9 research outputs found
Cannabidiol, â9 -Tetrahydrocannabinol, and Metabolites in Human Blood by Volumetric Absorptive Microsampling and LC-MS/MS Following Controlled Administration in Epilepsy Patients
Cannabidiol (CBD) exhibits anti-inflammatory, anxiolytic, antiseizure, and neuroprotective proprieties without addictive or psychotropic side effects, as opposed to Î9-tetrahydrocannabinol (THC). While recreational cannabis contains higher THC and lower CBD concentrations, medical cannabis contains THC and CBD in different ratios, along with minor phytocannabinoids, terpenes, flavonoids and other chemicals. A volumetric absorptive microsampling (VAMS) method combined with ultra-high-performance liquid chromatography coupled with mass spectrometry in tandem for quantification of CBD, THC and their respective metabolites: cannabidiol-7-oic acid (7-COOH-CBD); 7-hydroxy-cannabidiol (7-OH-CBD); 6-alpha-hydroxy-cannabidiol (6-Îą-OH-CBD); and 6-beta-hydroxycannabidiol (6-β-OH-CBD); 11- Hydroxy-Î9-tetrahydrocannabinol (11-OH-THC) and 11-Nor-9-carboxy-Î9-tetrahydrocannabinol (THCCOOH). After overnight enzymatic glucuronide hydrolysis at 37°C, samples underwent acidic along with basic liquid-liquid extraction with hexane: ethyl acetate (9:1, v/v). Chromatographic separation was carried out on a C18 column, with the mass spectrometer operated in multiple reaction monitoring mode and negative electrospray ionization. Seven patients with intractable epilepsy were dosed with various CBD-containing formulations and blood collected just before their daily morning administration. The method was validated following international guidelines in toxicology. Linear ranges were (ng/ml) 0.5-25 THC, 11-OH-THC, THCCOOH, 6-Îą-OH-CBD and 6-β-OH-CBD; 10-500 CBD and 7-OH-CBD; and 20-5000 7-COOH-CBD. 7-COOH-CBD was present in the highest concentrations, followed by 7-OH-CBD and CBD. This analytical method is useful for investigating CBD, THC and their major metabolites in epilepsy patients treated with CBD preparations employing a minimally invasive microsampling technique requiring only 30 ÂľL blood
Sviluppo e validazione di un metodo basato su microcampionamento e UHPLC-MS/MS per il monitoraggio terapeutico del Cannabidiolo.
Negli ultimi anni, lâanalisi dei principi attivi della cannabis per il monitoraggio terapeutico del farmaco (TDM), è divenuta sempre piĂš attraente alla luce della possibilitĂ di creare una terapia specifica per ciascun paziente. I metodi analitici impiegati per il monitoraggio terapeutico dei farmaci devono garantire elevata specificitĂ , accuratezza e riproducibilitĂ al fine di misurare una vasta gamma di analiti in diverse matrici complesse anche in presenza di interferenti, il tutto in tempistiche dâanalisi accettabili. Recentemente, la determinazione quantitativa dei cannabinoidi avviene mediante lâuso di metodi analitici basati sullâanalisi di cromatografia ad altissime prestazioni accoppiata alla spettrometria di massa tandem (UHPLC-MS/MS), strumentazione che garantisce specificitĂ , precisione e accuratezza nel risultato finale. Tale metodica dâanalisi è considerata come metodo âgold standardâ per la determinazione di tali analiti, e considerando le caratteristiche peculiari di tali composti, è necessaria unâalta specificitĂ metodologica.
Il TDM presenta numerosi vantaggi, pur portando con sĂŠ una serie di punti sfavorevoli. In primo luogo la necessitĂ di sottoporre periodicamente il paziente ad una trattamento invasivo: il prelievo ematico. Ponendo poi particolare attenzione ai pazienti pediatrici, questa procedura può addirittura risultare per certi versi âtraumaticaâ, per tale ragione potrebbe essere utile la valutazione dellâuso di un micro-campionamento non invasivo.
Lo scopo di questa tesi è stato quello di sviluppare e validare un metodo UHPLC-MS/MS, seguendo le linee guida dellâEuropean Medicines Agency, che permetta di determinare quantitativamente i principi attivi della cannabis, Î9-Tetraidrocannabinolo (THC) e Cannabibiolo (CBD) su, sangue intero campionato mediante dispositivi Volumetric Absorptive MicroSampling (VAMS).
