Infiltrating macrophages amplify doxorubicin-induced cardiac damage: role of catecholamines.

BACKGROUND: The functional contribution of non-myocyte cardiac cells, such as inflammatory cells, in the setup of heart failure in response to doxorubicin (Dox) is recently becoming of growing interest. OBJECTIVES: The study aims to evaluate the role of macrophages in cardiac damage elicited by Dox treatment. METHODS: C57BL/6 mice were treated with one intraperitoneal injection of Dox (20 mg/kg) and followed up for 5 days by cardiac ultrasounds (CUS), histological, and flow cytometry evaluations. We also tested the impact of Dox in macrophage-depleted mice. Rat cardiomyoblasts were directly treated with Dox (D-Dox) or with a conditioned medium from cultured murine macrophages treated with Dox (M-Dox). RESULTS: In response to Dox, macrophage infiltration preceded cardiac damage. Macrophage depletion prevents Dox-induced damage, suggesting a key role of these cells in promoting cardiotoxicity. To evaluate the crosstalk between macrophages and cardiac cells in response to DOX, we compared the effects of D-Dox and M-Dox in vitro. Cell vitality was lower in cardiomyoblasts and apoptosis was higher in response to M-Dox compared with D-Dox. These events were linked to p53-induced mitochondria morphology, function, and autophagy alterations. We identify a mechanistic role of catecholamines released by Dox-activated macrophages that lead to mitochondrial apoptosis of cardiac cells through ß-AR stimulation. CONCLUSIONS: Our data indicate that crosstalk between macrophages and cardiac cells participates in cardiac damage in response to Dox.

Platelet Inhibition with Ticagrelor 60 mg Versus 90 mg Twice Daily in Elderly Patients with Acute Coronary Syndrome: Rationale and Design of the PLINY THE ELDER Trial.

BACKGROUND: Elderly status is steadily increasing among patients with acute coronary syndrome (ACS). Dual antiplatelet therapy (DAPT) with aspirin and a potent P2Y12 receptor inhibitor is the cornerstone of treatment to prevent recurrent thrombotic complications in patients with ACS. However, DAPT in older patients is challenged by a concurrent heightened risk of ischemia and bleeding. The aim of this study is to evaluate the pharmacodynamic and pharmacokinetic profile of a lower dose of ticagrelor (60 mg twice daily) among elderly patients during the early phase of ACS. STUDY DESIGN: PLINY THE ELDER (PLatelet INhibition with two different doses of potent P2y12 inhibitors in THE ELDERly population) (NCT04739384) is a prospective, randomized, open-label, crossover trial to evaluate the non-inferiority of a lower dose of ticagrelor (60 mg twice daily) compared with a standard dose (90 mg twice daily) among elderly patients with ACS undergoing percutaneous coronary intervention (PCI). A total of 50 patients, aged 75 years or more, with indication to potent P2Y12 receptor inhibitors will be randomized within 3 days from PCI for the index ACS. Patients with indication to oral anticoagulant therapy, treatment with glycoprotein IIb/IIIa inhibitors, or active bleeding will be excluded. The primary endpoint is platelet reactivity determined by P2Y12 reaction units (PRU) (VerifyNow, Accumetrics, San Diego, CA, USA) after treatment with ticagrelor 60 or 90 mg twice daily for 14 days. Secondary endpoints will include other pharmacodynamic tests of ADP-induced aggregation (light transmittance aggregometry and multiple electrode aggregometry) and determination of pharmacokinetic profile (plasma levels of ticagrelor and its metabolite AR-C124910XX) by high performance liquid chromatography-tandem mass spectrometry. CONCLUSIONS: The PLINY THE ELDER trial will determine whether a lower dose of ticagrelor confers non-inferior platelet inhibition compared with the standard dose in the early phase of ACS among elderly patients undergoing PCI, informing future clinical investigation.

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.

Production in Escherichia coli of recombinant COVID-19 spike protein fragments fused to CRM197.

