Development of a UPLC-MS/MS Method to Simultaneously Measure 13 Antiepileptic Drugs with Deuterated Internal Standards.

BACKGROUND: The goal of this study was to develop and validate a UPLC-MS/MS method for simultaneous mea-surement of 13 AEDs, including carbamazepine, oxcarbazepine, lamotrigine, levetiracetam, topiramate, primidone, zonisamide, gabapentin, lacosamide, perampanel, pregabalin, rufinamide, and vigabatrin, in whole blood samples. METHODS: A UPLC-MS/MS method for simultaneous determination of 13 AEDs in whole blood was developed, and validation was conducted for accuracy, precision, limit of quantification (LOQ), matrix effect, and stability. Our method was compared to two different hospitals using UPLC-MS/MS. RESULTS: All AEDs exhibited linearity across the AMR (analytical measurement range), with R2 values ranging from 0.994 to 1.000. The imprecision and inaccuracy for low and high quality control (QC) levels were within an acceptable range, with the coefficient of variation (CV) < 15%. The LOQ was 0.62 µg/mL for carbamazepine, 1.61 µg/mL for oxcarbazepine, 1.30 µg/mL for lamotrigine, 13.20 µg/mL for levetiracetam, 1.26 µg/mL for topira-mate, 1.01 µg/mL for primidone, 1.59 µg/mL for zonisamide, 1.09 µg/mL for lacosamide, 1.61 µg/mL for gabapentin, 0.50 µg/mL for pregabalin, 0.07 ng/mL for perampanel, 3.00 µg/mL for rufinamide, and 2.06 µg/mL for vigabatrin. All AEDs demonstrated acceptable assay parameters for carryover, stability, and matrix effects. Moreover, the assay showed satisfactory results compared to two different hospitals with a bias of less than 15%. CONCLUSIONS: We successfully developed and validated a fast and robust UPLC-MS/MS method for routine therapeutic drug monitoring of thirteen antiepileptic drugs simultaneously.

Monitoring levetiracetam concentration in saliva during pregnancy is stable and feasible.

AIMS: This multicenter prospective cohort study (registration no. ChiCTR2000032089) aimed to investigate the relationship between saliva and plasma levetiracetam concentrations to determine whether saliva could be used for routine monitoring of levetiracetam during pregnancy. METHODS: The slot concentrations of levetiracetam in simultaneously obtained saliva and plasma samples were measured using UPLC-MS/MS. The correlations between saliva and plasma levetiracetam concentrations and the dose-normalized concentrations were compared among pregnant women in different stages and nonpregnant control participants with epilepsy. RESULTS: In total, 231 patients with 407 plasma and saliva sample pairs were enrolled from 39 centers. Linear relationships between salivary and plasma levetiracetam concentrations were reported in the enrolled population (r = 0.898, p < 0.001), including pregnant (r = 0.935, p < 0.001) and nonpregnant participants (r = 0.882, p < 0.001). Plasma concentrations were moderately higher than saliva concentrations, with ratios of saliva to plasma concentrations of 0.98 for nonpregnant women, 0.98, 1, and 1.12 for pregnant women during the first trimester, the second trimester, the and third trimester, respectively. The effective range of saliva levetiracetam concentration was found to be 9.98 µg/mL (lower limit) with an area under the curve (AUC) of 0.937 (95% confidence intervals, 0.915-0.959), sensitivity of 88.9%, specificity of 86.8%, and p < 0.001, to 24.05 µg/mL (upper limit) with an AUC of 0.952 (0.914-0.99), sensitivity of 100%, specificity of 92.3%, and p = 0.007. CONCLUSION: The saliva/plasma concentration ratio of levetiracetam remains constant during pregnancy and is similar to that in non-pregnant individuals. Monitoring levetiracetam concentration in saliva during pregnancy should be widely promoted.

Molecularly imprinted polymer-based extended-gate field-effect transistor chemosensors for selective determination of antiepileptic drug.

