Papers by Monica Mazzarino
ACS omega, Aug 29, 2022
We present a quick and simple multi-targeted analytical workflow based on ultra-high-performance ... more We present a quick and simple multi-targeted analytical workflow based on ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry for the screening in dried blood spots and dried plasma spots of a wide variety of drugs with different chemical properties. Seven different microsampling devices were evaluated in view of their application for the detection of the selected target analytes in the framework of doping control analysis. The extraction of the analytes was optimized by assessing the efficacy of protocols based on ultrasonication with aqueous buffers and/or organic solvents of different polarities. Optimal recoveries were obtained by using pure methanol or mixtures of methanol/acetonitrile and methanol/isopropanol, depending on both the device and the target analytes. The method was fully validated according to both ISO17025 and the requirements of the World Anti-Doping Agency: all the analytes were clearly distinguishable from the matrix, with limits of detection in the range of 0.1–3.0 ng mL–1. Stability studies simulating the storage of samples before the analysis and in view of a possible re-analysis showed that most of the analytes were stable for at least 24 h at 50 °C and for at least 3 weeks at 25 and at 4 °C. The real applicability of the method was assessed by analyzing the samples collected after the administration of two model drugs, acetazolamide and deflazacort. The performance of the method was confirmed to be fit for purpose, and data obtained in blood can also be used to complement those available in urine, allowing to refine the knowledge concerning the pharmacokinetic profiles.
Analytical and Bioanalytical Chemistry, Aug 14, 2008
This paper presents a general screening method, based on liquid chromatography/mass spectrometry ... more This paper presents a general screening method, based on liquid chromatography/mass spectrometry (LC/MS), for the simultaneous detection in human urine of 72 xenobiotics (21 diuretics, 16 synthetic glucocorticoids, 17 betaadrenergic drugs, 10 stimulants, 5 anti-oestrogens and 3 anabolic steroids), excreted free or as glucuro-conjugates in urine. Although the method has been specifically designed and evaluated in view of its potential application to antidoping analyses, it can also be effective in other areas of analytical toxicology. Sample preparation was based on two liquid/liquid separation steps (performed at alkaline and at acid pH, respectively) of hydrolyzed human urine, and then an assay by LC/MS-MS in positive and negative ionization mode using an electrospray ionization source (ESI) and multiple reaction monitoring (MRM) as the acquisition mode. The overall time needed for an LC run was less than 15 minutes. All compounds showed good reproducibility in terms of both the retention times (CV%<1) and the relative abundances of the diagnostic transitions (CV%<10). The limits of detection (LOD) were in the range of 1-50 ng/mL for glucocorticoids, anti-oestrogens and steroids, and 50-500 ng/mL for diuretics, beta-adrenergic drugs and stimulants, thus satisfying the minimum required performance limits (MRPL) set by the World Anti-Doping Agency (WADA) for the accredited anti-doping laboratories.
Analytica Chimica Acta, 2003
This work presents a complete method for the screening and confirmation analysis of diuretics in ... more This work presents a complete method for the screening and confirmation analysis of diuretics in human urine by gas chromatographymass spectrometry (GCMS). The method comprises a pretreatment stage (extraction, preconcentration and derivatization to form ...
Analytica Chimica Acta, Feb 1, 2006
This paper presents a gas chromatography–mass spectrometry (GC–MS) study carried out on human uri... more This paper presents a gas chromatography–mass spectrometry (GC–MS) study carried out on human urine to verify whether the administration of glucocorticoids can affect the urinary steroid profile, and especially the levels of endogenous glucocorticoids, androgens and their main metabolites.Betamethasone and beclomethasone, administered either systemically (per os or i.m.) or locally (by inhalation) have been studied. The determination of the urinary
Forensic Toxicology, May 27, 2015
A liquid chromatography–tandem mass spectrometry method was developed and used to simultaneously ... more A liquid chromatography–tandem mass spectrometry method was developed and used to simultaneously detect 19 small peptide hormones prohibited in sport and their main metabolites in urine after solid-phase extraction. Detection was achieved using a triple-quadrupole mass spectrometric detector coupled with an electrospray ionization interface after chromatographic separation with an octadecyl column based on fused-core particle technology. Sample pretreatment was performed by solid-phase extraction. The extraction procedure was optimized by comparison of different sorbents and washing/elution protocols. The best results were obtained using a mixed-mode weak cation exchange sorbent, two washing steps (ultrapurified water and methanol), and elution using 300 mM ammonium formate in 25 % ammonia/methanol (10/90) or 25 % ammonia/10 % formic acid/methanol (8/12/80). The procedure was validated in terms of sensitivity (lower limits of detection: 0.05–2.0 ng/ml depending on the target analyte), specificity, recovery [>60 %, coefficient of variation (CV) <15 % except for TB500 17–23 fragment, AOD9604, and ARA290 for which recovery was <50 %), ion suppression/enhancement (<35 %), robustness, carryover, stability of the target analytes [stable for at least for 2 days (25 °C, 2 weeks (4 °C), 2 months (−20 °C)], and repeatability of retention times (CV <0.1 %) and relative abundances of the selected ion transitions (CV <15 %). The suitability of the method was confirmed by analyzing spiked and excreted urines, the latter collected after intravenous injection of 0.1 mg of GHRP-2.
