► Screening of steroid profiling for anti-doping controls and for diagnostic tests. ► UHPLC/MS determination of prohibited steroids and their metabolites. ► Identification of unknown species by ...UHPLC/MS. ► Overview about the biological matrices suitable for steroid determination.
The use of doping agents, once restricted to professional athletes, has nowadays become a problem of public health, since it also concerns young people and non-competing amateurs in different sports. The use is also diffused in social life for improving physical appearance and enhancing performance and even dietary supplements assumed to improve performance often contain anabolic steroids. While decades ago the so-called “classical doping agents” (like stimulants and narcotics) were used, to-day anabolic steroids are more widely diffused. Anabolic steroids are synthetic substances prepared by introducing modifications in the molecular structure of testosterone, the main natural androgenic anabolic steroid that forms in testes interstitial cells. The first report concerning the use of anabolic steroids by an athlete who searched for increased weight and power dates 1954. In 1974 the misuse of anabolic steroids in sports was banned by the International Olympic Committee and control tests were implemented in 1976 Montreal Olympic Games through radioimmunoassay analysis: the technique, however, only allows for unspecific detection of a limited number of exogenous steroids. Over the years, always new doping substances are synthesized and, as a consequence, the list of prohibited compounds is continuously updated and new suitable analytical methods for their detection and determination in biological matrices are continuously required. In doping control analysis the knowledge of steroid metabolism pathway in human body is of primary importance and the analytical methods must permit the simultaneous detection and determination not only of the forbidden precursor agents but also of their metabolites. In addition, the potential presence and amount in the biological samples of species that can interfere in the analysis should be evaluated. Also the several anabolic steroids, specifically designed to circumvent doping control, put on the market have been incorporated in the list of the prohibited substances of the World Anti-Doping Agency (WADA). In WADA list steroids figure in three main classes, namely anabolic steroids, corticosteroids and substances with anti-estrogenic properties. It must be strongly reminded that assumption of doping agents not only leads to athletes the possible failing of doping tests but causes important health risk and WADA prohibited list establishes criteria to highlight the alteration of the natural steroid profile caused by exogenous administration. Doping control analyses are generally performed in urine, a matrix that provides a prolonged detection time window, and less often in blood, serum, plasma, hair, saliva, and nails. To identify the chemical structures of anabolic steroids the use of mass spectrometry detection is very advantageous. Gas chromatography–mass spectrometry (GC–MS) techniques allowed for the development of comprehensive screening methods. GC–MS methods are sensitive and robust but present the disadvantages of time-consuming sample pretreatment, that is often based on hydrolysis and derivatisation reactions. Liquid chromatography–mass spectrometry (LC–MS) methods have been successfully used to identify and determinate steroids in different matrices, as well as to study their metabolisms. Nowadays, automatic rapid ultra high performance liquid chromatography (UHPLC) tandem mass spectrometry has become the technique of choice for steroid analysis. Due to its generally higher speed, sensitivity, reproducibility and specificity with respect to HPLC, it can be used to simultaneously separate and determinate multi component steroid mixtures. The technique is of huge interest to separate conjugates anabolic androgenic steroids, as it allows efficiency enhancement due to the small particle (sub-2μm) column packing, which provides high peak capacity within analysis times even 5–10 fold shorter than conventional HPLC methods. Modern multiplex instruments can analyze thousands of samples per month so that, notwithstanding the generally high instrumental costs, the cost of the individual assay is affordable. In addition, the improved specificity and resolution offered by time-of-flight or quadrupole time-of-flight mass spectrometry allow their application in doping control analysis or in steroid profiling for accurate and sensitive full mass range acquisition. Aim of the present review is to consider, compare and discuss the applications of the UHPLC/MS methods present in literature for the identification and determination of forbidden steroids and their metabolites in human biological matrices.
Irinotecan (CPT-11) is a water-soluble anticancer drug widely used to treat several types of cancer.
