archivio digitale delle tesi discusse presso l'Università di Pisa


Tesi etd-10082018-175453

Tipo di tesi
Tesi di laurea magistrale
Determination of prostanoids and isoprostanoids in dried blood spots by Micro-Extraction by Packed Sorbent coupled to ultra-high performance liquid chromatography-tandem mass spectrometry
Corso di studi
relatore Dott. Lomonaco, Tommaso
correlatore Prof. Di Francesco, Fabio
controrelatore Dott.ssa Lucejko, Jeannette
Parole chiave
  • DBS
  • MEPS
  • isoprostanoids
  • prostanoids
Data inizio appello
Data di rilascio
Riassunto analitico
The analysis of dried blood spots (DBSs) provides several advantages over conventional venous blood analysis in terms of less invasive and painless sampling, minimum amount of blood needed (10-50 µL), lower risks of infection (both for the donor and the healthcare workers) and reduced creation of hazardous waste. Noteworthy, sampling is so easy that can be performed by the patient himself or untrained caregivers, and this would offer crucial advantages to patients requiring periodic monitoring over long periods of time (e.g. therapeutic drug monitoring). In fact, once the stability of the target chemical in the spot is ascertained, DBSs could be sampled at home and shipped to the hospital lab.<br>Oxidative stress is involved in many chronic diseases (e.g. cardiovascular diseases, cancer, diabetes, neurodegenerative diseases and chronic inflammatory diseases) and diseases of preterm newborns such as patent ductus arteriosus and retinopathy of prematurity. Various biomarkers have been proposed to assess oxidative stress, and products of lipid peroxidation like prostaglandins and isoprostanes are commonly determined in serum, plasma and urine samples to evaluate the oxidative damage. The complexity of these matrices, the low concentration levels (tens to hundreds of pg/mL) of these biomarkers and the poor stability caused by auto-oxidation of arachidonic acid (ex-vivo formation) make the analysis of these compounds quite challenging.<br>This thesis reports the development and validation of analytical procedures for the quantitative determination of isoprostanoids (8-iso-prostaglandin-F2α and 8-iso-prostaglandin-E2) and prostanoid (prostaglandin-E2) in DBS samples by an innovative approach based on micro-extraction by packed sorbent (MEPS) and ultra-high performance liquid chromatography-electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) analysis.<br>Micro-extraction by packed sorbent, a miniaturized version of solid-phase extraction, allowed the easy and rapid (&lt; 10 min) sample clean-up as well as the pre-concentration of analytes extracted from a single DBS (50 μL) by a 70:30 v/v methanol:water mixture before the highly sensitive and selective UHPLC-ESI-MS/MS analysis.<br>The analytical method was fully validated and showed for all the analytes limits of detection close to 20 pg/mL, linear calibration ranges (R2 &gt; 0.99) spanning over three orders of magnitude, almost quantitative recoveries (90-100%), and intra- and inter-day precisions close to 15%. The addition of a deuterated internal standard (8-iso-prostaglandin-F2α-d4) on the filter paper, instead of the common addition to the extraction solvent, guaranteed to correct for any analyte variability during sampling, drying, storage and extraction steps, thus improving the analytical performances of DBS assay.<br>The innovative analytical procedure developed in this thesis offers a reliable approach to determine these markers in DBSs and a broad applicability in medicine since alterations of prostanoids and isoprostanoids in blood are involved in the pathogenesis and progression of many chronic diseases.<br>