Rapid Communications in Mass Spectrometry, 2006
A fast liquid chromatographic/mass spectrometric (LC/MS/MS) screening method for the detection, i... more A fast liquid chromatographic/mass spectrometric (LC/MS/MS) screening method for the detection, in urine, of synthetic glucocorticoids, stimulants (formoterol, modafinil and mesocarb), anti-oestrogens (finasteride, exemestane, anastrozole, letrozole and formestane) and synthetic anabolic steroids (stanozolol, gestrinone and tetrahydrogestrinone) is described. All these drugs (and/or their urinary metabolites) can be simultaneously extracted by a single liquid/liquid extraction step, at alkaline pH, after enzymatic hydrolysis with beta-glucuronidase, and assayed in 7 min by LC/MS/MS using electrospray ionization in positive ion mode and multiple reaction monitoring as the acquisition mode. All compounds show good reproducibility of both the retention times (CV% &amp;amp;amp;amp;lt;2%) and the relative abundances (CV% &amp;amp;amp;amp;lt;10%). The limits of detection for the anti-oestrogens, glucocorticoids and steroids are in the range of 1-30 ng/mL, and for the stimulants are in the range of 100-200 ng/mL, thus satisfying the minimum required performance limits of the World Anti-Doping Agency.
Clinical Mass Spectrometry, Nov 1, 2020
Phthalates and bisphenol A interfere with the synthesis, secretion, transport, binding, metabolis... more Phthalates and bisphenol A interfere with the synthesis, secretion, transport, binding, metabolism, and excretion of endogenous hormones and, for this reason, are classified as endocrine disruptors.We are here presenting an analytical method for the simultaneous detection of six phthalates metabolites and bisphenol A in different biological fluids (urine, serum and follifular fluid) by liquid chromatography coupled to tandem mass spectrometry. The quantification was carried out in negative electrospray ionization mode using selected reaction monitoring as acquisition mode. Different extraction protocols, using either solid phase or liquid/liquid extraction, were comparatively evaluated to optimize the sample preparation procedure. Solid-phase extraction was chosen as it ensured the best recovery and overall sensitivity. The method was successfully validated: recovery varying in the range 71 ± 2%–107 ± 6% depending on the target analyte and the matrix considered, intra-assay and inter-assay precision ≤ 12% for follicular fluid, ≤11% for serum and ≤ 10% for urine and accuracy ≤ 115% for follicular fluid, ≤113% for serum ≤ 115% for urine , linearity with R2 > 0.99, with the exception of MEP (recovery 64 ± 8%, intra-assay precision ≤ 20%, inter-assay precision ≤ 16% for follicular fluid). The actual applicability of the method developed and validated in this study was assessed by the analysis of real samples, including 10 specimens of follicular fluid, serum and urine samples, that showed the presence of phthalates metabolites and Bisphenol A, and confirming that the newly developed method can be applied in the routine clinical laboratory for the identification and quantitation of these endocrine-disrupting chemicals.
