Is it possible to detect PEth 16:0/18:1 and PEth 18:1/18:1 in red blood cells after 20 years of storage in liquid nitrogen?

Phosphatidylethanol (PEth), as measured in freshly drawn blood samples, could be a promising new biomarker for habitual alcohol consumption, but it is still unknown whether PEth can also be determined from blood samples having been stored frozen for a longer period. PEth 16:0/18:1 and PEth 18:1/18:1 were determined by LC-MS/MS from red blood cells (RBC) derived from blood samples of (I) 20 healthy volunteers (after 1 month of storage at -80 A degrees C) and (II) 232 participants of the population-based European Prospective Investigation into Cancer and Nutrition (EPIC)-Heidelberg study (after 20 years of storage at -196 A degrees C). Analyses involved liquid-liquid extraction from 100 mu l aliquots with phosphatidylpropanol (PProp 18:1/18:1) as the internal standard. Extracts were subjected to a 10-min LC gradient separation, using multiple reaction monitoring of deprotonated molecules for quantification. After 1 month of storage at -80 A degrees C, PEth was detectable in all samples at mean concentrations of 393.6 +/- 12.4 ng/ml (PEth 16:0/18:1) and 43.3 +/- 1.1 ng/ml (PEth 18:1/18:1). In samples stored for 20 years at -196 A degrees C, PEth was detectable in 23.7% of all samples at mean concentrations of 412.2 +/- 655.5 ng/ml (PEth 16:0/18:1) and 38.0 +/- 74.8 ng/ml (PEth 18:1/18:1). PEth can be determined reliably from samples of moderate habitual alcohol consumers after 1 month of storage at -80 A degrees C. Our data suggest that PEth is generally also detectable in samples after 20 years of storage at -196 A degrees C. Further studies are needed to assess the still unknown impact of storage duration and temperature on different PEth specimen concentrations.