Fully-automated approach for online dried blood spot extraction and bioanalysis by two-dimensional-liquid chromatography coupled with high-resolution quadrupole time-of-flight mass spectrometry.

An integrated automated approach has been developed for the direct determination of drug concentrations using a SCAP DBS system for online extraction and analysis of dried blood spots (DBS) from DBS paper cards to a multidimensional liquid chromatography system coupled to a high-resolution QTOF mass spectrometry (LC-HRMS). An accurate, precise, selective, and sensitive two-dimensional liquid chromatography-high-resolution mass spectrometry (2D LC-HRMS) assay was developed and validated using small volumes of rat blood (approximately 1.25 μL) from a 2 mm DBS punch. The methodology was validated according to internationally accepted regulated bioanalysis acceptance criteria in order to establish the validity of the combination of online DBS assay and use of HRMS for quantitative bioanalysis. The fully automated procedure exhibited acceptable linearity (r(2) > 0.997) over the concentration range of 5 to 1000 ng/mL. Intra- and interday precision and accuracy runs indicated relative errors less than 20% at the LLOQ level and less than 15% at all other levels. The direct extraction and analysis of DBS samples resulted in a 5-fold improvement in assay sensitivity compared to conventional off-line extraction of punched DBS samples. In addition, the impact of blood hematocrit (Hct) on accurate quantification of the studied drugs also was evaluated, comparing Hct values of 30% and 60% against standards prepared at 45%. Hematocrit experiments show that Hct can influence the accuracy of drugs quantified by DBS and needs to be thoroughly evaluated prior to committing to validating a DBS assay. The online DBS system coupled to the LC-HRMS was then successfully applied to a pharmacokinetic study performed on male Sprague-Dawley rats after administration of a single dose of 5 and 10 mg/kg for midazolam and desipramine, respectively.