Journal of Pharmaceutical and Biomedical Analysis
Drug Testing and Analysis, 2021
Mexedrone is a synthetic cathinone structurally related to mephedrone, which belongs to the class... more Mexedrone is a synthetic cathinone structurally related to mephedrone, which belongs to the class of N‐alkyl cathinone derivatives, whose metabolic profile has not been fully clarified yet. This study considers the in vitro phase I metabolism of mexedrone, to pre‐select the most appropriate marker(s) of intake. Mexedrone was incubated in the presence of either human liver microsomes or single recombinant CYP450 isoforms. The metabolic profile was outlined by ultra‐high‐performance liquid chromatography coupled to both high‐ and low‐resolution mass spectrometry. In detail, the phase I metabolic profile of mexedrone was initially defined by a time‐of‐flight analyzer, while the chemical structures of the detected metabolites and the potential presence of minor metabolites were subsequently studied by tandem mass spectrometry, using a triple quadrupole analyzer. The main phase I metabolic reactions were hydroxylation and N‐ and O‐dealkylation. The CYP450 isoforms most involved were CYP2...
Drug Testing and Analysis, 2020
We have considered the urinary excretion profile of methiopropamine (MPA), a thiophene ring‐based... more We have considered the urinary excretion profile of methiopropamine (MPA), a thiophene ring‐based structural analog of methamphetamine with similar stimulant effects, with the aim of selecting the most appropriate marker(s) of intake that may be useful in forensic analysis. For this purpose, in vitro studies were preliminarily performed on human liver microsomes for tracing the phase I metabolic pathways of MPA, preselecting the best candidates as potential target analytes, and designing the optimal experimental strategy. In vivo studies were then conducted on mice, after the intraperitoneal administration of a 10‐mg/kg dose. Urine samples were collected every 3 h in the first 9 h and, subsequently, from 24 to 36 h, and stored at –80°C until further analysis. The measurements were performed using a targeted procedure based on liquid/liquid extraction followed by liquid chromatography–tandem mass spectrometry analysis. Our results show that in the time interval 0–9 h after administra...
Journal of Pharmaceutical and Biomedical Analysis, 2019
The present study aimed to design, develop, and optimize an analytical procedure to perform the q... more The present study aimed to design, develop, and optimize an analytical procedure to perform the quantitative determination of ecdysterone in commercially available dietary supplements. The newly developed procedure is based on the extraction of ecdysterone from the supplements and the subsequent analysis by an optimized UHPLC-MS/MS method. Chromatographic separation was performed on an Agilent Eclipse Plus C18 column (2.1 mm x 100 mm, particle size 1.8 m). The mass spectrometer was operated in positive ionization mode (ESI+) with acquisition in dynamic multiple reaction monitoring (dMRM) mode. Using the protonated molecular ion [M+H] + ecdysterone (target) and cortisol (internal reference) were detected at m/z 481 and 363, respectively. The assay was fully validated according to ICH guidelines and the method resulted to be fit for purpose in terms of accuracy and precision (CV% and RE% <15). Time-different intermediate precision was found within the reported range according to AOAC guideline for dietary supplements and botanicals. Quantitation has been performed using an external calibration considering the minimal matrix influences, preliminarily assessed following a cross comparison with an elaborate and time consuming standard addition method. The method was successfully applied to 12 different dietary supplements labelled to contain ecdysterone, showing an actual content generally much lower than the labelled one.
Journal of Endocrinological Investigation, 2009
To describe serum and urinary hormones, androgens metabolites and testosterone/epitestosterone ra... more To describe serum and urinary hormones, androgens metabolites and testosterone/epitestosterone ratio profiles after testosterone administration in male hypogonadal volunteers, and to evaluate their possible usefulness in detecting doping with testosterone in treated hypogonadal athletes. Controlled open label design vs placebo; pharmacokinetic study. Ten male volunteers affected by severe hypogonadism (serum testosterone &amp;amp;amp;amp;amp;amp;amp;amp;lt;2.31 ng/ml). Serum and urinary parameters were evaluated, by radioimmunoassay and gas chromatography-mass spectrometry, before and at different time points for 7/3 weeks after a single administration of testosterone enanthate (250 mg) or placebo, respectively. As partially known, testosterone administration increased, with great individual variability, urinary concentrations of glucuronide testosterone, androsterone, etiocholanolone, 5alpha-androstane- 3alpha,17beta-diol, 5beta-androstane-3alpha,17beta-diol, testosterone/ epitestosterone and testosterone/LH ratios; and decreased epitestosterone and 5alpha-androstane-3beta,17beta-diol/5beta-androstane- 3alpha,17beta-diol ratio. Serum testosterone and dihydrotestosterone increased in all volunteers, and concentrations higher than the upper reference limits were observed in many volunteers until 2 weeks after testosterone administration. Whereas the observed prolonged hyperandrogenism partially limited data interpretation, the report ed characteristics of variation of urinary parameters might be used to suspect testosterone misuse in hypogonadal athletes treated with testosterone enanthate. In this sense, while the actual threshold for tes tos terone/epites tos ter one ratio was confirmed to be of reduced usefulness, we suggest a contemporary evaluation of whole urinary androgen metabolites profile and serum androgens, at specific time points after testosterone enanthate administration. Moreover, an adequate tailoring of treatment, to avoid transitory hyperandrogenism, is highly advisable. Further studies on strategies for detecting doping with testosterone in hypogonadal athletes are warranted.
