Direct Solvent Extraction and Analysis of Biomarkers in Dried Blood Spots Using a Flow-Through Autosampler
A new flow-through autosampler designed to extract biologically important small molecules from dried blood spots (DBS) on filter paper for direct analysis by tandem mass spectrometry is described. The device included a microcomputer to control the timing and sequence of events within a cycle that was recognized by the mass spectrometer's datasystem, permitting a batch file to be programmed and executed automatically without operator intervention. Applications included the analysis of free acylcarnitines and amino acids from DBS, by the addition of the isotope-labeled internal standards from an in-line loop injector placed between the solvent pump and the DMPK card. The precision, accuracy, and recovery for most analytes were within acceptable limits, and were similar to results obtained using the more labor intensive punch-and-extract methods. We also describe preliminary results from a novel plasma assay for methylmalonic acid (MMA) using the DBS autosampler in conjunction with UPLC-MS/MS. In this case, the internal standard 2H3-MMA was added to plasma before spotting the samples onto a DMPK card and a linear solvent gradient was used for chromatographic separation of MMA from its normally much more abundant biological isomer, succinic acid. Based on the replicate analysis of calibrators, the assay was linear from 10 to 1000 μmol/L and both precision and accuracy were well within 15% (cv). In practice, we have found the DBS autosampler to be a robust and reliable device, with several hundred samples having been analyzed without any downstream clogging or maintenance. The time and labor saving features are attractive for high throughput applications such as newborn screening and therapeutic drug monitoring. By eliminating several steps that require sample transfers, potential laboratory errors are also minimized.
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