The objective of this study was to validate a novel assay using the volumetric absorptive microsampling (VAMS) technique combined with liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) for the determination of the antiseizure medication perampanel in saliva and its clinical applicability in patients with epilepsy. VAMS tips were loaded with 30 μL of saliva and dried for 60 min. Analytes were extracted with methanol. The supernatant was evaporated under a gentle stream of nitrogen and reconstituted with 60 μL of methanol. Separation and quantification were achieved on a monolithic column connected to a mass spectrometer. Calibration curves were linear between 0.5 and 300 ng/mL. Intra- and inter-day accuracy was within 85.6–103.2% and intra-day and inter-day precision did not exceed 12.1%. Perampanel was stable in samples collected by VAMS and stored under different storage conditions. The VAMS-LC-MS/MS method was validated according to internationally accepted criteria and tested in patients with epilepsy who were receiving a combination of perampanel and other antiseizure medications. The method showed adequate bioanalytical performances, holding great potential as an alternative strategy to support domiciliary TDM in patients with epilepsy treated with perampanel according to the simplicity of sample collection.

Heterozygous mutations in the GBA1 gene, encoding the enzyme glucocerebrosidase (GCase), are major risk factors for Parkinson’s Disease (PD). Ambroxol, a small chaperone originally used as a mucolytic agent, has been shown to cross the blood–brain barrier, enhance GCase activity, and reduce α-synuclein levels, making it a promising therapeutic candidate for disease-modifying effects in GBA1-associated PD (GBA1-PD). This study aimed to develop a method to quantify ambroxol levels in human plasma and cerebrospinal fluid (CSF) using liquid chromatography–tandem mass spectrometry (LC-MS/MS). Ambroxol was determined by online solid-phase extraction (SPE), coupled with LC-MS/MS, by gradient elution on a monolithic column. Detection employed a 3200 QTRAP tandem mass spectrometer in the positive electrospray ionization mode. Calibration curves exhibited linearity across the analyzed ranges in both plasma and CSF. The recovery rate ranged from 106.7% to 113.5% in plasma and from 99.0% to 103.0% in CSF. No significant matrix effect was observed. Intra-day and inter-day precisions were below 11.8% in both matrices, and accuracy ranged from 89.9% to 103.1% in plasma and from 96.3% to 107.8% in CSF. We evaluated and confirmed the stability of the analyte in plasma and CSF across various storage conditions. The method was successfully validated according to European Medicine Agency (EMA) guidelines and its applicability was confirmed in the context of a multicenter, randomized, double-blind, placebo-controlled, phase II study, designed to monitor the ambroxol levels in the plasma and CSF of GBA1-PD.

Electroencephalography (EEG) continues to be a pivotal investigation in children with epilepsy, providing diagnostic evidence and supporting syndromic classification. In the pediatric population, electroencephalographic recordings are frequently performed during sleep, since this procedure reduces the number of artifacts and activates epileptiform abnormalities. To date, no shared guidelines are available for sleep induction in EEG. Among the interventions used in the clinical setting, melatonin and sleep deprivation represent the most used methods. The main purpose of this study is to test the non-inferiority of 3–5 mg melatonin versus sleep deprivation in achieving sleep in nap electroencephalography in children and young adult patients with epilepsy. To test non-inferiority, a randomized crossover trial is proposed where 30 patients will be randomized to receive 3–5 mg melatonin or sleep deprivation. Each enrolled subject will perform EEG recordings during sleep in the early afternoon for a total of 60 EEGs. In the melatonin group, the study drug will be administered a single oral dose 30 min prior to the EEG recording. In the sleep deprivation group, parents will be required to subject the child to sleep deprivation the night before registration. Urinary and salivary concentrations of melatonin and of its main metabolite 6-hydroxymelatonin will be determined by using a validated LC-MS method. The present protocol aims to offer a standardized protocol for sleep induction to be applied to EEG recordings in those of pediatric age. In addition, melatonin metabolism and elimination will be characterized and its potential interference in interictal abnormalities will be assessed.