Forensic Toxicology, 2015
A liquid chromatography–tandem mass spectrometry method was developed and used to simultaneously ... more A liquid chromatography–tandem mass spectrometry method was developed and used to simultaneously detect 19 small peptide hormones prohibited in sport and their main metabolites in urine after solid-phase extraction. Detection was achieved using a triple-quadrupole mass spectrometric detector coupled with an electrospray ionization interface after chromatographic separation with an octadecyl column based on fused-core particle technology. Sample pretreatment was performed by solid-phase extraction. The extraction procedure was optimized by comparison of different sorbents and washing/elution protocols. The best results were obtained using a mixed-mode weak cation exchange sorbent, two washing steps (ultrapurified water and methanol), and elution using 300 mM ammonium formate in 25 % ammonia/methanol (10/90) or 25 % ammonia/10 % formic acid/methanol (8/12/80). The procedure was validated in terms of sensitivity (lower limits of detection: 0.05–2.0 ng/ml depending on the target analyte), specificity, recovery [>60 %, coefficient of variation (CV) <15 % except for TB500 17–23 fragment, AOD9604, and ARA290 for which recovery was <50 %), ion suppression/enhancement (<35 %), robustness, carryover, stability of the target analytes [stable for at least for 2 days (25 °C, 2 weeks (4 °C), 2 months (−20 °C)], and repeatability of retention times (CV <0.1 %) and relative abundances of the selected ion transitions (CV <15 %). The suitability of the method was confirmed by analyzing spiked and excreted urines, the latter collected after intravenous injection of 0.1 mg of GHRP-2.
Analytical and Bioanalytical Chemistry, Feb 9, 2008
This paper describes a liquid chromatographic/ tandem mass spectrometric (LC/MS-MS) method specif... more This paper describes a liquid chromatographic/ tandem mass spectrometric (LC/MS-MS) method specifically designed for the screening of synthetic glucocorticosteroids in human urine. The method is designed to recognize a common mass spectral fragment formed from the particular portion of the molecular structure that is common to all synthetic glucocorticosteroids and that is fundamental to their pharmacological activity. As such, the method is also suitable for detecting unknown substances, provided they contain the portion of the molecular structure selected as the analytical target. The effectiveness of this approach was evaluated on seventeen synthetic glucocorticosteroids. Urine samples, including blank urines spiked with one or more synthetic glucocorticosteroids, were treated according to a standard procedure (enzymatic hydrolysis, liquid/liquid extraction and evaporation to dryness) and analyzed using LC/MS-MS with electrospray ionization (ESI). MS-MS acquisition was carried out in a precursor ion scan, and the results were compared with those obtained by a previously developed reference technique based on acquisition in the multiple reaction monitoring (MRM) mode. All of the glucocorticosteroids considered in this study are clearly detectable in urine, with a limit of detection in the concentration range 5-20 ng/mL, depending on the glucocorticosteroid structure. The proposed method is therefore suitable for the detection of glucocorticosteroids in urine samples taken for "in competition" sport anti-doping control tests, matching the requirements of the World Anti-Doping Agency (WADA) for accredited anti-doping laboratories.
