Quantitative Analysis of Cenobamate and Concomitant Anti-Seizure Medications in Human Plasma via Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry.

Cenobamate (CNB) is a new anti-seizure medication (ASM) recently introduced in clinical practice after approval by the FDA and EMA for the add-on treatment of focal onset seizures in adult patients. Although its mechanism of action has not been fully understood, CNB showed promising clinical efficacy in patients treated with concomitant ASMs. The accessibility of CNB could pave a way for the treatment of refractory or drug-resistant epilepsies, which still affect at least one-third of the patients under pharmacological treatment. In this context, therapeutic drug monitoring (TDM) offers a massive opportunity for better management of epileptic patients, especially those undergoing combined therapy. Here, we describe the first fully validated ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the quantification of CNB and concomitant ASMs in human plasma, with samples extracted either manually or by means of a liquid handler. Our method was validated according to the most recent ICH International Guideline M10 for Bioanalytical Method Validation and Study Sample Analysis. The method proved to be selective for CNB and displayed a linear range from 0.8 to 80 mg/L; no matrix effect was found (98.2 ± 4.1%), while intra-day and inter-day accuracy and precision were within the acceptance range. Also, CNB short- and long-term stability in plasma under different conditions was assessed. Leftover human plasma samples were employed as study samples for method validation. Our method proved to be highly sensitive and selective to quantify CNB and concomitant ASMs in human plasma; therefore, this method can be employed for a routinely TDM-based approach to support physicians in the management of an epileptic patient.

Pharmacological Treatments and Therapeutic Drug Monitoring in Patients with Chronic Pain.

Pain is an unpleasant sensory and emotional experience that affects every aspect of a patient's life and which may be treated through different pharmacological and non-pharmacological approaches. Analgesics are the drugs most commonly used to treat pain, and in specific situations, the use of opioids may be considered with caution. These drugs, in fact, do not always induce optimal analgesia in patients, and several problems are associated with their use. The purpose of this narrative review is to describe the pharmacological approaches currently used for the management of chronic pain. We review several aspects, from the pain-scale-based methods currently available to assess the type and intensity of pain, to the most frequently administered drugs (non-narcotic analgesics and narcotic analgesics), whose pharmacological characteristics are briefly reported. Overall, we attempt to provide an overview of different pharmacological treatments while also illustrating the relevant guidelines and indications. We then report the strategies that may be used to reduce problems related to opioid use. Specifically, we focus our attention on therapeutic drug monitoring (TDM), a tool that could help clinicians select the most suitable drug and dose to be used for each patient. The actual potential of using TDM to optimize and personalize opioid-based pain treatments is finally discussed based on recent scientific reports.

Development and Validation of a New LC-MS/MS Bioanalytical Method for the Simultaneous Determination of Levodopa, Levodopa Methyl Ester, and Carbidopa in Human Plasma Samples.

Levodopa (L-DOPA) treatment, combined with the administration of dopa-decarboxylase inhibitors (DDCIs), is still the most effective symptomatic treatment of Parkinson's disease (PD). Although its efficacy in the early stage of the disease has been confirmed, its complex pharmacokinetics (PK) increases the variability of the intra-individual motor response, thus amplifying the risk of motor/non-motor fluctuations and dyskinesia. Moreover, it has been demonstrated that L-DOPA PK is strongly influenced by several clinical, therapeutic, and lifestyle variables (e.g., dietary proteins). L-DOPA therapeutic monitoring is therefore crucial to provide personalized therapy, hence improving drug efficacy and safety. To this aim, we have developed and validated an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method to quantify L-DOPA, levodopa methyl ester (LDME), and the DDCI carbidopa in human plasma. The compounds were extracted by protein precipitation and samples were analyzed with a triple quadrupole mass spectrometer. The method showed good selectivity and specificity for all compounds. No carryover was observed, and dilution integrity was demonstrated. No matrix effect could be retrieved; intra-day and inter-day precision and accuracy values met the acceptance criteria. Reinjection reproducibility was assessed. The described method was successfully applied to a 45-year-old male patient to compare the pharmacokinetic behavior of an L-DOPA-based medical treatment involving commercially available Mucuna pruriens extracts and an LDME/carbidopa (100/25 mg) formulation.

Standard addition method (SAM) in LC-MS/MS to quantify gluten-derived metabolites in urine samples.