Ultrathin molecularly imprinted polymer (MIP) films were deposited on the surfaces of ZnO nanorods (ZNRs) and nanosheets (ZNSs) by electropolymerization to afford extended-gate field-effect transistor sensors for detecting phenytoin (PHT) in plasma. Molecular imprinting efficiency was optimized by controlling the contents of functional monomers and the template in the precursor solution. PHT sensing was performed in plasma solutions with various concentrations by monitoring the drain current as a function of drain voltage under an applied gate voltage of 1.5 V. The reliability and reproducibility of the fabricated sensors were evaluated through a solution treatment process for complete PHT removal and PHT adsorption-removal cycling, while selectivity was examined by analyzing responses to chemicals with structures analogous to that of PHT. Compared with the ZNS/extracted-MIP sensor and sensors with non-imprinted polymer (NIP) films, the ZNR/extracted-MIP sensor showed superior responses to PHT-containing plasma due to selective PHT adsorption, achieving an imprinting factor of 4.23, detection limit of 12.9 ng/mL, quantitation limit of 53.0 ng/mL, and selectivity coefficients of 3-4 (against tramadol) and ~ 5 (against diphenhydramine). Therefore, we believe that the MIP-based ZNR sensing platform is promising for the practical detection of PHT and other drugs and evaluation of their proper dosages.

Simultaneous quantification of 11 antiepileptic drugs using limited isotope-labeled internal standards in LC-MS/MS: An accuracy assessment.

This study aims to establish an LC-MS/MS method to simultaneously analyze 11 antiepileptic drugs with a particular focus on maintaining accuracy while reducing the number of isotope-labeled internal standards employed for cost-effectiveness. By applying a water/acetonitrile gradient elution containing 0.1 % formic acid and 2 mM ammonium formate as the mobile phase, optimal sensitivity for the target drugs could be obtained in positive ESI mode in LC-MS/MS. After optimizing various extraction techniques, extraction with 70 % acetonitrile was selected as it provided good recoveries (>93 %) for all targets without matrix effects. Accuracies within 3 % were achieved from the combination of six internal standards, while accuracies of 5 % and 10 % were obtained by reducing the number of internal standards to four and two, respectively, for more economical analysis. The accuracy of the established method was maintained in hyperglycemia, hyperlipidemia, and hyperalbuminemia sera, suggesting that it can be successfully applied to individual serum samples with various properties.

Steroidal Saponins from Water Eggplant (Fruits of Solanum torvum) Exhibit Anti-Epileptic Activity against Pentylenetetrazole-Induced Seizure Model in Zebrafish.

The fruits of Solanum torvum Swartz, a wild relative of eggplant, are consumed as a wild vegetable in tropical regions of Africa, Asia, and South America. In traditional Chinese medicine, it is believed to have anti-inflammatory and sedative effects. In the Philippines, water decoction is used to treat hyperactivity disorder. Twenty-two steroidal saponins were isolated and purified from the fruits grown in Yunnan, China, including six new compounds: torvosides U-Z (1-6). During drying and cooking, the saponins may undergo transformation, resulting in small amounts of sapogenins. These transformations can include dehydration of hydroxyl groups at position C22, formation of double bonds at position 20, 22 or 22, 23, and even formation of peroxide products. Saponin compounds torvoside X (4), torvoside Y (5), torvoside A (7), and (25S)-3-oxo-5α-spirostan-6α-yl-O-ß-d-xylopyranoside (20), which are glycosylated at C-6, showed certain anti-epileptic activity in a pentylenetetrazole-induced zebrafish seizure model. No antiproliferative activity was detected when tested on the cancer cell line HepG2, and no hepatotoxic effect was noted on normal liver cell line LO2.

ICH Q14-inspired novel approach to establish an SFC-based purity method for carbamazepine.

