21 research outputs found
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 (<i>r</i><sup>2</sup> > 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