Comparative Performance Assessment of Novel Fluorescence Immunoassay POCTs for Measuring Circulating Levels of Vitamin-D.

Vitamin D (Vit D) is a fat-soluble molecule acting like a hormone, and it is involved in several biological mechanisms such as gene expression, calcium homeostasis, bone metabolism, immune modulation, viral protection, and neuromuscular functions. Vit D deficiency can lead to chronic hypocalcemia, hyperparathyroidism, and many other pathological conditions; in this context, low and very low levels of 25-hydroxy-vitamin D (25-OH-D) were found to be associated with an increased risk of COVID-19 infection and the likelihood of many severe diseases. For all these reasons, it is important to quantify and monitor 25-OH-D levels to ensure that the serum/blood concentrations are not clinically suboptimal. Serum concentration of 25-OH-D is currently the main indicator of Vit D status, and it is currently performed by different assays, but the most common quantitation techniques involve immunometric methods or chromatography. Nevertheless, other quantitation techniques and instruments are now emerging, such as AFIAS-1® and AFIAS-10® (Boditech and Menarini) based on the immunofluorescence analyzer, that guarantee an automated system with cartridges able to give quick and reliable results as a point-of-care test (POCT). This work aims to compare AFIAS-1® and AFIAS-10® (Boditech and Menarini) Vit D quantitation with Ultra High-Performance Liquid Chromatography coupled with tandem mass spectrometry that currently represents the gold standard technique for Vit D quantitation. The analyses were performed in parallel on 56 samples and in different conditions (from fresh and frozen plasma) to assess the reliability of the results. Any statistically significant differences in methods, the fixed error, and the error proportional to concentration were reported. Results obtained in all conditions showed a good correlation between both AFIAS® instruments and LC-MS/MS, and we can affirm that AFIAS-1® and AFIAS-10® are reliable instruments for measuring 25-OH-D with accuracy and in a fast manner.

Stability Study of Fosfomycin in Elastomeric Pumps at 4 °C and 34 °C: Technical Bases for a Continuous Infusion Use for Outpatient Parenteral Antibiotic Therapy.

BACKGROUND: Fosfomycin acts against aerobic Gram-/+ bacteria by blocking the synthesis of peptidoglycan. Its use has been currently re-evaluated for intravenous administration for the treatment of systemic infections by multidrug-resistant bacteria. Concentration-/time-dependent activity has been suggested, with potential clinical advantages from prolonged or continuous infusion. Nevertheless, little is known about Fosfomycin stability in elastomeric pumps. The aim of the present work was stability investigation before administration at 4 °C and during administration at 34 °C. METHODS: InfectoFos® (InfectoPharm s.r.l., Milan, Italy) preparation for intravenous use in elastomeric pumps at 4 °C and 34 °C was analyzed following EMA guidelines for drug stability. Samples were analyzed with an ultra-high performance liquid chromatography coupled with tandem mass spectrometry method on a LX50® UHPLC system equipped with a QSight 220® (Perkin Elmer, Milan, Italy) tandem mass spectrometer. RESULTS: Fosfomycin in elastomeric preparation is stable for at least 5 days at a storage temperature of 4 °C and 34 °C. CONCLUSIONS: The results suggest Fosfomycin eligibility for continuous infusion even in the context of outpatient parenteral antibiotic therapy. Therefore, this approach should be tested in clinical and pharmacokinetic studies, in order to evaluate the possible gains in the pharmacokinetic profile and the clinical effectiveness.

Analytical validation of a novel UHPLC-MS/MS method for 19 antibiotics quantification in plasma: Implementation in a LC-MS/MS Kit.