A tight adherence to a gluten-free diet (GFD), the most effective treatment currently available for celiac disease, is important to reduce symptoms, avoid nutritional deficiencies and improve quality of life in celiac patients. The development of analytical methods allowing detecting gluten exposure due to occasional or involuntary food transgressions could represent a useful tool to monitor patient habits and conditions and prevent long-term complications. The aim of this work was to develop and validate an approach based on the standard addition methodology (SAM) for the detection and quantification of two main metabolites of alkylresorcinols, 3,5-dihydroxybenzoic acid (DHBA) and 3-(3,5-dihydroxyphenyl)-propanoic acid (DHPPA), whose presence in urine samples is related to the intake of gluten-containing foods. Analytically, the method consisted of a protein precipitation step followed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis. The chromatographic method involved the use of a hydrophilic interaction liquid chromatography (HILIC) in a direct phase approach; LC-MS/MS analyses were performed in selected reaction monitoring (SRM) mode. Manipulation and instrumental errors were normalised using stable isotopic standards (ISs). The SAM approach here described requires less than 1 mL of urine per sample, thus greatly reducing the sample volume needed. Noteworthy, despite the small cohort of samples analysed, our data allowed to identify a potential "threshold" value, around 200 ng/mL for DHBA and 400 ng/mL for DHPPA, to discriminate between a GFD and a gluten rich diet (GRD).

A LC-MS/MS based methodology for the environmental monitoring of healthcare settings contaminated with antineoplastic agents.

Background: Adverse health events associated with the exposure of healthcare workers to antineoplastic drugs are well documented in literature and are often related to the chemical contamination of work surfaces. It is therefore crucial for healthcare professionals to validate the efficiency of safety procedures by periodic biological and environmental monitoring activities where the main methodological limitations are related to the complexity, in terms of chemical-physical features and chemical-biological stability, of the drugs analyzed. Materials and methods: Here we describe the evaluation and application of a UHPLC-MS/MS based protocol for the environmental monitoring of hospital working areas potentially contaminated with methotrexate, iphosphamide, cyclophosphamide, doxorubicin, irinotecan, and paclitaxel. This methodology was used to evaluate working areas devoted to the preparation of chemotherapeutics and combination regimens at the University Hospital "San Giovanni di Dio e Ruggi d'Aragona" in Salerno (Italy). Results: Our analyses allowed to uncover critical aspects in both working protocols and workspace organization, which highlighted, among others, cyclophosphamide and iphosphamide contamination. Suitable adjustments adopted after our environmental monitoring campaign significantly reduced the exposure risk for healthcare workers employed in the unit analyzed. Conclusion: The use of sensitive analytical approaches such as LC-MS/MS coupled to an accurate wiping procedure in routine environmental monitoring allows to effectively improve chemical safety for exposed workers.

Development and Validation of a UHPLC-MS/MS-Based Method to Quantify Cenobamate in Human Plasma Samples.

Cenobamate (CNB) is the newest antiseizure medication (ASM) approved by the FDA in 2019 to reduce uncontrolled partial-onset seizures in adult patients. Marketed as Xcopri in the USA or Ontozry in the EU (tablets), its mechanism of action has not been fully understood yet; however, it is known that it inhibits voltage-gated sodium channels and positively modulates the aminobutyric acid (GABA) ion channel. CNB shows 88% of oral bioavailability and is responsible for modifying the plasma concentrations of other co-administered ASMs, such as lamotrigine, carbamazepine, phenytoin, phenobarbital and the active metabolite of clobazam. It also interferes with CYP2B6 and CYP3A substrates. Nowadays, few methods are reported in the literature to quantify CNB in human plasma. The aim of this study was to develop and validate, according to the most recent guidelines, an analytical method using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) to evaluate CNB dosage in plasma samples. Furthermore, we provided a preliminary clinical application of our methodology by evaluating the pharmacokinetic parameters of CNB in two non-adult patients. Plasma levels were monitored for two months. Preliminary data showed a linear increase in plasma CNB concentrations, in both patients, in agreement with the increase in CNB dosage. A seizure-free state was reported for both patients at the dose of 150 mg per day.

The Effect of Plasma Protein Binding on the Therapeutic Monitoring of Antiseizure Medications.

Epilepsy is a widely diffused neurological disorder including a heterogeneous range of syndromes with different aetiology, severity and prognosis. Pharmacological treatments are based on the use, either in mono- or in polytherapy, of antiseizure medications (ASMs), which act at different synaptic levels, generally modifying the excitatory and/or inhibitory response through different action mechanisms. To reduce the risk of adverse effects and drug interactions, ASMs levels should be closely evaluated in biological fluids performing an appropriate Therapeutic Drug Monitoring (TDM). However, many decisions in TDM are based on the determination of the total drug concentration although measurement of the free fraction, which is not bound to plasma proteins, is becoming of ever-increasing importance since it correlates better with pharmacological and toxicological effects. Aim of this work has been to review methodological aspects concerning the evaluation of the free plasmatic fraction of some ASMs, focusing on the effect and the clinical significance that drug-protein binding has in the case of widely used drugs such as valproic acid, phenytoin, perampanel and carbamazepine. Although several validated methodologies are currently available which are effective in separating and quantifying the different forms of a drug, prospective validation studies are undoubtedly needed to better correlate, in real-world clinical contexts, pharmacokinetic monitoring to clinical outcomes.

