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Remdesivir is one of the most encouraging treatments against SARS‐CoV‐2 infection. After intravenous infusion, RDV is rapidly metabolized (T1/2 1h) within the cells to its active adenosine triphosphate analogue form (GS‐443902) and, then, it can be found in plasma in its nucleoside analogue form (GS‐441524). In this real life study we describe the Remdesivir and GS‐441524 concentrations at 3 time points in nine ICU patients, through a validated UHPLC‐MS/MS method. The observed data confirmed the very rapid conversion of RDV to its metabolite and the quite long half‐life of GS‐441524. The mean C min, C max, AUC0–24, were < 0.24 ng/mL and 122.3 ng/ml, 2637,3 ng/mL and 157,8 ng/ml, 5171.2 ng*h/mL and 3676.5 ng*h/ml respectively for RDV and GS‐441524. Three out of nine patients achieved a C max> 2610 ng/mL and 140 ng/mL and AUC0–24 > 1560 ng*h/mL and 2230 ng*h/mL for RDV and GS‐441524, respectively. The mean T1/2 value for GS‐441524 was 26.3 h. Although the low number of patients, these data can represent an interesting preliminary report of the variability of RDV and GS‐441524 concentrations in real‐life ICU setting.

Context Although current international guidelines recommend to avoid mineralocortcoid receptor antagonists in patients undergoing screening test for primary aldosteronism, a recent report suggested that mineralocorticoid receptor antagonist treatment can be continued without significant influence on screening results. Objective We aimed to evaluate the effect of mineralocorticoid receptor antagonists on the aldosterone to renin ratio in patients with primary aldosteronism. Methods We prospectively enrolled 121 patients with confirmed primary aldosteronism who started mineralocorticoid receptor antagonist (canrenone) treatment. Eighteen patients (11 with unilateral and 7 with bilateral primary aldosteronism) constituted the short-term study cohort and underwent aldosterone, renin, and potassium measurement after 2 and 8 weeks of canrenone therapy. The long-term cohort comprised 102 patients (16 with unilateral and 67 with bilateral primary aldosteronism, and 19 with undetermined subtype) who underwent hormonal and biochemical re-assessment after 2 to 12 months of canrenone therapy. Results Renin and potassium levels showed a significant increase, and the aldosterone to renin ratio displayed a significant reduction compared with baseline after both a short- and long-term treatment. These effects were progressively more evident with higher doses of canrenone and after longer periods of treatment. We demonstrated that canrenone exerted a deep impact on the diagnostic accuracy of the screening test for primary aldosteronism: the rate of false negative tests was raised to 16.7%, 38.9%, 54.5%, and 72.5% after 2 weeks, 8 weeks, 2 to 6 months, and 7 to 12 months of mineralocorticoid receptor antagonist treatment, respectively. Conclusion Mineralocorticoid receptor antagonists should be avoided in patients with hypertension before measurement of renin and aldosterone for screening of primary aldosteronism.

Nowadays the practice of therapeutic drug monitoring, consisting in the measurement of drugs concentrations in biological matrices in order to guide possible posological adjustments, is becoming more and more important for the management and optimization of several treatments, especially when drugs with narrow therapeutic indexes are administered. Although TDM on plasma samples is currently considered the gold standard, this practice shows some limitations: it requires venous blood sampling, centrifugation and, if necessary, shipment with refrigeration; moreover, drug concentrations in plasma or blood do not necessarily reflect the ones in the target tissues or cells. Therefore, in the recent years great attention has been given to alternative matrices for TDM purpose, in order to reduce invasiveness, costs or to obtain better information about drug concentrations at the active sites. This evolution is strongly sustained by the spreading use of liquid chromatography coupled with mass spectrometry (LC-MS) techniques for the analysis of small molecules, which is constantly increasing sensitivity and specificity of TDM assays. In this review, we present and summarize recently published LC-MS applications providing alternatives to plasma testing, in order to avoid blood withdrawal, plasma separation, refrigerated shipment or, if possible, to obtain better information about drug exposure in target cells. By analyzing the last 5 years of literature, reported in PubMed website, LC-MS/MS applications have been reported, with particular focus on the ones with higher probability to enter in the near future in clinical practice. Microsampling strategies and alternative biological matrices, from urine to tissue samples, have been included.

