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This work presents a strategy for the evaluation of differences in plasma phospholipid content between atherosclerotic and healthy mice from micro volumes (2 microL) spotted on filter paper. Dried plasma spots (DPS) were directly desorbed into a triple quadrupole linear ion trap mass spectrometer using a homemade prototype, ensuring high-throughput analysis of dried spots without any sample pretreatment. Multiple positive and negative neutral loss and precursor ion scans were simultaneously acquired in a single loop, allowing oriented fingerprinting until 2700 potential species including phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), and sphingomyelin (SM) classes. The phospholipidic variations between 15 healthy and 15 atherosclerotic mice were evaluated using t tests, matrix reduction and merging, and principal component analysis (PCA) as a chemometric statistical approach. The discriminating ions in PCA analysis were qualitatively identified in an information dependent acquisition (IDA) manner using enhanced resolution and enhanced product ion scans. PCA demonstrates a clear clustering between healthy and diseased mice. Regarding the most relevant variables identified, this procedure has confirmed the role of SM and PS classes in atherosclerosis and has established potential biomarkers shown to be significantly up- or down-regulated with the disease. The results presented in this work demonstrate the sample processing and analysis potential of the developed strategy to evaluate new biomarkers and the state of a disease.

Hematocrit (Hct) is one of the most critical issues associated with the bioanalytical methods used for dried blood spot (DBS) sample analysis. Because Hct determines the viscosity of blood, it may affect the spreading of blood onto the filter paper. Hence, accurate quantitative data can only be obtained if the size of the paper filter extracted contains a fixed blood volume. We describe for the first time a microfluidic-based sampling procedure to enable accurate blood volume collection on commercially available DBS cards. The system allows the collection of a controlled volume of blood (e.g., 5 or 10 μL) within several seconds. Reproducibility of the sampling volume was examined in vivo on capillary blood by quantifying caffeine and paraxanthine on 5 different extracted DBS spots at two different time points and in vitro with a test compound, Mavoglurant, on 10 different spots at two Hct levels. Entire spots were extracted. In addition, the accuracy and precision (n = 3) data for the Mavoglurant quantitation in blood with Hct levels between 26% and 62% were evaluated. The interspot precision data were below 9.0%, which was equivalent to that of a manually spotted volume with a pipet. No Hct effect was observed in the quantitative results obtained for Hct levels from 26% to 62%. These data indicate that our microfluidic-based sampling procedure is accurate and precise and that the analysis of Mavoglurant is not affected by the Hct values. This provides a simple procedure for DBS sampling with a fixed volume of capillary blood, which could eliminate the recurrent Hct issue linked to DBS sample analysis.