Even if the metabolites of CPT-11 are well-known and investigated, only limited information is ...available in the literature about the formation of photo-degradation products that can naturally originate from sunlight irradiation when the drug is released in aqueous systems.
CTP-11 solutions at 10.0 mg L−1 were irradiated by simulated sunlight. The intensity of the drug decreased by 90% after 7.5 days of irradiation and no significant reduction of absorbance values was observed after 13 days.
A sensitive UHPLC-MS/MS method was developed employing a hybrid triple quadrupole/linear ion trap mass spectrometer, that is able to work in data-dependent acquisition mode and to obtain information about the compounds formed during the photoirradiation. Moreover, a selected reaction monitoring method was built using the MS/MS fragmentation pattern of the compounds previously investigated. The method was validated considering LOD, LOQ, linearity, precision, selectivity, recovery and matrix effect. LOD and LOQ values were 0.02 and 0.05 ng mL−1, respectively, whereas MDL and MQL values in real water samples (river water, groundwater, well water, and wastewater) were lower than 0.05 and 0.2 ng mL−1, respectively.
Eight photodegradation products were identified, among which five for the first time. Based on the MS and MS/MS fragmentation, the chemical structures of the degradation products were proposed. Hydrolysis experiments were carried out on the same solutions preserved in the dark, but no formation of other species was highlighted.
The method was applied to several real samples: CPT-11 was detected and quantified only in a hospital effluent sample at the concentration of 0.41 ± 0.2 ng mL−1 together with the occurrence of PDP3.
The outcomes of this study may be useful for updating the pollutant screening in water samples.
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•The CPT-11 concentration decreases of 90% after 7.5 days of sunlight irradiation.•Identification of CPT-11 and its TPs by a developed and validated UHPLC-MS/MS method.•Eight photodegradation products were identified and five of them for the first time.•No contribution of hydrolysis reactions was highlighted.•The same TPs form both in river and ultrapure water.
The study deals with the identification of 8 irinotecan photodegradation products (5 of them unknown until now) in water samples by UHPLC-MS/MS using data-dependent acquisition mode.
In this review, we give an overview of the actual proteomic approaches used in the study of cancer cells secretome. In particular, we describe the proteomic strategies to decipher cancer cell ...secretome initially focusing on the different aspects of sample preparation. We examine the issues related to the presence of low abundant proteins, the analysis of secreted proteins in the conditioned media with or without the removal of fetal bovine serum and strategies developed to reduce intracellular protein contamination. As regards the identification and quantification of secreted proteins, we described the different proteomic approaches used, i.e. gel-based, MS-based (label-based and label-free), and the antibody and array-based methods, together with some of the most recent applications in the field of cancer research. Moreover, we describe the bioinformatics tools developed for the in silico validation and characterization of cancer cells secretome. We also discuss the most important available tools for protein annotation and for prediction of classical and non-classical secreted proteins. In summary in this review advances, concerns and challenges in the field of cancer secretome analysis are discussed.
Neural networks are rapidly gaining popularity in chemical modeling and Quantitative Structure–Activity Relationship (QSAR) thanks to their ability to handle multitask problems. However, outcomes of ...neural networks depend on the tuning of several hyperparameters, whose small variations can often strongly affect their performance. Hence, optimization is a fundamental step in training neural networks although, in many cases, it can be very expensive from a computational point of view. In this study, we compared four of the most widely used approaches for tuning hyperparameters, namely, grid search, random search, tree-structured Parzen estimator, and genetic algorithms on three multitask QSAR datasets. We mainly focused on parsimonious optimization and thus not only on the performance of neural networks, but also the computational time that was taken into account. Furthermore, since the optimization approaches do not directly provide information about the influence of hyperparameters, we applied experimental design strategies to determine their effects on the neural network performance. We found that genetic algorithms, tree-structured Parzen estimator, and random search require on average 0.08% of the hours required by grid search; in addition, tree-structured Parzen estimator and genetic algorithms provide better results than random search.
