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after Robert Guthrie [7]. He introduced the test in 1962, for the analysis of the analysis of phenylketonuria, a diseases which afflicted his 15 month old niece. This disorder occurs when there is an error in amino acid metabolism and can impair brain development initially. The test been expanded to allow for early detection of Cystic Fibrosis, Congenital hypothyroidism, Phenylketonuria, Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) and Sickle Cell Disease.
The application of this approach in a quantitative manner, within the pharmaceutical and clinical industries, has received much attention over the last decade [8-10]. This interest has been driven by the seminal paper by Spooner and Barfield from GSK [8] looking at using dried blood spots for the analysis of acetaminophen. The data from [8] demonstrated that quantitative results could be obtained using this approach with linearities covering a range of 0.1 to 50 µg/ mL from dog blood and recorded standard deviations on the QC’s and standards all being well below 15%. The sample preparation of the collected dried blood spot was simple (extraction with an organic solvent, described in detail below), aiding the benefits of this approach for routine sample analysis. Several reasons were given why this approach would have successful uptake within the pharmaceutical industry, the primary ones being;
• Alignment with the ethical doctrines of the 3R’s.
• Reduced financial costs associated with taking samples due to elimination of refrigerated storage and shipping conditions
• Improved sample stability
In this publication the approach to sample preparation taken was to punch a 3 mm diameter disk from the centre of the DBS into a clean tube. Methanol (100 µL) containing internal standard ([2H4]- acetaminophen) was added and the tube vortexed mixed for approximately 30 s. The tube was centrifuged for 5 min. at 3000 g and the supernatant transferred to a clean tube and a portion injected onto the HPLC- MS/MS system.
One of the greatest challenges to this approach is the Haematocrit (HCT) issue [12]. The viscosity of blood can differ from one sample to another, since the amount of red blood cells to plasma can vary, indeed one of the issues associated with serial
Figure 1 Diagram of the Mitra technology taking a microsample
sampling from rodents is that the blood thins throughout the sampling regime, since the animal is not able to produce red blood cells quickly enough to replenish the ones that have been taken during the sampling process. In a non-regulated or Discovery environment this is not an issue, since many of the animals will have undergone very similar environmental experiences resulting in a similar HCT, which in conjunction with the wider tolerances for assay validation mean that this approach is very favourable. Unfortunately, the approaches developed at this stage have to be transferrable to the clinical regime, where patient HCT can be very variable [13]. This is particularly the case for patients that are undergoing some form of therapy, where the drug will ultimately be targeted. The effect of the HCT on cellulose based substrates is three fold;
• Spot area bias which increase with increasing HCT
• Recovery bias which reduces with increasing HCT
• Suppression bias which is random
The nature of the relationship between the effect and the HCT results in scenarios where drug concentrations can be under or over predicted dependent on which of the three effects is dominant.
Some solutions have been suggested by researchers for reducing or eliminating the HCT effect including;
• Apply a constant volume of blood and extract the whole dried spot [13]
• Use of alternative substrates for the sorbent paper [14]
• Haemolyse the blood sample prior to spotting [15]
• Use a filter mechanism to separate the red blood cells and the plasma, and using the plasma as the primary sample [16]
The Mitra® technology, (Neoteryx LLC)
Figure 1, uses the constant volume approach, and uses a pipetting type device to accurately absorb a known amount of
fluid, in this case blood. The amount of blood that is taken up is consistent and does not vary for blood with differing HCT. A range of experiments have been performed using this technology to determine the robustness [13] investigating the drying time and also looked at the humidity effects which are known to be problematic for older style technologies. In all cases the recovery was found to be very good and the volume of blood that was absorbed was highly consistent.
Filtration Devices
Li [16] used technology that allows for the sample to undergo a pre-separation of the red blood cells and plasma, leaving the plasma in a single layer on an absorption pad, Figure 2. This approach is very effective at eliminating the HCT effect. The technology uses a two layer polymeric membrane for the formation of a dry plasma spot series on the bottom sheet, with the red blood cells left on the top sheet. The lower membrane surface is then physically separated from the upper membrane and dried. Li further processed the sample using online solid-phase extraction cartridge followed by liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS). The methodology was applied to the quantitative determination of guanfacine, although other compounds have been investigated by other authors [17]. This approach has the potential to eliminate the HCT as well capillary blood-to-plasma issues.
Stability of Blood at Room Temperature
One of the advantages that is often associated with the DBS approach is the stability of the samples. Several authors have investigated and substantiated this claim [18,19]. Ganz discovered that the analyte concentrations in human blood after storage for 2 days at 6 ± 4°C showed a decrease of 2.2% at the QClow level and a
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