The loss of 5-methyltetrahydrofolate in human serum under suboptimal preanalytical conditions can only partially be recovered by an oxidation product.

BACKGROUND Maintaining folate stability during sample handling is important, yet challenging. OBJECTIVE We investigated the effects of suboptimal preanalytical conditions on serum folate stability. METHODS By using an HPLC-tandem MS method we measured folates [5-methyltetrahydrofolate (5-methylTHF), folic acid, MeFox (5-methylTHF oxidation product, pyrazino-s-triazine derivative of 4α-hydroxy-5-methylTHF), and other minor folate forms at or below the limit of detection] in human serum exposed to suboptimal conditions. RESULTS Whole blood samples (n = 21) stored at 32°C for ≤ 3 d (Expt. 1: delayed processing) showed significant decreases in serum total folate (tFOL; sum of folate forms: 11-32%, 5.5-15.9 nmol/L) and 5-methylTHF (36-62%, 14.5-25.1 nmol/L) and a significant increase in MeFox (346-415%, 7.17-8.63 nmol/L). Serum samples (n = 21) stored at 11°C for 7-14 d (Expt. 2: delayed freezing) also showed significant decreases in tFOL (4.6-10.4%, 2.3-5.1 nmol/L) and 5-methylTHF (8.4-29%, 3.4-11.6 nmol/L) and significant increases in MeFox (88-320%, 1.82-6.62 nmol/L). The molar loss in 5-methylTHF exceeded the gain in MeFox in these 2 experiments. When we exposed 3 serum pools (tFOL: 16.7-58.3 nmol/L) for 24 h to an elevated temperature of 37°C (Expt. 3), the significant decrease in 5-methylTHF (33% on average) was compensated for by an equimolar gain in MeFox. Repeated freeze/thaw cycles (≤ 3 cycles) of serum [closed (Expt. 4) and open (Expt. 5) vials] showed generally stable folates with small (<1 nmol/L) changes. Long-term (≤ 12 mo) exposure of 3 serum pools (tFOL: 17.5-63.7 nmol/L) to a suboptimal (-20°C) freezing temperature (Expt. 6) showed significant decreases in tFOL (5% on average) already after 3 mo. The molar loss in 5-methylTHF exceeded the gain in MeFox. Folic acid generally showed good stability. CONCLUSIONS To avoid folate losses, unprocessed whole blood should be protected from elevated temperatures and serum should not be refrigerated for >2 d or for a long term stored at -20°C.