During 2020, the COVID-19 pandemic affected almost 108 individuals. Quite a number of vaccines against COVID-19 were therefore developed, and a few recently received authorization for emergency use. Overall, these vaccines target specific viral proteins by antibodies whose synthesis is directly elicited or indirectly triggered by nucleic acids coding for the desired targets. Among these targets, the receptor binding domain (RBD) of COVID-19 spike protein (SP) does frequently occur in the repertoire of candidate vaccines. However, the immunogenicity of RBD per se is limited by its low molecular mass, and by a structural rearrangement of full-length SP accompanied by the detachment of RBD. Here we show that the RBD of COVID-19 SP can be conveniently produced in Escherichia coli when fused to a fragment of CRM197, a variant of diphtheria toxin currently used for a number of conjugated vaccines. In particular, we show that the CRM197-RBD chimera solubilized from inclusion bodies can be refolded and purified to a state featuring the 5 native disulphide bonds of the parental proteins, the competence in binding angiotensin-converting enzyme 2, and a satisfactory stability at room temperature. Accordingly, our observations provide compulsory information for the development of a candidate vaccine directed against COVID-19.

Limonoids from Guarea guidonia and Cedrela odorata: Heat Shock Protein 90 (Hsp90) Modulator Properties of Chisomicine D.

Nine new limonoids (1-9) were isolated from the stem bark of Guarea guidonia (1-4) and Cedrela odorata (5-9). Their structures were elucidated using 1D and 2D NMR and MS data and chemical methods as three A2,B,D-seco-type limonoids (1-3), a mexicanolide (4), three nomilin-type (5-7) limonoids, and two limonol derivatives (8 and 9). A DFT/NMR procedure was used to define the relative configurations of 1 and 3. A surface plasmon resonance approach was used to screen the Hsp90 binding capability of the limonoids, and the A2,B,D-seco-type limonoid 8-hydro-(8S*,9S*)-dihydroxy-14,15-en-chisomicine A, named chisomicine D (1), demonstrated the highest affinity. By means of mass spectrometry data, biochemical and cellular assays, and molecular docking, 1 was found as a type of client-selective Hsp90 inhibitor binding to the C-terminus domain of the chaperone.

Pro-Inflammatory Effects of Indoxyl Sulfate in Mice: Impairment of Intestinal Homeostasis and Immune Response.

The intestines are recognized as the main source of chronic inflammation in chronic kidney disease (CKD) and, among other cells, macrophages are involved in modulating this process as well as in the impaired immune response which also occurs in CKD patients. In this study, we evaluated the effect of Indoxyl Sulfate (IS), a protein bound uremic toxin poorly eliminated by hemodialysis, on inflammatory, oxidative stress and pro-apoptotic parameters, at the intestinal level in mice, on intestinal epithelial cells (IEC-6) and on primary murine peritoneal macrophages. C57BL/6J mice were treated with IS (800 mg/kg i.p.) for 3 or 6 h and histopathological analysis showed that IS induced intestinal inflammation and increased cyclooxygenase-2 (COX-2), nitrotyrosine and Bax expression in intestinal tissue. In IEC-6 cells, IS (125-1000 µM) increased tumor necrosis factor-α levels, COX-2 and inducible nitric oxide synthase expression and nitrotyrosine formation. Moreover, IS increased pro-oxidant, pro-inflammatory and pro-apoptotic parameters in peritoneal macrophages from IS-treated mice. Also, the serum concentration of IS and pro-inflammatory levels of cytokines resulted increased in IS-treated mice. Our results indicate that IS significantly contributes to affect intestinal homeostasis, immune response, and to induce a systemic pro-inflammatory state thus highlighting its potential role as therapeutic target in CKD patients.

Outer Membrane Vesicles Derived from Klebsiella pneumoniae Are a Driving Force for Horizontal Gene Transfer.

