Development of ‘Ready to Use Kits’ for the simultaneous qualification and quantification of drugs in different matrices using Mass Spectrometry, expanded with the application of multimodal imaging techniques

This thesis presents several aspects and applications of mass spectrometry (MS) and mass spectrometry imaging (MSI) as diagnostic toolss. It describes the possibility of analyzing different sample types (solid, liquid and gas) and how these powerful tools iare used for quantification of substances, qualification of biologically relevant molecules within samples, locating molecules in tissue sections and combining MS with clinical tools to improve the outcomes of cancer patients. Techniques such as MS, MSI, Chromatography, MALDI, ESI and REIMS are addressed in this thesis

Quantification of 17 Endogenous and Exogenous Steroidal Hormones in Equine and Bovine Blood for Doping Control with UHPLC-MS/MS

A simple and fast analytical method able to simultaneously identify and quantify 17 endogenous and exogenous steroidal hormones was developed in bovine and equine blood using UHPLC-MS/MS. A total amount of 500 µL of sample was deproteinized with 500 µL of a mixture of methanol and zinc sulfate and evaporated. The mixture was reconstituted with 50 µL of a solution of 25% methanol and injected in the UHPLC-MS/MS triple quadrupole. The correlation coefficients of the calibration curves of the analyzed compounds were in the range of 0.9932-0.9999, and the limits of detection and quantification were in the range of 0.023-1.833 and 0.069-5.5 ppb, respectively. The developed method showed a high sensitivity and qualitative aspects allowing the detection and quantification of all steroids in equine and bovine blood. Moreover, the detection limit of testosterone (50 ppt) is half of the threshold admitted in plasma (100 ppt). Once validated, the method was used to quantify 17 steroid hormones in both bovine and equine blood samples. The primary endogenous compounds detected were corticosterone (range 0.28-0.60 ppb) and cortisol (range 0.44-10.00 ppb), followed by androstenedione, testosterone and 11-deoxycortisol

Development of an Untargeted LC-MS Metabolomics Method with Postcolumn Infusion for Matrix Effect Monitoring in Plasma and Feces

Untargeted metabolomics based on reverse phase LC-MS (RPLC-MS) plays a crucial role in biomarker discovery across physiological and disease states. Standardizing the development process of untargeted methods requires paying attention to critical factors that are under discussed or easily overlooked, such as injection parameters, performance assessment, and matrix effect evaluation. In this study, we developed an untargeted metabolomics method for plasma and fecal samples with the optimization and evaluation of these factors. Our results showed that optimizing the reconstitution solvent and sample injection amount was critical for achieving the balance between metabolites coverage and signal linearity. Method validation with representative stable isotopically labeled standards (SILs) provided insights into the analytical performance evaluation of our method. To tackle the issue of the matrix effect, we implemented a postcolumn infusion (PCI) approach to monitor the overall absolute matrix effect (AME) and relative matrix effect (RME). The monitoring revealed distinct AME and RME profiles in plasma and feces. Comparing RME data obtained for SILs through postextraction spiking with those monitored using PCI compounds demonstrated the comparability of these two methods for RME assessment. Therefore, we applied the PCI approach to predict the RME of 305 target compounds covered in our in-house library and found that targets detected in the negative polarity were more vulnerable to the RME, regardless of the sample matrix. Given the value of this PCI approach in identifying the strengths and weaknesses of our method in terms of the matrix effect, we recommend implementing a PCI approach during method development and applying it routinely in untargeted metabolomics

Development of a LC-MS-MS method for the simultaneous determination of natural steroidal hormones and synthetic anabolic steroids in equine and bovine blood matrix

Monitoring the hormones in equine matrix is crucial to understanding the health status of horses , and also to monitoring the abuse of these substances before a race or a horse transaction. Few analytical procedures exist which are “self made methods”, generally based on immunoassay analysis, often impossible to replicate in other laboratories or too complex for the staff. On that, it is necessary to develop new sensitive analytical methods, that can be diffused to external vets and give correct and comparable results. Furthermore, the few analytical procedures involving an HPLC-MS-MS system reported in literature allow for simultaneous quantification of no more than three or four hormones [1][2]. The proposed method makes it possible to detect and quantify seventeen hormones and metabolites in a single assay, in just ten minutes. Quantifiable hormones with the proposed method are: Pregnenolon, 17-OH-Pregnenolon, Progesteron, 17-OH-Progesteron, Androsteron, Androstenedion, DHEA, DHEAS, Testosteron, Cortisol, Corticosteron, Aldosteron, 11-Deossicortisol, 11-Deossicorticosteron, Diidrotestosteron, Estron, Estradiol. A deuterated hormone ( Testosteron-D3) has been used as internal standards in order to set a more accurate and precise procedure. The method was developed using the Agilent UHPLC chromatographic system (1290) using a Zorbax RRHD C18 – 1,8 μm column and a 6420 Agilent Mass Spectrometer . The procedure is fast and intuitive; the sample preparation is very easy, with the serum deproteinized in vials using a deproteinizing solution, centifuged and injected into the system. The mobile phases are made of water and acetonitrile, both containing formic acid. Overall, the method is very simple and robust and it brings about a remarkable saving of time and money with respect to previously reported methods; in fact, using the UHPLC–Tandem Mass spectrometer enables simultaneous quantification of seventeen Steroidal Hormones. The method has been applied to a number of equine and bovine blood samples supplied from different breeding farms and territorial vests

DEVELOPMENT OF A LC-MS-MS METHOD FOR THE SIMULTANEOUS DETERMINATION OF STEROIDAL HORMONES IN EQUINE SERUM.

