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This study compares the variability of relative response factors (RRFs) using Taguchi's multifactorial analysis for two internal standard (IS) methods in gas chromatography (GC) for quality control of alcoholic products. Methods where either ethanol or pentan-1-ol was used as an IS were compared. For ten volatile substances prescribed by legislation, the RRFs of both methods were compared under 27 different experimental conditions. The influence of parameters (control factors) such as ethanol content in the matrix, analyte concentration, injected volume, injector temperature, split ratio, and flame ionization detector temperature was evaluated. The selected control factors had values at one of the three levels to cover the commonly used ranges of their settings in the measuring system and to characterize the majority of alcoholic products commonly analyzed in practice. The obtained results showed that the biggest differences in the variability of the results between the two methods were found for the most strictly controlled substances in alcoholic products, acetaldehyde, and methanol, where the application of ethanol as an IS provides clearly better results. For both methods, the way control factors affect the repeatability of GC measurements expressed in the form of relative deviation was also evaluated.
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Many prey species change their antipredator defence during ontogeny, which may be connected to different potential predators over the life cycle of the prey. To test this hypothesis, we compared reactions of two predator taxa - spiders and birds - to larvae and adults of two invasive true bug species, Oxycarenus hyalinipennis and Oxycarenus lavaterae (Heteroptera: Oxycarenidae) with life-stage-specific chemical defence mechanisms. The reactions to larvae and adults of both true bug species strikingly differed between the two predator taxa. The spiders were deterred by the defences of adult bugs, but the larval defences were ineffective against them. By contrast, birds attacked the larvae considerably less often than the adult bugs. The results indicate a predator-specific ontogenetic change in defence effectiveness of both Oxycarenus species. The change in defence is likely linked to the life-stage-specific composition of secretions in both species: whereas secretions of larvae are dominated by unsaturated aldehydes, secretions of adults are rich in terpenoids, which probably serve dual function of defensive chemicals and pheromones. Our results highlight the variation in defence between different life stages and the importance of testing responses of different types of predators.
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Heterópteros , Animales , Heterópteros/fisiología , Larva , Aves , Aldehídos , Conducta PredatoriaRESUMEN
Riociguat is novel antihypertensive drug for treatment of pulmonary hypertension. As such, it is still being tested in many clinical and pharmacokinetic trials. Existing methods that determine serum riociguat and desmethylriociguat (DMR) are based solely on liquid chromatography with mass spectrometry. Therefore, we present a novel capillary electrophoresis with mass spectrometry method (CE-MS) for their determination in human serum as alternative method for ongoing trials. Complete resolution of both analytes was achieved by means of pH optimization of ammonium formate background electrolytes that are fully compatible with ESI/MS detection. Simple liquid-liquid extraction was used as sample pretreatment. The calibration dependence of the method was linear (in the range of 10-1000 ng/mL), with adequate accuracy (90.1-114.9%) and precision (13.4%). LOD and LOQ were arbitrarily set at 10 ng/mL for both analytes. Clinical applicability was validated using serum samples from patients treated with riociguat in pharmacokinetic study and the results corresponded with reference HPLC-MS/MS values. Capillary electrophoresis proved to be sensitive and selective tool for the analysis of riociguat and DMR.
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Electroforesis Capilar/métodos , Pirazoles/sangre , Pirimidinas/sangre , Espectrometría de Masa por Ionización de Electrospray/métodos , Electrólitos , Humanos , Límite de Detección , Modelos Lineales , Extracción Líquido-Líquido , Pirazoles/química , Pirazoles/aislamiento & purificación , Pirazoles/farmacocinética , Pirimidinas/química , Pirimidinas/aislamiento & purificación , Pirimidinas/farmacocinética , Reproducibilidad de los ResultadosRESUMEN
Riociguat is a novel antihypertensive drug for the treatment of pulmonary hypertension. We present electrophoretic characterization, i.e. migration behavior of riociguat and metabolite M1 as support for optimized CZE/MS assay. Fundamental separation parameters, such as peak width, symmetry, and resolution are studied in a series of ammonium formate buffers within pH range 2.60-5.61. The narrow region of peak symmetry lies close to pH 4.0 for both analytes. Accordingly, the value of resolution maximizes in a background electrolyte adjusted to pH 4.10. Basic calibration parameters estimated from CZE experiments with absorption photometric and mass spectrometric detection of riociguat and metabolite M1 were evaluated. More than three orders lower LOD was achieved with high resolution mass spectrometric detection. The observed difference in the sensitivity of both detection techniques gives priority to the utilization of CZE/MS in practice. The values of dissociation constants of riociguat and metabolite M1, pKBH , were determined from CZE measurements in lithium formate and lithium acetate background electrolytes with constant ionic strength. The value of pKBH = 4.30 ± 0.02 for riociguat corresponds well to the value already presented in the literature. According to our observation, metabolite M1 behaves like a slightly stronger base with estimated pKBH = 4.40 ± 0.02.
