via PubMed, Science Direct and Google Scholar. In all, 29 antiepileptic drugs were assayed from 6 different biological matrices. Forty-three of the reviewed articles estimated the concentration of only one antiepileptic drug, whilst 23 articles focused on simultaneous determination of two or more antiepileptic drugs. Thirty-four, 20, and 14 articles reported using liquid-liquid extraction, protein precipitation, or solid phase extraction for sample clean up, respectively. The ratio of reversed-phase to normal phase, LC-UV to LC-MS and isocratic elution to gradient elution were 61:3, 43:7 and 55:11, respectively. With the exception of one article the reported recoveries ranged from 60.3% to 109.6%. It is noteworthy, that, the performance metrics of high-performance liquid chromatography are better compared to other assays of antiepileptic drugs in biological matrices. This review describes the relevant liquid chromatographic method conditions over the past 30 years for the analysis of this class of drugs, which provides a basis for further method development and optimization.