Recently, large-scale screening for COVID-19 has presented a major challenge, limiting timely countermeasures. Therefore, the application of suitable rapid serological tests could provide useful information, however, little evidence regarding their robustness is currently available. In this work, we evaluated and compared the analytical performance of a rapid lateral-flow test (LFA) and a fast semiquantitative fluorescent immunoassay (FIA) for anti-nucleocapsid (anti-NC) antibodies, with the reverse transcriptase real-time PCR assay as the reference. In 222 patients, LFA showed poor sensitivity (55.9%) within two weeks from PCR, while later testing was more reliable (sensitivity of 85.7% and specificity of 93.1%). Moreover, in a subset of 100 patients, FIA showed high sensitivity (89.1%) and specificity (94.1%) after two weeks from PCR. The coupled application for the screening of 183 patients showed satisfactory concordance (K = 0.858). In conclusion, rapid serological tests were largely not useful for early diagnosis, but they showed good performance in later stages of infection. These could be useful for back-tracing and/or to identify potentially immune subjects.

Therapeutic options to treat HIV infection have widened in the past years, improving both effectiveness and tolerability, but nucleoside reverse transcriptase inhibitors (NRTIs) are still considered the standard backbone of the combination regimens. Therapeutic drug monitoring (TDM) can be useful for these drugs, due to concentration–effect relationship, with risk of ineffectiveness, toxicity or adherence concerns: in this scenario, robust and multiplexed methods are needed for an effective TDM activity. In this work, the first validated ultra-high spectrometry liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) method is described for the high-sensitive simultaneous quantification of all the currently used NRTIs in human plasma, including tenofovir alafenamide (TAF), following FDA and EMA guidelines. The automated sample preparation consisted in the addition of an internal standard (IS) working solution, containing stable-isotope-linked drugs, protein precipitation and drying. Dry extracts were reconstituted with water, then, these underwent reversed phase chromatographic separation: compounds were detected through electrospray ionization and multiple reaction monitoring. Accuracy, precision, recovery and IS-normalized matrix effect fulfilled guidelines’ requirements. The application of this method on samples from people living with HIV (PLWH) showed satisfactory performance, being capable of quantifying the very low concentrations of tenofovir (TFV) in patients treated with TAF.

Reduced or absent compliance to anti-hypertensive treatment is a major obstacle to the achievement of blood pressure target in patients with arterial hypertension. Current available methods for therapeutic adherence assessment display low accuracy, limited applicability in clinical practice and/or high costs. We designed a prospective study to evaluate the accuracy of serial measurement of aldosterone-to-renin ratio (ARR) to assess the therapeutic compliance to renin-angiotensin-aldosterone system (RAAS) inhibitors. We prospectively enrolled 80 subjects: 40 patients with arterial hypertension and 40 normotensive controls. The ARR was evaluated at baseline and 2 and 8 weeks after initiation of a RAAS inhibitor in patients with hypertension, and at baseline and 2 weeks for the control group. Adherence to the prescribed therapy was confirmed by therapeutic drug monitoring. We observed a significant increase of renin levels and reduction of aldosterone levels after RAAS inhibitors initiation, with consequent reduction of ARR. Delta ARR (ΔARR), defined as relative change in ARR before and after treatment initiation, provided high accuracy for determination of therapeutic compliance, with an area under the curve (AUC) of 0.900 at 2 weeks and 0.886 at 8 weeks. A cut-off of -48% of ΔARR provided 90% sensitivity and 75% specificity, at 2 and 8 weeks. In conclusion, the measurement of ΔARR is a powerful test, cheap and widely available to accurately identify the non-adherence to RAAS inhibitors treatment. Herein we propose the implementation of ΔARR in clinical practice through a multi-step flow-chart for the management of patients with uncontrolled blood pressure, with identification of those suspected of non-adherence, reserving therapeutic drug monitoring for non-adherence confirmation.