BACKGROUND: Therapeutic drug monitoring (TDM) for antibiotic drugs represents a consolidated practice to optimize the effectiveness and to limit the toxicity of specific drugs by guiding dosage adjustments. The comparison of TDM results with drug-specific pharmacokinetic/pharmacodynamic (PK/PD) parameters, based on killing dynamics and bacterial susceptibility, increases the probability of therapeutic success. PURPOSE: The aim of this study was the analytical validation of a new UHPLC-MS/MS assay for the quantification of 19 antibiotics divided in two different sets considering their chemical/pharmacological properties. This method has been implemented in an analytical LC-MS/MS Kit System by CoQua Lab s.r.l (Turin). METHODS: The analytical validation is developed in accordance with "ICH Harmonized Guideline M10 on bioanalytical method validation and study sample analysis" and "Guidelines for regulatory auditing of quality management system of medical device manufacturers". Method suitability in the clinical context was tested by analysing clinical samples from patients treated with antibiotic drugs. RESULTS: This method allows for simultaneous TDM of the following molecules: dalbavancin, daptomycin, linezolid, tedizolid, levofloxacin, moxifloxacin, meropenem, ertapenem, vaborbactam, avibactam, sulbactam, tazobactam, ceftazidime, ceftriaxone, ceftolozane, ceftobiprole, cefiderocol, ceftaroline and piperacillin. These drugs were quantified showing analytical performance parameters compliant with guidelines in terms of repeatability, reproducibility, robustness, bias, LOD, LOQ and linearity. The method was capable to successfully monitor drug concentrations in 65 samples from 52 patients undergoing treatment. CONCLUSION: The UHPLC-MS/MS method described in this work can be useful for TDM of the reported antimicrobial agents. The analytical protocol is rapid and suitable to be used in routine analysis.

A description of Cannabinoid levels in Cannabis oil by high-performance liquid chromatography-mass spectrometry in a reference laboratory of North-Italy.

BACKGROUND: Cannabis oils from FM2®, Bedica®, Bediol®, Bedrocan®, Bedrolite® and Pedanios 22/1® are largely used for medical purposes such as spasticity, chronic pain and appetite stimulating. Several studies showed cannabinoids action on CB1 and CB2 receptors reduces the hyperalgesic phase in inflammatory pain, leading to an improvement of conditions. The active compounds of these galenic preparations show a high variability making titration mandatory. For this reason, the exact oil composition knowledge is fundamental for personalizing therapy. This amis at adapting the correct dose to the patient, improving safety and efficacy of the galenic formulation, choosing the best preparation for each patient. PURPOSE: The aim of this study was to investigate oil preparations variability among different galenic laboratories in order to highlight the importance of titration activity. METHODS: Cannabis pharmacological active compounds titration has been performed in a large cohort of galenic laboratories in Italy. CBD, CBN, THC, THCA and CBDA quantification was carried out by a previous validated method in UHPLC-MS/MS. RESULTS: A number of 4318 samples of Cannabis oil from 83 pharmacies between January 2021 and February 2022 were evaluated. All galenic preparation specialities showed statistically significant differences among galenic laboratories (p-value < 0.001). THCA and CBDA concentrations were investigated as percentage of the extration yelds for total THC and CBD: these compounds had different values in the same specialities among distinct galenic laboratories. Moreover, seasonal variability in analytes concentrations was observed. CONCLUSION: This study described a wide range of oily samples from a large number of galenic laboratories, compared to published papers. In conclusion, knowledge of the exact oil composition is fundamental in the perspective of personalized therapy. Further studies aiming at the correlation between galenic composition and cannabinoids pharmacokinetics, clinical outcomes and toxic effects could be useful to improve our knowledge.

Cannabis-Based Oral Formulations for Medical Purposes: Preparation, Quality and Stability.

