Abstract：
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.
摘要：
干血微量采样由于其多重优点越来越多地用于家庭采样和流行病学研究，包括通常大大改善的分析物稳定性。然而,作为验证的一部分，应始终对现实条件下的稳定性进行严格评估。特别是不稳定的分子，如叶酸（维生素B9）。这里,目的是确定干血液微样品中的叶酸稳定性是否足以建立家庭采样研究，以监测例如，育龄妇女。进行了一系列广泛的稳定性实验，以评估主要叶酸维生素5-甲基四氢叶酸（5MTHF）的稳定性，其氧化产物MeFOX和次要的非甲基叶酸维生素10-甲酰叶酸（10FoFA），5,10-亚甲基四氢叶酸(5,10CH+THF)和四氢叶酸(THF)在干血微量样品中使用体积吸收微量取样(VAMS)或常规干血斑点(DBS)。评估包括（从单个供体收集EDTA抗凝血，每个条件和时间点重复四次）：（i）温度的影响（-20°C，4°C,环境温度和37°C)，(ii)光(干燥和储存期间)和湿度的影响，和(iii)真空储存和用稳定剂预处理微量样品对叶酸稳定性的影响。在-20°C和4°C时，在VAMS样品和DBS中,所有叶酸水平在基线值的85-115%范围内,直至储存2周.然而,在室温下，在VAMS样品中，分析的叶酸的稳定性仅持续观察到三天，并且在37°C下没有叶酸。湿度对5,10CH+THF的稳定性有重要影响，但这可以很容易地改善使用干燥剂。用0.1％DL-二硫苏糖醇和5％丁基化羟基甲苯对微量样品进行真空处理和预处理都提高了VAMS样品在室温下的稳定性，但这些影响在37°C和DBS中受到限制。总的来说,事实证明，个体叶酸维生素组的稳定性具有挑战性，并且强烈依赖于温度和时间。尽管如此，如果可以确保受控运输（温度和持续时间），使用干血液微样本评估叶酸状态的家庭采样研究仍然是有益的.