Background People living with human immunodeficiency virus are ageing under combination antiretroviral treatments but data on drug exposure in serum and cerebrospinal fluid are limited. Dolutegravir is a widely used second-generation integrase strand transfer inhibitor: conflicting data suggest that neuropsychiatric side effects may present at a higher frequency in patients with higher dolutegravir serum concentrations. Methods We performed a retrospective analysis of our therapeutic drug monitoring registry identifying patients receiving once-daily dolutegravir without concomitant interacting drugs and significant clinical conditions. Data were analysed stratifying time after drug dose intake (maximum concentration 0.5-4 and trough concentration 21-27 h). Cerebrospinal fluid samples from patients enrolled in neurological studies and receiving dolutegravir were analysed for dolutegravir cerebrospinal fluid concentrations and cerebrospinal fluid-to-plasma ratios. Serum and cerebrospinal fluid concentrations were measured through validated chromatographic methods. Results We included 207 (providing 457 serum samples) and 41 patients (providing 41 cerebrospinal fluid samples). Participants were mostly male (68.2-72.8%) of median age of 50 years (50-53 years). Non-significant changes in dolutegravir maximum concentration and trough concentration were observed with age at Spearman's test (pvalues > 0.05); linear logistic regression showed a significant effect of age on dolutegravir trough concentration (p = 0.0013) (Fig. 1). Dolutegravir maximum concentration [3830 ng/mL (2311-5057) vs 4230 ng/mL (2919-5272),p = 0.311] and trough concentration [838 ng/mL (362-1587) vs 966 ng/mL (460-2085),p = 0.056] were non-significantly or borderline higher in patients aged > 50 years. Cerebrospinal dolutegravir concentrations were associated with plasma concentrations (rho = 0.374,p = 0.016) and age (rho = 0.537,p = 0.003); cerebrospinal fluid dolutegravir concentrations (13.8 vs 7.3 ng/mL,p = 0.015) and cerebrospinal fluid-to-plasma ratios (0.57 vs 0.32%,p = 0.017] were higher in participants aged > 50 years. Conclusions We observed an increase in dolutegravir exposure in serum and in cerebrospinal fluid in older patients living with human immunodeficiency virus.

Recently, anti-HIV treatment has achieved high efficacy and tolerability. Nevertheless, few data are available about the intracellular penetration of antiretrovirals, partly due to the technical challenges related to intracellular quantification. This work aimed to validate an ultra-high performance liquid chromatography (UHPLC) tandem mass spectrometry (MS/MS) method for the simultaneous quantification of maraviroc, nevirapine, rilpivirine, dolutegravir, raltegravir, cobicistat, darunavir, ritonavir, atazanavir, efavirenz, elvitegravir, and etravirine within peripheral blood mononuclear cells (PBMCs) and apply it to samples from patients. PBMCs were isolated by density gradient on cell preparation tubes (CPT). Samples were prepared by addition of internal standards (IS), sonication, centrifugation, and drying. Reconstituted extracts underwent chromatographic separation by reversed phase UHPLC and detection was performed by electrospray ionization and multiple reaction monitoring. Method validation followed FDA and EMA guidelines, showing acceptable accuracy, precision, recovery and IS-normalized matrix effect. The application to 56 samples from patients undergoing antiretroviral treatment provided description of intracellular penetration, showing method eligibility for future studies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), with a clinical outcome ranging from mild to severe, including death. To date, it is unclear why some patients develop severe symptoms. Many authors have suggested the involvement of vitamin D in reducing the risk of infections; thus, we retrospectively investigated the 25-hydroxyvitamin D (25(OH)D) concentrations in plasma obtained from a cohort of patients from Switzerland. In this cohort, significantly lower 25(OH)D levels (p = 0.004) were found in PCR-positive for SARS-CoV-2 (median value 11.1 ng/mL) patients compared with negative patients (24.6 ng/mL); this was also confirmed by stratifying patients according to age >70 years. On the basis of this preliminary observation, vitamin D supplementation might be a useful measure to reduce the risk of infection. Randomized controlled trials and large population studies should be conducted to evaluate these recommendations and to confirm our preliminary observation.