Gram-negative bacteria release Outer Membrane Vesicles (OMVs) into the extracellular environment. Recent studies recognized these vesicles as vectors to horizontal gene transfer; however, the parameters that mediate OMVs transfer within bacterial communities remain unclear. The present study highlights for the first time the transfer of plasmids containing resistance genes via OMVs derived from Klebsiella pneumoniae (K. pneumoniae). This mechanism confers DNA protection, it is plasmid copy number dependent with a ratio of 3.6 times among high copy number plasmid (pGR) versus low copy number plasmid (PRM), and the transformation efficiency was 3.6 times greater. Therefore, the DNA amount in the vesicular lumen and the efficacy of horizontal gene transfer was strictly dependent on the identity of the plasmid. Moreover, the role of K. pneumoniae-OMVs in interspecies transfer was described. The transfer ability was not related to the phylogenetic characteristics between the donor and the recipient species. K. pneumoniae-OMVs transferred plasmid to Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa and Burkholderia cepacia. These findings address the pivotal role of K. pneumoniae-OMVs as vectors for antimicrobial resistance genes spread, contributing to the development of antibiotic resistance in the microbial communities.

Systematic Phytochemical Screening of Different Organs of Calotropis procera and the Ovicidal Effect of Their Extracts to the Foodstuff Pest Cadra cautella.

In developing countries, crop deterioration is mainly caused by inappropriate storage conditions that promote insect infestation. Synthetic pesticides are associated with serious adverse effects on humans and the environment. Thus, finding alternative "green" insecticides is a very pressing need. Calotropis procera (Aiton) Dryand (Apocynaceae) growing in Saudi Arabia was selected for this purpose. LC-MS/MS analysis was applied to investigate the metabolic composition of different C. procera extracts. Particularly, C. procera latex and leaves showed a high presence of cardenolides including calactin, uscharidin, 15ß-hydroxy-calactin, 16ß-hydroxy-calactin, and 12ß-hydroxy-calactin. The ovicidal activity of the extracts from different plant organs (flowers, leaves, branches, roots), and of the latex, against Cadra cautella (Walker) (Lepidoptera, Pyralidae) was assessed. Extracts of C. procera roots displayed the most potent activity with 50% of C. cautella eggs not hatching at 10.000 ppm (1%).

Substrate Activation of the Low-Molecular Weight Protein Tyrosine Phosphatase from Mycobacterium tuberculosis.

Mycobacterium tuberculosis is known to express a low-molecular weight protein tyrosine phosphatase. This enzyme, denoted as MptpA (Mycobacterium protein tyrosine phosphatase A), is essential for the pathogen to escape the host immune system and therefore represents a target for the search of antituberculosis drugs. MptpA was shown to undergo a conformational transition during catalysis, leading to the closure of the active site, which is by this means poised to the chemical step of dephosphorylation. Here we show that MptpA is subjected to substrate activation, triggered by p-nitrophenyl phosphate or by phosphotyrosine. Moreover, we show that the enzyme is also activated by phosphoserine, with serine being ineffective in enhancing MptpA activity. In addition, we performed assays under pre-steady-state conditions, and we show here that substrate activation is kinetically coupled to the closure of the active site. Surprisingly, when phosphotyrosine was used as a substrate, MptpA did not obey Michealis-Menten kinetics, but we observed a sigmoidal dependence of the reaction velocity on substrate concentration, suggesting the presence of an allosteric activating site in MptpA. This site could represent a promising target for the screening of MptpA inhibitors exerting their action independently of the binding to the enzyme active site.

Recombinant mussel protein Pvfp-5ß: A potential tissue bioadhesive.

During their lifecycle, many marine organisms rely on natural adhesives to attach to wet surfaces for movement and self-defense in aqueous tidal environments. Adhesive proteins from mussels are biocompatible and elicit only minimal immune responses in humans. Therefore these proteins have received increased attention for their potential applications in medicine, biomaterials, and biotechnology. The Asian green mussel Perna viridis secretes several byssal plaque proteins, molecules that help anchoring the mussel to surfaces. Among these proteins, protein-5ß (Pvfp-5ß) initiates interactions with the substrate, displacing interfacial water molecules before binding to the surface. Here, we established the first recombinant expression in Escherichia coli of Pvfp-5ß. We characterized recombinant Pvfp-5ß, finding that despite displaying a CD spectrum consistent with features of a random coil, the protein is correctly folded as indicated by MS and NMR analyses. Pvfp-5ß folds as a ß-sheet-rich protein as expected for an epidermal growth factor-like module. We examined the effects of Pvfp-5ß on cell viability and adhesion capacity in NIH-3T3 and HeLa cell lines, revealing that Pvfp-5ß has no cytotoxic effects at the protein concentrations used and provides good cell-adhesion strength on both glass and plastic plates. Our findings suggest that the adhesive properties of recombinant Pvfp-5ß make it an efficient surface-coating material, potentially suitable for biomedical applications including regeneration of damaged tissues.

