Is the stability of folates in dried blood microsamples sufficient to perform home-sampling studies?

Dried blood microsampling is increasingly used for home-sampling and epidemiological studies because of its multiple advantages, including an often greatly improved analyte stability. However, a critical assessment of the stability under realistic conditions should always be performed as part of the validation, especially for unstable molecules like folates (vitamin B9). Here, the objective was to determine whether folate stability in dried blood microsamples is sufficient to allow the set-up of home-sampling studies for the monitoring of folate status in e.g., women of reproductive age. An extensive set of stability experiments was performed to evaluate the stability of the main folate vitamer 5-methyltetrahydrofolate (5MTHF), its oxidation product MeFOX and the minor non-methyl folate vitamers 10-formylfolic acid (10FoFA), 5,10-methenyltetrahydrofolate (5,10CH+THF) and tetrahydrofolate (THF) in dried blood microsamples using volumetric absorptive microsampling (VAMS) or regular dried blood spots (DBS). The evaluations included (EDTA-anticoagulated blood was collected from a single donor measured in four replicates per condition and time point): (i) the effect of temperature (-20 °C, 4 °C, ambient temperature and 37 °C), (ii) the effect of light (during drying and storage) and humidity, and (iii) the effect of storage under vacuum and pretreatment of the microsamples with stabilizing agents on folate stability. At -20 °C and 4 °C, all folate levels were within 85 to 115% of the baseline value up till two weeks of storage in both VAMS samples and DBS. However, at room temperature the stability of the analyzed folates was only consistently observed up till three days in VAMS samples, and for none of the folates at 37 °C. Humidity had a major impact on 5,10CH+THF stability, but this could be easily improved by using desiccant. Both vacuum treatment and pretreatment of microsamples with 0.1% DL-dithiothreitol and 5% butylated hydroxytoluene improved the stability at room temperature in VAMS samples, but these effects were limited at 37 °C and in DBS. Overall, the stability of the individual folate vitamers proved to be challenging and strongly temperature- and time-dependent. Nonetheless, if controlled transport (temperature and duration) can be assured, the set-up of home-sampling studies to evaluate the folate status using dried blood microsamples can still be beneficial.

Quantitation of phosphatidylethanol in dried blood after volumetric absorptive microsampling.

BACKGROUND: Stimulated by the increased recognition of phosphatidylethanol (PEth) as sensitive direct marker of alcohol intake, the Ghent University's Laboratory of Toxicology and the National Institute of Criminalistics and Criminology combined their efforts to develop a quantitative method. To facilitate implementation the focus was on the use of a sampling technique which allows quick and easy blood collection, without the need of dedicated personnel at any place/any time. In the meantime the cooperation of the two labs should also allow to initiate a Belgian network of laboratories capable of quantifying PEth. METHODS: Dried blood microsamples were collected via volumetric absorptive microsampling (VAMS). PEth 16:0/18:1 was quantified after liquid-liquid extraction using two independent isotope dilution - liquid chromatography - tandem mass spectrometry methods. A systematic review of the entire process at both sites was performed before the final method comparison using samples from 59 routine toxicology cases collected within a one-year time interval. RESULTS: Initial differences between both laboratories were solved by focusing on important methodological aspects: (i) trueness verification of the calibration protocol focusing on the primary material, preparation of the stock solutions and adequate equilibration of calibrators and QCs, and (ii) verification of comparability of results obtained with different m/z transitions. Several of these aspects could only be verified by critically assessing spiked and native samples. After a final validation good average comparability of the two methods was observed. The average bias was -0.4%, with 85% of the differences within 20%. Moreover, the methods proved to be reproducible and robust within a one-year time interval. CONCLUSION: This study is the first to develop a quantitative volumetric absorptive microsampling based method for PEth measurements, in addition it is the first to perform a systematic comparison of PEth measurements between two laboratories. From the discussion on the encountered pitfalls it is clear that also on a global scale, more efforts are needed to improve interlaboratory agreement.