UHPLC-MS/MS method with protein precipitation extraction for the simultaneous quantification of ten antihypertensive drugs in human plasma from resistant hypertensive patients.

Today the management of resistant hypertension is a critical health problem: the main difficulty on this field is the discrimination of cases of poor therapeutic adherence from cases of real resistance. This gives rise to the need of high throughput and reliable quantification methods for the Therapeutic Drug Monitoring (TDM) of antihypertensive drugs. The aim of this work was the development and validation of a UHPLC-Tandem mass spectrometry assay for this application and its use in plasma from patients with resistant hypertension. The novelty of this method resides in the ability to simultaneously quantify a wide panel of antihypertensive drugs: amlodipine, atenolol, clonidine, chlortalidone, doxazosin, hydrochlorothiazide, nifedipine, olmesartan, ramipril and telmisartan. Moreover, this method stands out for its simplicity and cheapness, resulting feasible for clinical routine. Both standards and quality controls were prepared in human plasma. After the addition of internal standard, each sample underwent protein precipitation with acetonitrile and was then dried. Extracts were resuspended in water:acetonitrile 90:10 (0.05% formic acid) and then injected into the chromatographic system. Chromatographic separation was performed on an Acquity(®) UPLC HSS T3 1.8µm 2.1×150mm column, with a gradient of water and acetonitrile, both added with 0.05% formic acid. Accuracy, intra-day and inter-day precision fitted FDA guidelines for all analytes, while matrix effects and recoveries resulted stable between samples for each analyte. Finally, we tested this method by monitoring plasma concentrations in 22 hypertensive patients with good results. This simple analytical method could represent a useful tool for the management of antihypertensive therapy.

Entecavir plasma concentrations are inversely related to HBV-DNA decrease in a cohort of treatment-naïve patients with chronic hepatitis B.

The role of therapeutic drug monitoring (TDM) of entecavir (ETV) in the treatment of patients affected by chronic hepatitis B (CHB) has not yet been defined. Here we present an interim analysis regarding the role of ETV TDM in a prospective cohort of treatment-naïve patients with CHB who received this treatment. The results from 40 patients consecutively enrolled at our centre from 2010 to 2013 are described. The primary endpoint was the evaluation of the role of ETV plasma concentrations in the kinetics of hepatitis B virus (HBV) DNA decrease. Minimum ETV concentrations (Ctrough) were measured every month after the start of therapy for the first 3 months and then every 6 months. The main result of the pharmacokinetic analysis was the significant inverse correlation of ETV concentration after 1 month of treatment and HBV-DNA decrease after 3 months of treatment (r = -0.624; P <0.001). This correlation was also confirmed when stratifying patients on the basis of viral genotypes: A (r = -0.719; P = 0.003); C (r = -0.917; P = 0.007); and D (r = -0.760; P = 0.007). Possible explanations for this phenomenon could involve interpatient differences in liver conditions (tissue damage or inflammation) and/or genetic variability in specific drug transporters. Further investigations are needed to confirm these results quantifying ETV concentration in peripheral blood mononuclear cells as well as in a larger cohort.

UHPLC-MS/MS method with automated on-line solid phase extraction for the quantification of entecavir in peripheral blood mononuclear cells of HBV+ patients.

To date five nucleoside analogs are used in the treatment of chronic hepatitis B: among these, entecavir is the most used. Nevertheless a few information about its distribution in tissues is currently known. Since the determination of entecavir disposition in the hepatocytes is impracticable because of its invasiveness, the quantification in an "easier-to-obtain" cellular model could be a good choice. In this work, we developed and validated an ultra performance liquid chromatography-tandem mass spectrometry assay based on an automated on-line SPE, to quantify entecavir concentrations in peripheral blood mononucleated cells (PBMCs), in both its phosphorylated and un-phosphorylated forms. To achieve this, each PBMC isolate was divided in two aliquots, one was treated with acid phosphatase to convert entecavir phosphorylated metabolites into free form, the other one was not-treated. Standards and quality controls were prepared in PBMCs, isolated from healthy donors, and underwent the same process. 20 µL of the resulting solutions were injected in the on-line SPE system. Thymidine was used as internal standard. Calibration curves fitted a linear model for entecavir levels in a range from 0.039 ng to 5 ng (mean r(2)=0.998). Accuracy, intra-day and inter-day precision of the method fitted FDA guidelines recommendations. Moreover, recovery was consistent and matrix effect resulted low and reproducible. We tested this method by monitoring entecavir concentrations in PBMCs from 28HBV mono-infected patients, confirming its reliability and suitability for the evaluation of intracellular entecavir penetration.