The proposed ICH Q14 guideline "Analytical procedure development" describes science and risk-based approaches for development and maintenance of analytical procedures suitable for the assessment of the quality of drug substances and drug products. As a case study, the systematic development and validation of a supercritical fluid chromatography (SFC)-based purity method for carbamazepine is presented. Systematic analytical quality by design (AQbD) principles were applied using the software package Fusion QbD to the method development approach. The relationship between chromatographic parameters and the responses of interest were examined to improve the reliability of the method by understanding, reducing, and controlling sources of variability. Method performance qualification in terms of method robustness was finally carried out with the parameters that were classified as critical after method development and a validation study met previously set acceptance criteria. The developed SFC purity method for carbamazepine demonstrated readiness as a viable alternative to the official HPLC method published in the Ph.Eur. with improved peak resolution, improved peak symmetry, and faster analysis times (3 min vs. 80 min for the official method). Its inherent reliability illustrates the superiority of AQbD in method development and application for drug quality assurance.

Pharmacokinetics of the Recalcitrant Drug Lamotrigine: Identification and Distribution of Metabolites in Cucumber Plants.

Treated wastewater is an important source of water for irrigation. As a result, irrigated crops are chronically exposed to wastewater-derived pharmaceuticals, such as the anticonvulsant drug lamotrigine. Lamotrigine is known to be taken up by plants, but its plant-derived metabolites and their distribution in different plant organs are unknown. This study aimed to detect and identify metabolites of lamotrigine in cucumber plants grown for 35 days in a hydroponic solution by using LC-MS/MS (Orbitrap) analysis. Our data showed that 96% of the lamotrigine taken up was metabolized. Sixteen metabolites possessing a lamotrigine core structure were detected. Reference standards confirmed two; five were tentatively identified, and nine molecular formulas were assigned. The data suggest that lamotrigine is metabolized via N-carbamylation, N-glucosidation, N-alkylation, N-formylation, N-oxidation, and amidine hydrolysis. The metabolites LTG-N2-oxide, M284, M312, and M370 were most likely produced in the roots and were translocated to the leaves. Metabolites M272, M312, M314, M354, M368, M370, and M418 were dominant in leaves. Only a few metabolites were detected in the fruits. With an increasing exposure time, lamotrigine leaf concentrations decreased because of continuous metabolism. Our data showed that the metabolism of lamotrigine in a plant is fast and that a majority of metabolites are concentrated in the roots and leaves.

Transfer of anticonvulsants and lithium into amniotic fluid, umbilical cord blood & breast milk: A systematic review & combined analysis.

OBJECTIVE: Data on the ability of anticonvulsants and lithium to enter fetal and newborn circulation has become increasingly available; here we estimated penetration ratios in a series of matrices from combined samples of pregnant/breastfeeding women treated with anticonvulsants or lithium. METHODS: We conducted a systematic literature search in PubMed/EMBASE for studies with concentrations of anticonvulsants/lithium from maternal blood, amniotic fluid, umbilical cord blood and/or breast milk. Penetration ratios were calculated by dividing the concentrations in amniotic fluid, umbilical cord plasma or breast milk by the maternal concentrations. When data from multiple studies were available, we calculated combined penetration ratios, weighting studies' mean by study size. RESULTS: Ninety-one eligible studies for brivaracetam, carbamazepine, clonazepam, ethosuximide, gabapentin, lacosamide, lamotrigine, levetiracetam, lithium, oxcarbazepine, perampanel, phenobarbital, phenytoin, pregabalin, primidone, topiramate, valproate, vigabatrin and zonisamide were identified. For amniotic fluid, the highest penetration ratios were estimated for levetiracetam (mean 3.56, range 1.27-5.85, n = 2) and lowest for valproate (mean 0.11, range 0.02-1.02, n = 57). For umbilical cord plasma, oxcarbazepine had the highest ratio (mean 1.59, range 0.11-4.33, n = 12) with clonazepam having the lowest (mean 0.55, range 0.52-0.59, n = 2). For breast milk, the highest ratios were observed for oxcarbazepine (mean 3.75, range 0.5-7.0, n = 2), whereas the lowest were observed for valproate (mean 0.04, range 0.01-0.22, n = 121). DISCUSSION: We observed substantial variability between anticonvulsants and lithium regarding their ability to enter fetal/newborn circulation. Assessing concentrations of anticonvulsants and lithium in maternal samples can provide a surrogate of fetal/infant exposure, although patterns of concentration-dependent effects for maternal/neonatal safety are lacking.