Current legislation in Italy provides that medical Cannabis may be administered orally or by inhalation. One of the fundamental criteria for the administration of oral formulations is that they deliver a known consistent quantity of the active ingredients to ensure uniform therapies leading to the optimisation of the risks/benefits. In 2018, our group developed an improved Cannabis oil extraction technique. The objective of the present work was to carry out a stability study for the oil extracts obtained by this method. Furthermore, in order to facilitate the consumption of the prescribed medical Cannabis therapy by patients, a standard procedure was defined for the preparation of a single-dose preparation for oral use (hard capsules) containing the oil extract; thereafter, the quality and stability were evaluated. The hard capsules loaded with the oil extract were analysed and found to be uniform in content. The encapsulation process did not alter the quantity of the active molecule present in the oil. The stability tests yielded excellent results. Since the capsule dosage form is easily transported and administered, has pleasant organoleptic properties and is stable at room temperature for extended periods of time, this would facilitate the adherence to therapy by patients in treatment.

Correlation between Thiopurine S-Methyltransferase Genotype and Adverse Events in Inflammatory Bowel Disease Patients.

Background and Objectives: In patients with inflammatory bowel diseases (IBD), the use of azathioprine results in adverse events at a rate of 5% to 20%. The aim of the study was to assess a possible correlation between genetic variability of the enzyme thiopurine S-methyltransferase (TPMT) and the development of toxicity to azathioprine. Materials and Methods: A retrospective, single center, blind, case-control study was conducted on 200 IBD patients, of whom 60 cases suspended azathioprine due to toxicity (leukopenia, pancreatitis, hepatitis, and nausea or vomiting), and 140 controls continued treatment with the drug without adverse events. Results: In the entire cohort, only 8 cases of heterozygous mutations of TPMT were observed, corresponding to 4% mutated haplotype rate, much lower than that reported in literature (close to 10%). No homozygous mutation was found. Regarding the TPMT allelic variants, we did not find any statistically significant difference between patients who tolerated azathioprine and those who suffered from adverse events. (OR = 0.77, 95% CI = 0.08-7.72; p = 0.82). Conclusions: According to our study, in IBD patients, the search for TPMT gene mutations before starting treatment with azathioprine is not helpful in predicting the occurrence of adverse events. Importantly, patients with allelic variants should not be denied the therapeutic option of azathioprine, as they may tolerate this drug.

Development of Standard Operating Protocols for the Optimization of Cannabis-Based Formulations for Medical Purposes.

Under current legislation in Italy, Cannabis for medical purposes may be administered orally in the form of decoction or Cannabis oil extract. The scientific literature reports a number of preparation methods, mainly for oils, but no study is available that compares thoroughly, from a technological viewpoint, the Cannabis-based formulations currently administered to patients. With this in mind, this research work aimed to carry out specific formulation studies to design standard operating procedures for the preparation and optimization of Cannabis-based galenic formulations. Both decoctions and oils were prepared under different operating conditions to identify the most efficient process for the production of formulations with a high concentration of decarboxylated delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD). Regarding Cannabis oil, a new procedure has been developed that allows significantly higher recovery rates for THC and CBD compared with those for water-based extraction methods (decoction) and those for oil-based methods currently in use. Moreover, based on the results, it is possible to affirm that the prescription of Cannabis-based decoctions should not be the recommended first-choice solution for therapy, considering the low concentration of THC and CBD and, consequently, the high volume of decoction that the patient would have to ingest.

Cannabinoids concentration variability in cannabis olive oil galenic preparations.

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.

Atazanavir intracellular concentrations remain stable during pregnancy in HIV-infected patients.

