Development of a hematocrit-insensitive device to collect accurate volumes of dried blood spots without specialized skills for measuring clozapine and its metabolites as model analytes.

Dried blood spots have been used as alternatives to traditional plasma and serum samples. We have now developed new devices, named volumetric absorptive paper disc (VAPD) and mini-disc (VAPDmini), to collect accurate volumes of dried blood spots in a simple manner and without the need for additional instruments. VAPD consists of a filter paper disc and a filter paper sheet with holes slightly larger than the disc. The disc is fixed in one such hole without direct contact with the filter sheet. VAPDmini is a scaled-down version of the same device. When several drops of whole blood are applied, the disc becomes saturated and any excess sample is absorbed by the surrounding filter sheet. Accuracy and precision of sampling were assessed by determining the levels of clozapine and its metabolites as target analytes by liquid-liquid extraction and high-performance liquid chromatography with coulometric detection. In addition, differences in analyte recovery were within ±15% for all analytes in samples with 30-60% hematocrit, suggesting that VAPD and VAPDmini are insensitive to hematocrit for the analytes tested. The devices were also validated for analyte concentrations in the range 50-1000 ng/mL, and the limit of detection and lower limit of quantification were 5-17 ng/mL and 15-51 ng/mL, respectively. Intra- and inter-day precision ranged from 3% to 13%, whereas accuracy ranged from a -14% to 12% bias. Analytes were stable in the devices for at least 2 weeks at room temperature. Collectively, these results indicate that sampling using VAPD and VAPDmini is comparable to conventional hole punch sampling of entire dried blood spots, even for samples obtained from patients treated with clozapine. Importantly, the devices were also found to be suitable for sample self-collection.