Multiomics Analysis of Dried Plasma Spots Using Stable Isotope Internal Standards for Biomarker Discovery

A comprehensive analytical strategy for multiomics analysis of dried blood spots (DBS) has been developed, featuring prespotted stable isotope internal standards (SIIS) for enhanced quantitation. The method combines an optimized Folch extraction protocol for dried plasma spots with high-resolution mass spectrometry analysis using a Thermo Q Exactive Orbitrap mass spectrometer coupled to a Dionex Ultimate 3000 ultrahigh performance liquid chromatography system. This internal quantitative DBS approach addresses key analytical challenges in microsampling applications through strategic incorporation of SIIS directly into the sampling matrix, enabling reliable normalization with improved extraction efficiency. The analytical workflow successfully integrates the simultaneous analysis of metabolites and lipids, demonstrating broad molecular coverage from minimal sample volumes in the microliter range. Method applicability was demonstrated using fasting plasma samples from a cohort of persons with HIV, revealing distinct molecular profiles between diabetic and nondiabetic participants, with hundreds of metabolites and thousands of lipids identified. The analytical platform leverages the inherent advantages of DBS, including simplified sample collection, enhanced stability during storage, and reduced biohazard risk during transportation. This methodology represents a significant advancement in quantitative DBS analysis, particularly valuable for large-scale clinical studies where conventional biobanking presents logistical challenges. The integration of SIIS into the microsampling device establishes a robust foundation for reliable multiomics analysis in resource-limited settings where whole blood or plasma storage is infeasible.