BACKGROUND: Atazanavir (300 mg) boosted by ritonavir (100 mg) is the preferred third drug in pregnancy. However, there is still discordance on atazanavir dose increase during the third trimester. OBJECTIVES: To evaluate plasma and intracellular atazanavir and ritonavir concentrations in HIV-infected women during pregnancy and after delivery. METHODS: This was an observational study. HIV-infected pregnant patients treated with atazanavir/ritonavir plus either tenofovir/emtricitabine or abacavir/lamivudine had been prospectively enrolled after having signed a written informed consent form. Plasma and intracellular atazanavir and ritonavir Ctrough (24 ±âŸ3 h after drug intake) were measured at each visit during the first, second and third trimesters and post-partum using validated HPLC-MS and HPLC-photodiode array methods (with direct evaluation of cellular volume). Data are described as median (IQR) and compared through non-parametric tests. RESULTS: Twenty-five patients were enrolled; at baseline, the median age was 32 years (27-35). All patients had plasma HIV RNA <50 copies/mL; the median CD4+ count was 736 cells/mm3 (542-779). Atazanavir plasma concentrations were 441 ng/mL (261-1557), 710 ng/mL (338-1085), 556 ng/mL (334-1022) and 837 ng/mL (608-1757) during the first, second and third trimesters and post-partum, respectively; intracellular concentrations were 743 ng/mL (610-1928), 808 ng/mL (569-1620), 756 ng/mL (384-1074) and 706 ng/mL (467-2688), respectively. Atazanavir intracellular/plasma ratios were 1.32 (0.98-2.77), 1.34 (1.13-1.88), 1.38 (0.61-2.63) and 1.07 (0.56-2.69), respectively. Atazanavir intracellular concentrations and intracellular/plasma ratios showed non-significant changes over time (P > 0.05). CONCLUSIONS: This is the first demonstration that intracellular atazanavir exposure remains unchanged during pregnancy supporting the standard 300/100 mg atazanavir/ritonavir dosing throughout pregnancy.

UPLC-MS/MS method for the simultaneous quantification of three new antiretroviral drugs, dolutegravir, elvitegravir and rilpivirine, and other thirteen antiretroviral agents plus cobicistat and ritonavir boosters in human plasma.

Rilpivirine (RPV), dolutegravir (DTG) and elvitegravir (EVG) are the latest antiretroviral drugs approved for treatment of HIV infection. Currently, poor information is currently available concerning their pharmacokinetic and pharmacodynamic properties, thus making the use of therapeutic drug monitoring for these drugs not useful. This lack of information is partially due to the absence of an high-throughput method for their simultaneous quantification together with other antiretroviral drugs. In this work, we describe the development and validation of a new UPLC-MS/MS method to quantify these drugs, together with other fourteen antiretroviral agents, in human plasma. One hundred microliters of plasma samples were added with internal standard (6,7-Dimethyl- 2,3-di(2-pyridyl) quinoxaline), underwent a simple protein precipitation with methanol:acetonitrile (50:50v/v) followed by sample dilution with water. Chromatographic separation was performed on a Acquity® UPLC HSS T3 column (150mm x 2.1mm I.D) with a particle size of 1.8µm and compounds were detected with a tandem mass detector, monitoring two ion transitions for each drugs. The mean recovery of RPV, DTG and EVG were 101%, 87% and 112.3% respectively. Accuracy and precision inter/intra-day were below 15% for all drugs, in accordance to Food and Drug Administration guidelines requirements. The UPLC-MS/MS method reported here could be used routinely to monitor plasma concentrations of antiviral drugs, including RPV, DTG and EVG.

A UHPLC-MS/MS method for the quantification of direct antiviral agents simeprevir, daclatasvir, ledipasvir, sofosbuvir/GS-331007, dasabuvir, ombitasvir and paritaprevir, together with ritonavir, in human plasma.