Analytical and Bioanalytical Chemistry
ACS Omega
We present a quick and simple multi-targeted analytical workflow based on ultra-high-performance ... more We present a quick and simple multi-targeted analytical workflow based on ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry for the screening in dried blood spots and dried plasma spots of a wide variety of drugs with different chemical properties. Seven different microsampling devices were evaluated in view of their application for the detection of the selected target analytes in the framework of doping control analysis. The extraction of the analytes was optimized by assessing the efficacy of protocols based on ultrasonication with aqueous buffers and/or organic solvents of different polarities. Optimal recoveries were obtained by using pure methanol or mixtures of methanol/acetonitrile and methanol/ isopropanol, depending on both the device and the target analytes. The method was fully validated according to both ISO17025 and the requirements of the World Anti-Doping Agency: all the analytes were clearly distinguishable from the matrix, with limits of detection in the range of 0.1−3.0 ng mL −1. Stability studies simulating the storage of samples before the analysis and in view of a possible re-analysis showed that most of the analytes were stable for at least 24 h at 50°C and for at least 3 weeks at 25 and at 4°C. The real applicability of the method was assessed by analyzing the samples collected after the administration of two model drugs, acetazolamide and deflazacort. The performance of the method was confirmed to be fit for purpose, and data obtained in blood can also be used to complement those available in urine, allowing to refine the knowledge concerning the pharmacokinetic profiles.
Drug Testing and Analysis
Forensic Toxicology, 2016
We have investigated the influence of oral miconazole administration on the urinary concentration... more We have investigated the influence of oral miconazole administration on the urinary concentrations of endogenous anabolic androgenic steroids of doping relevance, specifically considering all these compounds routinely monitored in doping control analysis, in the framework of the steroidal module of the ''athlete biological passport'', and other steroids, including dehydroepiandrosterone, 5a-dihydrotestosterone, and the hydroxylated metabolites recently proposed as additional markers of the intake of testosteronerelated steroids (16a-hydroxy-androsterone, 16a-hydroxyetiocholanolone, 6b-hydroxy-androsterone, 6b-hydroxy-etiocholanolone, 7a-hydroxy-dehydroepiandrosterone, and 7bhydroxy-dehydroepiandrosterone). Urinary concentrations of the final metabolic products of the glucocorticoid biosynthetic pathways (11b-hydroxy-androsterone and 11b-hydroxy-etiocholanolone, the formerly used as an endogenous reference compound for the gas chromatography-combustion-isotope ratio mass spectrometry confirmation analysis) were also monitored. Two healthy Caucasian volunteers exhibiting physiologically high testosterone/epitestosterone ratios and elevated concentrations of the main target steroids were selected for the study. Miconazole was administered orally (500 mg/day) for 1 week. Multiple urine samples were collected for 1 week before and during the treatment, and analyzed according to a validated analytical procedure based on gas chromatography-electron ionization-mass spectrometry in selected ion monitoring mode. Our results indicated that oral administration of miconazole decreased the urinary concentrations of androsterone, and to a lesser extent, of etiocholanolone (both detected as the sum of free and glucuronated steroids), and consequently the androsterone/testosterone and androsterone/etiocholanolone ratios. Furthermore, the urinary concentrations of 16a-hydroxy-etiocholanolone, 16a-hydroxy-androsterone, 7b-hydroxy-dehydroepiandrosterone, 6b-hydroxy-etiocholanolone, 7a-hydroxy-dehydroepiandrosterone, 6b-hydroxy-androsterone, 11b-hydroxy-androsterone, and 11b-hydroxy-etiocholanolone were significantly suppressed. This evidence suggests the potential intake of miconazole whenever the urinary steroid profile is characterized by abnormally low concentrations of the above-mentioned steroids.
Toxicology in vitro : an international journal published in association with BIBRA, Jan 6, 2015
Liver in vitro systems that allow reliable prediction of major human in vivo metabolic pathways h... more Liver in vitro systems that allow reliable prediction of major human in vivo metabolic pathways have a significant impact in drug screening and drug metabolism research. In the present study, a novel porous scaffold composed of alginate was prepared by employing a gas-in-liquid foaming approach. Galactose residues were introduced on scaffold surfaces to promote cell adhesion and to enhance liver specific functions of the entrapped HepG2/C3A cells. Hepatoma cells in the gal-alginate scaffold showed higher levels of liver specific products (albumin and urea) and were more responsive to specific inducers (e.g. dexamethasone) and inhibitors (e.g. ketoconazole) of the CYP3A4 system than in conventional monolayer culture. HepG2/C3A cells were also more efficient in terms of rapid elimination of testosterone, used as a model substance, at rates comparable to those of in vivo excretion. In addition, an improvement in metabolism of testosterone, in terms of phase II metabolite formation, was...
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Papers by Monica Mazzarino