Arbuscular mycorrhizal fungi (AMF) colonize the roots of most terrestrial plant species, improving plant growth, nutrient uptake and biotic/abiotic stress resistance and tolerance. Similarly, plant ...growth promoting bacteria (PGPB) enhance plant fitness and production. In this study, three different AMF (
, and
) were used in combination with three different strains of
sp. (19Fv1t, 5Vm1K and Pf4) to inoculate plantlets of
×
var. Eliana F1. The effects of the different fungus/bacterium combinations were assessed on plant growth parameters, fruit production and quality, including health-promoting compounds. Inoculated and uninoculated plants were maintained in a greenhouse for 4 months and irrigated with a nutrient solution at two different phosphate levels. The number of flowers and fruits were recorded weekly. At harvest, fresh and dry weights of roots and shoots, mycorrhizal colonization and concentration of leaf photosynthetic pigments were measured in each plant. The following fruit parameters were recorded: pH, titratable acids, concentration of organic acids, soluble sugars, ascorbic acids, and anthocyanidins; volatile and elemental composition were also evaluated. Data were statistically analyzed by ANOVA and PCA/PCA-DA. Mycorrhizal colonization was higher in plants inoculated with
, followed by
and
. In general, AMF mostly affected the parameters associated with the vegetative portion of the plant, while PGPB were especially relevant for fruit yield and quality. The plant physiological status was differentially affected by inoculations, resulting in enhanced root and shoot biomass. Inoculation with Pf4 bacterial strain increased flower and fruit production per plant and malic acid content in fruits, while decreased the pH value, regardless of the used fungus. Inoculations affected fruit nutritional quality, increasing sugar and anthocyanin concentrations, and modulated pH, malic acid, volatile compounds and elements. In the present study, we show for the first time that strawberry fruit concentration of some elements and/or volatiles can be affected by the presence of specific beneficial soil microorganisms. In addition, our results indicated that it is possible to select the best plant-microorganism combination for field applications, and improving fruit production and quality, also in terms of health promoting properties.
Mass spectrometry (MS) is widely used for the identification of chemical compounds by matching the experimentally acquired mass spectrum against a database of reference spectra. However, this ...approach suffers from a limited coverage of the existing databases causing a failure in the identification of a compound not present in the database. Among the computational approaches for mining metabolite structures based on MS data, one option is to predict molecular fingerprints from the mass spectra by means of chemometric strategies and then use them to screen compound libraries. This can be carried out by calibrating multi-task artificial neural networks from large datasets of mass spectra, used as inputs, and molecular fingerprints as outputs. In this study, we prepared a large LC-MS/MS dataset from an on-line open repository. These data were used to train and evaluate deep-learning-based approaches to predict molecular fingerprints and retrieve the structure of unknown compounds from their LC-MS/MS spectra. Effects of data sparseness and the impact of different strategies of data curing and dimensionality reduction on the output accuracy have been evaluated. Moreover, extensive diagnostics have been carried out to evaluate modelling advantages and drawbacks as a function of the explored chemical space.
According to the 2021 World Drug Report, around 275 million people use drugs of abuse, and 36 million people suffer from addiction, fostering a thriving market for illicit substances. In Italy, ...30,083 people were reported to the Judicial Authority for offenses in violation of the Italian Law D.P.R. 309/1990. These offences are sentenced after a qualitative-quantitative analysis of seized materials. Given the large quantity of seized drugs and the need to perform accurate analytical determinations, Italian forensic laboratories struggle to complete analyses in a short time, delaying the entire reporting process needed to achieve sentencing. For this purpose, an UHPLC-MS/MS-based platform was developed at the University of Milano-Bicocca to support law-enforcement authorities. Software was designed to easily manage street seizure acquisition, documentation registration, and sampling. A sensitive UHPLC-MS/MS method was fully validated for the quantification of the traditional illicit substances (cocaine, heroin, 6-MAM, morphine, amphetamine, methamphetamine, MDMA, ketamine, GHB, GBL, LSD, trans-∆9-THC, and THCA) at the ppb level. The final report is relayed to the Prefecture in 3-4 days, even within 24 h for urgent requests. The platform allows for semi-automatic data handling to minimize erroneous results for an accurate report generation by standardized procedures.