Il presente lavoro di tesi è stato svolto grazie alla collaborazione con lâIstituto Giannina Gaslini, che è il centro di riferimento per la regione Liguria riguardo il trattamento di pazienti pediatrici mediante Cannabis terapeutica. Questi ultimi presentano spesso condizioni cliniche complesse, quali epilessie farmacoresistenti.
La possibilità dunque di poter effettuare un campionamento non invasivo, che sia inoltre di facile applicazione, può sicuramente garantire migliori condizioni di tollerabilità terapeutica nel paziente pediatrico
A novel LCâMS/MS-based method for the diagnosis of ADA2 deficiency from dried plasma spot
none10noAdenosine Deaminase 2 Deficiency (DADA2) (OMIM: 607575) is a monogenic, autoin-flammatory disease caused by the loss of functional homozygous or heterozygous mutations in the ADA 2 gene (previously CECR1, Cat Eye Syndrome Chromosome Region 1). A timely diagnosis is crucial to start Anti-TNF therapies that are efficacious in controlling the disease. The confirmation of DADA2 is based on DNA sequencing and enzymatic assay. It is, thus, very important to have robust and reliable assays that can be rapidly utilized in specialized laboratories that can centralize samples from other centers. In this paper, we show a novel enzymatic assay based on liquid chromatography-tandem mass spectrometry that allows the accurate determination of the ADA2 enzyme activity starting from very small amounts of plasma spotted on filter paper (dried plasma spot). The method allows significantly distinguishing healthy controls from affected patients and carriers and could be of help in implementing the diagnostic workflow of DADA2.openCafaro A.; Pigliasco F.; Barco S.; Penco F.; Schena F.; Caorsi R.; Volpi S.; Tripodi G.; Gattorno M.; Cangemi G.Cafaro, A.; Pigliasco, F.; Barco, S.; Penco, F.; Schena, F.; Caorsi, R.; Volpi, S.; Tripodi, G.; Gattorno, M.; Cangemi, G
Development and Validation of a Novel LC-MS/MS Method for a TDM-Guided Personalization of HSCT Conditioning with High-Dose Busulfan in Children
Personalization of busulfan (Bu) exposure via therapeutic drug monitoring (TDM) is recommended for patients treated with high-dose conditioning regimens. Several laboratoriesâ developed methods are available in the literature with a lack of standardization. The aim of this study is to develop a new standardized LC-MS/MS method and validate it according to the international ICH M10 (EMA) guidelines. Our method is based on rapid protein precipitation from 50 ÂľL plasma followed by separation on a reversed-phase C-18 UHPLC column after the addition of deuterated internal standard and has been tested on real samples from pediatric patients treated with myeloablative conditioning regimens, including Bu, before autologous or allogeneic hematopoietic stem cell transplantation (HSCT). The validated LC-MS/MS method is linear over wide concentration ranges (125â2000 ng/mL), accurate, and reproducible in the absence of matrix effects, allowing for the specific and rapid quantification of Bu and allowing next-dose recommendations to be made in a timely fashion to answer cliniciansâ needs. Given the lack of data on the stability of Bu in real clinical samples, stability was assessed both on quality controls and on real samples to set up a robust protocol in real-life conditions. This novel LC-MS/MS method is suitable for application to the TDM-guided personalization of conditioning treatments with high-dose busulfan in pediatric patients undergoing HSCT
A Novel LC-MS/MS Method for Therapeutic Drug Monitoring of Baricitinib in Plasma of Pediatric Patients
Background:
Janus kinase inhibitors are antirheumatic immunosuppressive drugs that target intracellular Janus kinases (JAKs). Baricitinib is a selective and reversible orally administered JAK1/JAK2 inhibitor approved for treating rheumatoid arthritis, atopic dermatitis, and alopecia areata in adult patients. Expanded access to baricitinib has been approved for treating pediatric patients affected by rare Mendelian autoinflammatory diseases with type I interferon-mediated damage. Knowledge of the pharmacokinetic properties and target plasma levels of baricitinib in pediatric patients is limited. In this study, a novel LC-MS/MS method for measuring baricitinib in plasma, validated according to the ICH M10 guidelines, is presented.
