Effects of long‐term storage and delayed hemolysis of whole blood on folate species by LC‐MS/MS

Whole blood (WB) folate stability during sample collection, hemolysis and analysis is critical. To facilitate logistics in the field, we investigated whether storage of intact WB at ‐70°C for up to 2 years is feasible. We used 13 WB samples each for subjects with C/C and T/T genotype of the MTHFR C677T polymorphism. Hemolysates were prepared in 1% ascorbic acid (1/11 dilution) at two pH values and incubated for 4‐h/37°C (pH 4.0) or 3‐h/37°C (pH 4.7), then extracted and analyzed for folate species by LC‐MS/MS at 0, 6, 20, and 24 months. Compared to baseline, total folate (TFOL) levels varied as follows: at pH 4.0, ‐4% to ‐13% for C/C samples and ‐7% to +10% for T/T samples; at pH 4.7, ‐11% to ‐12% for C/C samples and ‐6% to +1% for T/T samples. Interestingly, the variation from baseline appeared slightly larger for C/C compared to T/T samples and the drop in TFOL for C/C samples was mainly attributable to a decrease in 5‐methyltetrahydrofolate. At pH 4.7 we noted an interconversion of tetrahydrofolate to 5,10‐methenyltetrahydrofolate for T/T samples over the course of storage with no effect on TFOL levels. Maintaining a short thaw time (=1 h) to avoid folate degradation is very critical (1‐h thaw time: 1% loss of TFOL for C/C and 2% for T/T; 2‐h: 7% for C/C and 21% for T/T, respectively; 3‐h: 24% for C/C and 51% for T/T, respectively). These data show that intact WB can be stored frozen for up to 2 years with only small losses in TFOL.