Evaluation of a volumetric dried blood spot card using a gravimetric method and a bioanalytical method with capillary blood from 44 volunteers.

Dried blood spot (DBS) sampling is a promising method for collection of microliter blood samples. However, hematocrit-related bias in combination with subpunch analysis can result in inaccurate quantification of analytes in DBS samples. In this study we use a microfluidic DBS card, designed to automatically collect fixed volume DBS samples irrespective of the blood hematocrit, to measure caffeine concentration in normal finger prick samples obtained from 44 human individuals. Caffeine levels originating from blood drops of unknown volume collected on the volumetric microfluidic DBS card were compared to volume-controlled pipetted DBS samples from the same finger prick. Hematocrit independence and volumetric sampling performances were also verified on caffeine-spiked blood samples in vitro, using both LC-MS/MS and gravimetric methods, on hematocrits from 26 to 62%. The gravimetric measurements show an excellent metering performance of the microfluidic DBS card, with a mean blood sample volume of 14.25 mu L +/- 3.0% (n = 51). A measured mean bias below 2.9% compared to normal hematocrit (47%) demonstrates that there is no significant hematocrit-induced bias. LC-MS/MS measurements confirm low CV and hematocrit independence of the sampling system and exhibit no substantial mean bias compared to pipetted DBS. Tests with 44 individuals demonstrated applicability of the microfluidic DBS card for direct finger prick blood sampling, and measured caffeine concentrations show a good agreement with measurements of pipetted DBS. The presented concept demonstrates a good volumetric performance which can help to improve the accuracy of DBS analysis by analyzing a whole spot, equivalent to a defined volume of liquid blood.