Direct analysis of dried blood spot samples

The aim of the research reported herein was to identify and develop a dried blood spot (DBS) direct analysis technique that could support high sample throughput quantitative bioanalysis in a regulated drug development environment. An initial literature review, coupled with proof of concept testing of the most prominent direct analysis techniques coupled to mass spectrometers (MS), resulted in direct elution (direct extraction of DBS via a confined solvent, producing a liquid extract) being selected as the most suitable technique to develop for this application. Direct elution technology was then developed into fully automated techniques with sufficient functionality to enable compatibility with high sample throughput quantitative bioanalysis. Proof of concept robustness data demonstrated that direct elution, despite the lack of sample clean up, was a reliable technique which had no detrimental effects on detector or chromatographic performance compared to conventional wet plasma extraction and analysis. A proof of concept investigation also demonstrated that a method of improving internal standard (IS) performance by spraying IS solution onto DBS samples prior to extraction, allowed the analyte of interest and IS to be co-extracted, while retaining adequate analytical performance. The foregoing proof of concept data was then combined to produce a fully automated DBS direct elution instrument designed to introduce sample extracts into a LC-MS/MS system. This instrument incorporated a 500 DBS card capacity, an intelligent visual recognition system, a dynamic IS applicator module, and a highly effective wash system that virtually eliminates carryover. Ultimately, this work led to the production of a fully automated DBS direct elution system that is now commercially available. Subsequent research focused on optimising the system, and using this technology to address some of the issues that are currently inhibiting the development of DBS usage in drug development applications, namely haematocrit (HCT) based assay bias, and the decreased sensitivity on offer from DBS sampling. It was demonstrated that using the IS sprayer enabled the IS to integrate sufficiently with the DBS sample prior to extraction to nullify HCT based recovery bias. The direct elution mechanism was also optimised with a view to maximising assay sensitivity while retaining acceptable analytical and chromatographic (LC-MS/MS) performance. Generic optimised direct elution conditions were developed which demonstrated that increases in assay sensitivity of up to 30 fold (compared to conventional manual extraction methods) were possible using a set of representative small molecule compounds