To date, the new standard for treatment of chronic hepatitis C is based on the administration of novel direct acting antivirals. Among these, sofosbuvir, simeprevir, daclatasvir, ledipasvir, dasabuvir, ombitasvir and paritaprevir already entered the clinical use. Anyway, since few pharmacokinetic studies have been conducted on these drugs in a "real life" context poor knowledge is available about their optimal therapeutic range. Without this background, therapeutic drug monitoring is not applicable for treatment optimization. Up to now, a few methods are reported to quantify these drugs in human plasma, and none of them in a simultaneous way. The aim of this work was to develop and validate a simple, fast and cheap, but still reliable UHPLC-MS/MS method for the quantification of these drugs, feasible for a clinical routine use. Solid phase extraction was performed using HLB C18 96-well plates. Chromatographic separation was performed on a BEH C18 1.7µm, 2.1mm×50mm column, settled at 50°C, with a gradient run of two mobile phases: ammonium acetate 5mM (pH 9.5) and acetonitrile, with a flow rate of 0.4mL/min for 5min. Tandem-mass detection was carried out in positive electrospray ionization mode. Both inter and intraday imprecision and inaccuracy were below 15%, as required by FDA guidelines, while both recoveries and matrix effects resulted within the acceptance criteria. The method was tested on 80 patients samples with good performance. Being robust, simple and fast and requiring a low plasma volume, this method resulted eligible for a clinical routine use.

Efavirenz pharmacogenetics in a cohort of Italian patients.

The pharmacogenetics and pharmacokinetics of efavirenz (EFV) have been widely studied, although data in the Italian population are limited. Single nucleotide polymorphisms (SNPs) in the CYP2B6 gene have been associated with increased EFV plasma concentrations and central nervous system toxicity. The aim of this work was to evaluate EFV plasma exposure according to SNPs in genes involved in drug metabolism and elimination in a cohort of Italian HIV-1-positive patients treated with EFV. Plasma samples were used to measure EFV concentrations at 12h after intake (C12) by a validated HPLC/PDA system. Whole blood was used to identify SNPs in ABCB1, MRP2, CYP2B6, CYP2A6, UGT2B7, NR1I2 (PXR), NR1I3 (CAR) and HNF4α by real-time PCR. The association between SNPs and EFV plasma levels was evaluated through non-parametric tests. Among 201 patients, the median EFV C12 was 2618.5ng/mL. No significant associations were found for MRP2, CYP2A6, UGT2B7, PXR and CAR SNPs; conversely, an association of CYP2B6 516G>T, ABCB1 3435C>T and 2677G>T, and HNF4α 975C>G polymorphisms with EFV C12 was observed. In multivariate analysis, only CYP2B6 516 TT and ABCB1 3435 TT genotypes were independently associated with an EFV C12 of >4000ng/mL (toxicity cut-off). This study confirmed the role of CYP2B6 and ABCB1 polymorphisms, showed a relationship with HNF4α, and the lack of association of CYP2A6, UGT2B7, NR1I2 and NR1I3 SNPs on EFV plasma exposure. Data regarding some of the studied SNPs are the first obtained in an Italian cohort of HIV patients and lead to a global vision about EFV pharmacogenetics.

Tenofovir clearance is reduced in HIV-positive patients with subclinical tubular impairment.

OBJECTIVE: To assess if tenofovir (TFV) clearance is associated with urinary retinal-binding protein (RBP) in HIV-positive patients with normal estimated filtration rate. DESIGN: A cross-sectional diagnostic study. METHODS: HIV-positive patients with estimated creatinine clearance above 60 ml/min, on tenofovir disoproxil fumarate (TDF)-containing combination since at least 6 months, taking TDF at night, and without significant comorbidities (diabetes, untreated hypertension, known renal malformations, recurrent nephrolithiasis) and nephrotoxic drugs were included. TFV plasma and urinary concentrations were measured 12 h after drug intake (C12). RBP was measured through enzyme immunoassay kit on spot urines and corrected per urinary creatinine (uRBP/uCr); normality ranges were below 130 µg/g (in patients aged <50 years) and below 172 µg/g (in patients aged ≥50 years). RESULTS: Two hundred and eighty-nine patients were included (median age of 45.8 years, 71.6% male and 85.4% whites); patients were concomitantly treated with nonnucleoside reverse transcriptase inhibitors (155, 53.6%), protease inhibitors (118, 40.8%), or integrase inhibitors (16, 5.5%)-containing regimens. Estimated creatinine clearance was 89.4 ml/min (78.6-105.9). Urinary RBP (uRBP) and uRBP/uCr were 204.6 ng/ml (92-380) and 169.7 µg/g (85.8-318.3), respectively; abnormally high uRBP/uCr was observed in 157 patients (54.3%). A multivariate binary logistic regression confirmed that both ethnicity (P = 0.004, ß 2.93, 95% confidence interval 1.41-6.10) and TFV urinary C12 less than 21 mg/ml (P = 0.006, ß 2.04, 95% confidence interval 1.12-3.41) were significantly associated with abnormal uRBP/uCr. CONCLUSION: HIV-positive TDF-treated patients showed a high prevalence of proximal tubular impairment: ethnicity (whites) and low urinary TFV concentrations were significantly associated with elevated uRBP. SDC VIDEO:: http://links.lww.com/QAD/A852.