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Electroforesis Capilar/métodos , Espectrometría de Masas/métodos , Pirazoles/análisis , Pirazoles/metabolismo , Pirimidinas/análisis , Pirimidinas/metabolismo , Humanos , Límite de Detección , Modelos Lineales , Pirazoles/sangre , Pirazoles/química , Pirimidinas/sangre , Pirimidinas/química , Reproducibilidad de los ResultadosRESUMEN
This review follows up on Part 1, which focused on classification and evaluation of single drop and sorbent-based microextraction techniques. Membrane- and homogenous phase-based microextraction techniques are discussed and classified in Part 2. These techniques are more recent than those in Part 1 and considerable attention has been paid to their development. The new methodologies are more sensitive and, thanks to their miniaturization, they can be classified as "green", but no exhaustive classification is available. We hope that this review will contribute to better orientation in these methods.
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Sample pretreatment techniques or preconcentration constitute a very important step before the analysis of environmental, clinical, pharmaceutical, and other complex samples. Thanks to extraction techniques it is possible to achieve higher method sensitivities and selectivities. Miniaturization microextraction methods make them more environmentally friendly and only small amounts of samples are required. In the past 30 years, a number of microextraction methods have been developed and used and are documented in thousands of articles. Many reviews have been written focusing on their use in specified professional fields or on the latest trends. Unfortunately, no uniform nomenclature has been introduced for these methods. Therefore, this review attempts to classify all the essential microextraction techniques and describes their advantages, disadvantages, and the latest innovations. The methods are divided into two main groups: single drop and sorbent-based techniques according to the type of extraction phase.
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Natural organic additives such as eggs, lard, resins, and oils have been added to mortars since ancient times, because the ancient builders knew of their positive effect on the mortar quality. The tradition of adding organic materials to mortars was commonly handed down only verbally for thousands years. However, this practice disappeared in the nineteenth century, when the usage of modern materials started. Today, one of the most recent topics in the industry of building materials is the reusing of natural organic materials and searching for the forgotten ancient recipes. The research of the old technological approaches involves currently the most advanced analytical techniques and methods. This paper is focussed on testing the possibility of identification of proteinaceous additives in historical mortars and model mortar samples containing blood, bone glue, curd, eggs and gelatine, by Fourier transform infrared (FTIR) and Raman spectroscopy, gas chromatography - mass spectrometry (GC-MS), matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS), liquid chromatography-electrospray ionisation-quadrupole-time of flight mass spectrometry (LC-ESI-Q-TOF MS) and enzyme-linked immunosorbent assay (ELISA). All these methods were applied to the mortar sample taken from the interior of the medieval (sixteenth century) castle in Namest nad Oslavou in the Czech Republic and their comparison contributed to the rough estimation of the protein additive content in the mortar. The obtained results demonstrate that only LC-ESI-Q-TOF MS, MALDI-TOF MS and ELISA have the sufficiently low detection limits that enable the reliable identification of collagens in historical mortars. Graphical abstract Proteomics analyses of historical mortars.
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Materiales de Construcción/análisis , Materiales de Construcción/historia , Proteínas/análisis , Animales , Proteínas Sanguíneas/análisis , Colágeno/análisis , República Checa , Huevos/análisis , Ensayo de Inmunoadsorción Enzimática/métodos , Cromatografía de Gases y Espectrometría de Masas/métodos , Gelatina/análisis , Historia del Siglo XVI , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Espectrometría Raman/métodosRESUMEN
A simple method for the simultaneous derivatization of carbohydrates, polyols, amines and amino acids using hexamethyldisilazane and N,O-bis(trimethylsilyl)trifluoroacetamide was developed. This method allows the direct derivatization of urine samples without sample pretreatment before derivatization. The method was successfully used for analysis of the selected metabolites in urine samples of healthy individuals and neonates suffering from galactosemia. The limits of detection by positive chemical ionization gas chromatography with tandem mass spectrometry analysis were in the range of 1.0 mgL-1 for mannitol to 4.7 mg/L for glucose.