Methods:
Sample preparation was performed by adding 10 ÎźL of IS working solution (150 ng/mL) and 200 ÎźL of MeOH to each plasma sample. Chromatographic separation was conducted using a Thermo Scientific Accucore Polar Premium column (50 mm Ă 2.1 mm, i.d. 2.6 m). This method was applied to 7 real anonymous plasma samples obtained from pediatric patients treated with baricitinib at IRCCS Istituto Giannina Gaslini (Genoa, Italy). Patients of both sexes had a median age of 14 years (range, 10â17 years).
Results:
The LC-MS/MS method resulted linear over wide concentration ranges (1.024â100 ng/mL) and was accurate and reproducible in the absence of matrix effects, allowing for robust, specific, and rapid quantification of baricitinib from a low amount of plasma (50 ÎźL). The plasma concentration of baricitinib in the samples of the patients, expressed as mean Âą SD, was 11.25 Âą 10.86 ng/mL.
Conclusions:
This novel LC-MS/MS method is suitable for the therapeutic drug monitoring of baricitinib and can help guide therapy optimization in pediatric patients
A UHPLCâMS/MS Method for Therapeutic Drug Monitoring of Aciclovir and Ganciclovir in Plasma and Dried Plasma Spots
The role of therapeutic drug monitoring (TDM) of valaciclovir (VA)/aciclovir (A) and valganciclovir/ganciclovir (VG/G) in critically ill patients is still a matter of debate. More data on the doseâconcentration relationship might therefore be useful, especially in pediatrics where clinical practice is not adequately supported by robust PK studies. We developed and validated a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) micro-method to simultaneously quantify A and G from plasma and dried plasma spots (DPS). The method was based on rapid organic extraction from DPS and separation on a reversed-phase C-18 UHPLC column after addition of deuterated internal standards. Accurate analyte quantification using SRM detection was then obtained using a Thermo Fisher Quantiva triple-quadrupole MS coupled to an Ultimate 3000 UHPLC. It was validated following international (EMA) guidelines for bioanalytical method validation and was tested on samples from pediatric patients treated with A, VG, or G for cytomegalovirus infection following solid organ or hematopoietic stem cell transplantation. Concentrations obtained from plasma and DPS were compared using PassingâBablok and BlandâAltman statistical tests. The assay was linear over wide concentration ranges (0.01â20 mg/L) in both plasma and DPS for A and G, suitable for the expected therapeutic ranges for both Cmin and Cmax, accurate, and reproducible in the absence of matrix effects. The results obtained from plasma and DPS were comparable. Using an LC-MS/MS method allowed us to obtain a very specific, sensitive, and rapid quantification of these antiviral drugs starting from very low volumes (50 ÎźL) of plasma samples and DPS. The stability of analytes for at least 30 days allows for cost-effective shipment and storage at room temperature. Our method is suitable for TDM and could be helpful for improving knowledge on PK/PD targets of antivirals in critically ill pediatric patients
Simultaneous Quantification of Ivacaftor, Tezacaftor, and Elexacaftor in Cystic Fibrosis Patientsâ Plasma by a Novel LCâMS/MS Method
The new breakthrough cystic fibrosis (CF) drug combination of ivacaftor (IVA), tezacaftor (TEZ), and elexacaftor (ELX), namely âcaftorâ drugs, directly modulates the activity and trafficking of the defective CF transmembrane conductance regulator protein (CFTR) underlying the CF disease. The role of therapeutic drug monitoring (TDM) of caftor drugs in clinical settings has recently been established. The availability of reliable and robust analytical methods for the quantification of IVA, TEZ, and ELX is essential to support doseâconcentrationâeffect studies. We have developed and validated a new liquid chromatographyâtandem mass spectrometry (LCâMS/MS) for the rapid and simultaneous quantification of IVA, TEZ, and ELX from the plasma of CF patients. The method was based on a rapid extraction protocol from 50 ÎźL human plasma and separation on a reversed-phase C-18 HPLC column after the addition of deuterated internal standards. Accurate analyte quantification using multiple reaction monitoring (MRM) detection was then