Automated direct extraction and analysis of dried blood spots employing on-line SPE high-resolution accurate mass bioanalysis
|Summary||Online automated extraction of dried blood spots (DBS) via direct extraction to a solid- phase extraction (SPE) cartridge and bioanalysis by high-resolution accurate mass spectrometry was examined. The methodology was validated and used to investigate the effect of hematocrit on assay bias using partial and whole spot extractions from accurately dispensed blood samples. Results: The completed analysis of a DBS sample was accomplVol. 6, No. 15, Pages 2027-2041 , DOI 10.4155/bio.14.162 ished within 2 to 3 min using the online DBS-SPE platform. Hematocrit related bias was observed (>15%) for the partial DBS extractions, but not when the whole DBS was eluted. Conclusion: Results demonstrate successful implementation of automated online DBS-SPE high- resolution accurate mass spectrometry analysis and the remediation of hematocrit bias using a capillary micro dispenser for accurate spotting of blood samples.|
|Category||3. Symbiosis User Application
These are applications that have been made by Symbiosis customers. They are validated and have been published.
|Detection||Agilent 6540 QTOF high-resolution accurate mass spectrometer|
|Compounds||chloroquine, desipramine and midazolam|
|Contact||Regina V Oliveira, Jack Henion, & Enaksha R Wickremsinhe|
|Published||Vol. 6, No. 15, Pages 2027-2041 , DOI 10.4155/bio.14.162|
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|Remarks||DBS analysis techniques offer great potential as a micro sampling alternative with several advantages over traditional analysis of plasma samples. In this study we have
demonstrated the feasibility of analyzing a large number of DBS cards employing a fully automated DBS extraction procedure coupled with on-line SPE QTOF HRAMS for the quantitation of chloroquine, desipramine and midazolam, using 5 µl of rat blood collected on DMPK-C DBS cards. The method was validated to meet the current bioanalytical validation requirements for linearity, sensitivity, intra- and inter-day precision and accuracy, carryover, cycle time and stability. The methodology was also adopted to demonstrate accuracy and precision for the quantification of blood with Hct 30% as well as Hct 60% when analyzed against a calibration curve prepared with Hct 45% blood, thus negating the Hct effect. The described automated DBS-SPE system provides an attractive alternative for the challenges of off-line DBS sample preparation. It is capable of extracting up to 384 spots (96 DBS cards) without manual punching and with sample throughput of approximately 30 samples per hour. The degree of flexibility around the possibility to independently use different solvent compositions for the DBS extraction, SPE clean-up and analysis is particularly advantageous. Concerns of carryover during sample punching are eliminated and carryover on extraction clamps is reduced to acceptable levels or eliminated by flushing of the clamps with up two cleaning solvents followed by air drying. The SPE cartridges were robust and demonstrated chromatographic efficiency for sample clean-up and analysis, with no need for the use of LC columns.
The use of high-resolution accurate QTOF mass spectrometry showed excellent selectivity, linearity, precision and accuracy for quantitative analysis of chloroquine, desipramine and midazolam in DBS samples without the need to optimize MS/MS collision energies for each compound. Additionally, the use of HRAMS and the acquisition of full MS mass range acquisition data make it possible to subsequently reanalyze the data for the presence of interfering components that could produce ion suppression or metabolites that might be of interest.
The Hct experiments show that the Hct can influence the accuracy of drugs quantified by DBS and needs to be carefully investigated prior to validating a DBS assay for clinical or preclinical applications. However, typically Hct is not an issue for preclinical studies since these animals are carefully selected and bred for laboratory use and inter-animal variability is expected to be minimal. On the other hand, Hct becomes particularly important for special populations such as neonate studies, oncology patients, and so forth, as the interpatient variability of Hcts levels could be high [35,51].