Dried Matrix Spots Microsampling for Analysis of Clinical and Forensic Interest

Dried blood spot (DBS) microsampling has transformed bioanalysis by enabling minimally invasive, self-collected samples that can be shipped and stored without a cold chain. Its quantitative reliability, however, has historically been limited by uncontrolled sampling volume, the hematocrit eAect, incomplete extraction recovery and the mismatch between dried analytes and liquid internal standards. This thesis develops and validates analytical methods based on volumetric dried matrix spots (DMS), extending the DBS concept to plasma, serum and urine, for applications in human biomonitoring, clinical toxicology and forensic toxicology. The whole CapitainerÒ portfolio (B10, SEP10, B50 and DIP70 devices) was employed. The first part addresses environmental xenobiotics within the human exposome framework. A UHPLC-MS/MS method was developed for 25 per- and polyfluoroalkyl substances (PFAS) in 10 μL of blood, achieving limits of detection of 0.4-1.0 ng/mL, recoveries above 80% and precision and accuracy within 20%. For aflatoxin B1 (AFB1), a DBS-ELISA workflow on 50 μL of blood was optimized, with 94 ± 11% recovery, a working range of 2.4-24 ng/mL and analyte stability for up to 7 days at 4 °C and 25 °C, providing a low-cost screening tool for resource-limited and occupational settings. In a visiting period at Ghent University (Belgium), the same immunoassay was shown to detect the AFB1-albumin adduct, a marker of long-term exposure, in dried serum spots: in a preliminary set of real samples, positives and negatives were correctly discriminated. Glyphosate and its metabolite AMPA were determined by UHPLCMS/ MS in 70 μL dried urine spots (LODs 1.8 and 1.2 ng/mL), with stability demonstrated for 4 weeks at 25 °C. In collaboration with miQro Lab Solutions (Capitainer) in East Providence (RI, USA), ICP-MS was applied to 16 toxic and essential elements in dried blood and plasma spots; iron measurement in plasma obtained with SEP10 showed minimal hemolysis (<1%), and a proof of concept on authentic samples explored the Pb/Fe ratio as a candidate biomarker of lead exposure. The second part concerns lifestyle-related and forensic applications. A DUS-based UHPLCMS/ MS screening method for 131 new psychoactive substances was validated over 1.0-50 ng/mL (LOD 0.16-0.94 ng/mL), with stability up to 29 days at 25 °C and suitability for international shipment of samples. To overcome the internal standard limitation, an internal quantification DBS (iqDBS) strategy was designed during a 6-month visiting period at University of Florida (FL, USA); deuterated d5-PEth was incorporated directly into the sample disc of the Capitainer B10 card for quantification of phosphatidylethanol 16:0/18:1 (PEth), a direct alcohol biomarker. The method showed CV below 15% over 48 samples and three sessions, and device stability of up to 2.5 months under accelerated ageing. Moreover, additional experiments laid the groundwork for generating labelled PEth on the paper itself, which would help distinguish pre- from post-sampling formation. Overall, the work demonstrates the versatility of DMS across biological matrices (blood, plasma, serum, urine), detection platforms (LC-MS/MS, ELISA, ICP-MS), analyte classes and population groups, while aligning with green analytical chemistry principles. The main challenge was the recruitment of cohorts for authentic samples; the validated methods are now ready for application in biomonitoring campaigns. Future perspectives include integration with untargeted approaches and a participant-centered paradigm, in which self-sampling places the individual at the core of exposure assessment