Management of resistant hypertension (RHTN) is challenging and often implies the use of complex polypharmacy and interventional therapies. The main objectives of this study were (i) to describe the characteristics of patients with RHTN referred to two expert centres; (ii) to identify predictors of blood pressure (BP) control after intensive management. We reviewed electronic medical files of all patients referred for RHTN to the Brussels and Torino centres, and extracted detailed clinical data, informations on drug adherence and psychological profile. All patients with confirmed diagnosis of RHTN, according to office and ambulatory BP monitoring (ABPM) measurements, were considered eligible. 313 patients (51% men; age: 56 ± 12 years; office BP 177/98 mmHg; 24-hour ABPM 153/90 mmHg) were included. At the end of follow-up (median: 2 years [1-4]), only 26% of patients ( = 81) reached BP control. When compared to patients remaining resistant, patients eventually controlled had lower pulse pressure (71 vs. 82 mmHg

Objectives: Iron-burden-induced arrhythmia and heart failure are among the leading causes of morbidity and mortality in β-thalassaemia major patients. T2* cardiac magnetic resonance remains the only reliable noninvasive method for the heart iron excess assessment. We explored the role of single nucleotide polymorphisms involved in vitamin D metabolism, transport and activity and in deferasirox (DFX) metabolism on cardiac iron burden. Patients and methods: One hundred and five β-thalassaemia patients, treated with DFX, were enrolled in the present study. Drug plasma Ctrough was measured by a high-performance liquid chromatography-ultraviolet method. Allelic discrimination was carried out using the real-time PCR. Results: CYP1A1*1189 CC, ABCG2 421 GA, CYP24A1 8620 GG and VDR TaqI CC single nucleotide polymorphisms influenced T2* values. Age, serum ferritin, ABCG2 421 GA, ABCG2 1194 +928 TC/CC, CYP24A1 22776 TT and VDR TaqI TC/CC were retained in linear regression model. Conclusion: Our results suggested, for the first time, the role of DFX and vitamin D pharmacogenetics on cardiac iron overload.

Aim: We explored the role of SNPs within the SLCO1B3, SLCO1B1, SLC22A6, ABCB1, ABCG2, SLCO3A1, CYP2C19, ABCC2, SLC22A1, ABCB11 and NR1I2 genes on voriconazole pharmacokinetics. Patients & methods: 233 pediatric patients were enrolled. Drug plasma Ctrough was measured by a HPLC-MS method. Allelic discrimination was performed by qualitative real-time PCR. Results: SLCO1B3 rs4149117 c.334 GT/TT (p = 0.046), ABCG2 rs13120400 c.1194 + 928 CC (p = 0.029) and ABCC2 rs717620 c.-24 GA/AA (p = 0.025) genotype groups significantly influenced Ctrough. ethnicity (p = 0.042), sex (p = 0.033), SLCO1B3 rs4149117 c.334 GT/TT (p = 0.041) and ABCB1 rs1045642 c.3435 TT (p = 0.016) have been retained in linear regression model as voriconazole predictor factors. Conclusion: Understanding how some gene polymorphisms affect the voriconazole pharmacokinetic is essential to optimally dose this agent.

