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.
Bioanalytics 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.
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.[Page Break]
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. This 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.
The 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. The measured positioning error was below 0.1mm.
- Internal standard application.The 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 them in a sample loop/trapping column and further evaluating them in a LC-MS system. In collaboration with partners we have successfully performed direct extraction/analysis of several thousand samples.
- Wash station. Direct extraction parts are in contact with the actual blood spots and therefore possible carryover has to be observed carefully. An automatic wash station was constructed to control carryover. In all cases the desired carryover level of 10E-4 or around LOD has been reached. The automation of all above-mentioned steps will significantly reduce uncontrolled effects originating from the extraction process of DBS-cards. First tests confirmed that excellent reproducibility as well as minimal carryover can be achieved.
The automation of the entire extraction process for up to 500 DBS-cards is a very welcome simplification for performing DBS analysis and contributes to the improvements in reproducibility and reliability.[Page Break]
Conclusions
Our tests with semi-automated and automated DBS systems confirmed that with increasing automation of the DBS analysis the three factors addressed in this project gain in importance and should be considered as key issues:
- In practice we observed positioning variations of the blood spots relative to the DBS card boundaries of up to 3mm due to the blood spotting procedure and card tolerances. An automated system which does not detect those variations will miss part of the spot in such a situation. This would be unacceptable in routine analysis and accurate spot localization seems to be a must.
- All parts in contact with the DBS sample are potentially contributing to carryover. We experienced that for critical applications an automated wash system can be used to reduce or eliminate the remaining carryover after every sample.
- Normalisation of data by an internal standard has been found to be one of the key issues. Furthermore the application of the internal standard to the DBS card after blood spotting but prior to extraction seems to be favoured logistically and will have the potential to correct for extraction efficiencies.
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Matthias Loppacher, Christoph Fankhauser, Klaus Schetter and Urs Schranz are with CAMAG, Muttenz, Switzerland. www.camag.com. Manuel Altmeyer, Agathe Koller, Bettina Mueller and Silvio Walpen are with the Institute for Laboratory Technology, Rapperswil, Switzerland