Development and validation of an ultra performance liquid chromatography tandem mass method for sildenafil and N-desmethyl sildenafil plasma determination and quantification.

Sildenafil is a selective inhibitor of cGMP-specific type 5 phosphodiesterase (PDE5) used for the treatment of masculine erectile dysfunction and Pulmonary Arterial Hypertension (PAH). Sildenafil causes vasodilatation; relax of the smooth muscle and reduction of pulmonary arterial pressure. In the liver cytocrome P450 metabolizes sildenafil into its active metabolite, N-desmethyl sildenafil. The determination of plasma levels of sildenafil and N-desmethyl sildenafil could be useful for therapy optimization and pharmacokinetic studies. We have developed and validated a new method for the quantification of sildenafil and its metabolite in human plasma by rapid protein precipitation extraction, using an UPLC system, coupled with a tandem mass spectrometric detector (UPLC-MS/MS). The calibration range was fitted at least square model (r(2)≥0.999), with an accuracy and an intra- and inter-day RSD% (Relative Standard Deviation), both for sildenafil and N-desmethyl sildenafil, lower than 15%, as required by the FDA guidelines; LLOQ, LLOD, ULOQ were 3.9ng/mL, 1.95ng/mL and 1000ng/mL, respectively, for both analytes. Matrix effect, expressed as mean percent deviation of peak areas, was in the range between 2.6% and 5.8%, lower than 15% as required by guidelines. The mean recovery was 83.2 % for sildenafil and 84.5% for N-desmethyl sildenafil. This method has successfully been applied to a clinical pharmacokinetic study of sildenafil and N-desmethyl sildenafil in patients with PAH undergoing cardiac surgery.

Class-specific relative genetic contribution for key antiretroviral drugs.

OBJECTIVES: Antiretroviral pharmacokinetics is defined by numerous factors affecting absorption, distribution, metabolism and elimination. Biological processes underpinning drug distribution are only partially characterized and multiple genetic factors generate cumulative or antagonistic interactions, which complicates the implementation of pharmacogenetic markers. The aim of this study was to assess the degree to which heredity influences pharmacokinetics through the quantification of the relative genetic contribution (rGC) for key antiretrovirals. METHODS: A total of 407 patients receiving lopinavir/ritonavir, atazanavir/ritonavir, atazanavir, efavirenz, nevirapine, etravirine, maraviroc, tenofovir or raltegravir were included. Intra-patient variability (SDw) and inter-patient (SDb) variability were measured in patients with plasma concentrations available from more than two visits. The rGC was calculated using the following equation: 1 - (1 / F) where F = SDb(2) / SDw(2). RESULTS: Mean (95% CI) rGC was calculated to be 0.81 (0.72-0.88) for efavirenz, 0.74 (0.61-0.84) for nevirapine, 0.67 (0.49-0.78) for etravirine, 0.65 (0.41-0.79) for tenofovir, 0.59 (0.38-0.74) for atazanavir, 0.47 (0.27-0.60) for atazanavir/ritonavir, 0.36 (0.01-0.48) for maraviroc, 0.15 (0.01-0.44) for lopinavir/ritonavir and 0 (0-0.33) for raltegravir. CONCLUSIONS: The rank order for genetic contribution to variability in plasma concentrations for the study drugs was efavirenz > nevirapine > etravirine > tenofovir > atazanavir > atazanavir/ritonavir > maraviroc > lopinavir/ritonavir > raltegravir, indicating that class-specific differences exist. The rGC strategy represents a useful tool to rationalize future investigations as drugs with higher rGC scores may represent better candidates for pharmacogenetic-pharmacokinetic studies.