We measured ceftriaxone pharmacokinetics in patients' plasma and cerebrospinal fluid (CSF) and assessed the influence of biometric, demographic, genetic (ABCB1, ABCC2, ABCB11, ABCG2, and SLCO1A2 polymorphisms) and pathological features. Adult patients with signs and symptoms of central nervous system infections, receiving intravenous ceftriaxone, were enrolled. Ceftriaxone plasma and CSF concentrations were measured by high-precision liquid chromatographic methods; allelic discrimination was performed by real-time polymerase chain reaction. Forty-three patients were included: median ceftriaxone maximal concentration was 15,713 ng/mL in plasma and 3512 ng/mL in CSF with a CSF-to-plasma ratio of 0.3. ABCC2 1249 rs2273697 (P = .027) and ABCG2 1194+928 rs13120400 (P = .015) variants were significantly associated with CSF concentrations and CSF-to-plasma ratios. At linear regression analysis, CSF-to-serum albumin ratio was an independent predictor of ceftriaxone CSF concentrations (P = .001; also in those with intact blood-brain barrier: P = .031) and CSF-to-plasma ratio (P = .001; also in those with blood-brain barrier impairment: P = .040). We here report the role of transporters' genetic variants as well as of blood-brain barrier permeability in predicting ceftriaxone exposure in the central nervous system.

Dalbavancin (DBV) is an intravenous long-acting second-generation glycolipopeptide antibiotic with high efficacy and excellent tolerability, approved for use in the treatment of Gram-positive skin and skin structure infections (ABSSSI). Nevertheless, little is known about its pharmacokinetic/pharmacodynamic (PK/PD) properties in real life, which is also due to technical challenges in its quantification in human plasma, preventing an effective application of therapeutic drug monitoring (TDM). In fact, DBV has a high affinity to plasma proteins, possibly resulting in poor recovery after extraction procedure. The aim of this study was to validate a simple, cheap and reliable HPLC-MS method for use in TDM, in accordance with FDA and EMA guidelines. The optimized protein precipitation protocol required 50 μL of plasma, while chromatographic analysis could be performed in 12 min/sample. This method fulfilled the guidelines requirements and then, it was applied for routine DBV TDM in patients receiving off-label high doses (two 1500 + 1500 mg weekly infusions instead of 1000 + 500 mg), with normal renal function or undergoing hemodialysis: continuous hemodiafiltration caused a relevant reduction in DBV exposure, while intermittent dialysis showed comparable DBV concentrations with those of patients with normal renal function. This confirmed the eligibility of the presented method for use in TDM and its usefulness in clinical practice.

To date, vitamin D seems to have a significant role in affecting the prevention and immunomodulation in COVID-19 disease. Nevertheless, it is important to highlight that this pro-hormone has other several activities, such as affecting drug concentrations, since it regulates the expression of cytochrome P450 (CYP) genes. Efavirenz (EFV) pharmacokinetics is influenced by CYPs, but no data are available in the literature concerning the association among vitamin D levels, seasonality (which affects vitamin D concentrations) and EFV plasma levels. For this reason, the aim of this study was to evaluate the effect of 25-hydroxy vitamin D (25(OH)D3) levels on EFV plasma concentrations in different seasons. We quantified 25(OH)D3 by using chemiluminescence immunoassay, whereas EFV plasma concentrations were quantified with the HPLC–PDA method. A total of 316 patients were enrolled in Turin and Rome. Overall, 25(OH)D3levels resulted in being inversely correlated with EFV concentrations. Some patients with EFV levels higher than 4000 ng/mL showed a deficient 25(OH)D3 concentration in Turin and Rome cohorts and together. EFV concentrations were different in patients without vitamin D supplementation, whereas, for vitamin D-administered individuals, no difference in EFV exposure was present. Concerning seasonality, EFV concentrations were associated with 25(OH)D3 deficiency only in winter and in spring, whereas a significant influence was highlighted for 25(OH)D3 stratification for deficient, insufficient and sufficient values in winter, spring and summer. A strong and inverse association between 25(OH)D3and EFV plasma concentrations was suggested. These data suggest that vitamin D is able to affect drug exposure in different seasons, thus, the achievement of the clinical outcome could be improved by also considering this pro-hormone.