Monitoring the hormones in equine matrix is crucial to understanding the health status of horses , and also to monitoring the abuse of these substances before a race or a horse transaction. Few analytical procedures exist and those that do are “self made methods”, generally based on immunoassay analysis, often impossible to replicate in other laboratories or too complex for the staff. On that, it is necessary to develop new sensitive analytical methods, that can be diffused to external vets and giving correct and comparable results. Furthermore, the few analytical procedures involving an HPLC-MS-MS system reported in literature allow for simultaneous quantification of no more than three or four hormones [1][2]. The proposed method makes it possible to detect and quantify seventeen hormones and metabolites in a single assay, in just eleven minutes. Quantifiable hormones with the proposed method are: Pregnenolon, 17-OH-Pregnenolon, Progesteron, 17-OH-Progesteron, Androsteron, Androstenedion, DHEA, DHEAS, Testosteron, Cortisol, Corticosteron, Aldosteron, 11-Deossicortisol, 11-Deossicorticosteron, Diidrotestosteron, Estron, Estradiol. Three deuterated hormones (Cortisol-D4, Aldosteron-D7, Testosteron-D3) have been used as internal standards in order to set a more accurate and precise procedure. The method was developed using the Agilent UHPLC chromatographic system (1290) using a Zorbax RRHD C18 – 1,8 μm column and a 6420Agilent Mass Spectrometer . The procedure is fast and intuitive; the sample preparation is very easy, with the serum deproteinized in vials using a deproteinizing solution, centifuged and injected into the system. The mobile phases are made of water and acetonitrile, both containing formic acid. Overall, the method is very simple and robust and it brings about a remarkable saving of time and money with respect to previously reported methods; in fact, using the UHPLC–Tandem Mass spectrometer enables simultaneous quantification of seventeen Steroidal Hormones

Simultaneous Quantitation of 9 Anabolic and Natural Steroidal Hormones in Equine Urine by UHPLC-MS/MS Triple Quadrupole

A new fast and easy analytical procedure for the simultaneous detection and quantification of 9 anabolic steroids (deslorelin, dexamethasone sodium phosphate, prednisolone, methylprednisolone, stanozolol, boldenone, nandrolone, dexamethasone isonicotinate and altrenogest) in horse urine for doping control have been developed by using the ultra-high-performance liquid chromatography coupled with tandem mass spectrometry technique (UHPLC-MS/MS). A total amount of 400 μl of sample was evaporated, restored and injected in the UHPLC-MS/MS. The proposed method was fully validated showing a recovery higher than 89.12% and a coefficient of variation lower than 6.02%. The correlation coefficients range of the analyzed compound's calibration curves was 0.9955–0.9997, and the limits of detection and quantification were in the range of 0.1 and 0.25 μg/l, respectively

Detection of endocrine disrupting chemicals and evidence of their effects on the HPG axis of the European anchovy Engraulis encrasicolus

Natural/synthetic Endocrine Disrupting Chemicals (EDCs) may display estrogenic activity and a lower potency than 17β-estradiol. Nonetheless, their concentrations and additive effects can affect the endocrine system and reproductive processes related to the Hypothalamic-Pituitary-Gonadal (HPG) axis. Because of their persistence in both the environment and biological systems, they ultimately target multi-level predators, including humans. We detected presence and effects of xenobiotics on wild anchovy Engraulis encrasicolus in the Western Adriatic Sea. Twenty-one PCBs and five organochlorines were detected on the order of ng g(-1); vitellogenin, vitellogenin receptor and genes encoding for the zona radiata proteins were evaluated in gonad and/or liver and found transcribed in male specimens; in addition, intersex was histologically identified in the 13% of testis. Our results have developed the understanding of the European anchovy's reproductive toxicological risk and our approach could assist the comprehension of the complex dynamics of commercially relevant Teleost species

Quantification of 17 Endogenous and Exogenous Steroidal Hormones in Equine and Bovine Blood for Doping Control with UHPLC-MS/MS

A simple and fast analytical method able to simultaneously identify and quantify 17 endogenous and exogenous steroidal hormones was developed in bovine and equine blood using UHPLC-MS/MS. A total amount of 500 µL of sample was deproteinized with 500 µL of a mixture of methanol and zinc sulfate and evaporated. The mixture was reconstituted with 50 µL of a solution of 25% methanol and injected in the UHPLC-MS/MS triple quadrupole. The correlation coefficients of the calibration curves of the analyzed compounds were in the range of 0.9932–0.9999, and the limits of detection and quantification were in the range of 0.023–1.833 and 0.069–5.5 ppb, respectively. The developed method showed a high sensitivity and qualitative aspects allowing the detection and quantification of all steroids in equine and bovine blood. Moreover, the detection limit of testosterone (50 ppt) is half of the threshold admitted in plasma (100 ppt). Once validated, the method was used to quantify 17 steroid hormones in both bovine and equine blood samples. The primary endogenous compounds detected were corticosterone (range 0.28–0.60 ppb) and cortisol (range 0.44–10.00 ppb), followed by androstenedione, testosterone and 11-deoxycortisol