Perampanel dosage in plasma samples: development and validation of a novel HPLC method with combined UV-Fluorescence detection.

Therapeutic drug monitoring (TDM) is a recognized method to improve the quality of use of antiepileptic drugs, such as perampanel (PRP). It is the first compound in the class of selective non-competitive antagonists of AMPA receptors approved in 2012 in Europe and United States for adjunctive therapy of partial seizures. Although several studies have recently underlined that a general reference range for PRP plasmatic concentration might be difficult to propose, TDM of this drug is important in specific clinical situations, as hepatic or renal impairment or co-administration with enzyme-inducing antiepileptics. Several methods have been described in literature for the determination of PRP in different biological matrices, which include the use of liquid chromatography methods coupled with ultraviolet, fluorescence, mass or tandem-mass spectrometry detection. Here we describe the development and validation of a novel method for the measurement of PRP in plasma samples, based on a HPLC-UV/FL double detection approach and using ketoprofen as internal standard. PRP concentration in a small subset of plasma samples of treated patients was evaluated using both our approach and a commercially available CE-IVD LC-MS/MS method. The results obtained were compared, and confirmed the possibility to use our method as an alternative to LC-MS/MS in clinical routine.

Environmental and biological monitoring of formaldehyde inside a hospital setting: a combined approach to manage chemical risk in workplaces.

BACKGROUND: The safety of healthcare workers exposed to formaldehyde remains a great matter of concern for healthcare management units. This work aimed at describing the results of a combined monitoring approach (environmental and biological) to manage occupational exposure to formaldehyde in a hospital setting. DESIGN AND METHODS: Environmental monitoring of working spaces and biological monitoring of urinary formaldehyde in 16 exposed healthcare workers of the Anatomic Pathology Unit of a University Hospital in Southern Italy was performed on a four-year timescale (2016-2019). RESULTS: Values of aero-dispersed formaldehyde identified were on average low; although workers' urinary formaldehyde levels were also minimal, the statistical analysis highlighted a slight weekly accumulation. CONCLUSIONS: Our data confirm that both environmental and biological monitoring are important to identify risk situations, in particular when values of hazardous compounds are below the accepted occupational exposure levels.

Chemical risk in hospital settings: Overview on monitoring strategies and international regulatory aspects.

Chemical risk in hospital settings is a growing concern that health professionals and supervisory authorities must deal with daily. Exposure to chemical risk is quite different depending on the hospital department involved and might origin from multiple sources, such as the use of sterilizing agents, disinfectants, detergents, solvents, heavy metals, dangerous drugs, and anesthetic gases. Improving prevention procedures and constantly monitoring the presence and level of potentially toxic substances, both in workers (biological monitoring) and in working environments (environmental monitoring), might significantly reduce the risk of exposure and contaminations. The purpose of this article is to present an overview on this subject, which includes the current international regulations, the chemical pollutants to which medical and paramedical personnel are mainly exposed, and the strategies developed to improve safety conditions for all healthcare workers.

A Genotyping/Phenotyping Approach with Careful Clinical Monitoring to Manage the Fluoropyrimidines-Based Therapy: Clinical Cases and Systematic Review of the Literature.

Fluoropyrimidines (FP) are mainly metabolised by dihydropyrimidine dehydrogenase (DPD), encoded by the DPYD gene. FP pharmacogenetics, including four DPYD polymorphisms (DPYD-PGx), is recommended to tailor the FP-based chemotherapy. These polymorphisms increase the risk of severe toxicity; thus, the DPYD-PGx should be performed prior to starting FP. Other factors influence FP safety, therefore phenotyping methods, such as the measurement of 5-fluorouracil (5-FU) clearance and DPD activity, could complement the DPYD-PGx. We describe a case series of patients in whom we performed DPYD-PGx (by real-time PCR), 5-FU clearance and a dihydrouracil/uracil ratio (as the phenotyping analysis) and a continuous clinical monitoring. Patients who had already experienced severe toxicity were then identified as carriers of DPYD variants. The plasmatic dihydrouracil/uracil ratio (by high-performance liquid chromatography (HPLC)) ranged between 1.77 and 7.38. 5-FU clearance (by ultra-HPLC with tandem mass spectrometry) was measured in 3/11 patients. In one of them, it reduced after the 5-FU dosage was halved; in the other case, it remained high despite a drastic dosage reduction. Moreover, we performed a systematic review on genotyping/phenotyping combinations used as predictive factors of FP safety. Measuring the plasmatic 5-FU clearance and/or dihydrouracil/uracil (UH2/U) ratio could improve the predictive potential of DPYD-PGx. The upfront DPYD-PGx combined with clinical monitoring and feasible phenotyping method is essential to optimising FP-based chemotherapy.