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Aminas/orina , Carbohidratos/orina , Galactosemias/orina , Polímeros/análisis , Adulto , Algoritmos , Calibración , Congelación , Cromatografía de Gases y Espectrometría de Masas , Humanos , Recién Nacido , Límite de Detección , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem , Compuestos de Trimetilsililo/análisis , UrinálisisRESUMEN
A new, fast, selective, and reliable capillary electrophoresis method has been developed for analysis of selected phosphoesters (phosphoserine, phosphoethanolamine, phosphoglycerol) and phosphate. The method is based on separation of specific phosphate containing headgroups (phosphoesters) which are cleaved from the glycerol skeleton of a phospholipid by a regioselective enzyme (phospholipase C). Analysis of intact phospholipids with the same polar headgroup but different fatty acids shows that fatty acid composition has a high impact on separation of phospholipids, so analysis of separated polar headgroups, which avoids this influence, represents a much more suitable approach for phospholipid class research. Optimization of method parameters results in running buffers of relatively narrow pH interval (pH about 10) where all phosphoesters are separated. Further method validation has shown that direct UV detection has a sufficient detection limit for all analytes to perform suitable analyses of cell membrane lipids. The optimized method was tested on the lysate of cell membrane of Bacillus subtilis, where all analytes were determined.
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Bacillus subtilis/química , Membrana Celular/química , Electroforesis Capilar/métodos , Fosfatos/análisis , Fosfolípidos/análisis , Fosfolípidos/química , Fraccionamiento Celular , Fosfatos/química , Fosfolipasas de Tipo C/químicaRESUMEN
A new, rapid, sensitive, robust, and reliable method has been developed for the qualitative analysis of phosphoserine, phosphoethanolamine, phosphoglycerol, and phosphate using gas chromatography with mass spectrometry and two-step trimethylsilylation. The method employs hexamethyldisilazane for silylation of the phosphate and hydroxyl groups in the first phase and bis(trimethylsilyl)trifluoroacetamide for silylation of the less-reactive amino groups in the second phase. This order is of key importance for the method because of the different reactivities of the two reagents and the mechanism of derivatization of the active groups of the analytes. Trimethylsilylated derivatives of the analytes were identified on the basis of their retention times and mass spectra. The probable structures of the major fragments were identified in the spectra of the trimethylsilylated derivatives and characteristic m/z fragments were selected for each analyte. Fragments with m/z 73 and 299 occurred in the spectra of all the analytes. The characteristic retention data were employed to calculate the retention indices of the individual silylated phosphorylated substances in the hydrocarbon range C12-C19 for the DB-5ms column. The method was employed to measure the polar fraction of the hydrolysate of the cytoplasmic membrane of Bacillus subtilis. The detection limits vary between 5 µg/mL (trimethylsilylated phosphate) and 72 µg/mL (trimethylsilylated phosphoethanolamine).
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Bacillus subtilis/química , Cromatografía de Gases/métodos , Etanolaminas/análisis , Espectrometría de Masas/métodos , Fosfatos/análisis , Fosfoserina/análisis , Membrana Celular/químicaRESUMEN
Surfactin is a cyclic lipopeptide antibiotic that disturbs the integrity of the cytoplasmic membrane. In this study, the role of membrane lipids in the adaptation and possible surfactin tolerance of the surfactin producer Bacillus subtilis ATCC 21332 was investigated. During a 1-day cultivation, the phospholipids of the cell membrane were analyzed at the selected time points, which covered both the early and late stationary phases of growth, when surfactin concentration in the medium gradually rose from 2 to 84µmol·l(-1). During this time period, the phospholipid composition of the surfactin producer's membrane (Sf(+)) was compared to that of its non-producing mutant (Sf(-)). Substantial modifications of the polar head group region in response to the presence of surfactin were found, while the fatty acid content remained unaffected. Simultaneously with surfactin production, a progressive accumulation up to 22% of the stress phospholipid cardiolipin was determined in the Sf(+) membrane, whereas the proportion of phosphatidylethanolamine remained constant. At 24h, cardiolipin was found to be the second major phospholipid of the membrane. In parallel, the Laurdan generalized polarization reported an increasing rigidity of the lipid bilayer. We concluded that an enhanced level of cardiolipin is responsible for the membrane rigidification that hinders the fluidizing effect of surfactin. At the same time cardiolipin, due to its negative charge, may also prevent the surfactin-membrane interaction or surfactin pore formation activity.