Molecular Dissection of dH3w, A Fluorescent Peptidyl Sensor for Zinc and Mercury.

Previously, we reported that fluorescent peptide dansyl-HPHGHW-NH2 (dH3w), designed on the repeats of the human histidine-rich glycoprotein, shows a turn-on response to Zn(II) and a complex response to Hg(II) characterized by a turn-off phase at low Hg(II) concentrations and a turn-on phase at high concentrations. As Hg(II) easily displaces Zn(II), dH3w is a useful probe for the environmental monitoring of Hg(II). In order to investigate the molecular basis of the metal selectivity and fluorescence response, we characterized three variants, dH3w(H1A), dH3w(H3A), and dH3w(H5A), in which each of the three histidine residues was changed to alanine, and two variants with a single fluorescent moiety, namely dH3w(W6A), in which the tryptophan residue at the C-terminus was changed to alanine, and AcH3w, in which the N-terminal dansyl moiety was substituted by an acetyl group. These variants allowed us to demonstrate that all the histidine residues are essential for a strong interaction with Zn(II), whereas two histidine residues (in particular His5) and the dansyl group are necessary to bind Hg(II). The data reported herein shed light on the molecular behavior of dH3w, thus paving the way to the rational designing of further and more efficient fluorescent peptidyl probes for Hg(II).

Bioactive Ent-Kaurane Diterpenes Oridonin and Irudonin Prevent Cancer Cells Migration by Interacting with the Actin Cytoskeleton Controller Ezrin.

The ent-kaurane diterpene oridonin was reported to inhibit cell migration and invasion in several experimental models. However, the process by which this molecule exerts its anti-metastatic action has not been yet elucidated. In this article, we have investigated the anti-metastatic activity of Oridonin and of one homolog, Irudonin, with the aim to shed light on the molecular mechanisms underlying the biological activity of these ent-kaurane diterpenes. Cell-based experiments revealed that both compounds are able to affect differentiation and cytoskeleton organization in mouse differentiating myoblasts, but also to impair migration, invasion and colony formation ability of two different metastatic cell lines. Using a compound-centric proteomic approach, we identified some potential targets of the two bioactive compounds among cytoskeletal proteins. Among them, Ezrin, a protein involved in the actin cytoskeleton organization, was further investigated. Our results confirmed the pivotal role of Ezrin in regulating cell migration and invasion, and indicate this protein as a potential target for new anti-cancer therapeutic approaches. The interesting activity profile, the good selectivity towards cancer cells, and the lower toxicity with respect to Oridonin, all suggest that Irudonin is a very promising anti-metastatic agent.

Glycation affects fibril formation of Aß peptides.

Increasing evidence shows that ß-amyloid (Aß) peptides, which are associated with Alzheimer disease (AD), are heavily glycated in patients, suggesting a role of this irreversible nonenzymatic post-translational modification in pathology. Previous reports have shown that glycation increases the toxicity of the Aß peptides, although little is known about the mechanism. Here, we used the natural metabolic by-product methylglyoxal as a glycating agent and exploited various spectroscopic methods and atomic force microscopy to study how glycation affects the structures of the Aß40 and Aß42 peptides, the aggregation pathway, and the morphologies of the resulting aggregates. We found that glycation significantly slows down but does not prevent ß-conversion to mature fibers. We propose that the previously reported higher toxicity of the glycated Aß peptides could be explained by a longer persistence in an oligomeric form, usually believed to be the toxic species.

Identification of a novel esterase from the thermophilic bacterium Geobacillus thermodenitrificans NG80-2.