The octocoral family Alcyoniidae represents a rich source of bioactive substances with intriguing and unique structural features. This review aims to provide an updated overview of the compounds ...isolated from Alcyoniidae and displaying potential cytotoxic activity. In order to allow a better comparison among the bioactive compounds, we focused on molecules evaluated in vitro by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, by far the most widely used method to analyze cell proliferation and viability. Specifically, we surveyed the last thirty years of research, finding 153 papers reporting on 344 compounds with proven cytotoxicity. The data were organized in tables to provide a ranking of the most active compounds, to be exploited for the selection of the most promising candidates for further screening and pre-clinical evaluation as anti-cancer agents. Specifically, we found that (22
,24
)-24-methyl-22,25-epoxyfurost-5-ene-3β,20β-diol (
), 3β,11-dihydroxy-24-methylene-9,11-secocholestan-5-en-9-one (
), (24
)-ergostane-3β,5α,6β,25 tetraol (
), sinulerectadione (
), sinulerectol C (
), and cladieunicellin I (
) exhibited stronger cytotoxicity than their respective positive control and that their mechanism of action has not yet been further investigated.
Oleander is a spontaneous shrub widely occurring in Mediterranean regions. Poisoning is sporadically reported in livestock, mainly due to the ingestion of leaves containing toxic cardiac glycosides ...(primarily oleandrin). In this study, 50 lactating Fleckvieh cows were affected after being offered a diet containing dry oleander pruning wastes accidentally mixed with fodder. Clinical examination, electrocardiogram, and blood sampling were conducted. Dead animals were necropsied, and heart, liver, kidney, spleen, and intestine were submitted to histological investigation. Oleandrin detection was performed through ultra-high performance liquid chromatography-tandem mass spectrometry in blood, serum, liver, heart, milk, and cheese samples. Severe depression, anorexia, ruminal atony, diarrhea, serous nasal discharge, tachycardia, and irregular heartbeat were the most common clinical signs. The first animal died within 48 h, and a total of 13 cows died in 4 days. Disseminated hyperemia and hemorrhages, multifocal coagulative necrosis of the cardiac muscle fibers, and severe and diffuse enteritis were suggestive of oleander poisoning. The diagnosis was confirmed by the presence of oleandrin in serum, liver, heart, milk, and cheese. Our results confirm the high toxicity of oleander in cattle and report for the first time the transfer into milk and dairy products, suggesting a potential risk for the consumers.
A rapid on-line solid phase extraction ultra high performance liquid chromatography tandem mass spectrometry method was developed for the identification and quantitation of nine perfluorinated ...compounds in matrices of environmental, biological and food interest. Pre-treatment, solid phase extraction, chromatographic and mass detection conditions were optimised, in order to apply the whole methodology to the analysis of different matrices. Particular attention was devoted to the evaluation of matrix effect and the correlated phenomena of ion enhancement or suppression in mass spectrometry detection. LOD and LOQ range from 3 to 15
ng
L
−1 and from 10 to 50
ng
L
−1, respectively. Method detection limits (MDLs) were also calculated for each kind of matrix. The recovery, evaluated for each analyte, does not depend on analyte concentration in the explored concentration range: average
R
¯
%
values are always greater than 82.9%. In the whole, the results obtained for samples of river waters, blood serum, blood plasma, and fish confirm the ubiquitous presence of perfluorinated compounds, as recently denounced by many sources.
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