Monitoring and treating iron overload is crucial in transfusion-dependent thalassaemia patients. Liver stiffness measurement by transient elastography and T2* magnetic resonance imaging represent non-invasive ways to evaluate the adequacy of the iron chelation treatment. We explored the role of single nucleotide polymorphisms involved in vitamin D metabolism, transport and activity, and in deferasirox metabolism on liver iron burden parameters. One-hundred and five beta-thalassaemia patients, treated with deferasirox, have been enrolled. Drug plasma Ctrough and AUC were measured by a HPLC-UV method. Allelic discrimination was performed by real-time PCR. Age, UGT1A1-364 CT/TT and CYP27B1 -1260 GT/TT positively predicted liver stiffness values. Deferasirox dose and serum ferritin negatively predicted T2* data, whereas age and CYP2D6 1457 GG genotype positively influenced these values. The discoveries of this research may be useful for personalized medicine and the proposed method could be applied in patients with hereditary hemochromatosis and myelodysplastic syndromes.

Tenofovir disoproxyl fumarate (TDF) has been associated with renal tubular abnormalities, phosphaturia and proteinuria (retinol binding protein, RBP, loss): vitamin D (VD) and PTH affect these markers. Aim was to understand if some single nucleotide polymorphisms (SNPs) were predictors of renal abnormalities in an Italian cohort of HIV-affected patients. DNA was analyzed through real-time PCR, urinary RBP corrected by creatinine (uRBP/Cr). The majority of patients received TDF. Abnormal uRBP/Cr was more frequent in TDF recipients: eGFR

Aims Arterial hypertension is an important cardiovascular risk factor. A substantial proportion of patients show resistance to antihypertensive treatment but poor adherence to medication regimens is also a significant cause of treatment failure. In this context, therapeutic drug monitoring (TDM) could be useful. The objective of this study was to assess adherence to treatment in patients with resistant hypertension by TDM and to identify parameters that predict nonadherence. Methods Liquid chromatography tandem mass spectrometry was used to quantify a wide panel of antihypertensive drugs in human plasma to assess treatment compliance. Associations between TDM-determined adherence profiles, self-reported adherence and other patient-related clinical, anthropometric or demographic features were evaluated as potentially useful pre-TDM predictors of poor adherence. Results TDM was performed on 50 patients with suspected resistant hypertension: 24% of patients partially complied to treatment and 18% were nonadherent. No concordance was observed with questionnaire results, while nonadherence was associated with high diastolic blood pressure, high heart rate, previous onset of stroke and previous use of invasive treatments, including renal denervation or baroreceptor stimulation. Conclusions This evidence highlights the high prevalence of poor adherence in patients with resistant hypertension and the need for caution in using invasive approaches. These preliminary data require validation in a larger cohort, to confirm the need for TDM in routine clinical practice.

Background Sofosbuvir is a potent nucleotide HCV NS5B polymerase inhibitor that is also a P-glycoprotein (encoded by the ABCB1 gene) and breast cancer resistance protein (encoded by the ABCG2 gene) substrate. Concerning previous anti-HCV therapies, pharmacogenetics had a significant impact, particularly considering the association of interleukin28B polymorphisms with dual-therapy (ribavirin + pegylated IFN) outcomes. Objectives In this work, we investigated the association between sofosbuvir and its prevalent metabolite (GS-331007) plasma concentrations at 1 month of therapy and genetic variants (SNPs) in genes encoding transporters and nuclear factors (ABCB1, ABCG2 and HNF4α) related to sofosbuvir transport. Patients and methods Allelic discrimination was performed through real-time PCR, whereas plasma concentrations were evaluated through liquid chromatography. One hundred and thirteen patients were enrolled. Results Sofosbuvir concentrations were below the limit of quantification since the drug was converted into its GS-331007 metabolite. ABCB1 2677 G>T (P = 0.044) and HNF4α 975 C>G (P = 0.049) SNPs were associated with GS-331007 metabolite plasma concentrations. In linear multivariate analysis, liver stiffness, insulin resistance, baseline haemoglobin and haematocrit and SNPs in the ABCB1 gene (3435 CT/TT and 1236 TT genotypes) were significant predictors of GS-331007 concentrations. Furthermore, we performed sub-analyses considering the anti-HCV concomitant drug and HCV genotype, identifying specific polymorphisms associated with GS-331007 plasma concentrations: ABCB1 3435 C>T and HNF4α975 C>G in patients treated with daclatasvir, ABCB1 2677 G>T with ledipasvir and ABCB1 3435 C>T, ABCB1 2677 G>T, ABCG2 421 C>A and ABCG2 1194 + 928 C>A with ribavirin. Conclusions In this study we suggested sofosbuvir GS-331007 metabolite plasma levels were affected by variants in the ABCB1 and HNFα genes.