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Bacillus subtilis/metabolismo , Cardiolipinas/metabolismo , Citoplasma/metabolismo , Lipopéptidos/biosíntesis , Péptidos Cíclicos/biosíntesis , Bacillus subtilis/crecimiento & desarrollo , Secuencia de Bases , Membrana Celular/metabolismo , Cartilla de ADN , Ácidos Grasos/metabolismo , Lípidos de la Membrana/metabolismo , Reacción en Cadena de la PolimerasaRESUMEN
We report the forensic case of a 42-year-old man, a known drug user, who died at home and whose body was only discovered 2 months later. Autopsy was performed on a corpse in the late postmortem stage where no apparent cause of death was found. A toxicological screening of biological materials (blood, urine and gastric content) using liquid chromatography with different types of mass detection (ion trap and high-resolution) revealed the presence of methoxetamine (MXE), a ketamine analog, and its metabolites. MXE and a number of its metabolites (e.g., O-desmethyl, N-desethyl, hydroxy, glucuronides and sulfates) were identified in urine. Based on the results, a method using liquid chromatography with tandem mass spectrometry was developed and validated for the determination of MXE concentration in biological materials. The following values of MXE concentration were found: blood-3.6 ng/mL, urine-70.5 ng/mL and gastric content-18.0 ng/mL. Given the absence of other drugs, medications and poisons, it can be inferred that despite relatively low blood concentrations, MXE contributed to the victim's death. The present case demonstrates that even after 2 months, MXE and its several metabolites can be detected and determined in the human cadaver at a relatively advanced stage of decomposition.
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Líquidos Corporales , Ciclohexanonas , Ciclohexilaminas , Masculino , Humanos , Adulto , Ciclohexanonas/análisis , Ciclohexanonas/metabolismo , Autopsia , Cadáver , Líquidos Corporales/metabolismoRESUMEN
This paper focuses on the evaluation of variation of relative response factors (RRFs) obtained by two internal standard (IS) methods that are used to control the quality of alcoholic products. A standard IS method using 1-pentanol was compared with an "Ethanol as IS" method. The variation of RRF values for both methods was determined using standard solutions based on 20, 40 and 96% ethanol-water matrices. For this purpose, solutions of the ten most abundant volatile compounds were analysed at four different concentrations (250, 500, 1000 and 5000 mg L-1 absolute alcohol, AA) within these matrices. Each solution was measured four times by gas chromatography-mass spectrometry (GC-MS) in single ion monitoring (SIM) mode under repeatability conditions. Our results showed that for the 40% and 96% matrices, the ethanol and standard IS methods showed similar relative standard deviations (RSDs) variation of no more than 2% within the 250-1000 mg L-1 AA volatile compounds concentration range. For the 20% matrix as well as for the 250-5000 mg L-1 AA concentration range the resulting variation in calibration factors reaches 10% for both methods. As for the whole range of 20-96% alcohol by volume (ABV) and 250-5000 mg L-1 AA volatile concentration range, the resultant RRFs for the standard IS method (8% RSD) were more stable than those for the ethanol IS method (almost 40% RSD). Nonlinearity of the signal-to-amount dependence was also assessed with respect to the injection and detection processes.
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Etanol , Agua , Cromatografía de Gases y Espectrometría de Masas/métodos , Control de CalidadRESUMEN
Recently developed and validated simple and reliable quantitative method employing ethanol as an internal standard (IS) for GC-MS quantification of volatile compounds in alcoholic products was applied to 36 samples including commercially available world-famous brand spirits from 18 countries and homemade distillates. The GC-MS analyses were performed simultaneously by the suggested approach and official IS method that is prescribed in the legislation of EU and USA. The independent samples t-test was employed to evaluate the statistical difference between the results of these two methods. The test revealed no difference in the results and their repeatability. The main benefits of the suggested method are the elimination of the necessity of manual IS addition and samples density measurement thus making it more economical and productive.
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Bebidas Alcohólicas/análisis , Calidad de los Alimentos , Inocuidad de los Alimentos , Cromatografía de Gases y Espectrometría de Masas/métodosRESUMEN
The potential use of ethanol as an internal standard (IS) for GC-MS analysis was studied. The paper describes the analysis of spirit drinks and other alcoholic products which consist of a mixture of water, ethanol, and volatile compounds. In the suggested method, ethanol was employed as an IS for the common procedure of volatile compounds quantification. A number of standard solutions of nine compounds with different concentrations was prepared in a water-ethanol matrix and measured with GC-MS in the SIM mode. Two possible approaches were suggested to avoid detector saturation during ethanol detection. The first one consisted in using less abundant m/z 47 as quantifiers. These ions mainly correspond to unfragmented heavy ethanol molecules containing one 13 C isotope. The second method consisted in reduction of the voltage of MS electron multiplier. The experiment also included the preparation and subsequent dilution of the standard solution and ethanol with water, which determined the linearity of the modified MS response relative to the ethanol content. Analysis of the obtained results revealed that volatile compounds can be successfully accurately determined with GC-MS by employing ethanol as an IS. Application of the suggested method is not limited to the reported volatile compounds and alcoholic products.
