Microsampling is a technique for collecting samples in very small volumes and is most used
for the collection of biological material. As it represents a less invasive and more patient
friendly method of blood sampling, it is increasingly being implemented in various clinical
procedures. Malondialdehyde is an important biomarker of oxidative stress and an indicator
of lipid peroxidation and may serve as a useful marker of disease or the physiological state
of a patient. A developed method for malondialdehyde determination in blood microsamples
would enable less invasive and more efficient clinical monitoring of diseases. In this study,
we attempted to validate and optimize two methods for malondialdehyde determination: the
dried blood spot method and volumetric absorptive microsampling, and to compare them
with each other. Both approaches were based on an already established method for
determining malondialdehyde in plasma and dried blood samples, which we attempted to
adapt and validate for samples obtained by volumetric absorptive microsampling. In the final
stage, the method was further optimized using different antioxidants. Validation of both
methods was performed in the concentration range 10–90 μM. Validation of the dried blood
spot method was unsuccessful, as none of the validation parameters met the required criteria.
Validation of the volumetric absorptive microsampling method was partially successful, as
we confirmed satisfactory repeatability (coefficient of variation = 3,5–6,2 %), absence of
hematocrite effect and sample stability when using 5 % butylated hydroxytoluen at room
temperature and –20 ⁰C for up to 14 days and for up to 7 days at 40 ⁰C, with relative
concentrations ranging from –15,3 % to 14,9 %. The addition of butylated hydroxytoluen
reduced malondialdehyde formation, resulting in approximately 20 % lower concentrations,
and proved to be the most effective antioxidant. Linearity and accuracy of the method were
not satisfactory. Extraction recovery, process efficiency, and matrix effect were also
assessed; however, most of the calculated values were higher than 100%, which indicated
the inadequacy of the investigated method. For the volumetric absorptive microsampling
method, adjustment of the method's concentration range and the use of 5% butylated
hydroxytoluene as an antioxidant would be required, followed by revalidation of the method.
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