Simultaneous Estimation and Validation of Four Antiepileptic Drugs from Bulk and Formulations Using Reverse Phase HPLC

Abstract Epilepsy is a disorder of the central nervous system, in which the nerve cell activity in the brain is disturbed causing seizures. The objective was to develop an RP-HPLC method for consistent simultaneous quantitation of four antiepileptic drugs Levetiracetam (LVT), Lamotrigine (LTG), Phenobarbital (PBT) and Phenytoin (PTY). An isocratic method was developed on C18 column in JASCO HPLC using 5 mM potassium phosphate buffer (pH 6) and acetonitrile as the mobile phase at a flow rate of 1ml/min and detected at 230 nm using UV detector. The mean retention time for LVT, LTG, PBT and PTY were found as 2.55, 3.55, 4.65 and 5.99 minutes respectively. The method was validated as per ICH guidelines and was found to be acceptable. The %RSD value was <2.0 % thus stating the developed method was precise for the drugs in the given range. The accuracy values were within 85-115% of the recovery range. The specificity of the method was evaluated by an assay of marketed formulation, and it showed a percent content between 90-110% w/w for all the four drugs. The proposed analytical method was simple, accurate and robust and was precisely able to resolve the four major antiepileptic drugs. Hence, the current method can be applied successfully for routine examination of these drugs

Derivatization of γ-Amino Butyric Acid Analogues for Their Determination in the Biological Samples and Pharmaceutical Preparations: A Comprehensive Review.

γ-Aminobutyric acid (GABA) plays an important role in regulating neuronal excitability. Four structurally related drugs to GABA including pregabalin (PGB), gabapentin (GBP), vigabatrin (VGB), and baclofen are used for the treatment of central nervous system disorders. These drugs are small aliphatic molecules having neither fluorescent nor strong absorbance in the ultraviolet/visible region; therefore, direct determination of these analytes by optical methods is difficult. Additionally, their high boiling point makes gas chromatography impossible. Accordingly, the amine or acid moiety in these drugs is derivatized in order to improve their selectivity and sensitivity during determination in the biological samples. This review focuses on derivatization based methods and their different reactions for determination of PGB, GBP, VGB, and baclofen in the biological samples and pharmaceutical preparations reported between 1980 and 2020. High-performance liquid chromatography methods coupled with different detectors are a commonly used methods for determination of GABA analogs after derivatization. These methods cover 38.89% of all developed methods for determination of GABA analogs.

Interchangeability among carbamazepine formulations: the impact over epilepsy patients

Abstract The treatment of epilepsy is complex and a matter of concern is the interchangeability among different formulations available for antiepileptic drugs. To evaluate the effects of interchangeability among carbamazepine formulations on patients with epilepsy. This is a prospective cohort study that included adult outpatients diagnosed with epilepsy and under pharmacological treatment with carbamazepine. Before switching the brand/manufacturer, the "Interchangeable Pharmaceutical Product in the Treatment of Epilepsies" questionnaire was applied. The questionnaires "Adverse Events Profile" and Quality of Life in Epilepsy-31, so as the plasma carbamazepine concentrations, were evaluated before and after the brand/ manufacturer switch. Physical-chemical tests aiming to assess tablets quality were performed in accordance with the Brazilian Pharmacopoeia 5th edition. The study population was composed by 14 patients (mean age: 44.6 years), with 10 of females. From those interviewed, 10 had no knowledge about the three antiepileptic drugs formulations available. The frequency of adverse event "problems with skin" incresead (p=0.023) and "upset stomach" decreased (p=0.041) after the changeover. The adverse events profile was associated with only two quality of life domains: "energy/fatigue" (p=0.048) and "total score" (p=0.018). Divergent results between generic and reference formulations were observed in purity-water test (reference: 1.96%, generic: 4.84%) and dissolution test, in which the generic formulation presented 66.27 to 85.77% of carbamazepine dissolved after the third level. Conclusions: Objective differences before and after the brand/manufacturer switch were not observed, in spite of patients' perceptions. Despite that, more studies in the field are necessary, especially on the interchangeability among generic antiepileptics, in order to better elucidate switching consequences on patients' life.

pH-induced homogeneous liquid-liquid microextraction method based on new switchable deep eutectic solvent for the extraction of three antiepileptic drugs from breast milk.

