Preanalytical Investigation of Polyunsaturated Fatty Acids and Eicosanoids in Human Plasma by Liquid Chromatography–Tandem Mass Spectrometry

Abstract

Background:

Preanalytical variables have a great impact on sample matrices and are a source of laboratory errors. The effect of cryobanking, which is gaining great importance recently, requires systematic investigation. The arachidonic acid metabolism is useful as a quality marker since eicosanoids are easily subjected to in vitro oxidation processes.

Materials and Methods:

Polyunsaturated fatty acids (PUFAs) and related metabolites were analyzed by online solid-phase extraction coupled to liquid chromatography–tandem mass spectrometry. The influence of different plasma anticoagulants, as well as serum, freeze–thaw cycles (n = 5), short-term storage at 4°C, room temperature up to 120 minutes, and long-term storage at −20°C, −80°C, and −150°C up to 180 days, were investigated. We further investigated the influence of protein depletion, antioxidants, and shock-freezing on plasma.

Results:

PUFA metabolites were stable at 4°C in ethylenediaminetetraacetic acid (EDTA)-stabilized whole blood for 120 minutes and in EDTA-plasma for 30 minutes. Plasma stability at 4°C could be further increased up to 7 days after protein depletion, while addition of antioxidants such as butylated hydroxytoluene or coverage with nitrogen had no protective effects. Repeated freeze–thaw cycles (n > 1) resulted in eicosanoid formation up to 63%. Long-term storage at −20°C led to substantial eicosanoid increases after 30 days, which could be prevented by depleting proteins before storage. Cryobanking at −80°C and −150°C revealed decreased concentrations of eight eicosanoids after 180 days. An advantage of shock-freezing with liquid nitrogen could not be confirmed compared to conventional freezing.

Conclusion:

Defined preanalytical conditions for eicosanoid analysis in human matrices are required to minimize in vitro data variability.