Fully Automated Correction for the Hematocrit Bias of Non-Volumetric Dried Blood Spot Phosphatidylethanol Analysis

The quantitative analysis of substances in dried blood spots (DBS) has gained vast popularity in the past decade. The World Anti-Doping Agency (WADA) also recently committed to implementing DBS. Currently, DBS sampling mainly has focused on various volumetric sampling devices such as Hemaxis, Capitainer, and Mitra. These devices are designed to collect a specific sample volume, independent of the hematocrit (HCT), to enable quantitative DBS analysis. Here, we present an automated solution that makes the necessity of volumetric sampling for quantitative DBS analysis obsolete. Combining automated reflectance-based HCT correction in combination with fully automated DBS LC-MS/MS analysis, the novel strategy permits high-throughput analysis in combination with HCT independence. Studying the model compound phosphatidylethanol 16:0/18:1, which is HCT-dependent due to incorporation into red blood cells, an implementation of DBS HCT normalization is presented. First, the performance of the automated HCT module with DBS is demonstrated compared to standardized HCT analysis from whole blood using a centrifuge. Second, the HCT dependency of fully automated PEth analysis from DBS is evaluated. Third, a solution to correct for the HCT dependency of PEth using the HCT scanner is presented. The study demonstrates that as soon as the HCT dependence of an analyte is known, a correction factor can be applied for the normalization of HCT levels. In the context of PEth, a linear increase in PEth concentration was observed, as the analyte is primarily located within the cellular fraction. Based on the obtained results, the use of a common correction factor for PEth DBS is possible.