Aim: A pH-induced homogeneous liquid-liquid microextraction (HLLME) using a new switchable deep eutectic solvent has been used for the extraction of three antiepileptic drugs from breast milk samples. Methodology: This method is based on phase separation by changing pH. An ammonia solution and a phosphocholine chloride: hexanoic acid: p-aminophenol deep eutectic solvents were used as the phase separation agent and extraction solvent, respectively. Results: Significant factors were studied and the detection limits and enrichment factors were in the ranges of 0.009-0.19 ng ml-1 and 182-212 for the analytes, respectively. Also, linear ranges were wide (0.63-500 ng ml-1) and the method precision was acceptable. Conclusion: The introduced method was successfully applied for the determination of the analyte concentrations in breast milk samples.

Occurrence of the antiepileptic carbamazepine in water and bivalves from marine environments: A review.

A vast literature has already demonstrated that pharmaceutical drugs exert negative impacts on aquatic organisms but data is sparse on the occurrence of these contaminants in marine aquatic environments and their biota, particularly in comparison with freshwater systems. In marine environments, bivalves are known as good bioindicator species for environmental pollution monitoring. This review summarizes the current knowledge on carbamazepine (CBZ) concentrations in the marine environment (seawater and bivalves) and the analytical methods involved in the drug determination. Carbamazepine was chosen based on its ubiquitous occurrence and proven negative impacts on the aquatic organisms. Overall, CBZ is distributed in the marine environment with concentrations up to ∼ 1 µg/L, revealing its stability and high persistence. Also, CBZ was found in some species of marine bivalves, with concentrations up to 13 ng/g dry weight (DW), however, a bioaccumulation factor could not be calculated due to the absence of CBZ determination in seawater samples for most of the studies. CAPSULE: Carbamazepine is found in seawater up to the low µg/L level, and in bivalve tissue up to a few ng/g DW, with SPE and LC as the techniques of choice for drug extraction and identification.

Compounding and quality control of two pediatric topiramate pharmaceutical preparations.

In pediatric wards, topiramate is prescribed as an antiepileptic at non-licensed dosages. Compounding is the best way to obtain topiramate drug adapted to pediatric patients, but this practice requires to control the quality of batches and to manage a stability study to establish a beyond-use-date. With this objective, 6 mg. mL 1 topiramate oral suspension and 9 mg capsules were realized, and our laboratory was mandated for their quality control. Previously described dosing methods did not allow us to determine topiramate content in prescribed preparations. An original HPLC-UV derivatization dosing method of topiramate was validated and was proved to be stability indicating. This derivatization methodology, but also total aerobic microbial count (TAMC) and total combined yeasts and mold count (TYMC) allowed the quality control of topiramate capsules and topiramate suspension. Beyond-use-dates can be attributed with regards to United States Pharmacopoeia recommendations, and a stability study was performed on 6 mg. mL-1 topiramate suspension to confirm empirical data. Topiramate pediatric suspension was found to be stable for two months at +2/+8 °C, one month after opening and one day at ambient temperature.

Monitoring topiramate concentrations at delivery and during lactation.