In the framework of the discovery of new thermophilic enzymes of potential biotechnological interest, we embarked in the characterization of a new thermophilic esterase from the thermophilic bacterium Geobacillus thermodenitrificans. The phylogenetic analysis of the GTNG_0744 esterase indicated that the sequence belongs to the enterochelin/enterobactin esterase group, which have never been recognized as a family in the lipases/esterase classification. These enzymes catalyze the last step in the acquisition of environmental Fe3+ through siderophore hydrolysis. In silico analysis revealed, for the first time, that the machinery for the uptake of siderophores is present in G. thermodenitrificans. The purified recombinant enzyme, EstGtA3, showed different substrate specificity from known enterochelin/enterobactin esterases, recognizing short chain esters with a higher specificity constant for 4-NP caprylate. The enzyme does not require cofactors for its activity, is active in the pH range 7.0-8.5, has highest activity at 60 °C and is 100% stable when incubated for 16 h at 55 °C. DTT, ß-mercaptoethanol and Triton X-100 have an activating effect on the enzymatic activity. Organic solvents have in general a negative effect on the enzyme, but n-hexane is a strong activator up to 150, making EstGtA3 a good candidate for applications in biotechnology.

Fusicoccane Diterpenes from Hypoestes forsskaolii as Heat Shock Protein 90 (Hsp90) Modulators.

Ten new (1-10) and six known (11-16) fusicoccane diterpenes were isolated from the roots of Hypoestes forsskaolii. The structural characterization of 1-10 was performed by spectroscopic analysis, including 1D and 2D NMR, ECD, and HRESIMS experiments. From a perspective of obtaining potential Hsp90α inhibitors, the isolates were screened by surface plasmon resonance measurements and their cytotoxic activity was assayed using Jurkat and HeLa cancer cells. Compound 6, 18-hydroxyhypoestenone, was shown to be the most active compound against Hsp90, and its interactions were studied also by biochemical and cellular assays and by molecular docking.

Phytochemistry and Antioxidant Activity of Aerial Parts of Phagnalon sordidum L.

One new natural monoterpene, 5-O-ß-d-glucopyranosyl-2-hydroxy-p-cymene (1: ), and 11 known compounds were isolated through a biologically oriented approach from the aerial parts of Phagnalon sordidum L. The most active extract and fractions were selected using 3 complementary antioxidant activity assays. Results and the different methods were compared by relative antioxidant capacity index. In addition, the most active extract of P. sordidum was subjected to liquid chromatography coupled with electrospray ionization hybrid linear ion trap quadrupole Orbitrap mass spectrometry to quantify secondary metabolites. Antioxidant activities of ethyl acetate extract, and purified 3,4-dihydroxyacetophenone (3: ) and nebrodenside A (7: ) were demonstrated by in vitro cell free model assays, and their protective effect against H2O2-induced oxidative stress in a HepG2 (human hepatocellular carcinoma) cell line was established.

Manganese is a Deinococcus radiodurans growth limiting factor in rich culture medium.

To understand the effects triggered by Mn2+ on Deinococcus radiodurans, the proteome patterns associated with different growth phases were investigated. In particular, under physiological conditions we tested the growth rate and the biomass yield of D. radiodurans cultured in rich medium supplemented or not with MnCl2. The addition of 2.5-5.0 µM MnCl2 to the medium neither altered the growth rate nor the lag phase, but significantly increased the biomass yield. When higher MnCl2 concentrations were used (10-250 µM), biomass was again found to be positively affected, although we did observe a concentration-dependent lag phase increase. The in vivo concentration of Mn2+ was determined in cells grown in rich medium supplemented or not with 5 µM MnCl2. By atomic absorption spectroscopy, we estimated 0.2 and 0.75 mM Mn2+ concentrations in cells grown in control and enriched medium, respectively. We qualitatively confirmed this observation using a fluorescent turn-on sensor designed to selectively detect Mn2+in vivo. Finally, we investigated the proteome composition of cells grown for 15 or 19 h in medium to which 5 µM MnCl2 was added, and we compared these proteomes with those of cells grown in the control medium. The presence of 5 µM MnCl2 in the culture medium was found to alter the pI of some proteins, suggesting that manganese affects post-translational modifications. Further, we observed that Mn2+ represses enzymes linked to nucleotide recycling, and triggers overexpression of proteases and enzymes linked to the metabolism of amino acids.