Introduction Nowadays, the treatment of hypertension represents an important issue, particularly in developed countries. While in most cases the standard therapeutic approaches, consisting in the administration of 1 to 3 drugs, are adequate to reach adequate blood pressure levels, in some cases more drugs are needed: this condition is called “resistant hypertension”. In this context, the administration of a diuretic, such as spironolactone or canrenoate salts, represents a standard practice. Since a reliable discrimination of real cases of resistant hypertension from cases of poor therapeutic adherence is currently difficult to obtain, the adoption of therapeutic drug monitoring has been suggested as a useful tool for this purpose. In this work, the authors developed and validated a simple, cheap and fast dilute-and-shot method with UHPLC-PDA analysis for the quantification of spironolactone and its metabolite canrenone in human urine samples. Methods Standards and quality controls were prepared in urine. Only 100 μL of sample were added with 80 μL of internal standard (6,7-dimethyl-2,3-di(2-pyridyl)quinoxaline) working solution and 820 μL of phosphate buffer 10 mM pH 3.2 (phase A):acetonitrile (phase B) 90:10 v:v solution. Chromatographic separation was performed on an Acquity® UPLC HSS T3 1.8 μm 2.1 × 150 mm column, with a binary gradient for 11 min at 40 °C. Results Accuracy, intra-day and inter-day precision, selectivity and sensitivity fitted FDA guidelines for all analytes (LLOQ and LOD were 156.25 ng/mL and 78.12 ng/mL, respectively, for both analytes) and recovery resulted high and reproducible. Method performances were tested on urine samples from hypertensive patients with good results. Discussion This simple analytical method could represent a useful tool for the management of antihypertensive therapy.

Vitamin D levels and genetic variants were associated with drug outcome/toxicity and concentrations. The plasma exposure of GS-331007, the main sofosbuvir metabolite, has been related to SVR. We evaluated the impact of polymorphisms in genes (CYP27B1, CYP24A1, VDBP and VDR) related to vitamin D pathway on sofosbuvir and GS-331007 plasma levels in HCV mono-infected patients at one month of treatment. Polymorphisms were investigated through real-time PCR; drug plasma quantification was performed through a UHPLC-MS/MS method. GS-331007 levels were associated with CYP24A1rs2248359 and VDRCdx2 variants in all the analyzed patients and linear regression analysis showed that sex, body mass index, HCV genotype, baseline estimated glomerular filtration rate, VDRCdx2AG/GG and CYP27B1-1260TT genotypes significantly predict concentrations. We performed sub-analyses considering the HCV genotype and the concomitant drug, identifying polymorphisms associated with GS-331007 concentrations. This is the first study focusing on vitamin D pathway gene variants and DAAs concentrations, but further studies are required.

Objectives Knowledge of the exact concentration of active compounds in galenic preparations is crucial to be able to ensure their quality and to properly administer the prescribed dose. Currently, the need for titration of extracts is still debated. Considering this, together with the absence of a standard preparation method, the aim of this study was to evaluate cannabinoids concentrations variability in galenic olive oil extracts, to evaluate the interlot and interlaboratory variability in the extraction yield and in the preparation composition. Methods Two hundred and one extracts (123 (61.2%) from Bedrocan®, 54 (26.9%) from Bediol®, 11 (5.5%) from Bedrolite®, and 13 (6.5%) from mixed preparations) were analysed by liquid chromatography coupled with tandem mass spectrometry, quantifying cannabinoids (THC, CBD, THCA, CBDA and CBN) concentrations. Key findings The RSD% of THC and CBD concentrations resulted higher than 50%. Specifically for Bedrocan®, Bediol®, Bedrolite® (5 g/50 ml), these were THC 82%, THC 53% and CBD 91%, THC 58% and CBD 59%, respectively. The median extraction yields were greater than 75% for all preparations. Conclusions Our results highlighted a wide variability in THC and CBD concentrations that justify the need for titration and opens further questions about other pharmaceutical preparations without regulatory indication for this procedure.

