BIOTECHNOLOGY 41
Direct extraction/analysis of dried blood spots
A fully automated system has been developed for the extraction of DBS cards that can be coupled directly to the MS or LC-MS system.
B
ioanalytics of dried blood spot (DBS) cards by LC- MS/MS is a challenging
task and many aspects have to be considered in order to obtain reliable results.
Associated processes are, for example, standardised and reproducible blood spotting, compensation of different unwanted effects using an internal standard (IS) for normalisation, accurate spot localisation to precisely select the centre of the blood spots, reproducible and efficient extraction of analytes from DBS-cards, minimal carryover, and many issues more.
Fig. 1. The DBS-MS500: tests were carried out on hundreds of dried blood spot cards.
So far, those processes have neither been automated nor integrated into a fully automatic system. Today DBS-analysis still involves many cumbersome manual steps (spot centring, punching, etc) which
are neither well controlled nor automated and which generate a significant amount of manual work.
Methods and instruments Recently a semi-automatic system has been used by different partners from industry and academia, either for small studies or for general research on DBS direct extraction/ analysis. Automated DBS-card extraction and carryover has been studied and optimised intensively. Tis article is focussed on results obtained with a fully automatic system including automated spot localisation to precisely select the centre of the blood spots, subsequent application of the IS prior to the DBS extraction, a reproducible extraction of the analytes from the DBS-cards, and a wash station for minimal carryover.
Te following instrumentation has been used: CAMAG DBS-MS 500; CAMAG ATS 4 Automatic TLC Sampler 4; AB Sciex API 3000; Shimadzu LC 10AD pumps and different LC columns.
Results ■ Robotics for picking up and
replacing of DBS cards. For routine analysis DBS cards need to be handled very reliable during the entire process. For precise positioning a new gripping tool had to be developed which even keeps bent cardboard cards stable and accurate in position. Continuous handling tests with up to 1000 card transport cycles have been performed successfully.
■ Optical spot localisation. During tests with hundreds of DBS cards we observed that the manual process of spotting the blood onto the cards as well as the cards itself have large tolerances resulting in significant variations of the blood spot position. Uncertainties of the final blood spot position of up to 3mm have been observed, which need to be measured and corrected for by an optical spot analysis system. Te measured positioning error was below 0.1mm.
■ Internal standard application. Te addition of the internal standard is still a matter of debate. Applying the internal standard to the DBS card with the blood spots already applied seems to be favourable in terms of flexibility. Tests confirm the feasibility of this method, which is also capable to compensate for losses during the extraction process.
■ Direct extraction module. Direct extraction of samples from DBS cards has been studied intensively in the past. Sealing a defined area on the DBS card (without punching out this area) allows to directly extracting substances from sample spots, storing
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