Nutritional therapy reduces protein carbamylation through urea lowering in chronic kidney disease.

Background: Protein carbamylation is one of the non-enzymatic reactions involved in protein molecular ageing. We sought to investigate the relationship between urea levels and protein carbamylation, and whether a Mediterranean diet (MD) and a very low protein diet (VLPD) reduce protein carbamylation through reduction in urea levels in patients with chronic kidney disease (CKD). Methods: This is a prospective, randomized, crossover controlled trial that investigated 60 patients with CKD grades 3B-4 (46 males, mean age of 67 years). The enrolled CKD patients were randomly assigned (1:1) to two different nutritional treatment arms: (i) 3 months of free diet (FD), 6 months of VLPD, 3 months of FD and 6 months of MD; and (ii) 3 months of FD, 6 months of MD, 3 months of FD and 6 months of VLPD. Blood levels of lysine (Lys) and homocitrulline (Hcit) and their ratio were used as markers of cyanate levels. Due to a lack of pre-existing data on the potential effects of different dietary regimens and in light of the exploratory nature of the study, no formal sample size estimation was carried out. Results: At study completion, lower diastolic blood pressure and decreased serum levels of urea, sodium, phosphorus and parathyroid hormone, but higher serum levels of bicarbonate and haemoglobin, were noted with MD and VLPD. When compared with FD, both MD and VLPD were also associated with a decrease in serum Hcit levels and Hcit/Lys ratios (P < 0.001). Notably, reductions in urea levels correlated with substantial reductions in Hcit levels (R2 = 0.16 and 0.17 for VLPD and MD, respectively). Conclusion: In conclusion, nutritional treatments that significantly decrease serum levels of urea are associated with reduced protein carbamylation.

Purification of active recombinant human histone deacetylase 1 (HDAC1) overexpressed in Escherichia coli.

OBJECTIVE: We attempted to overexpress Human Histone Deacetylase 1 (HDAC1) in Escherichia coli. RESULTS: A synthetic gene coding for HDAC1, and optimised for E. coli codon usage, was cloned into pBADHisB, generating pBAD-rHDAC1. This construct was used to transform E. coli TOP10, and the target protein was overexpressed and partially purified. According to its elution volume from a Superdex 200 column, the partially purified rHDAC1 was obtained in aggregated form, i.e., as an octamer. The dissociation of octameric HDAC1 was tested using several agents, among which sodium dodecyl sulfate was competent in partially dissociating rHDAC1 aggregates. When the enzyme activity was tested in vitro using 3H-acetyl-labelled histones both protein samples, aggregated and dissociated, were active. Hence, our results suggest that E. coli represents an alternative system for the production of the recombinant HDAC1. CONCLUSIONS: We described a procedure for the overexpression in E. coli of recombinant HDAC1, the purification of which in active form can be successfully performed, although yielding an octameric aggregate.

Development of a novel ion-pairing HPLC-FL method for the separation and quantification of hydroxychloroquine and its metabolites in whole blood.

Hydroxychloroquine (HCQ) is an old antimalarial drug that has proven to be a safe and effective treatment for systemic lupus erythematosus (SLE) and other autoimmune diseases. Since hematic concentration of HCQ is closely related to the therapeutic response, monitoring the levels of the drug and its metabolites in the blood of HCQ-treated patients helps the clinician in the evaluation of partial or complete unresponsiveness to treatment. We developed and validated a novel ion-pairing HPLC-FL method for the simultaneous dosage of HCQ, and its major metabolites desethylhydroxychloroquine, desethylchloroquine and bisdesethylchloroquine, after extraction from whole blood. This methodological approach was used for the analysis of real samples obtained from patients affected by SLE and undergoing HCQ treatment. The same samples were also analyzed using a previously validated LC/MS/MS method and data obtained with the two approaches were in substantial agreement with each other. Results presented in this work indicate that this approach can be successfully used to monitor the level of HCQ and its metabolites in the blood of various categories of patients (i.e. low and high responders, or those not adhering to the therapy). Comparison of HPLC-FL and LC/MS/MS data confirmed the efficacy of the proposed method for routine clinical analyses.