Objective: Electroencephalography (EEG) plays a fundamental role in the diagnosis and classification of epilepsy, and inducing sleep during EEG can improve patient cooperation and enhance the detection of epileptiform activity. Despite its importance, there is currently no standardized approach for sleep induction in pediatric EEG recordings. Consequently, practices such as melatonin administration and sleep deprivation are commonly utilized. This study aimed to compare the effectiveness of 5 mg melatonin versus partial sleep deprivation in inducing sleep during nap‐time EEGs in children with epilepsy. Methods: A randomized crossover trial was conducted involving 33 participants (mean age 14.5 years), each undergoing EEG following either melatonin administration or partial sleep deprivation. In the melatonin arm, participants received an oral dose 30 min before the recording, while in the sleep deprivation arm, sleep was restricted the previous night. The primary outcome was sleep onset latency, defined as the time from relaxation to non‐REM stage 2 sleep on EEG. Additionally, melatonin and its metabolite, 6‐hydroxy‐melatonin, were measured using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). Results: The study showed a mean sleep onset latency of 8.5 min after sleep deprivation and 10.1 min after melatonin administration, with a mean difference of 1.5 min. The analysis of covariance conducted with stratification based on sleep onset latency (five classes) and considering all patients confirmed that melatonin is non‐inferior to sleep deprivation in sleep onset latency, with 97.5% lower confidence limits of −0.37. Melatonin levels in the treated group confirmed adequate absorption, while they were undetectable in the sleep‐deprived group. Significance: Melatonin is non‐inferior to partial sleep deprivation in reducing sleep onset latency, with comparable diagnostic yield and a favorable tolerability profile. The study demonstrates that 5 mg of melatonin is a safe, effective, and well‐tolerated alternative to partial sleep deprivation for sleep induction in pediatric EEG evaluations. Given its ease of use and consistent results, melatonin may be recommended as a practical standard for facilitating EEG recordings in children, particularly those with neurodevelopmental disorders. Plain Language Summary: This randomized crossover trial evaluated the effectiveness and tolerability of melatonin administered before an EEG recording to induce sleep, compared with partial sleep deprivation. The results showed that melatonin helped children achieve sleep and was non‐inferior to sleep deprivation. Moreover, melatonin demonstrated a favorable tolerability profile, representing a safe and easier alternative to support sleep during EEG, particularly in children with neurodevelopmental disorders, and could enhance the efficiency and quality of pediatric EEG recordings.

Perampanel (PER) is a third‐generation antiseizure medication (ASM) with a well‐established concentration–effect relationship. Saliva represents a non‐invasive alternative to plasma for therapeutic drug monitoring (TDM) of PER, as it reflects the free plasma fraction. Volumetric absorptive microsampling (VAMS) enables standardized, patient‐friendly saliva collection. This study aimed to clinically validate saliva‐VAMS by directly comparing it with conventional liquid saliva sampling for PER quantification. In a prospective study, saliva was collected from 11 people with epilepsy receiving PER therapy (dose range = 2–10 mg/day) using both VAMS and conventional saliva sampling. PER concentrations were measured by liquid chromatography coupled with tandem mass spectrometry and compared using repeated measures Bland–Altman analysis and Lin's concordance correlation coefficient to evaluate agreement between methods. PER concentrations measured in saliva‐VAMS showed excellent agreement with those obtained from conventional saliva sampling, with mean bias of −0.126 ng/mL (95% confidence interval = −0.582 to 0.330). No relevant constant or proportional bias was observed, and limits of agreement were within predefined acceptance criteria. Concordance between methods was high, supporting analytical equivalence. The demonstrated interchangeability between saliva‐VAMS and conventional saliva sampling supports adoption of saliva‐VAMS in clinical TDM workflows, enabling standardized fixed‐volume collection, simplified storage/transport, and potential home‐based sampling for remote dose optimization of PER and potentially other ASMs.

This study describes the development and validation of a fully automated workflow for serum sample preparation, enabling the quantitative determination of cannabidiol (CBD) and its active metabolite, 7-hydroxy-CBD, via liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) analysis. Implemented on an automated platform, the workflow performs key steps such as solvent dispensing, mixing, centrifugation, filtration, and supernatant transfer, producing 96-well plates ready for analysis. Human serum samples were obtained from patients with epilepsy treated with CBD. All samples were processed using both manual and automated methods to evaluate method agreement. Quantification was performed by LC–MS/MS with CBD-d₃ as the internal standard (IS). Method validation was conducted in accordance with European Medicine Agency (EMA) guidelines, confirming that the automated protocol meets the recommended acceptance criteria for both intraday and interday precision and accuracy. Calibration curves demonstrated excellent linearity across the concentration ranges. Comparative analysis using Passing–Bablok regression and Bland–Altman plots demonstrated strong agreement between the methods. These findings support the clinical applicability of the automated method for the therapeutic drug monitoring (TDM) of CBD and 7-hydroxy-CBD, and its robust performance and scalability provide a solid foundation for the development of an expanded analytical panel covering a broader range of antiseizure medications (ASMs), enabling more standardized TDM protocols in clinical practice.