OBJECTIVE: To determine transplacental passage of topiramate and its transport to colostrum, mature maternal milk and breastfed infants, we examined data from 27 women treated with topiramate from 2004 to 2020. METHODS: In this cohort study, maternal serum, umbilical cord serum, milk and infant serum levels were measured by gas chromatography in the delivery subgroup, the colostrum subgroup (3-4 days postpartum) and the mature milk subgroup (7-30 days postpartum). Paired umbilical cord serum, maternal serum, breastfed infant serum, and milk levels were used to assess the ratios of umbilical cord/maternal serum, milk/maternal serum and infant/maternal serum levels. RESULTS: Topiramate levels varied from 1.0 to 7.1 mg/L in maternal serum and from 0.8 to 6.2 mg/L in umbilical cord serum, and the mean umbilical cord/maternal serum ratio was 0.93 ± 0.11. At 3-4 days after delivery, topiramate concentrations were 1.4-8.4 mg/L in maternal serum, 1.5-8.6 mg/L in milk and 0.3-4.4 mg/L in infant serum. The mean milk/maternal serum ratio was 0.99 ± 0.45, and the mean infant/maternal serum ratio was 0.25 ± 0.15. At 7-30 days after delivery, maternal serum levels varied from 1.9 to 9.7 mg/L, milk levels ranged from 2.3 to 10.6 mg/L and infant serum levels ranged from 0.3 to 6.5 mg/L. The mean milk/maternal serum ratio was 1.07 ± 0.31, and the mean infant/maternal serum ratio was 0.51 ± 0.27. CONCLUSIONS: We extended information about free transplacental passage of topiramate and its extensive transport to maternal milk with lower serum concentrations in breastfed infants in the largest group of patients ever reported to our knowledge. DATA AVAILABILITY STATEMENT: Authors declare that take full responsibility for the data, the analyses and interpretation, and the conduct of the research; that they have full access to all of the data; and that they have the right to publish all data. Authors were not participations in industry-sponsored research and corporate activities for evaluation of a manuscript.

Sensing the Anti-Epileptic Drug Perampanel with Paper-Based Spinning SERS Substrates.

The applications of SERS in therapeutic drug monitoring, or other fields of analytical chemistry, require the availability of sensitive sensors and experimental approaches that can be implemented in affordable ways. In this contribution, we show the production of cost-effective SERS sensors obtained by depositing Lee-Meisel Ag colloids on filter paper either by natural sedimentation or centrifugation. We have characterized the morphological and plasmonic features of the sensors by optical microscopy, SEM, and UV-Vis spectroscopy. Such sensors can be used to quantify by SERS the anti-epileptic drug Perampanel (in the concentration range 1 × 10-4-5 × 10-6 M) by spinning them during the micro-Raman measurements on the top of a custom device obtained from spare part hard disk drives. This approach minimizes laser-induced heating effects and allows averaging over the spatial non-uniformity of the sensor.

Hair as a matrix in monitoring drug epilepsy therapy.

Hair is considered an efficient tool to investigate drug-related histories; thus, the selection of the method of sample preparation is important to obtain a reliable result. The aim of this study was to compare two methods of hair preparation (cutting and pulverizing) to analyse levetiracetam concentration in hair. An additional aim was to evaluate the potential usefulness of the levetiracetam concentration measured as an index of a dosing schedule. Four groups of 12 rats were included in the experiment. Depending on the group, the rats received 10 mg/kg of levetiracetam intraperitoneally every 24, 48 and 72 hours for 30 days. The control group was not treated. At the end of the drug administration, the rats' hair was shaved, cut or pulverized and analysed by the LC/MS-MS method to determine the concentration of levetiracetam. A stronger correlation between the mean hair levetiracetam concentration in hair and the number of drug doses was found in pulverized hair than in cut hair. A smaller standard deviation between the results was obtained in the case of pulverized hair. The results indicate that pulverization gives a more reliable result of drug concentration in hair than cutting and that drug concentration in hair can reflect the scheme of levetiracetam administration.

Supramolecular Solvent-Based Microextraction of Selected Anticonvulsant and Nonsteroidal Anti-Inflammatory Drugs from Sediment Samples.

