36 February / March 2016
major metabolites; desethylchloroquine and monodesethylamodiaquine.
Figure 2 Diagram showing how the filtration device works used by [16]
decrease of 11.2% at the QChigh level for the two compounds under investigation [18]. Anderson [19] reported that a series of drugs of abuse, including flunitrazepam, temazepam, oxazepam, lorazepam, nitrazepam, diazepam, and cocaine, could be stored at 4°C for up to a month on a Guthrie card 903 with less than 20% loss in all cases, which is less than that observed with the corresponding liquid blood samples.
Automation
In the early work on DBS, there was very little automation, however the growing interest from the pharmaceutical industry on the application of DBS has seen a growth of automation techniques that can be applied to this technology.
One of the approaches developed by Prolab/Agilent uses a modified CTC autosampler (CTC Analytics AG, Zwingen Switzerland), and allows for the manipulation of the spotted cards into a flow through device which clamps the card and allows liquid to pass through part of the dried blood spot, Figure 3. The use of different sized clamps allows the users to alter the size of the blood spot that is being analysed. As with previous technology several authors have reported positive experiences with this approach [20].
Another approach is delivered by CAMAG who offer a “state of the art technology to analyse DBS”. The DBS are directly eluted in a designed extraction chamber and guided online to an analysis system, typically a LC-MS/MS. The sample processing is fully automated, including the application of an internal standard through a high precision sprayer. An in-built camera takes a picture of every sample before and after extraction. The instrument allows for high throughput analysis of up to 500 DBS cards per run, with typical run times being a few minutes per sample. It utilises a robotic system to handle the DBS cards from 5 separate racks, each with a capacity of 100 cards. The gripping tool picks up cards and moves them to all of the subsequent processes in
the corresponding modules and back into their original position after the extraction. The extraction unit seals the DBS card and solvent is guided horizontally through the DBS into a sample loop. The flexibility in the solvent use also ensures that carryover is minimised.
Spark Holland, renowned for their automated on-line SPE system, have used a similar approach to that employed by ProLab in developing the DBS Autosampler. The technology uses a modified CTC autosampler to manipulate the DBS cards, which are then placed in between two clamps where the patented flow through desorption technology elutes the analytes either directly to a HPLC column or to an automated in-line SPE instrument. One of the big advantages of this approach is that the whole sample is analysed, meaning that the HCT issue is no longer relevant. The device is very flexible allowing for full control of solvent compositions and also flow rates through each of the individual components, autosampler and also the on-line SPE. As with other devices it comes supplied with a camera that allows for the blood spots to be pictured before and after the extraction process.
The SPE is taken from their Symbiosis model and is a unique cartridge design where SPE cartridges are placed between clamps allowing for up to 400 bar pressure to be applied to the cartridge. With just the SPE cartridge in this is an unrealistic pressure, however with a suitable analytical HPLC column it becomes necessary for the clamp to be able to maintain the fluid flow at elevated pressures.
A couple of applications have benefitted from this approach including [21] and also [22]. In [21] the HCT was compared using a traditional approach and also using the DBS autosampler. The data was very encouraging with the average recoveries at a high HCT of 0.7 increasing from about 70% for haloperidol, amitriptyline and verapamil to very close to 100%. With the second application of the technique two anti-malarial drugs; chloroquine and amodiaquine, were tested along with their
The very thorough investigation looked at several of the experimental parameters including the optimum card gradient elution solvent to achieve separation of the analytes, and also the optimal wash conditions for the SPE to effectively remove the phospholipids which are known to cause ion suppression.
Solutions have also been provided by a variety of other manufacturers including Hudson Robotics, Advion and Perkin Elmer.
Paper Spray
A similar approach to the use of the dry blood spotting has been employed by Cooks [23]. However, this approach uses the sampling device as part of the analytical process. The sample is placed onto a paper triangle and then this triangle is placed in front of a mass spectrometer. A solution is applied to the sample followed by the application of an electric voltage applied to the paper resulting in a soft form of ionisation of the analyte into the mass spectrometer. It has been proposed that the sample solution escapes from the surface or slowly migrates randomly through the fibres of the paper, with the sharpness of the tip of the triangular paper having a substantial effect on ionisation efficiency. Cooks et al. [23-25] suggested that two modes of spray operate during the paper spray process. Mode one occurs in solvent- rich systems, in which multiple Taylor cone jets are created, resulting in the production of a range of droplet sizes, whereas mode two occurs at low solvent flow rates and the higher currents that may be produced as the result of a corona discharge. Although this technology is clearly some way from being a commercialised product it has profound implications for the field of bioanalysis and potentially chromatography.
Application to Plasma Samples
The use of microsampling is not, however, restricted to the manipulation of blood samples. Many researchers are actively striving to utilise the microsampling techniques in the manipulation of plasma samples which do not suffer from the challenges associated with the HCT. Indeed most of the techniques previously discussed can be readily applied to the sampling of plasma samples.
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