obtained using a Thermofisher Quantiva triple-quadrupole MS coupled to an Ultimate 3000 UHPLC. The method has been validated following international (EMA) guidelines for bioanalytical method validation and has been tested on plasma samples from 62 CF patients treated with the three-drug combination IVA/TEZ/ELX, marketed as KaftrioÂŽ or TrikaftaÂŽ, in steady-state condition. The assay was linear over wide concentration ranges (0.008â12 mg/L) in plasma for IVA, TEZ, and ELX, suitable for a broad range of plasma concentrations, and accurate and reproducible in the absence of matrix effects. The stability of analytes for at least 30 days at room temperature could allow for cost-effective shipment and storage. On the same day of sample collection, a sweat test was evaluated for 26 associated patientsâ samples, FEV1 (%) for 58, and BMI was calculated for 62. However, Spearman correlation showed no correlation between Cthrough plasma concentrations of analytes (IVA, TEZ, ELX) and sweat test, FEV1 (%), or BMI. Our method proved to be suitable for TDM and could be helpful in assessing doseâconcentrationâresponse correlations in larger studies
UHPLC-MS/MS Analysis of Cannabidiol and Its Metabolites in Serum of Patients with Resistant Epilepsy Treated with CBD Formulations
Cannabidiol (CBD) is a promising therapeutic agent with analgesic, myorelaxant, and anti-epileptic actions. Recently, a purified form of CBD (EpidiolexÂŽ) has been approved by the European Medicines Agency (EMA) for the treatment of two highly-refractory childhood-onset epilepsies (Dravet and Lennox-Gastaut syndrome). Given the interindividual response and the relationship between the dose administered and CBD blood levels, therapeutic drug monitoring (TDM) is a valuable support in the clinical management of patients. We herein report for the first time a newly developed and validated method using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLCâMS/MS) to evaluate CBD and its metabolites (i.e., cannabidiol-7-oic acid (7-COOH-CBD), 7-hydroxycannabidiol (7-OH-CBD), 6-Îą-hydroxycannabidiol (6-ÎąâOHâCBD) and 6-β-hydroxycannabidiol (6-βâOHâCBD)) in serum samples. The method reached the sensitivity needed to detect minimal amounts of analytes under investigation with limits of quantification ranging from 0.5 to 20 ng/mL. The validation results indicated in this method were accurate (average inter/intra-day error, <15%), precise (inter/intra-day imprecision, <15%), and fast (8 min run time). The method resulted to be linear in the range of 1â10,000 ng/mL for CBD-COOH, 1â500 ng/mL for 7-OH-CBD and CBD and 1â25 ng/mL for 6-ÎąâOHâCBD and 6-βâOHâCBD. Serum levels of CBD (88.20â396.31 and 13.19â170.63 ng/mL) as well as of 7-OH-CBD (27.11â313.63 and 14.01â77.52 ng/mL) and 7-COOH-CBD (380.32â10,112.23 and 300.57â2851.82 ng/mL) were significantly higher (p < 0.05) in patients treated with GW pharma CBD compared to those of patients treated with galenic preparations. 6-ÎąâOHâCBD and 6-βâOHâCBD were detected in the first group and were undetectable in the second group. 7-COOH-CBD was confirmed as the most abundant metabolite in serum (5â10 fold higher than CBD) followed by 7-OH-CBD. A significant correlation (p < 0.05) between the dose administrated and a higher bioavailability was confirmed in patients treated with a GW pharma CBD preparation
Analysis of Cannabinoids Concentration in Cannabis Oil Galenic Preparations: Harmonization between Three Laboratories in Northern Italy
Medical cannabis is increasingly being used in the treatment and support of several diseases
and syndromes. The quantitative determination of active ingredients (delta-9 tetrahydrocannabinol,
THC, and cannabidiol, CBD) in galenic oily preparations is prescribed by law for each produced batch.
The aim of this work is to describe the organization of the titration activity centralized at three regional
reference laboratories in Northern Italy. Pre-analytical, analytical, and post-analytical phases have
been defined in order to guarantee high quality standards. A cross-validation between laboratories
allowed for the definition of the procedures that guarantee the interchangeability between reference
laboratories. The risk management protocol adopted can be useful for others who need to undertake
this activity