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The composition of defensive secretion produced by metathoracic scent glands was analysed in males and females of the milkweed bug Lygaeus equestris (Heteroptera) using gas chromatography with mass spectrometric detection (GC-MS). The bugs were raised either on cardenolide-containing Adonis vernalis or on control sunflower seeds in order to determine whether the possibility to sequester cardenolides from their host plants would affect the composition of defensive scent-gland secretion. Profiles of the composition of defensive secretions of males and females raised on sunflower were closely similar, with predominant presence of (E)-2-octenal, (E)-2-octen-1-ol, decanal and 3-octen-1-ol acetate. The secretion of bugs raised on A. vernalis was more sexually dimorphic, and some chemicals e.g. (E,E)-2,4-hexadienyl acetate and 2-phenylethyl acetate were dominant in males, but absent in females. Compared to bugs from sunflower, the scent-gland secretion of bugs raised on A. vernalis was characterized by lower overall intensity of the peaks obtained for detected chemicals and by absence of some chemicals that have supposedly antipredatory function ((E)-2-hexenal, (E)-4-oxo-hex-2-enal, 2,4-octadienal). The results suggest that there might be a trade-off between the sequestration of defensive chemicals from host plants and their synthesis in metathoracic scent-glands.
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Adonis/química , Heterópteros/química , Defensa de la Planta contra la Herbivoria , Acetatos/análisis , Aldehídos/análisis , Animales , Cromatografía de Gases , Femenino , Cromatografía de Gases y Espectrometría de Masas , Helianthus/química , Masculino , Alcohol Feniletílico/análogos & derivados , Alcohol Feniletílico/análisis , Feromonas/análisis , Glándulas Odoríferas/química , Caracteres Sexuales , Factores Sexuales , OlfatoRESUMEN
Pharmacokinetic data for riociguat in patients with chronic thromboembolic pulmonary hypertension (CTEPH) have previously been reported from randomized clinical trials, which may not fully reflect the population encountered in routine practice. The aim of the current study was to characterize the pharmacokinetic of riociguat and its metabolite M1 in the patients from routine clinical practice. A population pharmacokinetic model was developed in NONMEM 7.3, based on riociguat and its metabolite plasma concentrations from 49 patients with CTEPH. One sample with riociguat and M1 concentrations was available from each patient obtained at different time points after last dose. Age, bodyweight, sex, smoking status, concomitant medications, kidney and liver function markers were tested as potential covariates of pharmacokinetic of riociguat and its metabolite. Riociguat and M1 disposition was best described with one-compartment models. Apparent volume of distribution (Vd/F) for riociguat and M1 were assumed to be the same. Total bilirubin and creatinine clearance were the most predictive covariates for apparent riociguat metabolic clearance to M1 (CLf,M1/F) and for apparent riociguat clearance through remaining pathways (CLe,r/F), respectively. CLf,M1/F, CLe,r/F, Vd/F of riociguat and M1, and clearance of M1 (CLe,M1/F) for a typical individual with 70 mL/min creatinine clearance and 0.69 mg/dL total bilirubin were 0.665 L/h (relative standard error = 17%)), 0.66 (18%) L/h, 3.63 (15%) L and 1.47 (19%) L/h, respectively. Upon visual identification of six outlying individuals, an absorption lag-time of 2.95 (6%) h was estimated for these patients. In conclusion, the only clinical characteristics related to riociguat exposure in patients with CTEPH from routine clinical practice are total bilirubin and creatinine clearance. This confirms the findings of the previous population pharmacokinetic studies based on data from randomized clinical trials.
