Serum Folate Forms Are Stable during Repeated Analysis in the Presence of Ascorbic Acid and during Frozen Sample...

Serum folate forms, and particularly tetrahydrofolate, are sensitive to oxidation.

Using a repeated measures design, we investigated the stability of folate forms in convenience samples with added ascorbic acid (AA; 5 g/L) analyzed initially and after variable (approximately 1-33 weeks) storage time at -70 °C. We examined the recovery of tetrahydrofolate added at different spiking levels to serum with and without AA (5 g/L). We also assessed the long-term frozen storage stability of folate forms.

Repeat analysis produced consistent results with the initial analysis; the mean relative change (95% CI; Lin's concordance correlation between initial and repeat result; sample size) was 0.08% (-0.24% to 0.39%; r _c = 0.999; n = 301) for 5-methyltetrahydrofolate, 4.23% (2.44%-6.05%; r _c = 0.984; n = 211) for pyrazino-s-triazine derivative of 4α-hydroxy-5-methyltetrahydrofolate (MeFox), -0.22% (-1.90% to 1.49%; r _c = 0.986; n = 214) for folic acid, and 1.49% (-2.71% to 5.88%; r _c = 0.889; n = 81) for tetrahydrofolate. Linear regression testing for a time trend indicated an estimated average percent change of less than ±5% for samples retested after 4 months: 5-methyltetrahydrofolate P _trend = 0.0007, folic acid P _trend < 0.0001, MeFox P _trend = 0.38, and tetrahydrofolate P _trend = 0.0256. The mean ± SD tetrahydrofolate spiking recovery was 96.7% ± 9.4% for serum with added AA, but <50% for serum without added AA. We observed ≤10% loss for most serum folate forms during 4 years of storage at -70 °C.

Serum containing added AA showed acceptable stability of folate forms during repeat analysis from the same vial within 4 months, complete spiking recovery of tetrahydrofolate during sample processing, and long-term frozen storage stability of folate forms.