The increase in the production and consumption of pharmaceuticals increases their presence in the global environment, which may result in direct threats to living organisms. For this reason, there is a need for new methods to analyze drugs in environmental samples. Here, a new procedure for separating and determining selected drugs (diclofenac, ibuprofen, and carbamazepine) from bottom sediment and water samples was developed. Drugs were determined by ultra-high performance liquid chromatography coupled with an ultraviolet detector (UHPLC-UV). In this work, a universal and single-step sample treatment, based on supramolecular solvents (SUPRAS), was proposed to isolate selected anticonvulsants and nonsteroidal anti-inflammatory drugs (NSAIDs) from sediment samples. The following parameters were experimentally selected: composition of the supramolecular solvent (composition THF:H2O (v/v), amount of decanoic acid), volume of extractant, sample mass, extraction time, centrifugation time, and centrifugation speed. Finally, the developed procedure was validated. A Speedisk procedure was also developed to extract selected drugs from water samples. The recovery of analytes using the SUPRAS procedure was in the range of 88.8-115%, while the recoveries of the Speedisk solid-phase extraction procedure ranged from 81.0-106%. The effectiveness of the sorption of the tested drugs by sediment was also examined.

Simultaneous determination of barbiturates, phenytoin and topiramate in hair by LC-MS/MS and application to real samples.

INTRODUCTION: Hair analysis is useful for monitoring exposure to drugs such as antiepileptics owing to long-term therapy and a high possibility of abuse of drugs, which could be fatal. An effective and rapid analytical method for the simultaneous determination of six barbiturates, as well as phenytoin and topiramate in hair samples was developed and validated by liquid-chromatography-tandem mass spectrometry (LC-MS/MS). METHODS: Three different extraction methods were investigated for the development of an appropriate analytical method. Hair was finely cut and then extracted with methanol, methanol containing 1% hydrochloric acid, and liquid-liquid extraction in acidic condition. RESULTS: There was no significant difference in the matrix effects among these three methods. Recoveries clearly declined in the extraction involving both acidic methanol extraction and a LLE in acidic condition. Methanol incubation was chosen as the appropriate extraction method with acceptable matrix effects and recoveries. After validating the methanol incubation, the limit of detection (LOD) and limit of quantification (LOQ) were determined as 0.01 and 0.02 ng/mg for topiramate and 0.25-0.5 and 0.5-1 ng/mg for the others in hair. The LC-MS/MS method was precise and accurate with a dynamic linear range of 0.02-5 ng/mg for topiramate and 0.5 or 1-50 ng/mg for others. This method was applied to authentic hair samples of two drug users. The hair concentrations of phenobarbital were 0.2-17.1 ng/mg in segmental analysis in one female subject and those of topiramate were 0.19-0.93 ng/mg in another female subject. DISCUSSION: The quantitative method was developed to determine 8 antiepileptics using LC-MS/MS. This method performed hair segmental analysis to provide useful informative and chronological data in both of the forensic and clinical toxicology fields.

Salting-out assisted liquid-liquid extraction method optimized by design of experiments for the simultaneous high-performance liquid chromatography analysis of perampanel and stiripentol in mouse matrices.

We report a high-performance liquid chromatography method development able to simultaneously determine perampanel and stiripentol, two third-generation antiepileptics whose therapeutic spectrum can potentially be extended, in several mouse matrices. A salting-out assisted liquid-liquid extraction optimized by a design of experiments approach was adopted for sample preparations. Isopropanol and magnesium sulfate were the extraction solvent and salting-out agent, respectively. Both drugs and internal standard (terbinafine) were separated using a LiChroCART® Purospher Star column (C18 , 55 × 4 mm; 3 µm) isocratically pumped with mobile phase [1% triethylamine in water (pH 2.5) and acetonitrile (53:47, v/v)] at 1 mL/min. Stiripentol and terbinafine were detected by fluorescence at 254/372 nm and perampanel at 275/430 nm. Good linearity was demonstrated for perampanel at 1-500 ng/mL range in brain, 2-2000 ng/mL in liver and 1-2000 ng/mL in plasma and kidney (r2  ≥ 0.9922), and for stiripentol between 10 and 2000 ng/mL in brain and 10 and 20 000 ng/mL in the remaining matrices (r2  ≥ 0.9917). Precision (CV ≤ 15%) and accuracy (bias ±15%) were also verified, with obtained recovery values consistent with those predicted by the experimental design. This method was applied in preliminary pharmacokinetic studies to quantify perampanel or stiripentol after oral administration to mice, showing to be a promising bioanalytical tool to support future nonclinical in vivo pharmacokinetic studies.