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In the last decade there has been a dramatic increase in the availability and abuse of synthetic cathinones - new amphetamine-like stimulants. Even though their abuse during pregnancy could have serious adverse effects on the fetus, cathinones are not readily included in neonatal toxicological screenings. Meconium (first neonatal stool) is the specimen of choice to reveal long term drug exposure, however as it is a highly complex matrix, the sample preparation is a critical step before the instrumental analysis. The aim of this work was to develop a suitable meconium sample extraction technique using the advantages of salting-out assisted liquid-liquid extraction (SALLE) and using only MS-friendly organic ammonium salts. We further developed and validated liquid chromatography tandem-mass spectrometry method for the determination of 'traditional' stimulants (methamphetamine, amphetamine, MDMA) and cathinones (mephedrone, methylenedioxypyrovalerone (MDPV), α-pyrrolidinopentiophenone (α-PVP), methylone, butylone, flephedrone, and naphyrone). Matrix-matched calibration was prepared in the concentration range 10-2000 ng/g. The limits of quantification were determined as 10 ng/g, recoveries ranged from 48.2% to 94.3% and the matrix effect was between 60.2% and 101.4%. Accuracy (86.1-114.5%) and precision (4.9-14.9%) were determined and all validation criteria were met for all analytes except for naphyrone. Finally, our analytical method was tested on a set of real meconium samples, which were found positive for amphetamine, methamphetamine and methylone, thus demonstrating the validity of the method.
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Anfetaminas/análisis , Estimulantes del Sistema Nervioso Central/análisis , Extracción Líquido-Líquido/métodos , Meconio/química , Detección de Abuso de Sustancias/métodos , Compuestos de Amonio/química , Cromatografía Líquida de Alta Presión/métodos , Estudios de Factibilidad , Femenino , Humanos , Recién Nacido , Límite de Detección , Embarazo , Complicaciones del Embarazo/diagnóstico , Trastornos Relacionados con Sustancias/diagnóstico , Espectrometría de Masas en Tándem/métodosRESUMEN
Food contact materials (FCMs) may release their chemical components into food and thus raise safety concerns. This paper attempted to study the presence of four major groups of FCM-related endocrine disruptors in fatty food: dialkyl phthalates, bisphenols, printing ink photoinitiators, and polyfluoroalkyl substances. All 41 target compounds were analyzed simultaneously by means of liquid chromatography coupled to tandem mass spectrometry. The sample preparation was significantly streamlined to reduce analysis costs by employing acetonitrile extraction, extract modification by water, and refrigeration at 5 °C. The new method was validated and applied to 60 real samples, including edible oils, butter, and chocolate, where 16 target compounds were measured at levels ≤13000 ng/g. The study also described the blank level increase and sensitivity loss caused by impurities present in the HPLC methanol solvent.
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Cromatografía Líquida de Alta Presión/métodos , Análisis de los Alimentos/métodos , Contaminación de Alimentos/análisis , Espectrometría de Masas en Tándem/métodos , Compuestos de Bencidrilo/análisis , Fenoles/análisis , Ácidos Ftálicos/análisisRESUMEN
Perampanel is a novel antiepileptic drug used in paediatric patients. Existing methods that determine serum perampanel are of limited practicability. We developed a novel capillary electrophoresis (CE) method using a new version of acetonitrile stacking for on-line sample pre-concentration, and fluorescence detection (FD). CE separations were performed in a fused-silica capillary where the electroosmotic flow was reduced by coating the inner surface using a INST coating solution. The optimised background electrolyte composition was 50 mM chloroacetic acid with addition of 0.5% m/v polyvinylalcohol (pH 2.15) and separation was driven by application of positive voltage + 30 kV. Serum samples (25 µL) treated by the addition of acetonitrile in a ratio of 1:3 v/v were each injected into the capillary at a large volume that corresponded to the length (129 mm) of the sample zone (hydrodynamic pressure impulse 6000 mbars). Acetonitrile stacking is based on the forcing the sample zone out of the capillary with simultaneous application of the separation voltage. Under such conditions, the enhancing factor achieves the value 57 for peak area compared to the small sample injection length (3.2 mm, hydrodynamic pressure impulse 150 mbar.s). A fluorescence detector with a broad excitation filter (240-400 nm) and an emission filter (495 nm) was used for visualisation of the native fluorescence of perampanel. The calibration dependence of the method was linear (in the range of 10-1000 ng mL-1), with adequate accuracy (99.8-103.3 %) and precision (13.1%). LOD and LOQ for perampanel were 2.9 ng mL-1 and 9.5 ng mL-1, respectively. Clinical applicability was validated using serum samples from patients treated with perampanel and the results corresponded with reference LC-MS/MS values. Our method offers a promising alternative for determining serum perampanel with several advantages. In particular, the low quantity of serum (25 µL) required means that testing can be performed on samples obtained for monitoring other antiepileptic medications, and thus reduces the test-burden on paediatric patients.