Abstract
A two-layered polymeric membrane is employed for the formation of separated dried plasma spots from whole blood as an alternative to the direct analysis of whole dried blood spots (DBS). This dried plasma spot (DPS) analysis procedure precludes potential issues of hematocrit differences in whole blood samples while providing pharmokinetic data from plasma rather than whole blood. The described procedure is also semi-automated thus providing a simpler work flow for LC/MS/MS bioanalysis procedures. Molecular filtration of red blood cells (RBC) from applied microsamples of whole blood fortified with guanfacine and its stable isotope internal standard was accomplished with a two-layer polymeric membrane substrate. The lower membrane surface containing the separated plasma spot was physically separated from the upper membrane followed by semi-automated direct elution of the sample to an online solid-phase extraction (SPE) cartridge followed by liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS). A two-layer membrane sample preparation substrate produced plasma from whole blood without centrifugation which could be directly eluted for semi-automated LC/MS/MS bioanalysis. Standard curves were constructed by plotting peak area ratios between the analyte and the stable isotope labeled internal standard (SIL-IS) versus the nominal concentration in whole blood. A weighted 1/x(2) linear regression was applied to the data from DPS samples. Standard curves were linear over the range 0.25-250 ng/mL human whole blood. The representative regression equation was y = 0.0142x + 0.00248 (R(2) = 0.995) for the described DPS assay. The described work demonstrates proof-of-principle using membrane sample preparation techniques to form DPS samples from whole blood for subsequent bioanalysis by LC/MS/MS. This approach has the potential to eliminate the hematocrit issues from the current controversy surrounding validation of DBS assays.
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