Article pubs.acs.org/ac
Volumetric Absorptive Microsampling: A Dried Sample Collection Technique for Quantitative Bioanalysis Philip Denniff and Neil Spooner* Bioanalytical Science and Toxicokinetics, Drug Metabolism and Pharmacokinetics, GlaxoSmithKline Research and Development, Ware, Hertfordshire SG12 0DP, United Kingdom S Supporting Information *
ABSTRACT: Volumetric absorptive microsampling (VAMS) is a novel approach to obtaining a dried blood sample for quantitative bioanalysis that overcomes the area bias and homogeneity issues associated with conventional dried blood spot (DBS) sample when a subpunch is taken. The VAMS sampler absorbs a fixed volume of blood (∼10 μL) in 2−4 s with less than 5% volume variation across the hematocrit range of 20−70% with low tip-to-tip variability. There is no evidence of selective absorption by the tip of the plasma component over whole blood. Recommendations for best practice when collecting samples were developed based upon the results of tests examining a number of potential abuse scenarios.
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have to be understood in order to derive high-quality quantitative data. These center around the volume of blood spotted onto the card, the hematocrit (HCT) of the blood, and the homogeneity of the spot. The volume of blood taken for analysis contained within a fixed diameter subpunch cut from the DBS sample needs to be constant and independent of the volume of blood that is applied to the card. Thus, there needs to be a linear relationship between blood volume applied to the card and the area of the spot it produces. Over a small volume range this is true,16 but as the volume increases the relationship starts to break down.17 Thus, there is a need to control the volume of blood applied to the DBS card to be within limits that are known to give acceptable quantitative results and to ensure that both the sample and the calibration standards spots are prepared from similar blood volumes. It has also been demonstrated that HCT has an effect on the size of the blood spots that are created for a given volume of blood.16−18 This process has been described graphically elsewhere,19 but in essence, since blood with a low HCT has a lower viscosity relative to higher HCT blood, it creates a larger spot for a given volume of blood. Thus, a fixed diameter subpunch taken from a low HCT spot will contain less blood, and hence less analyte, than the same sized punch taken from a spot produced from higher HCT blood. This disparity in the volume of blood being analyzed leads to an assay bias. The assay bias reduces the closer the HCT of the samples are to the calibration standards.16 Vu et al. have derived a formula20 that compensates for the difference in HCT between sample and
ver the past few years there has been an upsurge of the interest in the use of dried blood spot (DBS)1−3 sampling for quantitative analyses of blood samples. Applications have included toxicokinetic animal studies,4 therapeutic drug monitoring,5 clinical drug development,6 heavy metals in blood,7 and forensics.8 These applications have built upon the routine use of the technique for over 50 years for the screening of newborn infants for a range of metabolite disorders.9,10 The interest in the technique is derived from the easy sample collection procedure of applying a drop of blood onto a filter card. A further benefit is that the volume of blood spotted does not have to be measured accurately. Instead, an approximate volume of blood can be spotted in the clinic or animal room with the accurate volume of blood being obtained by taking a fixed diameter subpunch of the spot in the analytical laboratory. A further advantage of the DBS approach over conventional sampling is the potential to be able to ship and store samples at ambient room temperature and their classification as nonhazardous, that allows shipping through the post.11 These factors make DBS sampling an ideal sample collection procedure for use in the field, particularly in locations where access to centrifuges to derive plasma, freezers, and dry ice to store and ship plasma samples is difficult. The fact that the technique can be used with blood volumes which are much smaller (26%) with a further contribution from the blood dried onto the handle (total bias >35%). The abuse scenarios examined indicate the need to avoid handling the tip before use or touching the tip against any surface after sample collection and the need to take care not to dip the tip too deeply into the blood pool. As these different situations can adversely affect the quality of the sample collected and hence the data derived from the sample, it is important that users of the VAMS samplers are suitably trained prior to their use for collection of study samples and are aware of the consequences of mistreating the samplers. Preloading VAMS Tip with EDTA. Blood samples collected from in-life studies of humans (finger or heel prick), or animals, using the VAMS samplers in their current form will not have EDTA incorporated into the sample. However, calibration standards and quality control (QC) samples used for quantitative bioanalysis can only be prepared from control blood containing an anticoagulant. In order to overcome this mismatch in matrixes between samples and calibrants/QCs, we investigated the pretreatment of the tips with EDTA prior to sample collection. Sampler tips were loaded with EDTA (0.02 mg of EDTA per tip, equivalent to a blood concentration of 2 mg/mL, which is comparable to levels obtained when using standard blood collection tubes), by dipping them into an aqueous solution of K2EDTA and drying overnight. The pretreated VAMS tips were dipped into pools of EDTA rat blood spiked with paracetamol (4 and 240 μg/mL), dried, extracted, and analyzed by LC−MS/MS. Acceptable precision (CV) and accuracy (bias) values were obtained for these samples (Table 2), indicating that this may be a viable approach to matching the matrix between samples and calibrants/QCs. As an alternative, the anticoagulant could be introduced into
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CONCLUSIONS The VAMS technique has the potential to supplant DBS for quantitative bioanalysis, since it retains all the recognized advantages of that technique, while overcoming the issues associated with HCT and homogeneity. Further, the VAMS approach makes the sample collection process easier and simplifies the work flow within the bioanalytical laboratory. It has been demonstrated that the device is capable of collecting an accurate volume of blood for analysis independent of HCT and that this is representative of the blood being sampled. However, as the abuse scenarios examined show, care needs to be taken when using the device and this will require suitable staff training before routine deployment in a busy animal house or clinical setting. Before the technique will be accepted as a mainstream bioanalytical procedure, full bioanalytical validations will be required across a wide range of analytes and bioanalytical laboratories. It is possible that this approach could become adopted as the sampling method of choice in such diverse areas as therapeutic drug monitoring, home sampling, and toxicokinetic and clinical studies including sampling from juvenile animals and young children, as well as other fields such as forensics and sports sampling. 8494
dx.doi.org/10.1021/ac5022562 | Anal. Chem. 2014, 86, 8489−8495
Analytical Chemistry
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Article
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare the following competing financial interest(s): The authors are employed by and have stock interests in GlaxoSmithKline.
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ACKNOWLEDGMENTS The authors thank Anna Williams and Claire Teague for assistance with the radiolabeled work, Aubrey Swain for hemoglobin measurements, and Phenomenex (Torrance, U.S.A.) for the gift of the VAMS samplers.
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