Triazoles chanced the prevention and treatment of invasive fungal infections, but their pharmacokinetic properties are still unclear. In particular, isavuconazole (ISC) is a new broad-spectrum antifungal triazole approved in 2015 as first-line treatment for intravenous and oral use against invasive aspergillosis and for mucormycosis. Nowadays, the optimal management of the treatments with triazoles requires the use of Therapeutic Drug Monitoring (TDM), in order to prevent sub-therapeutic or toxic concentrations. In turn, the routine use of TDM requires reliable quantification methods The aim of this work was the development and full validation of a HPLC-mass spectrometry assay for the simultaneous quantification of fluconazole, itraconazole, isavuconazole, posaconazole and voriconazole in human samples. Both standards and quality controls were prepared in human plasma. After the addition of internal standard (6,7-dimethyl-2,3-di(2-pyridyl)quinaxoline for voriconazole, posaconazole and itraconazole; stable isotope labeled compounds for fluconazole and isavuconazole), protein precipitation with acetonitrile and dilution with water were performed. Chromatographic separation was performed on Atlantis® T3 5μm 4.6×150mm column, with a gradient of water and acetonitrile, both added with 0.05% formic acid. Accuracy, intra-day and inter-day imprecision fitted FDA and EMA guidelines, while matrix effects and recoveries resulted stable between samples for each analyte. Stability results were in accordance with previously published data. Finally, we tested this method by monitoring plasma concentrations in real patients and using external quality controls with good results. This method resulted very simple, fast, cheap and very useful for TDM application, to improve clinical management of antifungal therapy in critically ill patients.

Background Daclatasvir is an inhibitor of HCV non-structural 5A protein and is a P-glycoprotein substrate. Pharmacogenetics has had a great impact on previous anti-HCV therapies, particularly considering the association of IL-28B polymorphisms with dual therapy outcome. Objectives We investigated the association between daclatasvir plasma concentrations at 2 weeks and 1 month of therapy and genetic variants (SNPs) in genes encoding transporters and nuclear factors (ABCB1, ABCB11 and HNF4α). Patients and methods Allelic discrimination was achieved through real-time PCR, whereas plasma concentrations were evaluated through LC-MS/MS. Results Fifty-two patients were analysed, all enrolled in the Kineti-C study. HNF4α 975 C > G polymorphism was found to be associated with the daclatasvir plasma concentrations at 2 weeks (P = 0.009) and 1 month of therapy (P = 0.006). Linear regression analysis suggested that, at 2 weeks of therapy, age, baseline BMI and haematocrit were significant predictors of daclatasvir concentrations, whereas at 1 month of therapy ABCB111131 CC and HNF4α CG/GG genotypes were significant predictors of daclatasvir concentrations. Conclusions These are the first and preliminary results from our clinical study focusing on daclatasvir pharmacogenetics, showing that this approach could have a role in the era of new anti-HCV therapies.

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.

In the adult brain, subsets of astrocytic cells residing in well-defined neurogenic niches constitutively generate neurons throughout life. Brain lesions can stimulate neurogenesis in otherwise non-neurogenic regions, but whether local astrocytic cells generate neurons in these conditions is unresolved. Here, through genetic and viral lineage tracing in mice, we demonstrate that striatal astrocytes become neurogenic following an acute excitotoxic lesion. Similar to astrocytes of adult germinal niches, these activated parenchymal progenitors express nestin and generate neurons through the formation of transit amplifying progenitors. These results shed new light on the neurogenic potential of the adult brain parenchyma.