UPLC-MS/MS method for the simultaneous quantification of anti-HBV nucleos(t)ides analogs: Entecavir, lamivudine, telbivudine and tenofovir in plasma of HBV infected patients.

Hepatitis B infection affects two billion people worldwide and 350 million of these are chronically infected. Chronic hepatitis B virus is one of the most important cause of mortality and morbidity worldwide. If it is left untreated, about one-third of affected people will develop progressive and possibly fatal liver disease, like hepatic cirrhosis and primary hepatocellular carcinoma. Currently, five nucleos(t)ide analogs are approved for the treatment of chronic HBV infection. They are: lamivudine, adefovir dipivoxil, telbivudine, entecavir and tenofovir disoproxil fumarate. In this work, we developed and validated an UPLC-Tandem mass spectrometry assay method capable of monitoring lamivudine, telbivudine, tenofovir and entecavir plasma concentrations. Both standards and quality controls (high, medium and low) were prepared in human plasma. Each sample was added with internal standard (5'amino-5'deoxy-thymidine) and then drugs were extracted through a protein precipitation protocol with acetonitrile+0.1% formic acid and then dried. The extracts were resuspended in water and then injected into the chromatographic system. The chromatographic separation was performed on an Acquity UPLC HSS T3 1.8 µm 2.1 × 150 mm column, with a gradient of water and acetonitrile, both added with formic acid (0.05%). Accuracy, intra-day and inter-day precision at quality controls levels fitted all 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 30 HBV+ patients with good results. This simple analytical method could represent a useful tool for the management of anti-HBV therapy.

A LC-MS method to quantify tenofovir urinary concentrations in treated patients.

Tenofovir disoproxil fumarate is a prodrug of tenofovir used in the treatment of HIV and HBV infections: it is the most used antiretroviral worldwide. Tenofovir is nucleotidic HIV reverse trascriptase inhibitor that showed excellent long-term efficacy and tolerability. However renal and bone complications (proximal tubulopathy, hypophosphatemia, decreased bone mineral density, and reduced creatinine clearance) limit its use. Tenofovir renal toxicity has been suggested as the consequence of drug entrapment in proximal tubular cells: measuring tenofovir urinary concentrations may be a proxy of this event and it may be used as predictor of tenofovir side effects. No method is currently available for quantifying tenofovir in this matrix: then, the aim of this work was to validate a new LC-MS method for the quantification of urinary tenofovir. Chromatographic separation was achieved with a gradient (acetonitrile and water with formic acid 0.05%) on an Atlantis 5 µm T3, 4.6 mm × 150 mm, reversed phase analytical column. Detection of tenofovir and internal standard was achieved by electrospray ionization mass spectrometry in the positive ion mode. Calibration ranged from 391 to 100,000 ng/mL. The limit of quantification was 391 ng/mL and the limit of detection was 195 ng/mL. Mean recovery of tenofovir and internal standard were consistent and stable, while matrix effect resulted low and stable. The method was tested on 35 urine samples from HIV-positive patients treated with tenofovir-based HAARTs and did not show any significant interference with antiretrovirals or other concomitantly administered drugs. All the observed concentrations in real samples fitted the calibration range, confirming the capability of this method for the use in clinical routine. Whether confirmed in ad hoc studies this method may be used for quantifying tenofovir urinary concentrations and help managing HIV-positive patients treated with tenofovir.